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Metabolic responses to recombinant bioprocesses in Escherichia coli

Metabolic responses to recombinant bioprocesses in Escherichia coli
Metabolic responses to recombinant bioprocesses in Escherichia coli

Journal of Biotechnology 164 (2013) 396–408

Contents lists available at SciVerse ScienceDirect

Journal of

Biotechnology

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /j b i o t e

c

Metabolic responses to recombinant bioprocesses in Escherichia coli

Sónia Carneiro ?,Eugénio C.Ferreira,Isabel Rocha

IBB –Institute for Biotechnology and Bioengineering,Centre of Biological,Engineering,Universidade do Minho,Campus de Gualtar,4710-057Braga,Portugal

a r t i c l e

i n f o

Article history:

Received 16January 2012

Received in revised form 9July 2012Accepted 8August 2012

Available online 26 September 2012

Keywords:

Recombinant proteins Metabolism

High-throughput methods Modelling

Systems biology

a b s t r a c t

Escherichia coli has been widely used for the production of recombinant proteins.However,the unbal-ances between host metabolism and recombinant biosynthesis continue to hamper the ef?ciency of these recombinant bioprocesses.The additional drainage of biosynthetic precursors toward recombinant processes burdens severely the metabolism of cells that,ultimately,elicits a series of stress responses,reducing biomass growth and recombinant protein production.

Several strategies to overcome these metabolic limitations have been implemented;however,in most cases,improvements in recombinant protein expression were achieved at the expense of biomass growth arrest,which signi?cantly hampers the ef?ciency of recombinant bioprocesses.With the advent of high throughput techniques and modelling approaches that provide a system-level understanding of the cellu-lar systems,it is now expected that new advances in recombinant bioprocesses are achieved.By providing means to deal with these systems,our understanding on the metabolic behaviour of recombinant cells will advance and can be further explored to the design of suitable hosts and more ef?cient and cost-effective bioprocesses.

Here,we review the major metabolic responses associated with recombinant processes and the engi-neering strategies relevant to overcome these stresses.Moreover,the advantages of applying systems levels engineering strategies to enhance recombinant protein production in E.coli cells are discussed and future perspectives on the advances of mathematical modelling approaches to study these systems are exposed.

? 2012 Elsevier B.V. All rights reserved.

1.Introduction

Escherichia coli is one of the most used microbial systems for the production of recombinant gene products.The relatively inexpen-sive scaling-up and the versatility as a host have established this cellular system an attractive alternative to the production of pro-tein drugs via non-microbial systems.Indeed,many products with pharmaceutical interest have been produced (under FDA approval)using E.coli ,such as:human insulin,somatotropin,human parathy-roid hormone or interferon alfa-2b (Ferrer-Miralles et al.,2009).The success of these products was essentially due to the ability to access a microbial platform that rapidly produces high-quality pro-teins in an ef?cient manner,maintaining the economic viability of these industrial processes.

However,some limitations still exist in these bioprocesses,par-ticularly concerning the metabolic performance of host cells (Enfors et al.,2001;Konstantinov et al.,1991;Chou,2007).The expression of recombinant products can challenge cells with different levels of toxicity and metabolic burden that,ultimately,can lead to the

?Corresponding author.

E-mail address:soniacarneiro@deb.uminho.pt (S.Carneiro).

decline of the biomass yield,productivity and cellular viability.Typ-ically,cells counteract these effects by triggering stress-response mechanisms that adapt and readjust the metabolism so that cells can restore functionality and viability.However,these cellular responses represent themselves a signi?cant deviation of cellu-lar resources from growth and recombinant protein production.Therefore,the metabolic load imposed by the recombinant protein production and its main consequences in the whole metabolism of E.coli are quite relevant to consider when optimizing recombinant bioprocesses.

Currently,the ability to engineer microbial cells using recom-binant DNA technology has provided us means to control the metabolism either by manipulating or introducing new enzymatic activities (Jeon et al.,2011;Zhu et al.,2011)or by interfering with regulatory interactions that ultimately in?uence metabolic activ-ities (Demain and Adrio,2008;Lee et al.,2005).Several reviews have been published summarizing the latest advances in genetic tools to enhance expression systems (Andersen and Krummen,2002;Sorensen and Mortensen,2005;Jana and Deb,2005)and also,the developments in culturing processes using different micro-bial systems,including E.coli (Shimizu et al.,1988;Konstantinov et al.,1990;Hsiao et al.,1990;Seo et al.,1992).However,with the advent of systems-level engineering approaches and the

0168-1656/$–see front matter ? 2012 Elsevier B.V. All rights reserved.https://www.wendangku.net/doc/1b11065581.html,/10.1016/j.jbiotec.2012.08.026

S.Carneiro et al./Journal of Biotechnology164 (2013) 396–408397

application of multiple omics technologies,a new perspective over the improvement of microbial strains has arisen.Now,it is possi-ble to characterize the recombinant systems at the system-level, which provide us with knowledge to engineer cells in a systematic manner,making these microbial strains more ef?cient and produc-tive.The targeted manipulation of strains at the molecular level, together with the application of enhanced expression systems and suitable culture processes,can improve considerably the productiv-ity of these microbial factories.In particular,by addressing the main metabolic limitations elicited during recombinant bioprocesses it would be possible to manipulate both the genetic background of the host cell and the culturing conditions in a controlled way to avoid any adverse activities.

This review will discuss the major metabolic responses associ-ated with recombinant processes and the engineering strategies relevant to enhance the metabolic performance of recombinant E.coli host cells.We?rst review all the potential factors that induce metabolic perturbations during recombinant protein pro-duction in E.coli cultures and the main consequences on the metabolism of host cells.Then,we will review the main engineer-ing strategies that have been successfully applied to manipulate the cellular metabolism to improve cells’physiology and the produc-tivity of recombinant proteins.The potential of omics technologies in uncovering the main metabolic responses to recombinant pro-cesses will be presented,as well as the application of novel strategies based on systems-level analyses to manipulate the metabolic behavior of cells in a holistic manner.

2.The metabolic burden in recombinant bioprocesses

During recombinant protein production,cells are subjected to several physiological stresses,including the metabolic burden caused by drainage of metabolic precursors to the biosynthesis of heterologous proteins that is very aggressive to the metabolism of the host.The drainage of energy and biomass precursors,including amino acids and nucleotides toward the synthesis of recombinant material imposes severe alterations in the metabolism of host cells. Since the main biomass precursors are generally associated to gly-colytic activities and the tricarboxylic acids(TCA)pathway,the disproportionate consumption of these metabolites leads to an imbalance in the central carbon metabolism.

According to several studies(Bentley et al.,1990;Corchero and Villaverde,1998;Rozkov et al.,2004),the over-expression of heterologous proteins provokes a reduction in the synthesis of biomass-related proteins,due to the unequal competition for the translation apparatus by the mRNA species synthesised from the high-level expression of recombinant material.Moreover,as in most cases the amino acids composition of the recombinant protein clearly differs from the average composition of biomass-related proteins,this will augment the metabolic imbalance in the host cells.Indeed,the effect of the amino acid content of recombinant proteins in E.coli growth rates has been evaluated by comparing the impact of the overexpression of peptides containing amino acids that are more and less abundant in the host(Bonomo and Gill,2005).It was shown that cells expressing the peptide with less abundant amino acids became limited for those amino acids,which reduced protein synthesis that ultimately resulted in a lower growth rate.Similar studies have demonstrated the impact of codon usage bias in the expression of proteins(Zhou et al.,2004;Angov et al.,2008;Gustafsson et al.,2004;Kane,1995). Heterologous genes containing codons that are rarely used in the host can cause ribosome stalling during translation at these spe-ci?c codons,as the concentration of tRNAs for less-used codons is remarkably lower.Alternatively,amino acid substitution and possibly frameshifting can occur,which may introduce signi?cant alterations in the structure of the recombinant protein.Either way, the effects on protein productivity and quality are critical.Further details are discussed in a recent review on the causes and conse-quences of codon bias(Plotkin and Kudla,2011).

Also,it has been suggested that the recombinant plasmid alone burdens the metabolic capacity of cells(Bentley et al.,1990; Birnbaum and Bailey,1991).Some of the building blocks and energy required to replicate plasmid DNA are intermediaries of the pentose phosphate(PP)pathway,e.g.ribose-5-phosphate and erythrose-4-phosphate,which would impair the generation of NADPH and many other metabolic precursors like amino acids.NADPH participates mostly in biosynthetic reactions and,under normal growth con-ditions,over60%is exclusively required for protein biosynthesis (Neidhardt et al.,1990).Therefore,the imbalance of this metabolic pathway would limit the availability of this reducing agent,and consequently reduce recombinant biosynthesis,as well as biomass formation.

Growth arrest and reduced protein synthesis are the most important effects of this metabolic stress(Bentley and Kompala, 1990;Glick,1995),but many other effects have been described, such as the accumulation of undesired by-products like acetate (Eiteman and Altman,2006).In general,the limitation or over-accumulation of some metabolic intermediaries changes the metabolic?uxes associated with these central pathways,which may induce cells to respond,either by restraining the activity of enzymes(either by enzymatic inhibition or transcriptional regula-tion)or by redirecting the metabolic?uxes through other pathways that lead to the accumulation of by-products.It has been reported that under recombinant protein producing conditions,cells lack immediate regulatory mechanisms for adjusting to a new bal-anced growth and can respond by destroying their ribosomes(Dong et al.,1995)or inducing the proteolysis of proteins(Rozkov et al., 2000;Rozkov and Enfors,1999).More recently,a study on the 13C-labeling patterns of amino acids from a heterologous protein expressed in E.coli,revealed that cells react by reducing biomass formation and increasing energy generation through glycolytic activities(Pinske et al.,2011).In addition,the up-regulation of genes involving the transport of amino acids and energy storing compounds has been described,as well as the induction of enzymes associated with the biosynthesis of amino acids(Bonomo and Gill, 2005).

The ppGpp-mediated stringent response has also been associ-ated with the metabolic burden caused by the over-expression of recombinant proteins(Bentley et al.,1990).This stress response is triggered by the accumulation of the alarmone ppGpp that controls gene expression by direct interaction with the RNA poly-merase(RNAP)upon the intracellular depletion of amino acids (Artsimovitch et al.,2004;Chatterji et al.,1998).The down-regulation of genes involved in the translational machinery and the up-regulation of stress-related proteins can explain the reduction of growth and protein synthesis frequently observed in recom-binant cultures.The discovery that this guanosine nucleotide is accumulated during recombinant bioprocesses put forward the hypothesis of using ppGpp-de?cient strains as hosts to improve the productivity of recombinant proteins(Dedhia et al.,1997; Carneiro et al.,2011b).Although some disadvantages in terms of the metabolic behaviour(e.g.failure to manage metabolic imbal-ances)exist,the enhanced protein production rate represents a major bene?t in recombinant bioprocesses.

3.Engineering strategies to overcome the metabolic

burden in recombinant processes

The latest advances in genetic engineering and fermentation methodologies allowed the optimisation of bioprocesses for the

398S.Carneiro et al./Journal of Biotechnology164 (2013) 396–408

production of recombinant proteins using different cellular sys-tems,especially bacteria like E.coli.This bacterium remains the primary choice for recombinant production,due to its simplicity, ease of manipulation,and low cost(Baneyx,1999),but the limited metabolic ef?ciency of this cellular system places the industrial bioprocesses far from optimal.Hence the importance of under-standing the impact of expressing heterologous proteins in the host cell metabolism.

Several groups have worked on developing strategies to over-come the metabolic burden in these biotechnological processes (Table1).So far,optimisation strategies have been based on genetic modi?cations,both in the host genetic background and in the recombinant material or,in some cases,changes in the fermen-tation conditions,like the culture medium composition.However, most of these strategies have been based on empirical approaches that are quite time consuming and do not always result in the expected outcomes.Therefore,new strategies based on the rational design of microbial cells need to be explored.Up to now,only few attempts were made to understand and manipulate recombinant cells at the systems-level in order to obtain even more ef?cient systems,but with the development of mathematical modelling approaches and high throughput technologies,further advances are expected.

In the next subsections,reported strategies to overcome the metabolic burden in recombinant host cells will be discussed, namely:the genetic manipulation of recombinant material and the host genome and the control of culture conditions.Several excel-lent reviews describing general strategies for improving protein productivity have already been published(Demain and Vaishnav, 2009;Gnoth et al.,2008;Chou,2007;Peti and Page,2007;Jana and Deb,2005;Sorensen and Mortensen,2005;Andersen and Krummen,2002).Therefore,we will focus primarily on the engi-neering of E.coli strains or the recombinant material that allowed to avoid(or overcome)the metabolic burden imposed by the pro-tein synthesis.Some cultivation strategies used to improve the metabolic performance of host cells will be also exposed.

3.1.Genetic engineering approaches

3.1.1.Engineering the recombinant material

In general,expression vectors are the primary target to con-trol recombinant production.Expression vectors are often based on naturally occurring plasmids that can be easily reconstructed to assemble parts from different genetic sources,such as control ele-ments(e.g.promoters,terminators and transcription/translation initiation sequences)and propagation elements(e.g.selection markers,replication origins).The recent developments in the opti-mization of these elements have been widely covered in previous reviews(Sorensen and Mortensen,2005;Chou,2007;Jana and Deb, 2005;Baneyx,1999),but their effects in the metabolism of host cells has been fairly discussed.

