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

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

Minimizing acetate formation in E. coli fermentations

Escherichia coli remains the best established production organisms in industrial biotechnology. However, during aerobic fermentation runs at high growth rates, considerable amounts of acetate are accumulated as by-product. This by-product has negative effects on growth and protein production. Over the last 20 years, substantial research efforts have been spent to reduce acetate accumulation during aerobic growth of E. coli on glucose. From the onset it was clear that this quest should not be a simple nor uncomplicated one. Simple deletion of the acetate pathway, reduced the acetate accumulation, but instead other by-products were formed. This minireview gives a clear outline of these research efforts and the outcome of them, including bioprocess level approaches and genetic approaches. Recently, the latter seems to have some promising results

Transport kinetics of ectoine, an osmolyte produced by Brevibacterium epidermis

Brevibacterium epidermis DSM 20659 is a halotolerant Gram-positive bacterium which can synthesize the osmolyte, ectoine, but prefers to take it up from its environment. The present study revealed that B. epidermis is equipped with at least one transport system for ectoine, with a maximal transport velocity of 15.7 +/- 4.3 nmol/g CDW center dot min. The transport requires energy (ATP) and is completely inhibited by the proton uncoupler, CCCP. The ectoine uptake system is constitutively expressed at a basal level of activity and its activity is immediately 10-fold increased by hyper-osmotic stress. Initial uptake rates are not influenced by the intensity of the hyper-osmotic shock but the duration of the increased activity of the uptake system could be directly related to the osmotic strength of the assay solution. Competition assays indicate that betaine, but not proline, is also transported by the ectoine uptake system

Comparison of protein quantification and extraction methods suitable for E-coli cultures

Many different extraction and analysis methods exist to determine the protein fraction of microbial cells. For metabolic engineering purposes it is important to have precise and accurate measurements. Therefore six different protein extraction protocols and seven protein quantification methods were tested and compared. Comparison was based on the reliability of the methods and boxplots of the normalized residuals. Some extraction techniques (SDS/chloroform and toluene) should never be used: the measurements are neither precise nor accurate. Bugbuster extraction combined with UV280 quantification gives the best results, followed by the combinations sonication-UV280 and EasyLyse-UV280. However, if one does not want to use the quantification method UV280, one can opt to use Bugbuster, EasyLyse or sonication extraction combined with any quantification method with exception of the EasyLyse-BCA_P and sonication-BCA_P combinations

High yield 1,3-propanediol production by rational engineering of the 3-hydroxypropionaldehyde bottleneck in Citrobacter werkmanii

Background: Imbalance in cofactors causing the accumulation of intermediates in biosynthesis pathways is a frequently occurring problem in metabolic engineering when optimizing a production pathway in a microorganism. In our previous study, a single knock-out Citrobacter werkmanii Delta dhaD was constructed for improved 1,3-propanediol (PDO) production. Instead of an enhanced PDO concentration on this strain, the gene knock-out led to the accumulation of the toxic intermediate 3-hydroxypropionaldehyde (3-HPA). The hypothesis was emerged that the accumulation of this toxic intermediate, 3-HPA, is due to a cofactor imbalance, i.e. to the limited supply of reducing equivalents (NADH). Here, this bottleneck is alleviated by rationally engineering cell metabolism to balance the cofactor supply. Results: By eliminating non-essential NADH consuming enzymes (such as lactate dehydrogenase coded by ldhA, and ethanol dehydrogenase coded by adhE) or by increasing NADH producing enzymes, the accumulation of 3-HPA is minimized. Combining the above modifications in C. werkmanii Delta dhaD resulted in the strain C. werkmanii Delta dhaD Delta ldhA.adhE::ChlFRT which provided the maximum theoretical yield of 1.00 +/- 0.03 mol PDO/mol glycerol when grown on glucose/glycerol (0.33 molar ratio) on flask scale under anaerobic conditions. On bioreactor scale, the yield decreased to 0.73 +/- 0.01 mol PDO/mol glycerol although no 3-HPA could be measured, which indicates the existence of a sink of glycerol by a putative glycerol dehydrogenase, channeling glycerol to the central metabolism. Conclusions: In this study, a multiple knock-out was created in Citrobacter species for the first time. As a result, the concentration of the toxic intermediate 3-HPA was reduced to below the detection limit and the maximal theoretical PDO yield on glycerol was reached