{"title":"酿酒酵母中 NADPH 供应策略的比较分析:以从二木糖生产二木糖醇为例进行研究","authors":"Priti Regmi , Melanie Knesebeck , Eckhard Boles , Dirk Weuster-Botz , Mislav Oreb","doi":"10.1016/j.mec.2024.e00245","DOIUrl":null,"url":null,"abstract":"<div><p>Enhancing the supply of the redox cofactor NADPH in metabolically engineered cells is a critical target for optimizing the synthesis of many product classes, such as fatty acids or terpenoids. In <em>S. cerevisiae</em>, several successful approaches have been developed in different experimental contexts. However, their systematic comparison has not been reported. Here, we established the reduction of xylose to xylitol by an NADPH-dependent xylose reductase as a model reaction to compare the efficacy of different NADPH supply strategies in the course of a batch fermentation, in which glucose and ethanol are sequentially used as carbon sources and redox donors. We show that strains overexpressing the glucose-6-phosphate dehydrogenase Zwf1 perform best, producing up to 16.9 g L<sup>−1</sup> xylitol from 20 g L<sup>−1</sup> xylose in stirred tank bioreactors. The beneficial effect of increased Zwf1 activity is especially pronounced during the ethanol consumption phase. The same notion applies to the deletion of the aldehyde dehydrogenase <em>ALD6</em> gene, albeit at a quantitatively lower level. Reduced expression of the phosphoglucose isomerase Pgi1 and heterologous expression of the NADP<sup>+</sup>-dependent glyceraldehyde-3-phosphate dehydrogenase Gdp1 from <em>Kluyveromyces lactis</em> acted synergistically with <em>ZWF1</em> overexpression in the presence of glucose, but had a detrimental effect after the diauxic shift. Expression of the mitochondrial NADH kinase Pos5 in the cytosol likewise improved the production of xylitol only on glucose, but not in combination with enhanced Zwf1 activity. To demonstrate the generalizability of our observations, we show that the most promising strategies – <em>ZWF1</em> overexpression and deletion of <em>ALD6</em> - also improve the production of <span>l</span>-galactonate from <span>d</span>-galacturonic acid. Therefore, we expect that these findings will provide valuable guidelines for engineering not only the production of xylitol but also of diverse other pathways that require NADPH.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":3.7000,"publicationDate":"2024-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030124000142/pdfft?md5=7ade0b7c412cf8487310e2ebc5404b91&pid=1-s2.0-S2214030124000142-main.pdf","citationCount":"0","resultStr":"{\"title\":\"A comparative analysis of NADPH supply strategies in Saccharomyces cerevisiae: Production of d-xylitol from d-xylose as a case study\",\"authors\":\"Priti Regmi , Melanie Knesebeck , Eckhard Boles , Dirk Weuster-Botz , Mislav Oreb\",\"doi\":\"10.1016/j.mec.2024.e00245\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Enhancing the supply of the redox cofactor NADPH in metabolically engineered cells is a critical target for optimizing the synthesis of many product classes, such as fatty acids or terpenoids. In <em>S. cerevisiae</em>, several successful approaches have been developed in different experimental contexts. However, their systematic comparison has not been reported. Here, we established the reduction of xylose to xylitol by an NADPH-dependent xylose reductase as a model reaction to compare the efficacy of different NADPH supply strategies in the course of a batch fermentation, in which glucose and ethanol are sequentially used as carbon sources and redox donors. We show that strains overexpressing the glucose-6-phosphate dehydrogenase Zwf1 perform best, producing up to 16.9 g L<sup>−1</sup> xylitol from 20 g L<sup>−1</sup> xylose in stirred tank bioreactors. The beneficial effect of increased Zwf1 activity is especially pronounced during the ethanol consumption phase. The same notion applies to the deletion of the aldehyde dehydrogenase <em>ALD6</em> gene, albeit at a quantitatively lower level. Reduced expression of the phosphoglucose isomerase Pgi1 and heterologous expression of the NADP<sup>+</sup>-dependent glyceraldehyde-3-phosphate dehydrogenase Gdp1 from <em>Kluyveromyces lactis</em> acted synergistically with <em>ZWF1</em> overexpression in the presence of glucose, but had a detrimental effect after the diauxic shift. Expression of the mitochondrial NADH kinase Pos5 in the cytosol likewise improved the production of xylitol only on glucose, but not in combination with enhanced Zwf1 activity. To demonstrate the generalizability of our observations, we show that the most promising strategies – <em>ZWF1</em> overexpression and deletion of <em>ALD6</em> - also improve the production of <span>l</span>-galactonate from <span>d</span>-galacturonic acid. Therefore, we expect that these findings will provide valuable guidelines for engineering not only the production of xylitol but also of diverse other pathways that require NADPH.</p></div>\",\"PeriodicalId\":18695,\"journal\":{\"name\":\"Metabolic Engineering Communications\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.7000,\"publicationDate\":\"2024-07-05\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.sciencedirect.com/science/article/pii/S2214030124000142/pdfft?md5=7ade0b7c412cf8487310e2ebc5404b91&pid=1-s2.0-S2214030124000142-main.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Metabolic Engineering Communications\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S2214030124000142\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Metabolic Engineering Communications","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2214030124000142","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
A comparative analysis of NADPH supply strategies in Saccharomyces cerevisiae: Production of d-xylitol from d-xylose as a case study
Enhancing the supply of the redox cofactor NADPH in metabolically engineered cells is a critical target for optimizing the synthesis of many product classes, such as fatty acids or terpenoids. In S. cerevisiae, several successful approaches have been developed in different experimental contexts. However, their systematic comparison has not been reported. Here, we established the reduction of xylose to xylitol by an NADPH-dependent xylose reductase as a model reaction to compare the efficacy of different NADPH supply strategies in the course of a batch fermentation, in which glucose and ethanol are sequentially used as carbon sources and redox donors. We show that strains overexpressing the glucose-6-phosphate dehydrogenase Zwf1 perform best, producing up to 16.9 g L−1 xylitol from 20 g L−1 xylose in stirred tank bioreactors. The beneficial effect of increased Zwf1 activity is especially pronounced during the ethanol consumption phase. The same notion applies to the deletion of the aldehyde dehydrogenase ALD6 gene, albeit at a quantitatively lower level. Reduced expression of the phosphoglucose isomerase Pgi1 and heterologous expression of the NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase Gdp1 from Kluyveromyces lactis acted synergistically with ZWF1 overexpression in the presence of glucose, but had a detrimental effect after the diauxic shift. Expression of the mitochondrial NADH kinase Pos5 in the cytosol likewise improved the production of xylitol only on glucose, but not in combination with enhanced Zwf1 activity. To demonstrate the generalizability of our observations, we show that the most promising strategies – ZWF1 overexpression and deletion of ALD6 - also improve the production of l-galactonate from d-galacturonic acid. Therefore, we expect that these findings will provide valuable guidelines for engineering not only the production of xylitol but also of diverse other pathways that require NADPH.
期刊介绍:
Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.