Co-expression of endoglucanase and cellobiohydrolase from yak rumen in lactic acid bacteria and its preliminary application in whole-plant corn silage fermentation

IF 4 2区生物学 Q2 MICROBIOLOGY Frontiers in Microbiology Pub Date : 2024-09-18 DOI:10.3389/fmicb.2024.1442797

Xuerui Wan, Yongjie SunKang, Yijun Chen, Zhao Zhang, Huitian Gou, Yu Xue, Chuan Wang, Yaqin Wei, Yuze Yang

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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, 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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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

方法为了构建能产生高活性 EG 和 CBH 的重组菌，本研究从牦牛瘤胃微生物基因组中克隆了 EG（内切葡聚糖酶）和 CBH（纤维素水解酶），并在乳球菌 NZ9000（L. lactococcus lactis NZ9000）中进行了独立表达和共表达。结果重组菌株 L. lactis NZ9000/pMG36e-usp45-cbh（L. lactis-cbh）、L. lactis NZ9000/pMG36e-usp45-eg（L. lactis-eg）和 L. lactis NZ9000/pMG36e-usp45-cbh（L. lactis-cbh）在牦牛瘤胃微生物基因组中分别独立表达和共同表达。成功构建了 L. lactis NZ9000/pMG36e-usp45-eg-usp45-cbh（L. lactis-eg-cbh），并证明了其分泌 EG、CBH 和 EG-CBH 的能力。重组酶 EG 的羧甲基纤维素钠活性最高，再生无定形纤维素（RAC）是重组酶 CBH 和 EG-CBH 的特异性底物。重组酶 CBH 的最佳反应温度为 60°C，而重组酶 EG 和 EG-CBH 可耐受更高的温度（80°C）。EG、CBH 和 EG-CBH 的最佳反应 pH 值为 6.0。Mn2+、Fe2+、Cu2+ 和 Co2+ 可促进 CBH 的活性。同样，Fe2+、Ba2+ 以及较高浓度的 Ca2+、Cu2+ 和 Co2+ 可促进 EG-CBH 的活性。在全株玉米青贮中添加工程菌株可改善饲料的营养质量，在 L. lactis-eg 组的青贮中观察到最低的 pH 值、酸性洗涤纤维（ADF）和中性洗涤纤维（NDF）含量（p &p;lt;0.05），在 L. lactis-eg + L. lactis-eg 组的青贮中观察到最低的氨氮（NH3-N）、最高的乳酸（LA）和粗蛋白（CP）含量。讨论成功构建了重组菌株 L. lactis-cbh、L. lactis-eg 和 L. lactis-eg-cbh，它们能成功表达 EG、CBH 和 EG-CBH。L.lactis-eg通过降解纤维素产生糖来促进青贮发酵，从而实现了EG、CBH和EG-CBH的分泌表达，在纤维素降解方面具有潜在的工业应用价值。

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来源期刊

Frontiers in Microbiology MICROBIOLOGY-

CiteScore

7.70

自引率

9.60%

发文量

4837

审稿时长

14 weeks

期刊介绍： 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.