Many optimization strategies disregard the in?uence of these vector elements on the metabolic state of host strains and,con-sequently in the global performance of the recombinant process. Properties of a typical expression vector,like the plasmid copy number,the size of the plasmid,the gene marker and promoter strength,can contribute to the metabolic load imposed by the main-tenance of recombinant DNA in host cells.Early studies on the effects of the plasmid content on the cellular growth(Jinho and Bailey,1985;Birnbaum and Bailey,1991;Bailey,1993;Peretti and Bailey,1987)have shown that the decrease in the biomass yield is one of the major responses to the large-scale production of plas-mid DNA in E.coli.Bailey and co-workers(1990)have reported some studies on the effects of the plasmid copy number in the growth of host strains,showing that the plasmid DNA content per cell increases with the decrease of the cellular growth rate,(Seo and Bailey,1985;Seo and Bailey,1986;Mason and Bailey, 1989).Although plasmid segregational instability could be impli-cated(Summers et al.,1993),in these studies no evidences were found,suggesting that these effects might be directly related with plasmid burden in host cells.

Another study with E.coli cells containing plasmids with the same size and expressing the same gene coding protein,but with increasing copy number,showed that the plasmid con-tent is inversely correlated with the speci?c growth rate(Jinho and Bailey,1985).Bentley and co-workers have also demon-strated that the plasmid copy number has a profound effect on the speci?c growth rates of the plasmid-bearing cultures,as well as the expression of plasmid-encoded proteins(i.e.,growth rates changed from0.45to0.23h?1when the expression of an plasmid-encoded protein,chloramphenicol acetyltransferases (CAT),increased approximately from500to1800U/mg)(Bentley et al.,1990).It was assumed that the additional drainage of biosyn-thetic precursors to replicate and express the recombinant material burdens the host metabolism,such that the metabolic changes decreased the growth rate of plasmid-bearing cells.Many other studies have also suggested that the plasmid copy number is the principal factor for the metabolic burden associated with plasmid maintenance,resulting in signi?cantly reduced growth rates(Jones et al.,2000;Corchero and Villaverde,1998;Flores et al.,2004).

The promoter strength is also an important factor affecting the amount of recombinant transcripts produced in cells and thereby the host metabolism.From different sources and with different speci?cities(https://www.wendangku.net/doc/1b11065581.html,c promoter from E.coli or T7promoter from a bacteriophage that is speci?c to only T7RNA polymerase),pro-moters have been chosen essentially by their transcription rates (Deuschle et al.,1986).Recently,engineering strategies that use promoter clusters consisting on multiple core-tac-promoters in the plasmid were tested to achieve high and stable gene overexpress-ion(Li et al.,2012).However,the most ef?cient promoters might not be the best choice to improve recombinant bioprocesses,as the disproportionate increase of recombinant transcripts can overload the translational machinery and/or burden the metabolism of host cells.In,general,multicopy plasmids with foreign genes under the control of strong promoters are used to obtain high levels of expres-sion of recombinant genes,but these two properties can induce detrimental effects on the host strain(Silva et al.,2012;Wang et al., 2006;Andersson et al.,1996;Rozkov et al.,2004;Neubauer and Winter,2001).

Other factors,like the selection of antibiotic resistance markers may also contribute to the metabolic burden.For instance,genes for the resistance to kanamycin,chloramphenicol and ampicillin are normally expressed at high levels,while tetracycline resis-tance genes are expressed at lower levels(Glick,1995).Antibiotic resistance markers can be also replaced by amino acid-auxotrophy complementation,i.e.by using auxotrophic strains transformed with a plasmid containing the gene that complements that auxotro-phy,transformants are able to grow on minimal media without that amino acid supplementation(Vidal et al.,2008).These selection markers based on the amino acids auxotrophy are actually bene?-cial for the host cell,since the addition of antibiotics is abolished. Either selecting low level expressing genes for antibiotic resis-tance or auxotrophy complementation,the impact of recombinant biosynthetic activities over the metabolism might decrease.

Another strategy that has been proposed is the integration of the target gene sequence alone into the chromosomal DNA of the host organism(Chen et al.,2008).This would prevent cells to waste energy and metabolic resources to synthesize unneeded products, once antibiotic resistance markers are essentially used to main-tain the stability of plasmid-bearing cultures.Though this seems promising,plasmid-based systems are still the most widely used, because they allow to obtain higher gene dosages and the genetic

S.Carneiro et al./Journal of Biotechnology164 (2013) 396–408399 Table1

Some examples of reported strategies to overcome the metabolic burden imposed by the expression of heterologous proteins in E.coli.

Strategies Descriptions Reference

Genetic modi?cations Low-copy number plasmids The use of plasmids with low copy number resulted in no signi?cant

changes in cell growth.

(Jones et al.,2000) Chromosomal insertion The chromosomal insertion of1-3copies of the lac Z gene revealed to

enhance the genetic stability compared to the plasmid expression

system under non-selective conditions(e.g.,medium without

antibiotics).

(Chen et al.,2008)

Alternative selection markers The replacement of antibiotic gene markers by amino acid-auxotrophy

complementation in an E.coli M15-derivated glycine-auxotrophic

strain was tested,allowing to obtain high cell density cultures and

high productivity levels comparable to those obtained with the

conventional systems.

(Vidal et al.,2008)

Alteration of transport systems(I)A mutation in the pts G gene encoding an enzyme of the glucose

phosphotransferase system(PTS)resulted in the reduction of acetate

excretion and,consequently biomass and recombinant protein

productivity were increased by more than50%compared to the

wild-type.

(Chou et al.,1994)

Alteration of transport systems(II)The replacement of the glucose phosphotransferase transport system

by galactose permease resulted in a reduced accumulation of acetate

and a concentration of recombinant protein almost four-fold higher.

(De Anda et al.,2006)

Engineering the PP pathway An E.coli strain carrying a high-copy number plasmid with the zwf

gene coding for the glucose-6-phosphate dehydrogenase enzyme that

participates in the pentose-phosphate(PP)pathway was used to

supply the extra demand of building blocks and energy required for

recombinant biosynthesis.The growth rate increased from0.46h?1

(uninduced)to0.64h?1(induced),which allowed to obtain a higher

recombinant productivity.

(Flores et al.,2004)

Introducing new enzymatic activities The als S gene from Bacillus subtilis encoding the enzyme acetolactate

synthase was introduced into E.coli cells using a multicopy plasmid.

By introducing this new metabolic activity the excess pyruvate was

redirected away from acetate to acetolactate and then acetoin,

reducing the acetate accumulation and improving the recombinant

protein production.

(Aristidou et al.,1995)

Knockout of host genes(I)The ack A-pta-nuo mutant strain,which is de?cient in acetate synthesis

(ack A-pta)and defective in the transmembrane NADH:ubiquinone

oxidoreductase(nuo)exhibited reduced acetate accumulation but also

signi?cantly lower ethanol and formate synthesis

(Yang et al.,1999b)

Knockout of host genes(II)The E.coli pta mutant defective in phosphotransacetylase excreted

unusual by-products such as pyruvate,D-lactate,and L-glutamate

instead of acetate.

(Chang et al.,1999)

Knockout of host genes(III)A recombinant E.coli strain with deleted pox B-ack A-pta genes and

overexpressing the pyruvate dehydrogenase produced less acetate and

more isoamyl acetate(a valuable by-product)than the wild-type

strain.

(Dittrich et al.,2005)

Overexpression of host enzymes An E.coli strain with a fad R::Tn10insertional mutation and

overexpressing the phosphoenolpyruvate carboxylase(PPC)enzyme

was used to test acetate accumulation.Results showed that acetate

yield is decreased more than fourfold compared to the control,while

the biomass yield is relatively unaffected

(Farmer and Liao,1997)

Culture conditions Amino acid supplementation The coordinated amino acid feeding was shown to increase the

heterologous protein yield.

(Harcum et al.,1992) Low glucose concentration Decreasing glucose concentration in the medium results in a reduced

accumulation of acetate and a higher recombinant productivity.

(Shiloach et al.,1996)

Control of the speci?c growth rate(I)Fed-batch and continuous cultures with E.coli JMI07carrying a

plasmid pQRI26with the?-amylase gene were evaluated at different

speci?c growth rates(dilution rates)Recombinant production was

shown to be maximum at an intermediate rate of0.2h?1.

(Turner et al.,1994b)

Control of the speci?c growth rate(II)Sophisticated feeding strategies in fed-batch cultures with

recombinant E.coli were developed to provide a?ne control of the

biomass formation at a constant speci?c growth rate and limiting

acetate formation.

(Gregory and Turner,1993;

Turner et al.,1994a)

manipulations are easier and less time consuming compared to methodologies where genes are integrated into the chromosome. This may explain why only few methodologies for the insertion of heterologous genes into the host chromosome have been devel-oped(Chiang et al.,2008;Martinez-Morales et al.,1999;Wei et al., 2010;Blaas et al.,2009;Chen et al.,2008).

Besides those strategies concerned with the engineering of expression vectors to reduce the metabolic burden in the host cells, together with the increase of protein expression levels,the redesign of coding-sequences of the target products has also been consid-ered.The sequence of the translation initiation region and biases in the codon usage can reduce dramatically the translation ef?ciency of recombinant transcripts.Therefore,foreign gene sequences can impair the translational machinery of the host cells and elicit sev-eral stress responses that will contribute to the metabolic burden (Jana and Deb,2005;Gustafsson et al.,2004;Kane,1995;Schweder et al.,2002).Different strategies have been applied either by per-forming codon-optimization of the heterologous genes,according to the frequency of codon usage in the host(Burgess-Brown et al., 2008;Han et al.,2010;Hale and Thompson,1998;Yang et al., 2004;Angov et al.,2008;Niemitalo et al.,2005),or through the co-expression of genes that encode the tRNAs that are scarce in

400S.Carneiro et al./Journal of Biotechnology164 (2013) 396–408

the host(Calderone et al.,1996;Spanjaard et al.,1990;Saxena and Walker,1992;Del et al.,1995).However,such approaches have shown some adverse effects,such as the lower speci?c activities of the target products due to unpredictable structural alterations (Gustafsson et al.,2004).

Other strategies targeting the gene sequence have also been exploited,but essentially related with protein stability,posttrans-lational modi?cations and the modulation of proteins activity. Although these are not directly related with the improvement of the metabolic performance of host cells,alterations can somehow in?uence the cell metabolism.For example,the design of a gene sequence without coding for any unnecessary amino acid residues reduces the drainage of biosynthetic resources.For protein stabil-ity,the replacement of cysteine by serine residues(O’Rourke et al., 1984;Doyle et al.,1985),as well as the deletion of the hydropho-bic regions like N-terminal amino acyl residues(Hsu et al.,2006) have been successfully tested.In all of these assays the modi?ed proteins showed increased stability and retention of activity.Other strategies have been applied to promote the secretion of proteins into the periplasmic space by fusion with coding sequences for a signal peptide(de Oliveira et al.,1999;Jonet et al.,2012).This signi?cantly improves the recovery yield and product quality of recombinant bioprocesses,but problems associated with proteases attack and incorrect protein folding,which typically elicit various stress responses affecting the metabolism,are also avoided(Choi and Lee,2004).Further approaches are discussed in(Kamionka, 2011).

The selection of the proper vector together with the use of codon-optimized genes in many instances may be adequate to allow the accumulation of the target protein at high levels,and, most importantly,reducing the impact of the metabolic burden in the host cell.However,the systematic application of differ-ent engineering approaches requires that the metabolic impact of heterologous gene expression can be predicted with a manage-able set of variables.The metabolic behavior of host cells during recombinant production remains unclear,but many tools are being developed.With the advent of synthetic biology and the ef?cient de novo DNA synthesis,it is now anticipated a great potential for the systematic engineering of recombinant systems(Gustafsson et al., 2012).

3.1.2.Engineering the host metabolism

Still more attractive is the possibility to modify the ef?ciency of the host cell’s metabolism by changing the expression of entire metabolic pathways or speci?c genes,either by increasing the gene dosage of rate-limiting enzymes or by deleting genes to convey the metabolic?ux toward the production of desired metabolic inter-mediaries to balance the extra drainage of metabolic precursors to recombinant biosynthesis.The modulation of the central carbon metabolism has been explored to overcome the effects of metabolic burden during recombinant production.Strategies are predomi-nantly related to carbon transport capacities(De Anda et al.,2006; Chou et al.,1994)and metabolic activities that supply for biosyn-thetic precursors,like amino acids,nucleotides and nucleosides (Flores et al.,2004).

