Heterologous phosphoketolase expression redirects flux towards acetate, perturbs sugar phosphate pools and increases respiratory demand in Saccharomyces cerevisiae

Abstract

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.

EngineeringYarrowia lipolyticaas a chassis forde novosynthesis of five aromatic-derived natural products and chemicals

“…In the pentose phosphate pathway, phosphoketolase splits fructose-6-phosphate into E4P and acetyl-phosphate (Figure 4a) 32, 55–57 . In a recent study, overexpression of heterologous phosphoketolase BbxfpK from Bifidobacterium breve led to a 5.4-fold increase of intracellular E4P concentration in S. cerevisiae 55 . We therefore introduced the codon-optimized phosphoketolase BbxfpK and AcxpkA (originating from Acidobacterium capsulatum) into our yeast chassis to generate strains YL27 and YL28 , respectively.…”

Rational engineering ofKluyveromyces marxianusto create a chassis for the production of aromatic products

“…As some PEPSes are exclusively used by their host organisms for either gluconeogenesis or PEP synthesis (Chen et al, 2019;Zhang and Anderson, 2013), a further investigation of more PEPSes with similar functions and kinetics may help us identify the best way to integrate this class of enzyme in yeast cell factories. In our efforts to increase E4P production, we overexpressed phosphoketolases that tap into K. marxianus' high fructose-6-phosphate levels and PPP flux (Bergman et al, 2019;Jung et al, 2016) to further boost available E4P.…”

“…An alternative strategy tested in S. cerevisiae was to express a non-native fructose-6-phosphate phosphoketolase (Xfpk, EC 4.1.2.22, which converts fructose-6-phosphate to E4P and acetyl phosphate (Fig. 3(A)) (Bergman et al, 2019). We tested four active phosphoketolases reported in the literature: three Xfpks from Bifidobacterium breve, Lactococcus lactis and Rhodotorula graminis (Bergman et al, 2016;Evans and Ratledge, 1984;Petrareanu et al, 2014), as well as a phosphoketolase from Clostridium acetobuytlicum which favours xylulose-5-phosphate as a substrate over fructose-6-phosphate (Fig 3(C)).…”

“…One possible explanation was that the acetyl phosphate produced by Xfpk is further converted to acetate, lowering the cytosolic pH and increasing metabolic burden (Bergman et al, 2019). Indeed, this appears to be the case because the overexpression of a phosphotransacetylase (Pta, EC 2.3.1.8) from Salmonella enterica (Septa) (Brinsmade and Escalante-Semerena, 2004) along with Bbxfpk alleviated the problem and generated strain KmASR.149 that produced over 4mM 2-PE.…”

Metabolic Engineering of Saccharomyces cerevisiae for Enhanced Carotenoid Production From Xylose-Glucose Mixtures

“…Meanwhile, overexpression of PK pathway exerted slight inhibition on the growth of strain SC103. The decreased cell growth might ascribe to that the expression of PK pathway was coupled with an increased acetate production ( Table 2; Bergman et al, 2019). The titers of carotenoid reached 64.9 and 75.9 mg L −1 from glucose and mixed sugars, respectively (Figure 3).…”