Average maximum growth rate, carbon balance and redox balance for batch cultures of the investigated strains.
Since the development of recombinant DNA technology (Cohen et al., 1973), it became possible to express heterologous genes in pro- or eukaryotic hosts,
It is clear that recombinant protein production has evolved to one of the most important branches in modern biotechnology, representing a billion-dollar business, both in the production of biopharmaceuticals and industrial enzymes.
A pivotal choice in the design of a recombinant protein bioprocess is the selection of a suitable host strain. This selection is influenced by different factors: (i) ease of cultivation and growth characteristics, (ii) ease of genetic manipulation and availability of molecular tools, (iii) ability of post-translational modifications (e.g. glycosylation patterns, disulfide bond formation), (iv) downstream processing, and (v) regulatory aspects (generally regarded as safe, SAFE (Lotti et al., 2004; Sahdev et al., 2008; Durocher & Butler, 2009).
These aspects will determine whether the designed recombinant protein bioprocess will end up in an economical viable bioprocess which can compete with the present process.
In contrast to biopharmaceuticals, industrial enzyme bioprocesses are only economical viable as a low production cost is assured. This implies that higher yields, titres and production rates are necessary which can only be obtained by fast growing organisms. This is reflected by the distribution of the most commonly used organisms in these two industries. Whereas slow growing organisms as plants and animals are used as host in half of the biopharmaceutical processes, they count only for 12% of the processes in the industrial enzyme market (Demain & Vaishnav, 2009; Ferrer-Miralles et al., 2009). Bacteria on the other hand, have a market share of 30% in both industries. However, yeasts and molds, which grow much faster in comparison with higher eukaryotes, are used in 58 % of the cases in the industrial enzyme market and only in 18% of the cases in the in the biopharmaceutical market.
Several bacteria have been explored as host for recombinant protein production. Recently, much interest is raised in the use of
Escherichia colifor recombinant protein production
Besides the advantage of many available molecular tools, the easily cultivable and genetically and metabolically well-known
The production of heterologous proteins to high titres concurs mostly with the initiation of a stress response and/or metabolic burden, both associated with the use of multi-copy plasmids, resulting in misfolding and degradation of the heterologous protein and formation of inclusion bodies (Noack et al., 1981; Parsell & Sauer, 1989; Bentley et al., 1990; Gill et al., 2000; Hoffmann & Rinas, 2004; Ventura & Villaverde, 2006).
Secretory production of recombinant proteins into the culture medium includes several advantages, especially in cases of toxic recombinant proteins. However, compared to other hosts,
The main difficulty when using
Many efforts have been made to overcome these hurdles and hence to increase recombinant protein production in
The primarily used approach to produce recombinant proteins is to clone the gene of interest on a multi-copy plasmid under the control of a strong promoter in order to achieve high transcription rates and hence high recombinant protein concentrations. However, problems such as metabolic burden, segregational instability, misfolding and proteolytic breakdown or aggregation in inclusion bodies, and difficulties in controlling gene expression are usually associated with multi-copy plasmids and the use of strong promoters (Noack et al., 1981; Parsell & Sauer, 1989; Bentley et al., 1990; Dong et al., 1995; Kurland & Dong, 1996; Gill et al., 2000; Hoffmann & Rinas, 2004; Ventura & Villaverde, 2006). Most engineering strategies to tackle these problems focus on prevention of misfolding, neutralisation of increased protease activity or stress response (Chou, 2007). An elaborated review of these efforts is given in (Waegeman & Soetaert, 2011).
Two post-translational modifications which are pivotal for the stability and activity of many more complex eukaryotic proteins are disulfide bonds and glycosylation. The former is being facilitated in
Besides the proper formation of disulfide bonds,
The secretory production of recombinant proteins into the fermentation broth includes several advantages compared to cytoplasmic production. Although
3. An alternative approach to reduce acetate production and improve recombinant protein production in
Throughout the years, various
Many different strategies have been applied to increase recombinant protein formation and decrease acetate formation in
The first, rational effort to decrease acetate production is to block the acetate pathway by knocking out genes that encode for acetate pathway enzymes, e.g.