Flores and co-workers(2004)showed that by increasing the expression of the zwf gene,which codes for the?rst enzyme in the oxidative branch of the PP pathway,i.e.glucose-6-phosphate dehydrogenase,it is possible to overcome the bottleneck for the supply of building blocks(e.g.nucleotides)and reducing power (NADPH)that are required for plasmid replication and plasmid-encoded protein expression.On the other hand,the replacement of the glucose phosphotransferase transport system(PTS)with an alternate glucose transport system,reduces the glucose uptake rate and,consequently,the levels of acetate excretion and improves the recombinant protein production over the wild-type strain(Lara et al.,2008;De Anda et al.,2006;Chou et al.,1994).Additionally, it was found that the bene?ts of replacing the phosphotrans-ferase system(PTS)by other glucose transporters(e.g.glk and gal P genes)can be augmented in arc A phenotypes,allowing to enhance the glycolytic and respiratory capacities of the engineered strain(Flores et al.,2007).The problem of acetate accumulation in the medium during recombinant bioprocesses,especially under high cell density cultivations,has long been identi?ed(Eiteman and Altman,2006;Suarez and Kilikian,2000).Acetate negative effects on growth of different recombinant E.coli strains have been tested(Koh et al.,1992)drawing attention to the importance of selecting suitable host strains and growth conditions that minimize acetate accumulation.In E.coli,acetate is synthesized mainly by the phosphotranscetylase acetate kinase pathway(Pta-AckA)using acetyl coenzyme A(AcCoA)as the substrate.Since AcCoA is pri-marily consumed via the tricarboxylic acid(TCA)cycle,only when an imbalance in this pathway occurs or the TCA capabilities are exceeded by the carbon?ux through the glycolysis pathway(so-called over?ow metabolism),the AcCoA is channelled to acetate biosynthesis(Xu et al.,1999;Castano-Cerezo et al.,2009;Chang et al.,1999).Although the phenomena that result in the accumu-lation of acetate during the production of recombinant proteins is not entirely clear,it is likely that the drainage of metabolic pre-cursors to recombinant biosynthesis imbalances the TCA activity, increasing the intracellular accumulation of AcCoA and resulting in the production of acetate.It should be also mentioned that the amino acid composition of recombinant proteins has a major effect on the metabolic imbalances that may arise.If the recombinant protein sequence is rich in less abundant amino acids in E.coli,the precursors used to produce those amino acids will be the?rst to be depleted and?uxes around those precursors will change sig-ni?cantly.Thus,depending on the precursor becoming depleted, the channelling of?uxes through the accumulation of acetate will change.To exemplify,it is likely that if?-ketoglutarate becomes deprived,TCA?uxes will decrease,accumulating AcCoA that will be channelled to the synthesis of acetate.The accumulation of acetate not only represents a deviation of carbon that might otherwise be used to generate energy and precursors for biosynthetic purposes, but it disrupts the proton motive force and impairs the cellular growth and the production of recombinant protein(Luli and Strohl, 1990;Eiteman and Altman,2006).Thus,various strategies have been developed to overcome this metabolic phenomenon(Table1) (De Anda et al.,2006;Eiteman and Altman,2006;Kim and Cha, 2003;Shiloach et al.,1996;Suarez and Kilikian,2000;Turner et al., 1994;Van de Walle and Shiloach,1998;Wong et al.,2008).Genetic engineering methods like the direct knockout of genes encoding for enzymes in the biosynthetic pathway for acetate(e.g.,ack A,pta and pox B)(Yang et al.,1999b;Chang et al.,1999;Dittrich et al.,2005;Tao et al.,2012)or the alteration of the level of gene expression in order to enforce the carbon?ux through alternative pathways reducing acetate formation(Farmer and Liao,1997)have been applied.For example,the over-expression of enzymes from anaplerotic path-ways,like the phosphoenolpyruvate carboxylase(PPC),coupled with the deregulation of the glyoxylate bypass by using a fad R strain was investigated(Farmer and Liao,1997).The reduction in acetate accumulation was also achieved by expressing a heterol-ogous gene from B.subtilis,the als A that codes for an acetolactate synthase,which is capable to channel the excess of carbon to ace-toin,a by-product less toxic than acetate(Aristidou et al.,1995).

Unfortunately,these engineered strains have shown to improve recombinant protein production still with low biomass growth associated,with large amounts of resources being driven to the for-mation of other by-products,such as lactate,pyruvate,acetoin and ethanol.This has still an impact on the product and biomass yields, making these strategies yet far from obtaining effective production systems.

S.Carneiro et al./Journal of Biotechnology164 (2013) 396–408401

Since the effects of these genetic alterations were not consid-ered at the systems-level,local perturbations can be propagated through the metabolic network in an unpredicted way.For instance, the increase of?uxes through the PP pathway may provide larger amounts of building blocks(e.g.nucleotides)toward the recombi-nant production,but it can also induce the accumulation of other metabolic intermediaries that exert an enzymatic control over cen-tral metabolic activities(Usui et al.,2012;Nicolas et al.,2007).As an example,the6-phospho-D-gluconate that is an intermediary of the PP pathway can be over-accumulated and then exert an enzy-matic inhibition over the enzyme phosphoglucose isomerase that catalyses the interconversion of glucose-6-phosphate and fructose-6-phosphate(Schreyer and Bock,1980).This can severely decrease the growth rate of E.coli cultures,as this activity is an essential step of glycolysis and gluconeogenesis pathways.When altering the metabolism of cells,it is important to consider the complex inter-connectivity between pathways and,more importantly,to take into account that slight alterations in metabolite ratios induce immedi-ate changes in the activity of enzymes.

3.2.Culturing conditions

The optimisation towards an ef?cient and robust recombinant production platform,includes the implementation of culturing conditions that attempts to minimize stresses during recombi-nant processes and further eliminate potential bottlenecks in the metabolism.One of the most simplistic approaches is the supple-mentation of amino acids that are limiting during the recombinant process(Harcum et al.,1992).However,this only solves the prob-lem of the limitation of building blocks,while the limitation of energy generation persists.In addition,the supplementation of amino acids may exert unpredictable metabolic effects over the amino acid biosynthetic activities,since the enzymatic regulation of these pathways is primarily performed by the end-product, i.e.the amino acids(Lourenc?o et al.,2011).Only with a system-atic approach that allows a holistic overview over these complex systems,it would be possible to fully understand the metabolic implications of these supplementation strategies.

Acetate formation is known to be strain-dependent(Son et al., 2011;Phue and Shiloach,2004;Phue et al.,2005),i.e.for exam-ple E.coli BL21reacts more ef?ciently to lower concentrations of acetate than the strain JM109,by reducing its speci?c glu-cose uptake.The choice of a more suitable strain can therefore be considered.Besides this,the accumulation of acetate is primar-ily connected to growth and carbon source uptake rates(Shiloach et al.,1996;Van de Walle and Shiloach,1998).Therefore,high ini-tial glucose concentrations will promote acetate formation,as well as cultivations at high speci?c growth rates.In turn,this results in the decrease of the speci?c production rate of recombinant pro-tein(Turner et al.,1994).Accordingly,methodologies based on the control of nutrient feeding are valuable to prevent the over-feeding of media components that contribute to the metabolic over?ow and the consequent accumulation of by-products that have an inhibitory effect in biomass growth(e.g.acetate),or to avoid the underfeeding in which cells become starved for limiting-nutrients(e.g.carbon source).Feeding strategies can consist on the application of constant feeding rates,stepwise-increasing feed-ing rates or exponential feeding rates(with or without feedback control).The last one provides the advantage that by controlling the speci?c growth rate,acetate production can be minimized or even avoided(Rocha et al.,2008;Carneiro et al.,2011b;Gregory and Turner,1993).However,by using advanced continuous cul-tivation methods(e.g.A-stat and D-stat),it was found that the speci?c growth rate is not the only mechanism controlling the cen-tral carbon metabolism in cultures(Valgepea et al.,2010);although most of the fermentation strategies are concerned to avoid acetate over?ow by manipulating the speci?c growth rate,due to the detri-mental effects on the carbon?ow and energy spilling that leads to decreased protein productivity(as discussed above).

During recombinant bioprocesses,cellular responses like nutri-ent starvation can be elicited,which contribute to the metabolic stress of the host cells.Some of these responses may be associated with the cultivation mode.For example,high cell density cultures have long been preferred for recombinant fermentations with E.coli (Choi et al.,2006),but they can cause severe problems to the cul-ture,such as substrate or oxygen limitations.These conditions may further contribute to the metabolic stress induced by recom-binant processes and induce the expression of stress-responsive proteins that will compete for the translation apparatus and,more importantly for energy and metabolic sources,resulting in high productivity losses.

A major problem in large-scale recombinant E.coli cultures is the existence of gradients in dissolved oxygen tension(DOT). The effects of DOT gradients on the metabolic responses of cells and in the production of recombinant proteins have been stud-ied(Sandoval-Basurto et al.,2005;Lara et al.,2006a).Losses in the biomass and protein yields and the accumulation of by-products like acetate,lactate,formate,and succinate,indicate that in oscil-latory DOT cultures the deviation of carbon?ow to fermentative pathways is signi?cant.Moreover,the transcriptional responses showed that E.coli cells can respond very fast to intermittent DOT conditions,which suggests that rational scale-up criteria and strain design strategies should be established for improved cul-ture performance at large scales.Further studies by Lara and co-workers(Lara et al.,2006b),have shown that fermentative pathways can be obviated as they are not necessary for bacterial survival during the short circulation times typical of large-scale cultures.By using different engineered E.coli strains(e.g.a pox

B single mutant that eliminated the conversion of pyruvate into acetate;a ldh A p?B double mutant unable to produce lactate and formate;and a ldh A p?B pox B triple mutant)grown under oscillating DOT conditions it was found that the engineered cells were able to continue growing with improved speci?c growth rates and reduced by-product formation when compared to the parental strain.

Still concerning the improvement of fermentation processes to prevent oxygen limitation during growth in high cell density cul-tures,oxygen carriers like liquid per?uorochemicals that increase the oxygen transfer into cell cultures have been tested(Pilarek et al., 2011).In recombinant E.coli cultures,the cell density increased by40%compared to control cultures and the amount of protein increased from1.5to4.5units.Many other recent fermentation strategies have been developed to provide high cell densities,high protein productivity per cell and good protein quality(Krause et al., 2010;Siurkus and Neubauer,2011;Ukkonen et al.,2011);and most of them concern the improvement of the metabolic performance of host cells,even if indirectly.From conventional cultivations meth-ods to new the systematically designed approaches,the ability to manipulate different variables has provided a faster and straight-forward development of recombinant bioprocess.

Finally,the use of inducible expression systems and the moment of induction of the recombinant expression have been pointed as important factors to minimize the metabolic burden(Curless et al., 1990;Neubauer et al.,1992).The physiological state of the culture at the time of induction can affect the metabolic response to the recombinant process and,more importantly,its ef?ciency.If the induction is performed at the mid-logarithmic growth phase,cells can provide suf?cient levels of energy and metabolic precursors for the recombinant biosynthesis,since the cell is at its maximum catabolic capacity.However,if induced at the late-logarithmic or stationary phase there is a higher cell density for product forma-tion,but the metabolic state of the cells is unfavourable and the

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presence of stress-related proteins like proteases can reduce the yield of foreign proteins.

Hence,it is important to?ne-tune the expression of recombi-nant proteins,the metabolic capabilities of the host cell metabolism and the cultivation conditions.The selection of cultivation modes that allow prolonging the production phase,at controlled growth rates and minimizing the induction of stress events is a suitable strategy to attain maximal yields of recombinant protein.

4.Omics technologies as tools to elucidate the impact of recombinant processes

Typically,the characterization of metabolic stresses during recombinant bioprocesses is done through the determination of physiological parameters,like growth yields or acetate accumula-tion.Although these are good indicators of the metabolic state of the cells,they do not reveal the extent of the metabolic perturbations in the entire biochemical network.

With the advent of omics technologies,such as transcriptomics, proteomics and metabolomics,it is now possible to evaluate the abundance of the main components involved in metabolic path-ways.Changes in metabolite levels or alterations in the expression of enzymes can now be evaluated in a high-throughput manner.As metabolic networks are usually composed by numerous and highly interconnected components,a slight perturbation in a metabolite level affects the entire network and often these effects are dif-?cult to predict.Therefore,a system-level perspective is needed to understand metabolic responses to different conditions,in par-ticular during recombinant biosynthesis,and to identify potential metabolic targets that can be modi?ed allowing an ef?cient supply for energy and metabolic resources toward recombinant protein synthesis.

The DNA microarray technology(i.e.,transcriptomics)has been widely used to characterize changes in the transcriptional pat-terns when inducing the recombinant protein expression in E.coli cells(Choi et al.,2003;Duerrschmid et al.,2008;Haddadin and Harcum,2005;Oh and Liao,2000).In most studies,a large number of transcripts displayed statistically signi?cant changes upon the induction of the recombinant protein,from which those involved in key metabolic activities,such as amino acid biosynthesis,acetate synthesis and transport(in particular,the glucose phosphotrans-ferase system)were found to be up-regulated.Glycolytic and TCA genes were not affected signi?cantly,but presented consistent down-regulated expression levels,which explains the decrease in the metabolic activity of cells encountered during recombinant protein overproduction.The scrutiny of these results has already led to the implementation of genetic engineering strategies to improve recombinant protein production.For example,Choi et al. (2003)co-expressed the prs A gene to further supply precursors for the synthesis of purines,pyrimidines and amino acids.This decreased the speci?c growth rate of the recombinant culture, but the protein content was2.3times higher than that obtained without the co-expression of the prs A gene.This indicates that transcriptome pro?ling can assist in the selection of target genes to be overexpressed or deleted to achieve a desired goal.However,the evaluation of results from these analyses should consider the com-plexity of these biological systems,as it is often dif?cult to predict how the system will react to certain genetic changes.

