Identification of the citrate exporter Cex1 of Yarrowia lipolytica.

Yarrowia lipolytica is a yeast with many talents, one of them being the production of citric acid. Although the citrate biosynthesis is well studied, little is known about the transport mechanism by which citrate is exported. To gain better insight into this mechanism, we set out to identify a transporter involved in citrate export of Y. lipolytica. A total of five proteins were selected for analysis based on their similarity to a known citrate exporter, but neither a citrate transport activity nor any other phenotypic function could be attributed to them. Differential gene expression analysis of two strains with a distinct citrate productivity revealed another three putative transporters, one of which is YALI0D20196p. Disrupting YALI0D20196g in Y. lipolytica abolished citrate production, while extrachromosomal expression enhanced citrate production 5.2-fold in a low producing wildtype. Furthermore, heterologous expression of YALI0D20196p in the non-citrate secreting yeast Saccharomyces cerevisiae facilitated citrate export. Likewise, expression of YALI0D20196p complemented the ability to secrete citrate in an export-deficient strain of Aspergillus niger, confirming a citrate export function of YALI0D20196p. This report on the identification of the first citrate exporter in Y. lipolytica, termed Cex1, represents a valuable starting point for further investigations of the complex transport processes in yeasts.

Engineered E. coli W enables efficient 2,3-butanediol production from glucose and sugar beet molasses using defined minimal medium as economic basis.

BACKGROUND: Efficient microbial production of chemicals is often hindered by the cytotoxicity of the products or by the pathogenicity of the host strains. Hence 2,3-butanediol, an important drop-in chemical, is an interesting alternative target molecule for microbial synthesis since it is non-cytotoxic. Metabolic engineering of non-pathogenic and industrially relevant microorganisms, such as Escherichia coli, have already yielded in promising 2,3-butanediol titers showing the potential of microbial synthesis of 2,3-butanediol. However, current microbial 2,3-butanediol production processes often rely on yeast extract as expensive additive, rendering these processes infeasible for industrial production. RESULTS: The aim of this study was to develop an efficient 2,3-butanediol production process with E. coli operating on the premise of using cost-effective medium without complex supplements, considering second generation feedstocks. Different gene donors and promoter fine-tuning allowed for construction of a potent E. coli strain for the production of 2,3-butanediol as important drop-in chemical. Pulsed fed-batch cultivations of E. coli W using microaerobic conditions showed high diol productivity of 4.5 g l-1 h-1. Optimizing oxygen supply and elimination of acetoin and by-product formation improved the 2,3-butanediol titer to 68 g l-1, 76% of the theoretical maximum yield, however, at the expense of productivity. Sugar beet molasses was tested as a potential substrate for industrial production of chemicals. Pulsed fed-batch cultivations produced 56 g l-1 2,3-butanediol, underlining the great potential of E. coli W as production organism for high value-added chemicals. CONCLUSION: A potent 2,3-butanediol producing E. coli strain was generated by considering promoter fine-tuning to balance cell fitness and production capacity. For the first time, 2,3-butanediol production was achieved with promising titer, rate and yield and no acetoin formation from glucose in pulsed fed-batch cultivations using chemically defined medium without complex hydrolysates. Furthermore, versatility of E. coli W as production host was demonstrated by efficiently converting sucrose from sugar beet molasses into 2,3-butanediol.

Characterizing the effect of expression of an acetyl-CoA synthetase insensitive to acetylation on co-utilization of glucose and acetate in batch and continuous cultures of E. coli W.

BACKGROUND: Due to its high stress tolerance and low acetate secretion, Escherichia coli W is reported to be a good production host for several metabolites and recombinant proteins. However, simultaneous co-utilization of glucose and other substrates such as acetate remains a challenge. The activity of acetyl-CoA-synthetase, one of the key enzymes involved in acetate assimilation is tightly regulated on a transcriptional and post-translational level. The aim of this study was to engineer E. coli W for overexpression of an acetylation insensitive acetyl-CoA-synthetase and to characterize this strain in batch and continuous cultures using glucose, acetate and during co-utilization of both substrates. RESULTS: Escherichia coli W engineered to overexpress an acetylation-insensitive acetyl-CoA synthetase showed a 2.7-fold increase in acetate uptake in a batch process containing glucose and high concentrations of acetate compared to a control strain, indicating more efficient co-consumption of glucose and acetate. When acetate was used as the carbon source, batch duration could significantly be decreased in the overexpression strain, possibly due to alleviation of acetate toxicity. Chemostat cultivations with different dilution rates using glucose revealed only minor differences between the overexpression and control strain. Accelerostat cultivations using dilution rates between 0.20 and 0.70 h-1 indicated that E. coli W is naturally capable of efficiently co-utilizing glucose and acetate over a broad range of specific growth rates. Expression of acetyl-CoA synthetase resulted in acetate and glucose accumulation at lower dilution rates compared to the control strain. This observation can possibly be attributed to a higher ratio between acs and pta-ackA in the overexpression strain as revealed by gene expression analysis. This would result in enhanced energy dissipation caused by an imbalance in the Pta-AckA-Acs cycle. Furthermore, yjcH and actP, genes co-transcribed with acetyl-CoA synthetase showed significant down-regulation at elevated dilution rates. CONCLUSIONS: Escherichia coli W expressing an acetylation-insensitive acetyl-CoA synthetase was shown to be a promising candidate for mixed feed processes using glucose and acetate. Comparison between batch and continuous cultures revealed distinct differences in glucose-acetate co-utilization behavior, requiring additional investigations such as multi-omics analysis and further engineering towards even more efficient co-utilization strains of E. coli W.

Utilizing yeasts for the conversion of renewable feedstocks to sugar alcohols - a review.

Sugar alcohols are widely marketed compounds. They are useful building block chemicals and of particular value as low- or non-calorigenic sweeteners, serving as sugar substitutes in the food industry. To date most sugar alcohols are produced by chemical routes using pure sugars, but a transition towards the use of renewable, non-edible feedstocks is anticipated. Several yeasts are naturally able to convert renewable feedstocks, such as lignocellulosic substrates, glycerol and molasses, into sugar alcohols. These bioconversions often face difficulties to obtain sufficiently high yields and productivities necessary for industrialization. This review provides insight into the most recent studies on utilizing yeasts for the conversion of renewable feedstocks to diverse sugar alcohols, including xylitol, erythritol, mannitol and arabitol. Moreover, metabolic approaches are highlighted that specifically target shortcomings of sugar alcohol production by yeasts from these renewable substrates.