A second widely followed approach to minimise acetate formation during high cell density fermentations is to limit rapid uptake of glucose causing overflow metabolism. Overflow metabolism occurs when high glycolytic fluxes, due to rapid glucose uptake, are not further processed in the TCA cycle developing a bottleneck at the pyruvate node and consequently pyruvate is converted to acetate.
Strategies based on optimising the bioprocess conditions to reduce the glucose uptake rate comprise applying specific glucose feeding patterns, the application of alternative substrates, the addition of supplements to the medium, the control of a range of fermentation parameters and the application of systems to remove acetate from the fermentation broth (Farmer & Liao, 1997; Nakano et al., 1997; Akesson et al., 1999; Akesson et al., 2001b; Akesson et al., 2001a; Fuchs et al., 2002 ; Chen et al., 2005; Eiteman & Altman, 2006). Although all these attempts were in many cases successful to reduce acetate production, they imply a severe lower growth rate and they do not utilise the full potential of the microbial host.
Engineering of the glucose uptake system is being successfully applied as well to overcome overflow metabolism. By deleting one of the phosphotransferase system genes, e.g.
A third approach to overcome overflow metabolism is to redirect the fluxes around the bottleneck, the phosphoenolpyruvate-pyruvate-oxaloacetate node, instead of restricting the glucose uptake. Farmer & Liao (1997) increased anaplerotic and glycolate fluxes by overexpressing phosphenolpyruvate carboxylase (encoded by
An alternative approach to enhance recombinant protein production is mimicking the
3.1. Influence of transcriptional regulators ArcA and IclR on
Regulation of gene expression is very complex and transcriptional regulators can be subdivided in global and local regulators depending on the number of operons they control. Global regulators control a vast number of genes, which must be physically separated on the genome and belong to different metabolic pathways (Gottesman, 1984). According to EcoCyc (Keseler et al., 2011)
The global regulator ArcA (anaerobic redox control) was first discovered in 1988 by Iuchi and Lin and the regulator seemed to have an inhibitory effect on expression of aerobic TCA cycles genes under anaerobic conditions (Iuchi & Lin, 1988). Later on, it was unravelled that ArcA is a component of the dual-component regulator ArcAB, in which ArcA is the regulatory protein and ArcB acts as sensory protein (Iuchi et al., 1990).
Acording to EcoCyc (Keseler et al., 2011) ArcA is involved in the regulation of 168 genes and itself is regulated by 2 regulators (FnrR, RpoD). Statistical analysis of gene expression data (Salmon et al., 2005) showed that ArcA regulates the expression of a wide variety of genes involved in the biosynthesis of small macromolecules, transport, carbon and energy metabolism, cell structure, etc. The regulatory activity of ArcA is dependent on the oxygen concentration in the environment. The most profound effects of ArcA are noticed under microaerobic conditions (Alexeeva et al., 2003) but recently it was reported that also under aerobic conditions ArcA has an effect on central metabolic fluxes (Perrenoud & Sauer, 2005).
Similarly to the global transcriptional regulator ArcA, the local transcriptional regulator isocitrate lyase regulator IclR has a reductive effect on the flux through the TCA cycle (Rittinger et al., 1996). IclR represses the expression of the
As both transcriptional regulators, ArcA and IclR, are involved in controlling the flux through the TCA cycle and glyoxylate pathway, they are interesting targets for metabolic engineering for mimicking the
To investigate their effect, single knockouts as a knockout combination were made in
|E. coli strain||μmax(h -1)||Carbon (%)||Redox (%)|
|MG1655||0.66 ± 0.02||97||101|
|MG1655 ΔarcA||0.60 ± 0.01||96||94|
|MG1655 ΔiclR||0.61 ± 0.02||95||95|
|MG1655 ΔarcA ΔiclR||0.44 ± 0.03||99||101|
|BL21(DE3)||0.59 ± 0.02||93||99|
Product yields in c-mole/c-mole glucose for
The deletion of local transcriptional regulator
13C-metabolic flux analysis confirmed our hypothesis that the deletion of both
Moreover, similar central metabolic fluxes were observed in the combined
Thus, by deletion of a local and global transcriptional regulator, ArcA and IclR respectively, we could mimic the physiological and metabolic properties of
Escherichia coliMG1655 Δ arcAΔ iclRas potential candidate for recombinant protein production
Our previous research has shown that similar metabolic and physiological characteristics as
To investigate whether these metabolic alterations in
Batch cultures were performed in 2L stirred tank bioreactors. Yields are calculated by dividing GFP and biomass concentrations during the cultivation phase when biomass concentrations are higher than 2 gL-1. The values represented in the graph are the average of at least two separate experiments and the errors are the standard deviations calculated on the yields.