Similarly,proteomics studies have been explored to examine the global physiological changes in E.coli during the production of recombinant proteins(Duerrschmid et al.,2008;Lee et al., 2007;Wang et al.,2005;Aldor et al.,2005).As expected,differen-tial expression levels of proteins were detected when comparing producing and nonproducing cells,in particular the down-regulation of proteins involved in the central carbon metabolism (e.g.glycolysis,PP pathway and TCA cycle)and the up-regulation of cell protection proteins and some sugar transport proteins.In an attempt to investigate the PP pathway as a potential metabolic bottleneck for the improvement of recombinant protein produc-tion,Wang et al.(2005)have engineered E.coli BL21(DE3)to overproduce the phosphogluconolactonase(PGL)enzyme from Pseudomonas aeruginosa,in order to increase the metabolic?ux through the PP pathway.As a result,a higher growth rate and biomass yield were observed,as well as the up-regulation of pro-teins participating in the tricarboxylic acid(TCA)cycle,suggesting that the supplementary PGL activity was valuable to overcome lim-itations in the supply of reducing power and precursors for high level protein synthesis.

Proteomic pro?ling can also provide a valuable tool to hypoth-esize metabolic targets to make these systems more robust,but as referred before,these results only offer information relative to changes of a single level of the system under speci?c conditions. The combination of different levels of information and predictive computational tools has been started to provide valuable insights to understand these systems.

As proteomics data correlates to a certain degree with tran-scriptomics data,the combination of these two technologies can be included in integrative approaches(Duerrschmid et al.,2008;Yoon et al.,2003).For example,the protein pro?les obtained from differ-ence gel electrophoresis(Ettan(TM)DIGE)and the transcriptome pro?les from total microarrays were evaluated to monitor stress responses during recombinant protein expression in E.coli chemo-stat cultures.This study showed fairly good correlations between data(approximately35%to56%),whereby seven proteins showed consistent expression levels,such as:the DnaK and IbpA chaper-ones;the proteins involved in the cell movement and chemotaxis FlgK and FliC;the TCA cycle enzymes AceA and IdH;and the cell division protein FtsZ.

Metabolomics,one of the newest omics technologies being developed,has provided the determination of the abundance of hundreds of metabolites simultaneously.Since metabolite levels are the closest link between the metabolic status and the phenotype of cells,information provided by these analyses is useful to examine the metabolic and cellular changes in cells during the production of recombinant proteins.Metabolomics studies have revealed that, besides increased?uxes of over?ow pathways,especially acetate synthesis,the glyoxylate shunt,not active during growth,was uti-lized during recombinant production,probably to minimize the uncoupling of the glycolytic and TCA activities(Wittmann et al., 2007).Surprisingly,other studies revealed that upon the induction of recombinant protein expression there is a rapid accumulation of unexpected metabolites outside of the cell,such as malonate and cis-aconitate(Carneiro et al.,2011b).The immediate secre-tion of isocitrate lyase inhibitors,such as cis-aconitate,suggests that the anaplerotic glyoxylate shunt is activated to replenish the TCA intermediaries that were withdrawn toward the formation of recombinant protein.

The metabolic burden was also quanti?ed through the deter-mination of intracellular?uxes using13C-based metabolic?ux analysis(Heyland et al.,2011).Though this methodology is still limited by the information available on the organism’s metabolic network,for recombinant E.coli it was possible to determine?uxes through the TCA cycle,which were fairly constant,and acetate syn-thesis that increased with the increasing amount of the inducer added to the culture.

The information extracted from omics analyses is invaluable to examine the physiological changes occurring during these processes(Oh and Liao,2000;Haddadin and Harcum,2005; Duerrschmid et al.,2008;Lee et al.,2007;Wittmann et al.,2007). In Fig.1,the main metabolic responses covered by reported omics analyses are illustrated,together with some examples of genetic

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403

Fig.1.Representation of some of the main results from omics analyses on the metabolic responses of E.coli cells to the induction of the expression of recombinant proteins. The metabolic map represents the central carbon metabolism of E.coli and results obtained essentially from transcriptomic and proteomic studies are represented by up-arrows,if the metabolic activity is up-regulated after the induction of the recombinant protein expression,and down-arrows,if the metabolic activity is down-regulated after the induction of the recombinant protein expression.Some genetic engineering strategies affecting speci?c metabolic activities are also depicted by grey text boxes pointing to the metabolic pathway being engineered.

engineering strategies that were implemented in the last years to overcome metabolic limitations.However,the potential of these tools to rationally design microorganisms with desired metabolic activities has rarely been explored,in particular for the produc-tion of recombinant proteins in microbial cells(Park et al.,2005; Han et al.,2011).It is important to render these omics analyses into integrated tools of existing systematic approaches to predict new engineering targets or to re-design fermentation strategies that otherwise would be much harder.

5.Advancing recombinant bioprocesses through systems biology

As previously presented,the majority of the reported strategies has enabled the improvement of protein production at reason-able levels,but often at the expense of biomass growth.This makes recombinant bioprocesses less ef?cient and pro?table,as the biomass yield and the volumetric productivity are major factors in the production and downstream processing of these microbial fer-mentations.Although some strategies have been focused on the adjustment of the metabolic capacities of microbial cells to over-come some limitations during recombinant processes,this has not been done at a systematic level,which implies that detrimental changes can be provoked in the cellular metabolism.

Typically,these adjustments were achieved by simplistic trial-and-error methodologies(or empirical approaches)that were carried either by introducing or replacing metabolic activities from other organism sources(De Anda et al.,2006)or by enforcing or removing existing metabolic activities(Eiteman and Altman,2006; Wong et al.,2008).These methodologies allowed to increase the formation of metabolic precursors that become limiting during recombinant processes,but in some cases the growth rate recov-ery was only partial,with the carbon?ux being channelled to the formation of metabolic by-products(Yang et al.,1999a;Aristidou et al.,1995).To avoid this metabolic waste it is crucial to employ new strategies that account for the structure and functionality of the entire metabolic network,therefore overcoming the negative impact of the genetic changes performed(the so-called systematic approaches)(see Fig.2).

Metabolic engineering has emerged as a successful tool to direct the metabolic behaviour of cells into more ef?cient sys-tems(Yu et al.,2011;Bulter et al.,2003;Aristidou et al.,1995; Covert et al.,2001).In this approach the metabolic capabilities of cells are considered an integrated system and their functional properties,like enzymatic and regulatory interactions and even the network topology are taken into account when re-designing metabolic networks.This way,promising targets that maximize the formation of the desired products and prevent ineffective alter-ations in the metabolism can be found.

Stoichiometric-based models have been explored to study the metabolic behaviour of microbial cells and to predict phen-otypes that optimize the production of target metabolites.In more detail,metabolic networks can be represented by stoichio-metric models that are usually simulated by constrained-based approaches,termed?ux balance analysis(FBA),assuming a steady-state of the metabolism and a metabolic objective(e.g.growth rate

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408

Fig.2.Schematic representation of the empirical and new systematic strategies being implemented at the three stages in the optimisation of recombinant bioprocesses.Systematic strategies are enclosed in a unique level,since often the design of these strategies considers the tree steps in the optimisation process as an integrated https://www.wendangku.net/doc/1b11065581.html,putational tools that assist in the design of these strategies are also indicated:JCat (Grote et al.,2005);Optimizer (Puigbo et al.,2007);OptFlux (Rocha et al.,2010);OptGene (Patil et al.,2005),OptKnock (Burgard et al.,2003),OptStrain (Pharkya et al.,2004),OptReg (Pharkya and Maranas,2006);OptORF (Kim and Reed,2010);OptFerm (Rocha et al.,2009);GAME.opt (Link and Weuster-Botz,2006).

maximization)(Edwards et al.,2002;Kauffman et al.,2003;Price et al.,2003;Raman and Chandra,2009;Varma and Palsson,1994).FBA explores the metabolic capabilities of cells and predicts their phenotype under de?ned environmental conditions (e.g.aerobio-sis/anaerobiosis or with different carbon sources).Additionally,these stoichiometric-based models can also be used to predict gene deletions in the wild-type strain that would maximize the produc-tion rate of a desired metabolic product.To ?nd feasible metabolic ?ux distributions that ful?l these conditions it is possible to use sev-eral optimization methods:OptGene (Patil et al.,2005),OptKnock (Burgard et al.,2003),OptStrain (Pharkya et al.,2004),OptReg (Pharkya and Maranas,2006)and more recently OptORF (Kim and Reed,2010).Although the availability of computational tools is increasing,the successful application of these stoichiometric mod-els has been limited to the design of strains for the production of metabolic end-products,such as:the overproduction of purine nucleosides,ribo?avin,and folic acid in Bacillus subtilis (Sauer et al.,1998);the enhancement of the biosynthesis of sesquiterpenes in the yeast Saccharomyces cerevisiae (Asadollahi et al.,2009);the improved bioethanol production in Saccharomyces cerevisiae (Bro et al.,2006);the overproduction of threonine in E.coli (De Atauri et al.,2009);or the production of vanillin in baker’s yeast (Brochado et al.,2010).These in silico approaches are still inadequate for the optimization of recombinant protein production,since the dynam-ics behind recombinant processes makes dif?cult to simulate and predict the behaviour of the system.For this reason,most modelling approaches to study recombinant bioprocesses have been based on kinetic descriptions that reproduce the physiological behaviour of cells.However,it is unfeasible to represent all levels of a sys-tem using this type of models.Not only because there is a large number of cellular components that would be needed to be repre-sented in the model,but also because it is impossible to describe the mathematical relationships between components participating

in such cellular activities,at least in the current state-of-art.Despite all,E.coli strains have been metabolically engineered using approaches to obtain optimal phenotypes for the production of end-products,which can be of interest to the improvement of the production of recombinant products,by increasing precursors and cofactors availability (Chemler et al.,2010;Fowler et al.,2009;Park et al.,2007).The metabolically engineered strain carrying pgi , ppc and pld A deletions is one of the best examples that demon-strate the advantages of using stoichiometric models to identify combinations of gene knockouts to improve the NADPH availabil-ity in E.coli (Chemler et al.,2010).A constraint-based metabolic model combined with an evolutionary-based optimization method was used to investigate the phenotype of knockout candidates that maximizes the production rate of NADPH while also maximizing for growth,i.e.biomass product coupled yield (BPCY).The BPCY objective function avoids selecting mutants with high speci?c pro-duction rates but that are not viable.Though the overexpression of a recombinant protein was not accounted in the model,this approach showed some advantages because it improves the overall NADPH production in the mutant strain,which has been identi?ed as a limiting resource during recombinant bioprocesses.

Another example is the rationally engineered E.coli strain to overproduce L-valine by introducing the following genetic mod-i?cations: ace F, mdh ,and pfk A.This strain can also be interesting for the overproduction of recombinant proteins that have a sequence rich in L-valine.As previously discussed,the amino acid sequence of the recombinant protein in?uences the physiolog-ical behaviour of the host strain due to shortages in the intracellular amino acid pools,especially for least abundant amino acids.How-ever,this is not always so straightforward and modelling strategies to predict the amino acid shortages during recombinant processes should be ?rst implemented.Sarkandy et al.(2010)developed an amino acid supplementation strategy based on the simulation of

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a stoichiometric model that could predict which amino acids are most needed to enhance the production of IL-2(interleukin-2as a model protein)in a fed-batch high-cell-density culture.The most effective amino acid mixture was found to be leucine,aspartic acid and glycine,which increased the production of IL-2almost two-fold(Sarkandy et al.,2010).Seemingly,a modelling approach based on a stoichiometric model combined with kinetic-based descrip-tions for the production of a recombinant protein and cell growth was proposed(Carneiro et al.,2011a).This model allows to follow the withdrawn of amino acids for biomass and recombinant pro-tein formation along the cultivation process and to estimate which amino acids are the?rst to become limited.More importantly,these models enable to couple the modelling of cultivation strategies with the metabolic engineering of cells.These systematic approaches might be some of the earliest attempts to overcome the metabolic burden of recombinant processes by combining different aspects of the bioprocesses optimization problem.

6.Concluding remarks

The success of recombinant bioprocesses depends on the under-standing of the metabolic responses to the burden imposed by additional drainages of metabolic resources toward the production of recombinant material.In this review,strategies that have been developed,either to cope with the shortage of metabolic resources or the control of the metabolism over?ow,were addressed. Although most of these strategies were able to improve the recombinant protein production,reduced biomass yields and the accumulation of metabolic by-products were also observed. Therefore,alternative strategies that allow to manipulate these recombinant systems in a more ef?cient way are in great need.

Systems biology and particularly metabolic engineering have been valuable to rationally design microbial strains for the produc-tion of many products of interest(Sauer et al.,1998;Asadollahi et al.,2009;Bro et al.,2006;De Atauri et al.,2009;Brochado et al.,2010).So far,modeling approaches have mostly applied constrained-based models,which seem to work well in the design of strains for the improved production of chemicals that belong to the central carbon metabolism.Recombinant processes are far more complex,since the expression of recombinant proteins is gen-erally plasmid-based,meaning that the interaction with the central carbon metabolism is not straightforward to understand and to model.However,the combination of dynamic and stoichiometric models can be useful for the simulation of these cellular processes, since the dynamics of the recombinant synthesis can be easily cap-tured,as well as the growth-dependent metabolic behavior of cells. Metabolic models,like those reconstructed for E.coli,e.g.the iJR904 (Reed et al.,2003)and the iAF1260(Feist et al.,2007),when com-bined with mechanistic models can offer a more comprehensive representation of the cellular organization.

