Xuerui Wan, Yongjie SunKang, Yijun Chen, Zhao Zhang, Huitian Gou, Yu Xue, Chuan Wang, Yaqin Wei, Yuze Yang
{"title":"牦牛瘤胃中的内切葡聚糖酶和纤维素水解酶在乳酸菌中的共表达及其在全株玉米青贮发酵中的初步应用","authors":"Xuerui Wan, Yongjie SunKang, Yijun Chen, Zhao Zhang, Huitian Gou, Yu Xue, Chuan Wang, Yaqin Wei, Yuze Yang","doi":"10.3389/fmicb.2024.1442797","DOIUrl":null,"url":null,"abstract":"IntroductionEndoglucanase (EG) and cellobiohydrolase (CBH) which produced by microorganisms, have been widely used in industrial applications.MethodsIn order to construct recombinant bacteria that produce high activity EG and CBH, in this study, <jats:italic>eg</jats:italic> (endoglucanase) and <jats:italic>cbh</jats:italic> (cellobiohydrolase) were cloned from the rumen microbial genome of yak and subsequently expressed independently and co-expressed within <jats:italic>Lactococcus lactis</jats:italic> NZ9000 (<jats:italic>L. lactis</jats:italic> NZ9000).ResultsThe recombinant strains <jats:italic>L. lactis</jats:italic> NZ9000/pMG36e-usp45-<jats:italic>cbh</jats:italic> (<jats:italic>L. lactis</jats:italic>-<jats:italic>cbh</jats:italic>), <jats:italic>L. lactis</jats:italic> NZ9000/pMG36e-usp45-<jats:italic>eg</jats:italic> (<jats:italic>L. lactis</jats:italic>-<jats:italic>eg</jats:italic>), and <jats:italic>L. lactis</jats:italic> NZ9000/pMG36e-usp45-<jats:italic>eg</jats:italic>-usp45-<jats:italic>cbh</jats:italic> (<jats:italic>L. lactis</jats:italic>-<jats:italic>eg</jats:italic>-<jats:italic>cbh</jats:italic>) were successfully constructed and demonstrated the ability to secrete EG, CBH, and EG-CBH. The sodium carboxymethyl cellulose activity of the recombinant enzyme EG was the highest, and the regenerated amorphous cellulose (RAC) was the specific substrate of the recombinant enzyme CBH, and EG-CBH. The optimum reaction temperature of the recombinant enzyme CBH was 60°C, while the recombinant enzymes EG and EG-CBH were tolerant to higher temperatures (80°C). The optimum reaction pH of EG, CBH, and EG-CBH was 6.0. Mn<jats:sup>2+</jats:sup>, Fe<jats:sup>2+</jats:sup>, Cu<jats:sup>2+</jats:sup>, and Co<jats:sup>2+</jats:sup> could promote the activity of CBH. Similarly, Fe<jats:sup>2+</jats:sup>, Ba<jats:sup>2+</jats:sup>, and higher concentrations of Ca<jats:sup>2+</jats:sup>, Cu<jats:sup>2+</jats:sup>, and Co<jats:sup>2+</jats:sup> could promote the activity of EG-CBH. The addition of engineered strains to whole-plant corn silage improved the nutritional quality of the feed, with the lowest pH, acid detergent fiber (ADF), and neutral detergent fiber (NDF) contents observed in silage from the <jats:italic>L. lactis-eg</jats:italic> group (<jats:italic>p</jats:italic> &lt; 0.05), and the lowest ammonia nitrogen (NH<jats:sub>3</jats:sub>-N), and highest lactic acid (LA) and crude protein (CP) contents in silage from the <jats:italic>L. lactis-eg</jats:italic> + <jats:italic>L. lactis-cbh</jats:italic> group (<jats:italic>p</jats:italic> &lt; 0.05), while the silage quality in the <jats:italic>L. lactis</jats:italic>-<jats:italic>cbh</jats:italic> group was not satisfactory.DiscussionConsequently, the recombinant strains <jats:italic>L. lactis-cbh</jats:italic>, <jats:italic>L. lactis-eg</jats:italic>, and <jats:italic>L. lactis-eg-cbh</jats:italic> were successfully constructed, which could successfully expressed EG, CBH, and EG-CBH. <jats:italic>L. lactis-eg</jats:italic> promoted silage fermentation by degrading cellulose to produce sugar, enabling the secretory expression of EG, CBH, and EG-CBH for potential industrial applications in cellulose degradation.","PeriodicalId":12466,"journal":{"name":"Frontiers in