To our regret, the combined
Proteases play an important role in the degradation of foreign proteins (Gottesman & Maurizi, 1992) and it is generally believed that recombinant proteins are better produced in
Although also other proteases are known for the degradation of proteins, but in a lesser extent towards recombinant proteins (Jürgen et al., 2010) and since
The additional deletion of the proteases Lon and OmpT, resulting in the quadruple knockout strain
To date, recombinant protein production has evolved to one of the most important branches in modern biotechnology, representing a billion-dollar business, both in the production of biopharmaceuticals and industrial enzymes. Although many organisms have been used as host,
Logically, many endeavours have been reported to decrease acetate formation and increase recombinant protein production in this host. However, among the different
Traditionally, acetate formation in
In conclusion, by deleting only four genes, i.e.
The research of Hendrik Waegeman was financially supported by the Special Research Fund (BOF) of Ghent University
Abdallah A. M. van Pittius N. C. G. Champion P. A. D. Cox J. Luirink J. Vandenbroucke-Grauls C. M. J. E. Appelmelk B. J. Bitter W. 2007Type VII secretion-mycobacteria show the way.
Abu-Qarn M. Eichler J. Sharon N. 2008Not just for Eukarya anymore: protein glycosylation in Bacteria and Archaea.
Akesson M. Hagander P. Axelsson J. P. 2001aAvoiding acetate accumulation in Escherichia coli cultures using feedback control of glucose feeding.
Akesson M. Hagander P. Axelsson J. P. 2001bProbing control of fed-batch cultivations: analysis and tuning.
Akesson M. Karlsson E. N. Hagander P. Axelsson J. P. Tocaj A. 1999On-line detection of acetate formation in Escherichia coli cultures using dissolved oxygen responses to feed transients.
Alexeeva S. Hellingwerf K. J. de Mattos M. J. T. 2003Requirement of ArcA for redox regulation in Escherichia coli under microaerobic but not anaerobic or aerobic conditions.
Andersen K. B. von Meyenburg K. 1980Are growth rates of Escherichia coli in batch cultures limited by respiration?
Bentley W. E. Mirjalili N. Andersen D. C. Davis R. H. Kompala D. S. 1990Plasmid-encoded protein: the principal factor in the "metabolic burden" associated with recombinant bacteria.
Chen X. Cen P. Chen J. 2005Enhanced production of human epidermal growth factor by a recombinant Escherichia coli integrated with in situ exchange of acetic acid by macroporous ion-exchange resin.
Choi J. H. Lee S. Y. 2004Secretory and extracellular production of recombinant proteins using Escherichia coli.
Chou C. H. Bennett G. N. San K. Y. 1994Effect of modified glucose uptake using genetic engineering techniques on high-level recombinant protein production in Escherichia coli dense cultures.
Chou C. P. 2007Engineering cell physiology to enhance recombinant protein production in Escherichia coli.
Cohen S. N. Chang A. C. Boyer H. W. Helling R. B. 1973Construction of biologically functional bacterial plasmids in vitro.
Contiero J. Beatty C. Kumari S. De Santi C. Strohl W. Wolfe A. 2000Effects of mutations in acetate metabolism on high-cell-density growth of Escherichia coli.