Acknowledgements

This work was partially supported by the MIT-Portugal Pro-gram in Bioengineering(MIT-Pt/BS-BB/0082/2008),the research project HeliSysBio-Molecular Systems Biology Helicobacter pylori (FCT PTDC/EBB-EBI/104235/2008)and a PhD grant from Portuguese FCT(Fundac??o para a Ciência e Tecnologia)(SFRH/BD/22863/2005). References

Aldor,I.S.,Krawitz,D.C.,Forrest,W.,Chen,C.,Nishihara,J.C.,Joly,J.C.,Champion,K.M., 2005.Proteomic pro?ling of recombinant Escherichia coli in high-cell-density fermentations for improved production of an antibody fragment biopharma-ceutical.Applied and Environmental Microbiology71,1717–1728. Andersen,D.C.,Krummen,L.,2002.Recombinant protein expression for therapeutic applications.Current Opinion in Biotechnology13,117–123.Andersson,L.,Yang,S.,Neubauer,P.,Enfors,S.-O.,1996.Impact of plasmid pres-ence and induction on cellular responses in fed batch cultures of Escherichia coli.

Journal of Biotechnology46,255–263.

Angov,E.,Hillier,C.J.,Kincaid,R.L.,Lyon,J.A.,2008.Heterologous protein expression is enhanced by harmonizing the codon usage frequencies of the target gene with those of the expression host.PLoS One3,e2189.

Aristidou, A.A.,San,K.Y.,Bennett,G.N.,1995.Metabolic engineering of Escherichia coli to enhance recombinant protein production through acetate reduction.Biotechnology Progress11,475–478.

Artsimovitch,I.,Patlan,V.,Sekine,S.I.,Vassylyeva,M.N.,Hosaka,T.,Ochi,K., Yokoyama,S.,Vassylyev,D.G.,2004.Structural basis for transcription regulation by alarmone ppGpp.Cell117,299–310.

Asadollahi,M.A.,Maury,J.,Patil,K.R.,Schalk,M.,Clark,A.,Nielsen,J.,2009.Enhancing sesquiterpene production in Saccharomyces cerevisiae through in silico driven metabolic engineering.Metabolic Engineering11,328–334.

Bailey,J.E.,1993.Host-vector interactions in Escherichia coli.Advances in Biochem-ical Engineering Biotechnology48,29–52.

Baneyx,F.,1999.Recombinant protein expression in Escherichia coli.Current Opinion in Biotechnology10,411–421.

Bentley,W.E.,Kompala,D.S.,1990.Optimal induction of protein synthesis in recom-binant bacterial cultures.Annals of the New York Academy of Sciences589, 121–138.

Bentley,W.E.,Mirjalili,N.,Andersen,D.C.,Davis,R.H.,Kompala,D.S.,1990.Plasmid-encoded protein–the principal factor in the metabolic burden associated with recombinant bacteria.Biotechnology and Bioengineering35,668–681. Birnbaum,S.,Bailey,J.E.,1991.Plasmid presence changes the relative levels of many host cell proteins and ribosome components in recombinant Escherichia coli.

Biotechnology and Bioengineering37,736–745.

Blaas,L.,Musteanu,M.,Eferl,R.,Bauer,A.,Casanova,E.,2009.Bacterial arti?cial chromosomes improve recombinant protein production in mammalian cells.

BMC Biotechnology9.

Bonomo,J.,Gill,R.T.,2005.Amino acid content of recombinant proteins in?uences the metabolic burden response.Biotechnology and Bioengineering90,116–126. Bro,C.,Regenberg,B.,Forster,J.,Nielsen,J.,2006.In silico aided metabolic engineer-ing of Saccharomyces cerevisiae for improved bioethanol production.Metabolic Engineering8,102–111.

Brochado,A.R.,Matos,C.,Moller,B.L.,Hansen,J.,Mortensen,U.H.,Patil,K.R.,2010.

Improved vanillin production in baker’s yeast through in silico design.Microbial Cell Factories9.

Bulter,T.,Bernstein,J.R.,Liao,J.C.,2003.A perspective of metabolic engineering strategies:moving up the systems hierarchy.Biotechnology and Bioengineering 84,815–821.

Burgard,A.P.,Pharkya,P.,Maranas,C.D.,2003.OptKnock:A bilevel programming framework for identifying gene knockout strategies for microbial strain opti-mization.Biotechnology and Bioengineering84,647–657.

Burgess-Brown,N.A.,Sharma,S.,Sobott,F.,Loenarz,C.,Oppermann,U.,Gileadi, O.,2008.Codon optimization can improve expression of human genes in Escherichia coli.A multi-gene study.Protein Expression and Puri?cation59,94–102.

Calderone,T.L.,Stevens,R.D.,Oas,T.G.,1996.High-level misincorporation of lysine for arginine at AGA codons in a fusion protein expressed in Escherichia coli.

Journal of Molecular Biology262,407–412.

Carneiro,S.,Ferreira,E.C.,Rocha,I.,2011a.A systematic modeling approach to elu-cidate the triggering of the stringent response in recombinant E.coli systems.

Advances in Intelligent and Soft Computing93,313–320.

Carneiro,S.,Villas-Boas,S.G.,Ferreira,E.C.,Rocha,I.,2011b.Metabolic footprint analysis of recombinant Escherichia coli strains during fed-batch fermentations.

Molecular Biosystems7,899–910.

Castano-Cerezo,S.,Pastor,J.M.,Renilla,S.,Bernal,V.,Iborra,J.L.,Canovas,M.,2009.An insight into the role of phosphotransacetylase(pta)and the acetate/acetyl-CoA node in Escherichia coli.Microbial Cell Factories8,54.

Chang,D.E.,Shin,S.,Rhee,J.S.,Pan,J.G.,1999.Acetate metabolism in a pta mutant of Escherichia coli W3110:importance of maintaining acetyl coenzyme A?ux for growth and survival.Journal of Bacteriology181,6656–6663.

Chatterji,D.,Fujita,N.,Ishihama,A.,1998.The mediator for stringent control,ppGpp, binds to the beta-subunit of Escherichia coli RNA polymerase.Genes to Cells3, 279–287.

Chemler,J.A.,Fowler,Z.L.,McHugh,K.P.,Koffas,M.A.,2010.Improving NADPH availability for natural product biosynthesis in Escherichia coli by metabolic engineering.Metabolic Engineering12,96–104.

Chen,H.T.,Lin,M.S.,Hou,S.Y.,2008.Multiple-copy-gene integration on chromosome of Escherichia coli for beta-galactosidase production.Korean Journal of Chemical Engineering25,1082–1087.

Chiang,C.J.,Chen,P.T.,Chao,Y.P.,2008.Replicon-free and markerless methods for genomic insertion of DNAs in phage attachment sites and controlled expression of chromosomal genes in Escherichia coli.Biotechnology and Bioengineering101, 985–995.

Choi,J.H.,Keum,K.C.,Lee,S.Y.,2006.Production of recombinant proteins by high cell density culture of Escherichia coli.Chemical Engineering Science61,876–885. Choi,J.H.,Lee,S.J.,Lee,S.J.,Lee,S.Y.,2003.Enhanced production of insulin-like growth factor I fusion protein in Escherichia coli by coexpression of the down-regulated genes identi?ed by transcriptome pro?ling.Applied Environment and Microbi-ology69,4737–4742.

Choi,J.H.,Lee,S.Y.,2004.Secretory and extracellular production of recombinant proteins using Escherichia coli.Applied Microbiology and Biotechnology64, 625–635.

406S.Carneiro et al./Journal of Biotechnology164 (2013) 396–408

Chou,C.H.,Bennett,G.N.,San,K.Y.,1994.Effect of modi?ed glucose uptake using genetic engineering techniques on high-level recombinant protein produc-tion in Escherichia coli dense cultures.Biotechnology and Bioengineering44, 952–960.

Chou,C.P.,2007.Engineering cell physiology to enhance recombinant protein pro-duction in Escherichia coli.Applied Microbiology and Biotechnology76,521–532. Corchero,J.L.,Villaverde,A.,1998.Plasmid maintenance in Escherichia coli recombi-nant cultures is dramatically,steadily,and speci?cally in?uenced by features of the encoded proteins.Biotechnology and Bioengineering58,625–632. Covert,M.W.,Schilling,C.H.,Famili,I.,Edwards,J.S.,Goryanin,I.I.,Selkov,E.,Palsson,

B.O.,2001.Metabolic modeling of microbial strains in silico.Trends in Biochem-

ical Sciences26,179–186.

Curless,C.,Pope,J.,Tsai,L.,1990.Effect of preinduction speci?c growth rate on recombinant alpha consensus interferon synthesis in Escherichia coli.Biotech-nology Progress6,149–152.

De Anda,R.,Lara,A.R.,Hernandez,V.,Hernandez-Montalvo,V.,Gosset,G.,Bolivar,F., Ramirez,O.T.,2006.Replacement of the glucose phosphotransferase transport system by galactose permease reduces acetate accumulation and improves pro-cess performance of Escherichia coli for recombinant protein production without impairment of growth rate.Metabolic Engineering8,281–290.

De Atauri,P.,Rodriguez-Prados,J.C.,Maury,J.,Ortega,F.,Portais,J.C.,Chassagnole,

C.,Acerenza,L.,Lindley,N.

D.,Cascante,M.,2009.In silico strategy to rationally

engineer metabolite production:a case study for threonine in Escherichia coli.

New Biotechnology25,S358.

de Oliveira,J.E.,Soares,C.R.,Peroni,C.N.,Gimbo,E.,Camargo,I.M.,Morganti,L., Bellini,M.H.,Affonso,R.,Arkaten,R.R.,Bartolini,P.,Ribela,M.T.,1999.High-yield puri?cation of biosynthetic human growth hormone secreted in Escherichia coli periplasmic space.Journal of Chromatography A852,441–450.

Dedhia,N.,Richins,R.,Mesina,A.,Chen,W.,1997.Improvement in recombinant protein production in ppGpp-de?cient Escherichia coli.Biotechnology and Bio-engineering53,380–386.

Del Jr.,T.B.,Ward,J.M.,Hodgson,J.,Gershater,C.J.,Edwards,H.,Wysocki,L.A.,Watson,

F.A.,Sathe,

G.,Kane,J.F.,1995.Effects of a minor isoleucyl tRNA on heterologous

protein translation in Escherichia coli.Journal of Bacteriology177,7086–7091. Demain,A.L.,Adrio,J.L.,2008.Strain improvement for production of pharmaceuticals and other microbial metabolites by fermentation.Progress in Drug Research65, 251,253-251,289.

Demain,A.L.,Vaishnav,P.,2009.Production of recombinant proteins by microbes and higher organisms.Biotechnology Advances27,297–306.

Deuschle,U.,Kammerer,W.,Gentz,R.,Bujard,H.,1986.Promoters of Escherichia coli:

a hierarchy of in vivo strength indicates alternate structures.EMBO Journal5,

2987–2994.

Dittrich,C.R.,Vadali,R.V.,Bennett,G.N.,San,K.Y.,2005.Redistribution of metabolic ?uxes in the central aerobic metabolic pathway of E.coli mutant strains with deletion of the ack A-pta and pox B pathways for the synthesis of isoamyl acetate.

Biotechnology Progress21,627–631.

Dong,H.J.,Nilsson,L.,Kurland,C.G.,1995.Gratuitous overexpression of genes in Escherichia coli leads to growth inhibition and ribosome destruction.Journal of Bacteriology177,1497–1504.

Doyle,M.V.,Lee,M.T.,Fong,S.,https://www.wendangku.net/doc/1b11065581.html,parison of the biological activities of human recombinant interleukin-2(125)and native interleukin-2.Journal of Biological Response Modi?ers4,96–109.

Duerrschmid,K.,Reischer,H.,Schmidt-Heck,W.,Hrebicek,T.,Guthke,R.,Rizzi,A., Bayer,K.,2008.Monitoring of transcriptome and proteome pro?les to inves-tigate the cellular response of E.coli towards recombinant protein expression under de?ned chemostat conditions.Journal of Biotechnology135,34–44. Edwards,J.S.,Covert,M.,Palsson,B.,2002.Metabolic modelling of microbes:the ?ux-balance approach.Environmental Microbiology4,133–140.

Eiteman,M.A.,Altman,E.,2006.Overcoming acetate in Escherichia coli recombinant protein fermentations.Trends in Biotechnology24,530–536.

Enfors,S.O.,Jahic,M.,Rozkov,A.,Xu,B.,Hecker,M.,Jurgen,B.,Kruger,E.,Schweder, T.,Hamer,G.,O’Beirne,D.,Noisommit-Rizzi,N.,Reuss,M.,Boone,L.,Hewitt,

C.,McFarlane,C.,Nienow,A.,Kovacs,T.,Tragardh,C.,Fuchs,L.,Revstedt,J.,

Friberg,P.C.,Hjertager,B.,Blomsten,G.,Skogman,H.,Hjort,S.,Hoeks,F.,Lin,H.Y., Neubauer,P.,van der Lans,R.,Luyben,K.,Vrabel,P.,Manelius,A.,2001.Physio-logical responses to mixing in large scale bioreactors.Journal of Biotechnology 85,175–185.

Farmer,W.R.,Liao,J.C.,1997.Reduction of aerobic acetate production by Escherichia coli.Applied and Environmental Microbiology63,3205–3210. Feist,A.M.,Henry,C.S.,Reed,J.L.,Krummenacker,M.,Joyce,A.R.,Karp,P.D.,Broadbelt, L.J.,Hatzimanikatis,V.,Palsson,B.O.,2007.A genome-scale metabolic recon-struction for Escherichia coli K-12MG1655that accounts for1260ORFs and thermodynamic information.Molecular Systems Biology3,121.