Microbiology","volume":null,"pages":null},"PeriodicalIF":4.0000,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Co-expression of endoglucanase and cellobiohydrolase from yak rumen in lactic acid bacteria and its preliminary application in whole-plant corn silage fermentation\",\"authors\":\"Xuerui Wan, Yongjie SunKang, Yijun Chen, Zhao Zhang, Huitian Gou, Yu Xue, Chuan Wang, Yaqin Wei, Yuze Yang\",\"doi\":\"10.3389/fmicb.2024.1442797\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"IntroductionEndoglucanase (EG) and cellobiohydrolase (CBH) which produced by microorganisms, have been widely used in industrial applications.MethodsIn order to construct recombinant bacteria that produce high activity EG and CBH, in this study, <jats:italic>eg</jats:italic> (endoglucanase) and <jats:italic>cbh</jats:italic> (cellobiohydrolase) were cloned from the rumen microbial genome of yak and subsequently expressed independently and co-expressed within <jats:italic>Lactococcus lactis</jats:italic> NZ9000 (<jats:italic>L. lactis</jats:italic> NZ9000).ResultsThe recombinant strains <jats:italic>L. lactis</jats:italic> NZ9000/pMG36e-usp45-<jats:italic>cbh</jats:italic> (<jats:italic>L. lactis</jats:italic>-<jats:italic>cbh</jats:italic>), <jats:italic>L. lactis</jats:italic> NZ9000/pMG36e-usp45-<jats:italic>eg</jats:italic> (<jats:italic>L. lactis</jats:italic>-<jats:italic>eg</jats:italic>), and <jats:italic>L. lactis</jats:italic> NZ9000/pMG36e-usp45-<jats:italic>eg</jats:italic>-usp45-<jats:italic>cbh</jats:italic> (<jats:italic>L. lactis</jats:italic>-<jats:italic>eg</jats:italic>-<jats:italic>cbh</jats:italic>) were successfully constructed and demonstrated the ability to secrete EG, CBH, and EG-CBH. The sodium carboxymethyl cellulose activity of the recombinant enzyme EG was the highest, and the regenerated amorphous cellulose (RAC) was the specific substrate of the recombinant enzyme CBH, and EG-CBH. The optimum reaction temperature of the recombinant enzyme CBH was 60°C, while the recombinant enzymes EG and EG-CBH were tolerant to higher temperatures (80°C). The optimum reaction pH of EG, CBH, and EG-CBH was 6.0. Mn<jats:sup>2+</jats:sup>, Fe<jats:sup>2+</jats:sup>, Cu<jats:sup>2+</jats:sup>, and Co<jats:sup>2+</jats:sup> could promote the activity of CBH. Similarly, Fe<jats:sup>2+</jats:sup>, Ba<jats:sup>2+</jats:sup>, and higher concentrations of Ca<jats:sup>2+</jats:sup>, Cu<jats:sup>2+</jats:sup>, and Co<jats:sup>2+</jats:sup> could promote the activity of EG-CBH. The addition of engineered strains to whole-plant corn silage improved the nutritional quality of the feed, with the lowest pH, acid detergent fiber (ADF), and neutral detergent fiber (NDF) contents observed in silage from the <jats:italic>L. lactis-eg</jats:italic> group (<jats:italic>p</jats:italic> &lt; 0.05), and the lowest ammonia nitrogen (NH<jats:sub>3</jats:sub>-N), and highest lactic acid (LA) and crude protein (CP) contents in silage from the <jats:italic>L. lactis-eg</jats:italic> + <jats:italic>L. lactis-cbh</jats:italic> group (<jats:italic>p</jats:italic> &lt; 0.05), while the silage quality in the <jats:italic>L. lactis</jats:italic>-<jats:italic>cbh</jats:italic> group was not satisfactory.DiscussionConsequently, the recombinant strains <jats:italic>L. lactis-cbh</jats:italic>, <jats:italic>L. lactis-eg</jats:italic>, and <jats:italic>L. lactis-eg-cbh</jats:italic> were successfully constructed, which could successfully expressed EG, CBH, and EG-CBH. <jats:italic>L. lactis-eg</jats:italic> promoted silage fermentation by degrading cellulose to produce sugar, enabling the secretory expression of EG, CBH, and EG-CBH for potential industrial applications in cellulose degradation.