Cortay J. C. Nègre D. Galinier A. Duclos B. Perrière G. Cozzone A. J. 1991Regulation of the acetate operon in Escherichia coli: purification and functional characterization of the IclR repressor.
Cozzone A. J. 1998Regulation of acetate metabolism by protein phosphorylation in enteric bacteria.
De Anda R. Lara A. R. Hernández V. Hernández-Montalvo V. Gosset G. Bolívar F. Ramírez O. T. 2006Replacement of the glucose phosphotransferase transport system by galactose permease reduces acetate accumulation and improves process performance of Escherichia coli for recombinant protein production without impairment of growth rate.
de Marco A. 2009Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli.
De Mey M. Lequeux G. J. Beauprez J. J. Maertens J. Van Horen E. Soetaert W. K. Vanrolleghem P. A. Vandamme E. J. 2007aComparison of different strategies to reduce acetate formation in Escherichia coli.
De Mey M. Lequeux G. J. Beauprez J. J. Maertens J. Waegeman H. J. Van Bogaert I. N. Foulquie-Moreno M. R. Charlier D. Soetaert W. K. Vanrolleghem P. A. Vandamme E. J. 2010Transient metabolic modeling of Escherichia coli MG1655 and MG1655 DeltaackA-pta, DeltapoxB Deltapppc ppc-p37 for recombinant beta-galactosidase production.
De Mey M. Maeseneire S. D. Soetaert W. Vandamme E. 2007bMinimizing acetate formation in E. coli fermentations.
Demain A. L. Vaishnav P. 2009Production of recombinant proteins by microbes and higher organisms.
Diaz-Ricci J. C. Regan L. Bailey J. E. 1991Effect of alteration of the acetic acid synthesis pathway on the fermentation pattern of Escherichia coli.
Dittrich C. R. Vadali R. V. Bennett G. N. San K.-Y. 2005Redistribution of metabolic fluxes in the central aerobic metabolic pathway of E. coli mutant strains with deletion of the ackA-pta and poxB pathways for the synthesis of isoamyl acetate.
Dong H. Nilsson L. Kurland C. G. 1995Gratuitous overexpression of genes in Escherichia coli leads to growth inhibition and ribosome destruction.
Durocher Y. Butler M. 2009Expression systems for therapeutic glycoprotein production.
Eiteman M. A. Altman E. 2006Overcoming acetate in Escherichia coli recombinant protein fermentations.
El-Mansi E. M. Holms W. H. 1989Control of carbon flux to acetate excretion during growth of Escherichia coli in batch and continuous cultures.
El-Mansi M. Cozzone A. J. Shiloach J. Eikmanns B. J. 2006Control of carbon flux through enzymes of central and intermediary metabolism during growth of Escherichia coli on acetate.
Farmer W. R. Liao J. C. 1997Reduction of aerobic acetate production by Escherichia coli.
Ferrer-Miralles N. Domingo-Espín J. Corchero J. L. Vázquez E. Villaverde A. 2009Microbial factories for recombinant pharmaceuticals.
Fischer E. Sauer U. 2003A novel metabolic cycle catalyzes glucose oxidation and anaplerosis in hungry Escherichia coli.
Fisher A. C. Haitjema C. H. Guarino C. Çelik E. Endicott C. E. Reading C. A. Merritt J. H. Ptak A. C. Zhang S. De Lisa M. P. 2011Production of secretory and extracellular N-linked glycoproteins in Escherichia coli.
Fuchs C. Koster D. Wiebusch S. Mahr K. Eisbrenner G. Markl H. 2002Scale-up of dialysis fermentation for high cell density cultivation of Escherichia coli.
Gill R. T. Valdes J. J. Bentley W. E. 2000A comparative study of global stress gene regulation in response to overexpression of recombinant proteins in Escherichia coli.
Global Industry Analysts, I. 2011Global Industrial Enzymes, In:
Gottesman S. 1984Bacterial regulation: global regulatory networks.
