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)

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

Modulating transcription through development of semi-synthetic yeast core promoters

Altering gene expression regulation by promoter engineering is a very effective way to finetune heterologous pathways in eukaryotic hosts. Typically, pathway building approaches in yeast still use a limited set of long, native promoters. With the today's introduction of longer and more complex pathways, an expansion of this synthetic biology toolbox is necessary. In this study we elucidated the core promoter structure of the well-characterized yeast TEF1 promoter and determined the minimal length needed for sufficient protein expression. Furthermore, this minimal core promoter sequence was used for the creation of a promoter library covering different expression strengths. This resulted in a group of short, 69 bp promoters with an 8.0-fold expression range. One exemplar had a two and four times higher expression compared to the native CYC1 and ADH1 promoter, respectively. Additionally, as it was described that the protein expression range could be broadened by upstream activating sequences (UASs), we integrated earlier described single and multiple short, synthetic UASs in front of the strongest yeast core promoter. This approach resulted to further variation in protein expression and an overall promoter library spanning a 20-fold activity range and covering a length from 69 bp to maximally 129 bp. Furthermore, the robustness of this library was assessed on three alternative carbon sources besides glucose. As such, the suitability of short yeast core promoters for metabolic engineering applications on different media, either in an individual context or combined with UAS elements, was demonstrated

Assembly and optimization of synthetic pathways in Saccharomyces cerevisiae: aurones as case study

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