Use of sigma factor M from Bacillus subtilis in the development of an orthogonal expression system in Escherichia coli

Background: Technological advances in synthetic biology, systems biology, and metabolic engineering have boosted applications of industrial biotechnology for an increasing number of complex and high added-value molecules. In general, the transfer of multi- gene or poorly understood heterologous pathways into the production host leads to imbalances due to lack of adequate regulatory mechanisms. Hence, fine-tuning expression of synthesis pathways in specific conditions is mandatory. Objectives: Here we develop a new genetic circuit for regulated expression specifically in stationary phase due to clear advantages during this period (reduction of toxicity, competition). Methods: This circuit consists of a heterologous sigma factor () recognizing specific promoter sequences, which are not recognised by the native factors of E. coli and is expressed upon entering the stationary phase. First, several factors of B. subtilis were tested for their orthogonality in E. coli on the level of promoter recognition, by using a red-fluorescent reporter system. Secondly, the potential of factors of B. subtilis to work together with the E. coli core RNA polymerase was tested, by expressing these proteins together with their promoters. Based on the results a specific factor will be chosen for further optimalisation and the corresponding gene can be cloned in the S factor operon of E. coli, which is most abundantly expressed in stationary conditions. Conclusions: Combining all these elements should allow us to create an orthogonal genetic circuit that is able to transcribe specific genes under stationary phase with a limited influence on the host cell’s metabolism

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

Metabolic characterisation of E. coli citrate synthase and phosphoenolpyruvate carboxylase mutants in aerobic cultures

E. coli is still one of the most commonly used hosts for protein production. However, when it is grown with excess glucose, acetate accumulation occurs. Elevated acetate concentrations have an inhibitory effect on growth rate and recombinant protein yield, and thus elimination of acetate formation is an important aim towards industrial production of recombinant proteins. Here we examine if over-expression of citrate synthase (gltA) or phosphoenolpyruvate carboxylase (ppc) can eliminate acetate production. Knock-out as well as over-expression mutants were constructed and characterized. Knocking out ppc or gltA decreased the maximum cell density by 14% and increased the acetate excretion by 7%, respectively decreased it by 10%. Over-expression of ppc or gltA increased the maximum cell dry weight by 91% and 23%, respectively. No acetate excretion was detected at these increased cell densities (35 and 23 g/l, respectively)

Automated design of ligand responsive RNA devices

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