IntroductionPhosphoketolases (Xfpk) are a non-native group of enzymes in yeast, which can be expressed in combination with other metabolic enzymes to positively influence the yield of acetyl-CoA derived products by reducing carbon losses in the form of CO2. In this study, a yeast strain expressing Xfpk from Bifidobacterium breve, which was previously found to have a growth defect and to increase acetate production, was characterized.ResultsXfpk-expression was found to increase respiration and reduce biomass yield during glucose consumption in batch and chemostat cultivations. By cultivating yeast with or without Xfpk in bioreactors at different pHs, we show that certain aspects of the negative growth effects coupled with Xfpk-expression are likely to be explained by proton decoupling. At low pH, this manifests as a reduction in biomass yield and growth rate in the ethanol phase. Secondly, we show that intracellular sugar phosphate pools are significantly altered in the Xfpk-expressing strain. In particular a decrease of the substrates xylulose-5-phosphate and fructose-6-phosphate was detected (26% and 74% of control levels) together with an increase of the products glyceraldehyde-3-phosphate and erythrose-4-phosphate (208% and 542% of control levels), clearly verifying in vivo Xfpk enzymatic activity. Lastly, RNAseq analysis shows that Xfpk expression increases transcription of genes related to the glyoxylate cycle, the TCA cycle and respiration, while expression of genes related to ethanol and acetate formation is reduced. The physiological and transcriptional changes clearly demonstrate that a heterologous phosphoketolase flux in combination with endogenous hydrolysis of acetyl-phosphate to acetate increases the cellular demand for acetate assimilation and respiratory ATP-generation, leading to carbon losses.ConclusionOur study shows that expression of Xfpk in yeast diverts a relatively small part of its glycolytic flux towards acetate formation, which has a significant impact on intracellular sugar phosphate levels and on cell energetics. The elevated acetate flux increases the ATP-requirement for ion homeostasis and need for respiratory assimilation, which leads to an increased production of CO2. A majority of the negative growth effects coupled to Xfpk expression could likely be counteracted by preventing acetate accumulation via direct channeling of acetyl-phosphate towards acetyl-CoA.Electronic supplementary materialThe online version of this article (10.1186/s12934-019-1072-6) contains supplementary material, which is available to authorized users.

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Microbial conversion of biomass to fatty acids (FA) and products derived thereof is an attractive alternative to the traditional oleochemical production route from animal and plant lipids. This study examined if NADPH-costly FA biosynthesis could be enhanced by overexpressing the transcription factor Stb5 in Saccharomyces cerevisiae. Stb5 activates expression of multiple genes encoding enzymes within the pentose phosphate pathway (PPP) and other NADPH-producing reactions. Overexpression of STB5 led to a decreased growth rate and an increased free fatty acid (FFA) production during growth on glucose. The improved FFA synthetic ability in the glucose phase was shown to be independent of flux through the oxidative PPP. RNAseq analysis revealed that STB5 overexpression had wide-ranging effects on the transcriptome in the batch phase, and appeared to cause a counterintuitive phenotype with reduced flux through the oxidative PPP. During glucose limitation, when an increased NADPH supply is likely less harmful, an overall induction of the proposed target genes of Stb5 (eg. GND1/2, TAL1, ALD6, YEF1) was observed. Taken together, the strategy of utilizing STB5 overexpression to increase NADPH supply for reductive biosynthesis is suggested to have potential in strains engineered to have strong ability to consume excess NADPH, alleviating a potential redox imbalance.