{"title":"Identifying promoters to enhance heterologous gene expression in recombinant Saccharomyces cerevisiae strains cultivated on non-native substrates","authors":"Jordan Fortuin, Riaan den Haan","doi":"10.1007/s00253-025-13563-6","DOIUrl":null,"url":null,"abstract":"<p>Efficient bioconversion of lignocellulosic biomass (LCB) to ethanol by <i>Saccharomyces cerevisiae</i> requires its engineering to express heterologous enzymes at titres high enough to make significant impacts on industrial consolidated bioprocessing (CBP). Promoters are required for this purpose, but are reportedly influenced by various environmental factors as well as the protein specific nature of expression, warranting the need for assessment under the conditions for which they are intended. Heterologous xylosidase- and xylanase-encoding genes (<i>xln43_SED1</i> and <i>xyn2</i>) were individually cloned under transcriptional control of the <i>SED1</i><sub>P</sub> and <i>TDH3</i><sub>P</sub> promoters, and <i>DIT1</i><sub>T</sub> terminator, and integrated into the genome of an a <i>S. cerevisiae</i> strain engineered for xylose utilization. Enzymatic assays were used to quantify the performance of the promoters when strains were cultivated on glucose (aerobically and micro-aerobically) and xylose. Additional strains containing both <i>xln43_SED1</i> and <i>xyn2</i> under different promoter combinations were then used to allow direct fermentation of beechwood xylan to ethanol in a CBP. The <i>SED1</i><sub>P</sub>/<i>DIT1</i><sub>T</sub> and <i>TDH3</i><sub>P</sub>/<i>DIT1</i><sub>T</sub> combinations significantly outperformed the benchmark <i>ENO1</i><sub>P/T</sub> under all of the tested cultivation conditions, as well as with regard to growth trials on non-native substrates (xylo-oligosaccharides/XOS and beechwood xylan) and fermentations of beechwood xylan to ethanol. Overall, <i>TDH3</i><sub>P</sub> was the best-performing promoter. This study demonstrates that heterologous metabolic pathways and CBP can be significantly enhanced by employing carefully selected promoters tailored to specific conditions.</p><p>• <i>Promoters are unpredictable and must be tested under their intended conditions.</i></p><p>• <i>TDH3</i><sub><i>P</i></sub>, <i>SED1</i><sub><i>P</i></sub>, <i>and DIT1</i><sub><i>T</i></sub> <i>were effective in enhancing heterologous xylanase activity.</i></p><p>• <i>Optimized xylanolytic enzyme expression improved CBP of xylan to ethanol.</i></p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":"109 1","pages":""},"PeriodicalIF":4.3000,"publicationDate":"2025-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12296805/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Applied Microbiology and Biotechnology","FirstCategoryId":"5","ListUrlMain":"https://link.springer.com/article/10.1007/s00253-025-13563-6","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Efficient bioconversion of lignocellulosic biomass (LCB) to ethanol by Saccharomyces cerevisiae requires its engineering to express heterologous enzymes at titres high enough to make significant impacts on industrial consolidated bioprocessing (CBP). Promoters are required for this purpose, but are reportedly influenced by various environmental factors as well as the protein specific nature of expression, warranting the need for assessment under the conditions for which they are intended. Heterologous xylosidase- and xylanase-encoding genes (xln43_SED1 and xyn2) were individually cloned under transcriptional control of the SED1P and TDH3P promoters, and DIT1T terminator, and integrated into the genome of an a S. cerevisiae strain engineered for xylose utilization. Enzymatic assays were used to quantify the performance of the promoters when strains were cultivated on glucose (aerobically and micro-aerobically) and xylose. Additional strains containing both xln43_SED1 and xyn2 under different promoter combinations were then used to allow direct fermentation of beechwood xylan to ethanol in a CBP. The SED1P/DIT1T and TDH3P/DIT1T combinations significantly outperformed the benchmark ENO1P/T under all of the tested cultivation conditions, as well as with regard to growth trials on non-native substrates (xylo-oligosaccharides/XOS and beechwood xylan) and fermentations of beechwood xylan to ethanol. Overall, TDH3P was the best-performing promoter. This study demonstrates that heterologous metabolic pathways and CBP can be significantly enhanced by employing carefully selected promoters tailored to specific conditions.
• Promoters are unpredictable and must be tested under their intended conditions.
• TDH3P, SED1P, and DIT1Twere effective in enhancing heterologous xylanase activity.
• Optimized xylanolytic enzyme expression improved CBP of xylan to ethanol.
期刊介绍：
Applied Microbiology and Biotechnology focusses on prokaryotic or eukaryotic cells, relevant enzymes and proteins; applied genetics and molecular biotechnology; genomics and proteomics; applied microbial and cell physiology; environmental biotechnology; process and products and more. The journal welcomes full-length papers and mini-reviews of new and emerging products, processes and technologies.
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号