Ferrer-Miralles,N.,Domingo-Espin,J.,Corchero,J.L.,Vazquez,E.,Villaverde,A.,2009.

Microbial factories for recombinant pharmaceuticals.Microbial Cell Factories8,

17.

Flores,N.,Leal,L.,Sigala,J.C.,de,A.R.,Escalante,A.,Martinez,A.,Ramirez,O.T., Gosset,G.,Bolivar,F.,2007.Growth recovery on glucose under aerobic condi-tions of an Escherichia coli strain carrying a phosphoenolpyruvate:carbohydrate phosphotransferase system deletion by inactivating arc A and overexpressing the genes coding for glucokinase and galactose permease.Journal of Molecular Microbiology and Biotechnology13,105–116.

Flores,S.,de Anda-Herrera,R.,Gosset,G.,Bolivar,F.G.,2004.Growth rate recov-ery of Escherichia coli cultures carrying a multicopy plasmid,by engineering of the pentose-phosphate pathway.Biotechnology and Bioengineering87,485–494.Fowler,Z.L.,Gikandi,W.W.,Koffas,M.A.,2009.Increased malonyl coenzyme A biosynthesis by tuning the Escherichia coli metabolic network and its appli-cation to?avanone production.Applied and Environmental Microbiology75, 5831–5839.

Glick,B.R.,1995.Metabolic load and heterologous gene expression.Biotechnology Advances13,247–261.

Gnoth,S.,Jenzsch,M.,Simutis,R.,Lubbert,A.,2008.Control of cultivation processes for recombinant protein production:a review.Bioprocess and Biosystems Engi-neering31,21–39.

Gregory,M.E.,Turner,C.,1993.Open-loop control of speci?c growth rate in fed-batch cultures of recombinant E.coli.Biotechnology Techniques7,889–894.

Grote,A.,Hiller,K.,Scheer,M.,Munch,R.,Nortemann,B.,Hempel,D.C.,Jahn,D., 2005.JCat:a novel tool to adapt codon usage of a target gene to its potential expression host.Nucleic Acids Research33,W526–W531.

Gustafsson,C.,Govindarajan,S.,Minshull,J.,2004.Codon bias and heterologous protein expression.Trends in Biotechnology22,346–353.

Gustafsson,C.,Minshull,J.,Govindarajan,S.,Ness,J.,Villalobos,A.,Welch,M.,2012.

Engineering genes for predictable protein expression.Protein Expression and Puri?cation83,37–46.

Haddadin,F.T.,Harcum,S.W.,2005.Transcriptome pro?les for high-cell-density recombinant and wild-type Escherichia coli.Biotechnology and Bioengineering 90,127–153.

Hale,R.S.,Thompson,G.,1998.Codon optimization of the gene encoding a domain from human type1neuro?bromin protein results in a threefold improvement in expression level in Escherichia coli.Protein Expression and Puri?cation12, 185–188.

Han,J.H.,Choi,Y.S.,Kim,W.J.,Jeon,Y.H.,Lee,S.K.,Lee,B.J.,Ryu,K.S.,2010.Codon optimization enhances protein expression of human peptide deformylase in E.

coli.Protein Expression and Puri?cation70,224–230.

Han,M.J.,Lee,J.W.,Lee,S.Y.,2011.Understanding and engineering of microbial cells based on proteomics and its conjunction with other omics studies.Proteomics 11,721–743.

Harcum,S.W.,Ramirez,D.M.,Bentley,W.E.,1992.Optimal nutrient feed policies for heterologous protein production.Applied Biochemistry and Biotechnology 34-5,161–173.

Heyland,J.,Blank,L.M.,Schmid,A.,2011.Quanti?cation of metabolic limitations dur-ing recombinant protein production in Escherichia coli.Journal of Biotechnology 155,178–184.

Hsiao,J.,Ahluwalia,M.,Kaufman,J.,Clem,T.R.,Shiloach,J.,1990.Adaptive con-trol strategy for maintaining dissolved oxygen concentration in high density growth of recombinant E.coli.Annals of New York Academy of Sciences,321–333.

Hsu,E.,Osslund,T.,Nybo,R.,Chen,B.L.,Kenney,W.C.,Morris,C.F.,Arakawa,T., Narhi,L.O.,2006.Enhanced stability of recombinant keratinocyte growth factor by mutagenesis.Protein Engineering Design and Selection19,147–153. Jana,S.,Deb,J.K.,2005.Strategies for ef?cient production of heterologous proteins in Escherichia coli.Applied Microbiology and Biotechnology67, 289–298.

Jeon,E.,Lee,S.,Won,J.I.,Han,S.O.,Kim,J.,Lee,J.,2011.Development of Escherichia coli MG1655strains to produce long chain fatty acids by engineering fatty acid synthesis(FAS)metabolism.Enzyme and Microbial Technology49,44–51. Jinho,S.,Bailey,J.E.,1985.Effects of recombinant plasmid content on growth prop-erties and cloned gene product formation in Escherichia coli.Biotechnology and Bioengineering27,1668–1674.

Jones,K.L.,Kim,S.W.,Keasling,J.D.,2000.Low copy plasmids can perform as well as or better than high copy plasmids for metabolic engineering of bacteria.

Metabolic Engineering2,328–338.

Jonet,M.A.,Mahadi,N.M.,Murad, A.M.A.,Rabu, A.,Bakar, F.D.A.,Rahim,R.A., Low,K.O.,Illias,R.,2012.Optimization of a heterologous signal peptide by site-directed mutagenesis for improved secretion of recombinant proteins in Escherichia coli.Journal of Molecular Microbiology and Biotechnology22,48–58. Kamionka,M.,2011.Engineering of therapeutic proteins production in Escherichia coli.Current Pharmaceutical Biotechnology12,268–274.

Kane,J.F.,1995.Effects of rare codon clusters on high-level expression of heterolo-gous proteins in Escherichia coli.Current Opinion in Biotechnology6,494–500. Kauffman,K.J.,Prakash,P.,Edwards,J.S.,2003.Advances in?ux balance analysis.

Current Opinion in Biotechnology14,491–496.

Kim,J.,Reed,J.L.,2010.OptORF:Optimal metabolic and regulatory perturbations for metabolic engineering of microbial strains.BMC Systems Biology4,53.

Kim,J.Y.,Cha,H.J.,2003.Down-regulation of acetate pathway through antisense strategy in Escherichia coli:improved foreign protein production.Biotechnology and Bioengineering83,841–853.

Koh,B.T.,Nakashimada,U.,Pfeiffer,M.,Yap,M.G.S.,https://www.wendangku.net/doc/1b11065581.html,parison of acetate inhibition on growth of host and recombinant Escherichia coli K12strains.

Biotechnology Letters14,1115–1118.

Konstantinov,K.,Kishimoto,M.,Seki,T.,Yoshida,T.,1990.A balanced DO-stat and its application to the control of acetic acid excretion by recombinant Escherichia coli.Biotechnology and Bioengineering36,750–758.

Konstantinov,K.,Nishio,N.,Seki,T.,Yoshida,T.,1991.Physiologically motivated strategies for control of the fed-batch cultivation of recombinant Escherichia coli for phenylalanine production.Journal of Fermentation and Bioengineering71, 350–355.

Krause,M.,Ukkonen,K.,Haataja,T.,Ruottinen,M.,Glumoff,T.,Neubauer, A., Neubauer,P.,Vasala,A.,2010.A novel fed-batch based cultivation method pro-vides high cell-density and improves yield of soluble recombinant proteins in shaken cultures.Microbial Cell Factories9,11.

S.Carneiro et al./Journal of Biotechnology164 (2013) 396–408407

Lara,A.R.,Caspeta,L.,Gosset,G.,Bolivar,F.,Ramirez,O.T.,2008.Utility of an Escherichia coli strain engineered in the substrate uptake system for improved culture performance at high glucose and cell concentrations:an alternative to fed-batch cultures.Biotechnology and Bioengineering99,893–901.

Lara,A.R.,Leal,L.,Flores,N.,Gosset,G.,Bolivar,F.,Ramirez,O.T.,2006a.Transcrip-tional and metabolic response of recombinant Escherichia coli to spatial dissolved oxygen tension gradients simulated in a scale-down system.Biotechnology and Bioengineering93,372–385.

Lara,A.R.,Vazquez-Limon,C.,Gosset,G.,Bolivar,F.,Lopez-Munguia,A.,Ramirez, O.T.,2006b.Engineering Escherichia coli to improve culture performance and reduce formation of by-products during recombinant protein production under transient intermittent anaerobic conditions.Biotechnology and Bioengineering 94,1164–1175.

Lee,D.H.,Kim,S.G.,Park,Y.C.,Nam,S.W.,Lee,K.H.,Seo,J.H.,2007.Proteome analysis of recombinant Escherichia coli producing human glucagon-like peptide-1.Jour-nal of Chromatography B:Analytical Technologies in the Biomedical and Life Sciences849,323–330.

Lee,S.Y.,Lee,D.Y.,Kim,T.Y.,2005.Systems biotechnology for strain improvement.

Trends in Biotechnology23,349–358.

Li,M.,Wang,J.,Geng,Y.,Li,Y.,Wang,Q.,Liang,Q.,Qi,Q.,2012.A strategy of gene over-expression based on tandem repetitive promoters in Escherichia coli.Microbial Cell Factories11.

Link,H.,Weuster-Botz,D.,2006.Genetic algorithm for multi-objective experimental optimization.Bioprocess and Biosystems Engineering29,385–390.

Lourenc?o,A.,Carneiro,S.,Pinto,J.P.,Rocha,M.,Ferreira,E.C.,Rocha,I.,2011.A study of the short and long-term regulation of E.coli metabolic pathways.Journal of Integrative Bioinformatics8,183.

Luli,G.W.,Strohl,W.R.,https://www.wendangku.net/doc/1b11065581.html,parison of growth,acetate production,and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations.

Applied and Environmental Microbiology56,1004–1011.

Martinez-Morales,F.,Borges,A.C.,Martinez,K.,Shanmugam,K.T.,Ingram,L.O.,1999.

Chromosomal integration of heterologous DNA in Escherichia coli with precise removal of markers and replicons used during construction.Journal of Bacteri-ology181,7143–7148.

Mason,C.A.,Bailey,J.E.,1989.Effects of plasmid presence on growth and enzyme activity of Escherichia coli DH5alpha.Applied Microbiology and Biotechnology 32,54–60.

Neidhardt,F.C.,Ingraham,J.L.,Schaechter,M.,1990.Physiology of the Bacterial Cell—A Molecular Approach,1st ed.Sinauer Associates,Sunderland,USA. Neubauer,P.,Hofmann,K.,Holst,O.,Mattiasson,B.,Kruschke,P.,1992.Maximiz-ing the expression of a recombinant gene in Escherichia coli by manipulation of induction time using lactose as inducer.Applied Microbiology and Biotechnol-ogy36,739–744.

Neubauer,P.,Winter,J.,2001.Expression and fermentation strategies for recom-binant protein production in Escherichia coli.Recombinant Protein Production with Prokaryotic and Eukaryotic Cells.A Comparative View on Host Physi-ology.,O.-W.Merten et al.(Ed.).Kluwer Academic Publisher,Dortrecht,The Netherlands,pp.196–260.

Nicolas,C.,Kiefer,P.,Letisse,F.,Kromer,J.,Massou,S.,Soucaille,P.,Wittmann,

C.,Lindley,N.

D.,Portais,J.C.,2007.Response of the central metabolism of

Escherichia coli to modi?ed expression of the gene encoding the glucose-6-phosphate dehydrogenase.FEBS Letters581,3771–3776.

Niemitalo,O.,Neubauer,A.,Liebal,U.,Myllyharju,J.,Juffer,A.H.,Neubauer,P.,2005.

Modelling of translation of human protein disul?de isomerase in Escherichia coli-A case study of gene optimisation.Journal of Biotechnology120,11–24. O’Rourke,E.C.,Drummond,R.J.,Creasey,A.A.,1984.Binding of125I-labeled recom-binant beta interferon(IFN-beta Ser17)to human cells.Molecular and Cellular Biology4,2745–2749.

Oh,M.K.,Liao,J.C.,2000.DNA microarray detection of metabolic responses to protein overproduction in Escherichia coli.Metabolic Engineering2,201–209.

Park,J.H.,Lee,K.H.,Kim,T.Y.,Lee,S.Y.,2007.Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico gene knockout simulation.Proceedings of National Academy of Science of the United States of America104,7797–7802.

Park,S.J.,Lee,S.Y.,Cho,J.,Kim,T.Y.,Lee,J.W.,Park,J.H.,Han,M.J.,2005.Global physi-ological understanding and metabolic engineering of microorganisms based on omics studies.Applied Microbiology and Biotechnology68,567–579.

Patil,K.R.,Rocha,I.,Forster,J.,Nielsen,J.,2005.Evolutionary programming as a platform for in silico metabolic engineering.BMC Bioinformatics6.

Peretti,S.W.,Bailey,J.E.,1987.Simulations of host-plasmid interactions in Escherichia coli–copy number,promoter strength,and ribosome binding site strength effects on metabolic activity and plasmid gene expression.Biotechnol-ogy and Bioengineering29,316–328.

