Anna Ylinen , Laura Salusjärvi , Mervi Toivari , Merja Penttilä
{"title":"合成基因表达系统控制酿酒酵母P(LA-3HB)共聚物中d -乳酸含量","authors":"Anna Ylinen , Laura Salusjärvi , Mervi Toivari , Merja Penttilä","doi":"10.1016/j.mec.2022.e00199","DOIUrl":null,"url":null,"abstract":"<div><p>The fully biobased polyhydroxyalkanoate (PHA) polymers provide interesting alternatives for petrochemical derived plastic materials. The mechanical properties of some PHAs, including the common poly(3-hydroxybutyrate) (PHB), are limited, but tunable by addition of other monomers into the polymer chain. In this study we present a precise synthetic biology method to adjust lactate monomer fraction of a polymer by controlling the monomer formation <em>in vivo</em> at gene expression level, independent of cultivation conditions. We used the modified doxycycline-based Tet-On approach to adjust the expression of the stereospecific D-lactate dehydrogenase gene (<em>ldhA</em>) from <em>Leuconostoc mesenteroides</em> to control D-lactic acid formation in yeast <em>Saccharomyces cerevisiae</em>. The synthetic Tet-On transcription factor with a VP16 activation domain was continuously expressed and its binding to a synthetic promoter with eight transcription factor specific binding sites upstream of the <em>ldhA</em> gene was controlled with the doxycycline concentration in the media. The increase in doxycycline concentration correlated positively with <em>ldhA</em> expression, D-lactic acid production, poly(D-lactic acid) (PDLA) accumulation <em>in vivo</em>, and D-lactic acid content in the poly(D-lactate-<em>co</em>-3-hydroxybutyrate) P(LA-3HB) copolymer. We demonstrated that the D-lactic acid content of the P(LA-3HB) copolymer can be adjusted linearly from 6 mol% to 93 mol% <em>in vivo</em> in <em>S. cerevisiae</em>. These results highlight the power of controlling gene expression and monomer formation in the tuning of the polymer composition. In addition, we obtained 5.6% PDLA and 19% P(LA-3HB) of the cell dry weight (CDW), which are over two- and five-fold higher accumulation levels, respectively, than reported in the previous studies with yeast. We also compared two engineered PHA synthases and discovered that in <em>S. cerevisiae</em> the PHA synthase PhaC1437<sub>Ps6-19</sub> produced P(LA-3HB) copolymers with lower D-lactic acid content, but with higher molecular weight, in comparison to the PHA synthase PhaC1Pre.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":3.7000,"publicationDate":"2022-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030122000086/pdfft?md5=46cba499ea94cf885917415684ffaf00&pid=1-s2.0-S2214030122000086-main.pdf","citationCount":"0","resultStr":"{\"title\":\"Control of D-lactic acid content in P(LA-3HB) copolymer in the yeast Saccharomyces cerevisiae using a synthetic gene expression system\",\"authors\":\"Anna Ylinen , Laura Salusjärvi , Mervi Toivari , Merja Penttilä\",\"doi\":\"10.1016/j.mec.2022.e00199\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>The fully biobased polyhydroxyalkanoate (PHA) polymers provide interesting alternatives for petrochemical derived plastic materials. The mechanical properties of some PHAs, including the common poly(3-hydroxybutyrate) (PHB), are limited, but tunable by addition of other monomers into the polymer chain. In this study we present a precise synthetic biology method to adjust lactate monomer fraction of a polymer by controlling the monomer formation <em>in vivo</em> at gene expression level, independent of cultivation conditions. We used the modified doxycycline-based Tet-On approach to adjust the expression of the stereospecific D-lactate dehydrogenase gene (<em>ldhA</em>) from <em>Leuconostoc mesenteroides</em> to control D-lactic acid formation in yeast <em>Saccharomyces cerevisiae</em>. The synthetic Tet-On transcription factor with a VP16 activation domain was continuously expressed and its binding to a synthetic promoter with eight transcription factor specific binding sites upstream of the <em>ldhA</em> gene was controlled with the doxycycline concentration in the media. The increase in doxycycline concentration correlated positively with <em>ldhA</em> expression, D-lactic acid production, poly(D-lactic acid) (PDLA) accumulation <em>in vivo</em>, and D-lactic acid content in the poly(D-lactate-<em>co</em>-3-hydroxybutyrate) P(LA-3HB) copolymer. We demonstrated that the D-lactic acid content of the P(LA-3HB) copolymer can be adjusted linearly from 6 mol% to 93 mol% <em>in vivo</em> in <em>S. cerevisiae</em>. These results highlight the power of controlling gene expression and monomer formation in the tuning of the polymer composition. In addition, we obtained 5.6% PDLA and 19% P(LA-3HB) of the cell dry weight (CDW), which are over two- and five-fold higher accumulation levels, respectively, than reported in the previous studies with yeast. We also compared two engineered PHA synthases and discovered that in <em>S. cerevisiae</em> the PHA synthase PhaC1437<sub>Ps6-19</sub> produced P(LA-3HB) copolymers with lower D-lactic acid content, but with higher molecular weight, in comparison to the PHA synthase PhaC1Pre.</p></div>\",\"PeriodicalId\":18695,\"journal\":{\"name\":\"Metabolic Engineering Communications\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.7000,\"publicationDate\":\"2022-06-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.sciencedirect.com/science/article/pii/S2214030122000086/pdfft?md5=46cba499ea94cf885917415684ffaf00&pid=1-s2.0-S2214030122000086-main.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Metabolic Engineering Communications\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S2214030122000086\",\"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/S2214030122000086","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
Control of D-lactic acid content in P(LA-3HB) copolymer in the yeast Saccharomyces cerevisiae using a synthetic gene expression system
The fully biobased polyhydroxyalkanoate (PHA) polymers provide interesting alternatives for petrochemical derived plastic materials. The mechanical properties of some PHAs, including the common poly(3-hydroxybutyrate) (PHB), are limited, but tunable by addition of other monomers into the polymer chain. In this study we present a precise synthetic biology method to adjust lactate monomer fraction of a polymer by controlling the monomer formation in vivo at gene expression level, independent of cultivation conditions. We used the modified doxycycline-based Tet-On approach to adjust the expression of the stereospecific D-lactate dehydrogenase gene (ldhA) from Leuconostoc mesenteroides to control D-lactic acid formation in yeast Saccharomyces cerevisiae. The synthetic Tet-On transcription factor with a VP16 activation domain was continuously expressed and its binding to a synthetic promoter with eight transcription factor specific binding sites upstream of the ldhA gene was controlled with the doxycycline concentration in the media. The increase in doxycycline concentration correlated positively with ldhA expression, D-lactic acid production, poly(D-lactic acid) (PDLA) accumulation in vivo, and D-lactic acid content in the poly(D-lactate-co-3-hydroxybutyrate) P(LA-3HB) copolymer. We demonstrated that the D-lactic acid content of the P(LA-3HB) copolymer can be adjusted linearly from 6 mol% to 93 mol% in vivo in S. cerevisiae. These results highlight the power of controlling gene expression and monomer formation in the tuning of the polymer composition. In addition, we obtained 5.6% PDLA and 19% P(LA-3HB) of the cell dry weight (CDW), which are over two- and five-fold higher accumulation levels, respectively, than reported in the previous studies with yeast. We also compared two engineered PHA synthases and discovered that in S. cerevisiae the PHA synthase PhaC1437Ps6-19 produced P(LA-3HB) copolymers with lower D-lactic acid content, but with higher molecular weight, in comparison to the PHA synthase PhaC1Pre.
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.