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

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          Abstract

          Introduction

          Phosphoketolases (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 CO 2. 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.

          Results

          Xfpk-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.

          Conclusion

          Our 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 CO 2. 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 material

          The online version of this article (10.1186/s12934-019-1072-6) contains supplementary material, which is available to authorized users.

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          Most cited references25

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          Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories

          Sustainable production of oleochemicals requires establishment of cell factory platform strains. The yeast Saccharomyces cerevisiae is an attractive cell factory as new strains can be rapidly implemented into existing infrastructures such as bioethanol production plants. Here we show high-level production of free fatty acids (FFAs) in a yeast cell factory, and the production of alkanes and fatty alcohols from its descendants. The engineered strain produces up to 10.4 g l−1 of FFAs, which is the highest reported titre to date. Furthermore, through screening of specific pathway enzymes, endogenous alcohol dehydrogenases and aldehyde reductases, we reconstruct efficient pathways for conversion of fatty acids to alkanes (0.8 mg l−1) and fatty alcohols (1.5 g l−1), to our knowledge the highest titres reported in S. cerevisiae. This should facilitate the construction of yeast cell factories for production of fatty acids derived products and even aldehyde-derived chemicals of high value.
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            Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae.

            The widely used pESC vector series (Stratagene, La Jolla, CA, USA) with the bidirectional GAL1/GAL10 promoter provides the possibility of simultaneously expressing two different genes from a single vector in Saccharomyces cerevisiae. This system can be induced by galactose and is repressed by glucose. Since S. cerevisiae prefers glucose as a carbon source, and since its growth rate is higher in glucose than in galactose-containing media, we compared and evaluated seven different promoters expressed during growth on glucose (pTEF1, pADH1, pTPI1, pHXT7, pTDH3, pPGK1 and pPYK1) with two strong galactose-induced promoters (pGAL1 and pGAL10), using lacZ as a reporter gene and measuring LacZ activity in batch and continuous cultivation. TEF1 and PGK1 promoters showed the most constant activity pattern at different glucose concentrations. Based on these results, we designed and constructed two new expression vectors which contain the two constitutive promoters, TEF1 and PGK1, in opposite orientation to each other. These new vectors retain all the features from the pESC-URA plasmid except that gene expression is mediated by constitutive promoters. Copyright © 2010 John Wiley & Sons, Ltd.
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              Role of transcriptional regulation in controlling fluxes in central carbon metabolism of Saccharomyces cerevisiae. A chemostat culture study.

              In contrast to batch cultivation, chemostat cultivation allows the identification of carbon source responses without interference by carbon-catabolite repression, accumulation of toxic products, and differences in specific growth rate. This study focuses on the yeast Saccharomyces cerevisiae, grown in aerobic, carbon-limited chemostat cultures. Genome-wide transcript levels and in vivo fluxes were compared for growth on two sugars, glucose and maltose, and for two C2-compounds, ethanol and acetate. In contrast to previous reports on batch cultures, few genes (180 genes) responded to changes of the carbon source by a changed transcript level. Very few transcript levels were changed when glucose as the growth-limiting nutrient was compared with maltose (33 transcripts), or when acetate was compared with ethanol (16 transcripts). Although metabolic flux analysis using a stoichiometric model revealed major changes in the central carbon metabolism, only 117 genes exhibited a significantly different transcript level when sugars and C2-compounds were provided as the growth-limiting nutrient. Despite the extensive knowledge on carbon source regulation in yeast, many of the carbon source-responsive genes encoded proteins with unknown or incompletely characterized biological functions. In silico promoter analysis of carbon source-responsive genes confirmed the involvement of several known transcriptional regulators and suggested the involvement of additional regulators. Transcripts involved in the glyoxylate cycle and gluconeogenesis showed a good correlation with in vivo fluxes. This correlation was, however, not observed for other important pathways, including the pentose-phosphate pathway, tricarboxylic acid cycle, and, in particular, glycolysis. These results indicate that in vivo fluxes in the central carbon metabolism of S. cerevisiae grown in steadystate, carbon-limited chemostat cultures are controlled to a large extent via post-transcriptional mechanisms.
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                Author and article information

                Contributors
                aleber@chalmers.se
                johnhe@chalmers.se
                thomas.moritz@slu.se
                siewers@chalmers.se
                nielsenj@chalmers.se
                +46 31772 31 40 , yunc@chalmers.se
                Journal
                Microb Cell Fact
                Microb. Cell Fact
                Microbial Cell Factories
                BioMed Central (London )
                1475-2859
                1 February 2019
                1 February 2019
                2019
                : 18
                : 25
                Affiliations
                [1 ]ISNI 0000 0001 0775 6028, GRID grid.5371.0, Department of Biology and Biological Engineering, , Chalmers University of Technology, ; Kemivägen 10, 41296 Gothenburg, Sweden
                [2 ]ISNI 0000 0001 0775 6028, GRID grid.5371.0, Novo Nordisk Foundation Center for Biosustainability, , Chalmers University of Technology, ; 41296 Gothenburg, Sweden
                [3 ]ISNI 0000 0000 8578 2742, GRID grid.6341.0, Department of Forest Genetics and Plant Physiology, , Swedish University of Agricultural Sciences, Umeå Plant Science Center (UPSC), ; 901 83 Umeå, Sweden
                [4 ]ISNI 0000 0004 0613 9724, GRID grid.467081.c, Swedish Metabolomics Centre, Umeå Plant Science Center (UPSC), ; 901 83 Umeå, Sweden
                [5 ]ISNI 0000 0001 2181 8870, GRID grid.5170.3, Novo Nordisk Foundation Center for Biosustainability, , Technical University of Denmark, ; 2800 Kongens Lyngby, Denmark
                Article
                1072
                10.1186/s12934-019-1072-6
                6359841
                30709397
                6f3df562-12bc-40f3-80ad-1b82da41b38a
                © The Author(s) 2019

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 21 December 2018
                : 23 January 2019
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100009708, Novo Nordisk Fonden;
                Award ID: NNF10CC1016517
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100004063, Knut och Alice Wallenbergs Stiftelse;
                Funded by: FundRef http://dx.doi.org/10.13039/501100001729, Stiftelsen för Strategisk Forskning;
                Funded by: FundRef http://dx.doi.org/10.13039/501100001862, Svenska Forskningsrådet Formas;
                Funded by: FundRef http://dx.doi.org/10.13039/501100002805, Carl Tryggers Stiftelse för Vetenskaplig Forskning;
                Categories
                Research
                Custom metadata
                © The Author(s) 2019

                Biotechnology
                saccharomyces cerevisiae,phosphoketolase,acetyl-phosphate,acetate,acetyl-coa,physiology,sugar phosphate,rnaseq

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