Peti,W.,Page,R.,2007.Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost.Protein Expression and Puri?cation51,1–10. Pharkya,P.,Burgard,A.P.,Maranas,C.D.,2004.OptStrain:A computational frame-work for redesign of microbial production systems.Genome Research14, 2367–2376.

Pharkya,P.,Maranas,C.D.,2006.An optimization framework for identifying reaction activation/inhibition or elimination candidates for overproduction in microbial systems.Metabolic Engineering8,1–13.

Phue,J.N.,Noronha,S.B.,Hattacharyya,R.,Wolfe,A.J.,Shiloach,J.,2005.Glucose metabolism at high density growth of E.coli B and E.coli K:differences in metabolic pathways are responsible for ef?cient glucose utilization in E.coli

B as determined by microarrays and Northern blot analyses.Biotechnology and

Bioengineering90,805–820.Phue,J.N.,Shiloach,J.,2004.Transcription levels of key metabolic genes are the cause for different glucose utilization pathways in E.coli B(BL21)and E.coli K(JM109).

Journal of Biotechnology109,21–30.

Pilarek,M.,Glazyrina,J.,Neubauer,P.,2011.Enhanced growth and recombinant protein production of Escherichia coli by a per?uorinated oxygen carrier in minia-turized fed-batch cultures.Microbial Cell Factories10,50.

Pinske,C.,Bonn,M.,Kruger,S.,Lindenstrauss,U.,Sawers,R.G.,2011.Metabolic de?ciences revealed in the biotechnologically important model bacterium Escherichia coli BL21(DE3).Plos One6.

Plotkin,J.B.,Kudla,G.,2011.Synonymous but not the same:the causes and conse-quences of codon bias.Nature Reviews Genetics12,32–42.

Price,N.D.,Papin,J.A.,Schilling,C.H.,Palsson,B.O.,2003.Genome-scale microbial in silico models:the constraints-based approach.Trends in Biotechnology21, 162–169.

Puigbo,P.,Guzman,E.,Romeu,A.,Garcia-Vallve,S.,2007.OPTIMIZER:a web server for optimizing the codon usage of DNA sequences.Nucleic Acids Research35, W126–W131.

Raman,K.,Chandra,N.,2009.Flux balance analysis of biological systems:applica-tions and challenges.Brie?ngs in Bioinformatics10,435–449.

Reed,J.L.,Vo,T.D.,Schilling,C.H.,Palsson,B.O.,2003.An expanded genome-scale model of Escherichia coli K-12(iJR904GSM/GPR).Genome Biology4,R54. Rocha,I.,Maia,P.,Evangelista,P.,Vilaca,P.,Soares,S.,Pinto,J.P.,Nielsen,J.,Patil,K.R., Ferreira,E.C.,Rocha,M.,2010.OptFlux:an open-source software platform for in silico metabolic engineering.BMC Systems Biology4,45.

Rocha,I.,Veloso,A.C.A.,Carneiro,S.,Costa,R.,Ferreira,E.C.,2008.Implementation of a speci?c rate controller in a fed-batch E.coli fermentation.In:Proceedings of the17th IFAC(International Federation of Automatic Control)World Congress, Seoul,Korea,pp.15565–15570.

Rocha,O.,Maia,P.,Rocha,I.,Rocha,M.,2009A Computational Platform for the Optimization of Fermentation Processes.Proc.of the European Simulation and Modelling Conference(ESM2009).

Rozkov,A.,Enfors,S.O.,1999.Stabilization of a proteolytically sensitive cytoplasmic recombinant protein during transition to downstream processing.Biotechnol-ogy and Bioengineering62,730–738.

Rozkov,A.,Schweder,T.,Veide,A.,Enfors,S.O.,2000.Dynamics of proteolysis and its in?uence on the accumulation of intracellular recombinant proteins.Enzyme and Microbial Technology27,743–748.

Rozkov,A.,vignone-Rossa,C.A.,Ertl,P.F.,Jones,P.,O’Kennedy,R.D.,Smith,J.J., Dale,J.W.,Bushell,M.E.,2004.Characterization of the metabolic burden on Escherichia coli DH1cells imposed by the presence of a plasmid containing a gene therapy sequence.Biotechnology and Bioengineering88,909–915. Sandoval-Basurto, E.A.,Gosset,G.,Bolivar, F.,Ramirez,O.T.,2005.Culture of Escherichia coli under dissolved oxygen gradients simulated in a two-compartment scale-down system:metabolic response and produc-tion of recombinant protein.Biotechnology and Bioengineering89,453–463.

Sarkandy,S.Y.,Khalilzadeh,R.,Shojaosadati,S.A.,Sadeghizadeh,M.,Farnoud,A.M., Babaeipour,V.,Maghsoudi,A.,2010.A novel amino acid supplementation strat-egy based on a stoichiometric model to enhance human IL-2(interleukin-2) expression in high-cell-density Escherichia coli cultures.Biotechnology and Applied Biochemistry57,151–156.

Sauer,U.,Cameron,D.C.,Bailey,J.E.,1998.Metabolic capacity of Bacillus subtilis for the production of purine nucleosides,ribo?avin,and folic acid.Biotechnology and Bioengineering59,227–238.

Saxena,P.,Walker,J.R.,1992.Expression of arg U,the Escherichia coli gene coding for

a rare arginine tRNA.Journal of Bacteriology174,1956–1964.

Schreyer,R.,Bock,A.,1980.Phosphoglucose isomerase from Escherischia coli K10: puri?cation,properties and formation under aerobic and anaerobic condition.

Archives of Microbiology127,289–298.

Schweder,T.,Lin,H.Y.,Jurgen,B.,Breitenstein,A.,Riemschneider,S.,Khalameyzer, V.,Gupta,A.,Buttner,K.,Neubauer,P.,2002.Role of the general stress response during strong overexpression of a heterologous gene in Escherichia coli.Applied Microbiology and Biotechnology58,330–337.

Seo,Jin D.,Chung,B.H.,Hwang,Y.B.,Park,Y.H.,1992.Glucose-limited fed-batch culture of Escherichia coli for production of recombinant human interleukin-2with the DO-stat method.Journal of Fermentation and Bioengineering74, 196–198.

Seo,J.H.,Bailey,J.E.,1986.Continuous cultivation of recombinant Escherichia coli: Existence of an optimum dilution rate for maximum plasmid and gene product concentration.Biotechnology and Bioengineering28,1590–1594.

Seo,J.H.,Bailey,J.E.,1985.Effects of recombinant plasmid content on growth prop-erties and cloned gene product formation in Escherichia coli.Biotechnology and Bioengineering27,1668–1674.

Shiloach,J.,Kaufman,J.,Guillard,A.S.,Fass,R.,1996.Effect of glucose supply strat-egy on acetate accumulation,growth,and recombinant protein production by Escherichia coli BL21(?DE3)and Escherichia coli JM109.Biotechnology and Bio-engineering49,421–428.

Shimizu,N.,Fukuzono,S.,Fujimori,K.,Nishimura,N.,Odawara,Y.,1988.Fed-batch cultures of recombinant Escherichia coli with inhibitory substance concentration monitoring.Journal of Fermentation Technology66,187–191.

Silva,F.,Queiroz,J.A.,Domingues,F.C.,2012.Evaluating metabolic stress and plasmid stability in plasmid DNA production by Escherichia coli.Biotechnology Advances 30,691–708.

Siurkus,J.,Neubauer,P.,2011.Heterologous production of active ribonuclease inhibitor in Escherichia coli by redox state control and chaperonin coexpression.

Microbial Cell Factories10,65.

408S.Carneiro et al./Journal of Biotechnology164 (2013) 396–408

Son,Y.J.,Phue,J.N.,Trinh,L.B.,Lee,S.J.,Shiloach,J.,2011.The role of Cra in regulating acetate excretion and osmotic tolerance in E.coli K-12and E.coli B at high density growth.Microbial Cell Factories10,52.

Sorensen,H.P.,Mortensen,K.K.,2005.Advanced genetic strategies for recom-binant protein expression in Escherichia coli.Journal of Biotechnology115, 113–128.

Spanjaard,R.A.,Chen,K.,Walker,J.R.,van,D.J.,1990.Frameshift suppression at tan-dem AGA and AGG codons by cloned tRNA genes:assigning a codon to argU tRNA and T4tRNA(Arg).Nucleic Acids Research18,5031–5036.

Suarez,D.C.,Kilikian,B.V.,2000.Acetic acid accumulation in aerobic growth of recombinant Escherichia coli.Process Biochemistry35,1051–1055. Summers,D.K.,Beton,C.W.,Withers,H.L.,1993.Multicopy plasmid instability:the dimer catastrophe hypothesis.Molecular Microbiology8,1031–1038.

Tao,Y.,Cheng,Q.,Kopatsis,A.D.,2012.Metabolic engineering for acetate control in large scale fermentation.Methods in Molecular Biology834,283–303. Turner,C.,Gregory,M.E.,Turner,M.K.,1994.A study of the effect of speci?c growth rate and acetate on recombinant protein production of Escherichia coli JM107.

Biotechnology Letters16,891–896.

Ukkonen,K.,Vasala,A.,Ojamo,H.,Neubauer,P.,2011.High-yield production of bio-logically active recombinant protein in shake?ask culture by combination of enzyme-based glucose delivery and increased oxygen transfer.Microbial Cell Factories10,107.

Usui,Y.,Hirasawa,T.,Furusawa,C.,Shirai,T.,Yamamoto,N.,Mori,H.,Shimizu,H., 2012.Investigating the effects of perturbations to pgi and eno gene expression on central carbon metabolism in Escherichia coli using13C metabolic?ux analysis.

Microbial Cell Factories11,87.

Valgepea,K.,Adamberg,K.,Nahku,R.,Lahtvee,P.J.,Arike,L.,Vilu,R.,2010.Systems biology approach reveals that over?ow metabolism of acetate in Escherichia coli is triggered by carbon catabolite repression of acetyl-CoA synthetase.BMC Sys-tems Biology4,166.

Van de Walle,M.,Shiloach,J.,1998.Proposed mechanism of acetate accumulation in two recombinant Escherichia coli strains during high density fermentation.

Biotechnology and Bioengineering57,71–78.

Varma,A.,Palsson,B.O.,1994.Metabolic?ux balancing–basic concepts,scienti?c and practical use.Bio-Technology12,994–998.

Vidal,L.,Pinsach,J.,Striedner,G.,Caminal,G.,Ferrer,P.,2008.Development of an antibiotic-free plasmid selection system based on glycine auxotrophy for recom-binant protein overproduction in Escherichia coli.Journal of Biotechnology134, 127–136.Wang,Y.H.,Wu,S.L.,Hancock,W.S.,Trala,R.,Kessler,M.,Taylor,A.H.,Patel,P.S., Aon,J.C.,2005.Proteomic pro?ling of Escherichia coli proteins under high cell density fed-batch cultivation with overexpression of phosphogluconolactonase.

Biotechnology Progress21,1401–1411.

Wang,Z.J.,Li,X.A.,Shao,J.J.,Wegrzyn,A.,Wegrzyn,G.,2006.Effects of the presence of CoIEI plasmid DNA in Escherichia coli on the host cell metabolism.Microbial Cell Factories5.

Wei,X.X.,Shi,Z.Y.,Li,Z.J.,Cai,L.,Wu,Q.,Chen,G.Q.,2010.A mini-Mu transposon-based method for multiple DNA fragment integration into bacterial genomes.

Applied Microbiology and Biotechnology87,1533–1541.

Wittmann,C.,Weber,J.,Betiku,E.,Kromer,J.,Bohm,D.,Rinas,U.,2007.Response of?uxome and metabolome to temperature-induced recombinant protein syn-thesis in Escherichia coli.Journal of Biotechnology132,375–384.

Wong,M.S.,Wu,S.,Causey,T.B.,Bennett,G.N.,San,K.Y.,2008.Reduction of acetate accumulation in Escherichia coli cultures for increased recombinant protein pro-duction.Metabolic Engineering10,97–108.

Xu,B.,Jahic,M.,Enfors,S.O.,1999.Modeling of over?ow metabolism in batch and fed-batch cultures of Escherichia coli.Biotechnology Progress15,81–90. Yang,B.,Guo,Z.,Huang,Y.,Zhu,S.,2004.Codon optimization of MTS1and its expression in Escherichia coli.Protein Expression and Puri?cation36,307–311.

Yang,Y.T.,Aristidou,A.A.,San,K.Y.,Bennett,G.N.,1999a.Metabolic?ux analysis of Escherichia coli de?cient in the acetate production pathway and expressing the Bacillus subtilis acetolactate synthase.Metabolic Engineering1,26–34.

Yang,Y.T.,Bennett,G.N.,San,K.Y.,1999b.Effect of inactivation of nuo and ack A-pta on redistribution of metabolic?uxes in Escherichia coli.Biotechnology and Bioengineering65,291–297.

Yoon,S.H.,Han,M.J.,Lee,S.Y.,Jeong,K.J.,Yoo,J.S.,https://www.wendangku.net/doc/1b11065581.html,bined transcriptome and proteome analysis of Escherichia coli during high cell density culture.Biotech-nology and Bioengineering81,753–767.

Yu,C.,Cao,Y.J.,Zou,H.B.,Xian,M.,2011.Metabolic engineering of Escherichia coli for biotechnological production of high-value organic acids and alcohols.Applied Microbiology and Biotechnology89,573–583.