Gottesman S. 1989Genetics of proteolysis in Escherichia coli.
Gottesman S. 1996Proteases and their targets in Escherichia coli.
Gottesman S. Maurizi M. R. 1992Regulation by proteolysis: energy-dependent proteases and their targets.
Hodgson J. 1994The changing bulk biocatalyst market.
Hoffmann F. Rinas U. 2004Stress induced by recombinant protein production in Escherichia coli.
Holms W. H. 1986The central metabolic pathways of Escherichia coli: relationship between flux and control at a branch point, efficiency of conversion to biomass, and excretion of acetate.
Iuchi S. Lin E. C. 1988arcA (dye), a global regulatory gene in Escherichia coli mediating repression of enzymes in aerobic pathways.
Iuchi S. Matsuda Z. Fujiwara T. Lin E. C. 1990The arcB gene of Escherichia coli encodes a sensor-regulator protein for anaerobic repression of the arc modulon.
Jensen E. B. Carlsen S. 1990Production of recombinant human growth hormone in Escherichia coli: expression of different precursors and physiological effects of glucose, acetate, and salts.
Jong W. S. P. Saurí A. Luirink J. 2010Extracellular production of recombinant proteins using bacterial autotransporters.
Jürgen B. Breitenstein A. Urlacher V. Büttner K. Lin H. Hecker M. Schweder T. Neubauer P. 2010Quality control of inclusion bodies in Escherichia coli.
Keseler I. M. Collado-Vides J. Santos-Zavaleta A. Peralta-Gil M. Gama-Castro S. Muniz-Rascado L. Bonavides-Martinez C. Paley S. Krummenacker M. Altman T. Kaipa P. Spaulding A. Pacheco J. Latendresse M. Fulcher C. Sarker M. Shearer A. G. Mackie A. Paulsen I. Gunsalus R. P. Karp P. D. 2011EcoCyc: a comprehensive database of Escherichia coli biology.
Ko C.-H. Tsai C.-H. Lin P.-H. Chang K.-C. Tu J. Wang Y.-N. Yang C.-Y. 2010 Characterization and pulp refining activity of a Paenibacillus campinasensis cellulase expressed in Escherichia coli.
Kurland C. G. Dong H. 1996Bacterial growth inhibition by overproduction of protein.
Lotti M. Porro D. Srienc F. 2004Recombinant proteins and host cell physiology.
Maharjan R. P. Yu P.-L. Seeto S. Ferenci T. 2005The role of isocitrate lyase and the glyoxylate cycle in Escherichia coli growing under glucose limitation.
Makrides S. C. 1996Strategies for achieving high-level expression of genes in Escherichia coli.
March J. C. Eiteman M. A. Altman E. 2002Expression of an anaplerotic enzyme, pyruvate carboxylase, improves recombinant protein production in Escherichia coli.
Martinez-Antonio A. Collado-Vides J. 2003Identifying global regulators in transcriptional regulatory networks in bacteria.
Nakano K. Rischke M. Sato S. Märkl H. 1997Influence of acetic acid on the growth of Escherichia coli K12 during high-cell-density cultivation in a dialysis reactor.
Noack D. Roth M. Geuther R. Muller G. Undisz K. Hoffmeier C. Gaspar S. 1981Maintenance and genetic stability of vector plasmids pBR322 and pBR325 in Escherichia coli K12 strains grown in a chemostat.
Noronha S. B. Yeh H. J. Spande T. F. Shiloach J. 2000Investigation of the TCA cycle and the glyoxylate shunt in Escherichia coli BL21 and JM109 using (13)C-NMR/MS.
Parsell D. A. Sauer R. T. 1989Induction of a heat shock-like response by unfolded protein in Escherichia coli: dependence on protein level not protein degradation.
Perrenoud A. Sauer U. 2005Impact of global transcriptional regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on glucose catabolism in Escherichia coli.
Phue J.-N. Noronha S. B. Ritabrata Wolfe A. J. Shiloach J. 2005Glucose metabolism at high density growth of E. coli B and E. coli K: differences in metabolic pathways are responsible for efficient glucose utilization in E. coli B as determined by microarrays and Northern blot analyses.
