Increasing recombinant protein production in Escherichia coli K12 by increasing the biomass yield of the host cell

摘要

For more than three decades micro-organisms have been employed as hosts for recombinant protein production, with the most popular organisms being Escherichia coli and Saccharomyces cerevisiae (1). One of the crucial factors to obtain high product yields in recombinant protein bioprocesses is the biomass yield of the host cell. High biomass yields not only result in less carbon loss and higher conversion to recombinant protein due to a potential higher drain of precursors, but are also accompanied by lower conversion to growth inhibiting byproducts, such as acetate (2). Furthermore, acidic byproducts hinder the expression of heterologous proteins (3) and consequently decrease protein yield in a direct and indirect manner. Many strategies have been tested to decrease the amount of acetate produced, including optimal feeding, choice of other carbon sources and metabolic engineering (4). Fed-batch and continuous feeding strategies result in low residual glucose concentrations and minimize overflow metabolism (’Crabtree effect’) (5; 6). Aristidou and coworkers improved biomass yield and protein production by using fructose as a primary carbon source without greatly affecting the fermentation cost (7). A third strategy is to alter the genetic machinery. Knocking out genes that code for acetate producing pathways, i.e. acetate kinase-phosphate acetyltransferase (ackA-pta) and pyruvate oxidase (poxB ) decrease acetate yield dramatically, but at the expense of lactate and pyruvate (8). The objective of this study was to focus on the combined effect of a global and a local regulator to increase biomass yield and hence recombinant protein production using GFP as a biomarker. Deletion of arcA reduces the repression on expression of TCA cycle genes (9) while deletion of iclR removes the repression on the aceBAK operon and opens the glyoxylate pathway (10; 11) in aerobic batch cultivations. This metabolic engineering approach simultaneously decreased the acetate yield with 70% and increased the biomass yield of the host cell with 50%. Due to a lower carbon loss and a lower inhibition of protein production by acetate, the GFP production of the ∆arcA∆iclR double knockout strain increased with 100% as opposed to the wild type E. coli K12. Further deletion of genes lon and ompT encoding for non-specific proteases even further increases GFP-production (3 times the wild type value). The effect of a deletion of arcA and iclR was also evaluated in a E. coli BL21 genetic background. However in this industrial strain the deletion had no effect on protein production. References [1] Ferrer-Miralles N, Domingo-Esp ́ J, Corchero JL, V ́zquez E, Villaverde A: Microbial factories for recombinant pharmaceuticals. Microb Cell Fact 2009, 8:17 [2] El-Mansi EM, Holms WH: Control of carbon flux to acetate excretion during growth of Escherichia coli in batch and continuous cultures. J Gen Microbiol 1989, 135(11):2875–2883. [3] Jensen EB, Carlsen S: Production of recombinant human growth hormone in Escherichia coli: expression of different precursors and physiological effects of glucose, acetate, and salts. Biotechnol Bioeng 1990, 36:1–11 [4] De Mey M, Maeseneire SD, Soetaert W, Vandamme E: Minimizing acetate formation in E. coli fermentations. J. Ind. Microbiol. Biotechnol. 2007, 34:689–700. [5] Babaeipour V, Shojaosadati SA, Khalilzadeh R, Maghsoudi N, Tabandeh F: A proposed feeding strategy for the overproduction of recombinant proteins in Escherichia coli. Biotechnol Appl Biochem 2008, 49(Pt 2):141–147. [6] San KY, Bennett GN, Aristidou AA, Chou CH: Strategies in high-level expression of recombinant protein in Escherichia coli. Ann N Y Acad Sci 1994, 721:257–267. [7] Aristidou AA, San KY, Bennett GN: Improvement of biomass yield and recombinant gene expression in Escherichia coli by using fructose as the primary carbon source. Biotechnol Prog 1999, 15:140–145. [8] De Mey M, Lequeux GJ, Beauprez JJ, Maertens J, Horen EV, Soetaert WK, Vanrolleghem PA, Vandamme EJ: Comparison of different strategies to reduce acetate formation in Escherichia coli. Biotechnol Prog 2007. [9] Perrenoud A, Sauer U: Impact of global transcriptional regulation by ArcA, ArcB, Cra,Crp, Cya, Fnr, and Mlc on glucose catabolism in Escherichia coli . J. Bacteriol. 2005, 187:3171–3179. [10] van de Walle M, Shiloach J: Proposed mechanism of acetate accumulation in two recombinant Escherichia coli strains during high density fermentation. Biotechnol Bioeng 1998, 57:71–78. [11] Maharjan RP, Yu PL, Seeto S, Ferenci T: The role of isocitrate lyase and the glyoxylate cycle in Escherichia coli growing under glucose limitation. Res Microbiol 2005, 156(2):178–183.