\",\"PeriodicalId\":12466,\"journal\":{\"name\":\"Frontiers in Microbiology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.0000,\"publicationDate\":\"2024-09-18\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Frontiers in Microbiology\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.3389/fmicb.2024.1442797\",\"RegionNum\":2,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Frontiers in Microbiology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.3389/fmicb.2024.1442797","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"MICROBIOLOGY","Score":null,"Total":0}
Co-expression of endoglucanase and cellobiohydrolase from yak rumen in lactic acid bacteria and its preliminary application in whole-plant corn silage fermentation
IntroductionEndoglucanase (EG) and cellobiohydrolase (CBH) which produced by microorganisms, have been widely used in industrial applications.MethodsIn order to construct recombinant bacteria that produce high activity EG and CBH, in this study, eg (endoglucanase) and cbh (cellobiohydrolase) were cloned from the rumen microbial genome of yak and subsequently expressed independently and co-expressed within Lactococcus lactis NZ9000 (L. lactis NZ9000).ResultsThe recombinant strains L. lactis NZ9000/pMG36e-usp45-cbh (L. lactis-cbh), L. lactis NZ9000/pMG36e-usp45-eg (L. lactis-eg), and L. lactis NZ9000/pMG36e-usp45-eg-usp45-cbh (L. lactis-eg-cbh) were successfully constructed and demonstrated the ability to secrete EG, CBH, and EG-CBH. The sodium carboxymethyl cellulose activity of the recombinant enzyme EG was the highest, and the regenerated amorphous cellulose (RAC) was the specific substrate of the recombinant enzyme CBH, and EG-CBH. The optimum reaction temperature of the recombinant enzyme CBH was 60°C, while the recombinant enzymes EG and EG-CBH were tolerant to higher temperatures (80°C). The optimum reaction pH of EG, CBH, and EG-CBH was 6.0. Mn2+, Fe2+, Cu2+, and Co2+ could promote the activity of CBH. Similarly, Fe2+, Ba2+, and higher concentrations of Ca2+, Cu2+, and Co2+ could promote the activity of EG-CBH. The addition of engineered strains to whole-plant corn silage improved the nutritional quality of the feed, with the lowest pH, acid detergent fiber (ADF), and neutral detergent fiber (NDF) contents observed in silage from the L. lactis-eg group (p < 0.05), and the lowest ammonia nitrogen (NH3-N), and highest lactic acid (LA) and crude protein (CP) contents in silage from the L. lactis-eg + L. lactis-cbh group (p < 0.05), while the silage quality in the L. lactis-cbh group was not satisfactory.DiscussionConsequently, the recombinant strains L. lactis-cbh, L. lactis-eg, and L. lactis-eg-cbh were successfully constructed, which could successfully expressed EG, CBH, and EG-CBH. L. lactis-eg promoted silage fermentation by degrading cellulose to produce sugar, enabling the secretory expression of EG, CBH, and EG-CBH for potential industrial applications in cellulose degradation.
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Frontiers in Microbiology is a leading journal in its field, publishing rigorously peer-reviewed research across the entire spectrum of microbiology. Field Chief Editor Martin G. Klotz at Washington State University is supported by an outstanding Editorial Board of international researchers. This multidisciplinary open-access journal is at the forefront of disseminating and communicating scientific knowledge and impactful discoveries to researchers, academics, clinicians and the public worldwide.
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