Rittinger K. Negre D. Divita G. Scarabel M. Bonod-Bidaud C. Goody R. S. Cozzone A. J. Cortay J. C. 1996Escherichia coli isocitrate dehydrogenase kinase/phosphatase.
Ryu K. Kim K.-H. Yoo S.-Y. Lee E.-Y. Lim K.-H. Min M.-K. Kim H. Choi S. I. Seong B. L. 2010Production and characterization of active hepatitis C virus RNA-dependent RNA polymerase.
Sahdev S. Khattar S. K. Saini K. S. 2008Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies.
Salmon K. A. Hung S.-p. Steffen N. R. Krupp R. Baldi P. Hatfield G. W. Gunsalus R. P. 2005Global gene expression profiling in Escherichia coli K12: effects of oxygen availability and ArcA.
Shiloach J. Kaufman J. Guillard A. S. Fass R. 1996Effect of glucose supply strategy on acetate accumulation, growth, and recombinant protein production by Escherichia coli BL21 ($\lambda$DE3) and Escherichia coli JM109.
Siguenza R. Flores N. Hernandez G. Martinez A. Bolivar F. Valle F. 1999Kinetic characterization in batch and continuous culture of Escherichia coli mutants affected in phosphoenolpyruvate metabolism: differences in acetic acid production.
Striedner G. Pfaffenzeller I. Markus L. Nemecek S. Grabherr R. Bayer K. 2010Plasmid-free T7-based Escherichia coli expression systems.
Szymanski C. M. Yao R. Ewing C. P. Trust T. J. Guerry P. 1999Evidence for a system of general protein glycosylation in Campylobacter jejuni.
Terpe K. 2006Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems.
Tseng T.-T. Tyler B. M. Setubal J. C. 2009Protein secretion systems in bacterial-host associations, and their description in the Gene Ontology.
Varma A. Boesch B. W. Palsson B. O. 1993aBiochemical production capabilities of Escherichia coli.
Varma A. Boesch B. W. Palsson B. O. 1993bStoichiometric interpretation of Escherichia coli glucose catabolism under various oxygenation rates.
Ventura S. Villaverde A. 2006Protein quality in bacterial inclusion bodies.
Wacker M. Linton D. Hitchen P. G. Nita-Lazar M. Haslam S. M. North S. J. Panico M. Morris H. R. Dell A. Wren B. W. Aebi M. 2002N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli.
Waegeman H. Beauprez J. Moens H. Maertens J. De Mey M. Foulquie-Moreno M. R. Heijnen J. J. Charlier D. Soetaert W. 2011aEffect of iclR and arcA knockouts on biomass formation and metabolic fluxes in Escherichia coli K12 and its implications on understanding the metabolism of Escherichia coli BL21 (DE3).
Waegeman H. De Lausnay S. Beauprez J. Maertens J. De Mey M. Soetaert W. 2011bIncreasing recombinant protein production in Escherichia coli K12 through metabolic engineering.
Waegeman H. Maertens J. Beauprez J. De Mey M. Soetaert W. 2011c Effect of iclR and arcA deletion on physiology and metabolic fluxes in Escherichia coli BL21(DE3).
Waegeman H. Soetaert W. 2011 Increasing recombinant protein production in Escherichia coli through metabolic and genetic engineering.
Walsh G. 2010Post-translational modifications of protein biopharmaceuticals.
Wong M. S. Wu S. Causey T. B. Bennett G. N. San K.-Y. 2008Reduction of acetate accumulation in Escherichia coli cultures for increased recombinant protein production.
Yamamoto K. Ishihama A. 2003Two different modes of transcription repression of the Escherichia coli acetate operon by IclR.
Yang Y. T. Aristidou A. A. San K. Y. Bennett G. N. 1999Metabolic flux analysis of Escherichia coli deficient in the acetate production pathway and expressing the Bacillus subtilis acetolactate synthase.