Zhou,Z.,Schnake,P.,Xiao,L.,Lal,A.A.,2004.Enhanced expression of a recombi-nant malaria candidate vaccine in Escherichia coli by codon optimization.Protein Expression and Puri?cation34,87–94.

Zhu,J.F.,Sanchez,A.,Bennett,G.N.,San,K.Y.,2011.Manipulating respiratory levels in Escherichia coli for aerobic formation of reduced chemical products.Metabolic Engineering13,704–712.

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曹操《短歌行》其二翻译及赏析

曹操《短歌行》其二翻译及赏析 引导语:曹操(155—220),字孟德,小名阿瞒,《短歌行 二首》 是曹操以乐府古题创作的两首诗, 第一首诗表达了作者求贤若渴的心 态,第二首诗主要是曹操向内外臣僚及天下表明心迹。 短歌行 其二 曹操 周西伯昌,怀此圣德。 三分天下,而有其二。 修奉贡献,臣节不隆。 崇侯谗之,是以拘系。 后见赦原,赐之斧钺,得使征伐。 为仲尼所称,达及德行, 犹奉事殷,论叙其美。 齐桓之功,为霸之首。 九合诸侯,一匡天下。 一匡天下,不以兵车。 正而不谲,其德传称。 孔子所叹,并称夷吾,民受其恩。 赐与庙胙,命无下拜。 小白不敢尔,天威在颜咫尺。 晋文亦霸,躬奉天王。 受赐圭瓒,钜鬯彤弓, 卢弓矢千,虎贲三百人。 威服诸侯,师之所尊。 八方闻之,名亚齐桓。 翻译 姬昌受封为西伯,具有神智和美德。殷朝土地为三份,他有其中两分。 整治贡品来进奉,不失臣子的职责。只因为崇侯进谗言,而受冤拘禁。 后因为送礼而赦免, 受赐斧钺征伐的权利。 他被孔丘称赞, 品德高尚地位显。 始终臣服殷朝帝王,美名后世流传遍。齐桓公拥周建立功业,存亡继绝为霸 首。

聚合诸侯捍卫中原,匡正天下功业千秋。号令诸侯以匡周室,主要靠的不是 武力。 行为磊落不欺诈,美德流传于身后。孔子赞美齐桓公,也称赞管仲。 百姓深受恩惠,天子赐肉与桓公,命其无拜来接受。桓公称小白不敢,天子 威严就在咫尺前。 晋文公继承来称霸,亲身尊奉周天王。周天子赏赐丰厚,仪式隆重。 接受玉器和美酒,弓矢武士三百名。晋文公声望镇诸侯,从其风者受尊重。 威名八方全传遍,名声仅次于齐桓公。佯称周王巡狩,招其天子到河阳,因 此大众议论纷纷。 赏析 《短歌行》 (“周西伯昌”)主要是曹操向内外臣僚及天下表明心 迹,当他翦灭群凶之际,功高震主之时,正所谓“君子终日乾乾,夕惕若 厉”者,但东吴孙权却瞅准时机竟上表大说天命而称臣,意在促曹操代汉 而使其失去“挟天子以令诸侯”之号召, 故曹操机敏地认识到“ 是儿欲据吾著炉上郁!”故曹操运筹谋略而赋此《短歌行 ·周西伯 昌》。 西伯姬昌在纣朝三分天下有其二的大好形势下, 犹能奉事殷纣, 故孔子盛称 “周之德, 其可谓至德也已矣。 ”但纣王亲信崇侯虎仍不免在纣王前 还要谗毁文王,并拘系于羑里。曹操举此史实,意在表明自己正在克心效法先圣 西伯姬昌,并肯定他的所作所为,谨慎惕惧,向来无愧于献帝之所赏。 并大谈西伯姬昌、齐桓公、晋文公皆曾受命“专使征伐”。而当 今天下时势与当年的西伯、齐桓、晋文之际颇相类似,天子如命他“专使 征伐”以讨不臣,乃英明之举。但他亦效西伯之德,重齐桓之功,戒晋文 之诈。然故作谦恭之辞耳,又谁知岂无更讨封赏之意乎 ?不然建安十八年(公元 213 年)五月献帝下诏曰《册魏公九锡文》,其文曰“朕闻先王并建明德, 胙之以土,分之以民,崇其宠章,备其礼物,所以藩卫王室、左右厥世也。其在 周成,管、蔡不静,惩难念功,乃使邵康公赐齐太公履,东至于海,西至于河, 南至于穆陵,北至于无棣,五侯九伯,实得征之。 世祚太师,以表东海。爰及襄王,亦有楚人不供王职,又命晋文登为侯伯, 锡以二辂、虎贲、斧钺、禾巨 鬯、弓矢,大启南阳,世作盟主。故周室之不坏, 系二国是赖。”又“今以冀州之河东、河内、魏郡、赵国、中山、常 山,巨鹿、安平、甘陵、平原凡十郡,封君为魏公。锡君玄土,苴以白茅,爰契 尔龟。”又“加君九锡,其敬听朕命。” 观汉献帝下诏《册魏公九锡文》全篇,尽叙其功,以为其功高于伊、周,而 其奖却低于齐、晋,故赐爵赐土,又加九锡,奖励空前。但曹操被奖愈高,心内 愈忧。故曹操在曾早在五十六岁写的《让县自明本志令》中谓“或者人见 孤强盛, 又性不信天命之事, 恐私心相评, 言有不逊之志, 妄相忖度, 每用耿耿。

2008年浙师大《外国文学名著鉴赏》期末考试答案

(一)文学常识 一、古希腊罗马 1.(1)宙斯(罗马神话称为朱庇特),希腊神话中最高的天神,掌管雷电云雨,是人和神的主宰。 (2)阿波罗,希腊神话中宙斯的儿子,主管光明、青春、音乐、诗歌等,常以手持弓箭的少年形象出现。 (3)雅典那,希腊神话中的智慧女神,雅典城邦的保护神。 (4)潘多拉,希腊神话中的第一个女人,貌美性诈。私自打开了宙斯送她的一只盒子,里面装的疾病、疯狂、罪恶、嫉妒等祸患,一齐飞出,只有希望留在盒底,人间因此充满灾难。“潘多拉的盒子”成为“祸灾的来源”的同义语。 (5)普罗米修斯,希腊神话中造福人间的神。盗取天火带到人间,并传授给人类多种手艺,触怒宙斯,被锁在高加索山崖,受神鹰啄食,是一个反抗强暴、不惜为人类牺牲一切的英雄。 (6)斯芬克司,希腊神话中的狮身女怪。常叫过路行人猜谜,猜不出即将行人杀害;后因谜底被俄底浦斯道破,即自杀。后常喻“谜”一样的人物。与埃及狮身人面像同名。 2.荷马,古希腊盲诗人。主要作品有《伊利亚特》和《奥德赛》,被称为荷马史诗。《伊利亚特》叙述十年特洛伊战争。《奥德赛》写特洛伊战争结束后,希腊英雄奥德赛历险回乡的故事。马克思称赞它“显示出永久的魅力”。 3.埃斯库罗斯,古希腊悲剧之父,代表作《被缚的普罗米修斯》。6.阿里斯托芬,古希腊“喜剧之父”代表作《阿卡奈人》。 4.索福克勒斯,古希腊重要悲剧作家,代表作《俄狄浦斯王》。5.欧里庇得斯,古希腊重要悲剧作家,代表作《美狄亚》。 二、中世纪文学 但丁,意大利人,伟大诗人,文艺复兴的先驱。恩格斯称他是“中世纪的最后一位诗人,同时又是新时代的最初一位诗人”。主要作品有叙事长诗《神曲》,由地狱、炼狱、天堂三部分组成。《神曲》以幻想形式,写但丁迷路,被人导引神游三界。在地狱中见到贪官污吏等受着惩罚,在净界中见到贪色贪财等较轻罪人,在天堂里见到殉道者等高贵的灵魂。 三、文艺复兴时期 1.薄迦丘意大利人短篇小说家,著有《十日谈》拉伯雷,法国人,著《巨人传》塞万提斯,西班牙人,著《堂?吉诃德》。 2.莎士比亚,16-17世纪文艺复兴时期英国伟大的剧作家和诗人,主要作品有四大悲剧——《哈姆雷特》、《奥赛罗》《麦克白》、《李尔王》,另有悲剧《罗密欧与朱丽叶》等,喜剧有《威尼斯商人》《第十二夜》《皆大欢喜》等,历史剧有《理查二世》、《亨利四世》等。马克思称之为“人类最伟大的戏剧天才”。 四、17世纪古典主义 9.笛福,17-18世纪英国著名小说家,被誉为“英国和欧洲小说之父”,主要作品《鲁滨逊漂流记》,是英国第一部现实主义长篇小说。10.弥尔顿,17世纪英国诗人,代表作:长诗《失乐园》,《失乐园》,表现了资产阶级清教徒的革命理想和英雄气概。 25.拉伯雷,16世纪法国作家,代表作:长篇小说《巨人传》。 26.莫里哀,法国17世纪古典主义文学最重要的作家,法国古典主义喜剧的创建者,主要作品为《伪君子》《悭吝人》(主人公叫阿巴公)等喜剧。 五、18世纪启蒙运动 1)歌德,德国文学最高成就的代表者。主要作品有书信体小说《少年维特之烦恼》,诗剧《浮士德》。 11.斯威夫特,18世纪英国作家,代表作:《格列佛游记》,以荒诞的情节讽刺了英国现实。 12.亨利·菲尔丁,18世纪英国作家,代表作:《汤姆·琼斯》。 六、19世纪浪漫主义 (1拜伦, 19世纪初期英国伟大的浪漫主义诗人,代表作为诗体小说《唐璜》通过青年贵族唐璜的种种经历,抨击欧洲反动的封建势力。《恰尔德。哈洛尔游记》 (2雨果,伟大作家,欧洲19世纪浪漫主义文学最卓越的代表。主要作品有长篇小说《巴黎圣母院》、《悲惨世界》、《笑面人》、《九三年》等。《悲惨世界》写的是失业短工冉阿让因偷吃一片面包被抓进监狱,后改名换姓,当上企业主和市长,但终不能摆脱迫害的故事。《巴黎圣母院》 弃儿伽西莫多,在一个偶然的场合被副主教克洛德.孚罗洛收养为义子,长大后有让他当上了巴黎圣母院的敲钟人。他虽然十分丑陋而且有多种残疾,心灵却异常高尚纯洁。 长年流浪街头的波希米亚姑娘拉.爱斯梅拉达,能歌善舞,天真貌美而心地淳厚。青年贫诗人尔比埃尔.甘果瓦偶然同她相遇,并在一个更偶然的场合成了她名义上的丈夫。很有名望的副教主本来一向专心于"圣职",忽然有一天欣赏到波希米亚姑娘的歌舞,忧千方百计要把她据为己有,对她进行了种种威胁甚至陷害,同时还为此不惜玩弄卑鄙手段,去欺骗利用他的义子伽西莫多和学生甘果瓦。眼看无论如何也实现不了占有爱斯梅拉达的罪恶企图,最后竟亲手把那可爱的少女送上了绞刑架。 另一方面,伽西莫多私下也爱慕着波希米亚姑娘。她遭到陷害,被伽西莫多巧计救出,在圣母院一间密室里避难,敲钟人用十分纯朴和真诚的感情去安慰她,保护她。当她再次处于危急中时,敲钟人为了援助她,表现出非凡的英勇和机智。而当他无意中发现自己的"义父"和"恩人"远望着高挂在绞刑架上的波希米亚姑娘而发出恶魔般的狞笑时,伽西莫多立即对那个伪善者下了最后的判决,亲手把克洛德.孚罗洛从高耸入云的钟塔上推下,使他摔的粉身碎骨。 (3司汤达,批判现实主义作家。代表作《红与黑》,写的是不满封建制度的平民青年于连,千方百计向上爬,最终被送上断头台的故事。“红”是将军服色,指“入军界”的道路;“黑”是主教服色,指当神父、主教的道路。 14.雪莱,19世纪积极浪漫主义诗人,欧洲文学史上最早歌颂空想社会主义的诗人之一,主要作品为诗剧《解放了的普罗米修斯》,抒情诗《西风颂》等。 15.托马斯·哈代,19世纪英国作家,代表作:长篇小说《德伯家的苔丝》。 16.萨克雷,19世纪英国作家,代表作:《名利场》 17.盖斯凯尔夫人,19世纪英国作家,代表作:《玛丽·巴顿》。 18.夏洛蒂?勃朗特,19世纪英国女作家,代表作:长篇小说《简?爱》19艾米丽?勃朗特,19世纪英国女作家,夏洛蒂?勃朗特之妹,代表作:长篇小说《呼啸山庄》。 20.狄更斯,19世纪英国批判现实主义文学的重要代表,主要作品为长篇小说《大卫?科波菲尔》、《艰难时世》《双城记》《雾都孤儿》。21.柯南道尔,19世纪英国著名侦探小说家,代表作品侦探小说集《福尔摩斯探案》是世界上最著名的侦探小说。 七、19世纪现实主义 1、巴尔扎克,19世纪上半叶法国和欧洲批判现实主义文学的杰出代表。主要作品有《人间喜剧》,包括《高老头》、《欧也妮·葛朗台》、《贝姨》、《邦斯舅舅》等。《人间喜剧》是世界文学中规模最宏伟的创作之一,也是人类思维劳动最辉煌的成果之一。马克思称其“提供了一部法国社会特别是巴黎上流社会的卓越的现实主义历史”。

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