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

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

JOURNAL=Frontiers in Microbiology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2024.1442797

DOI=10.3389/fmicb.2024.1442797

ISSN=1664-302X

ABSTRACT=<sec id="sec1001"><title>Introduction</title><p>Endoglucanase (EG) and cellobiohydrolase (CBH) which produced by microorganisms, have been widely used in industrial applications.</p></sec><sec id="sec2001"><title>Methods</title><p>In order to construct recombinant bacteria that produce high activity EG and CBH, in this study, <italic>eg</italic> (endoglucanase) and <italic>cbh</italic> (cellobiohydrolase) were cloned from the rumen microbial genome of yak and subsequently expressed independently and co-expressed within <italic>Lactococcus lactis</italic> NZ9000 (<italic>L. lactis</italic> NZ9000).</p></sec><sec id="sec3001"><title>Results</title><p>The recombinant strains <italic>L. lactis</italic> NZ9000/pMG36e-usp45-<italic>cbh</italic> (<italic>L. lactis</italic>-<italic>cbh</italic>), <italic>L. lactis</italic> NZ9000/pMG36e-usp45-<italic>eg</italic> (<italic>L. lactis</italic>-<italic>eg</italic>), and <italic>L. lactis</italic> NZ9000/pMG36e-usp45-<italic>eg</italic>-usp45-<italic>cbh</italic> (<italic>L. lactis</italic>-<italic>eg</italic>-<italic>cbh</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<sup>2+</sup>, Fe<sup>2+</sup>, Cu<sup>2+</sup>, and Co<sup>2+</sup> could promote the activity of CBH. Similarly, Fe<sup>2+</sup>, Ba<sup>2+</sup>, and higher concentrations of Ca<sup>2+</sup>, Cu<sup>2+</sup>, and Co<sup>2+</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 <italic>L. lactis-eg</italic> group (<italic>p</italic> &lt; 0.05), and the lowest ammonia nitrogen (NH<sub>3</sub>-N), and highest lactic acid (LA) and crude protein (CP) contents in silage from the <italic>L. lactis-eg</italic> + <italic>L. lactis-cbh</italic> group (<italic>p</italic> &lt; 0.05), while the silage quality in the <italic>L. lactis</italic>-<italic>cbh</italic> group was not satisfactory. </p></sec><sec id="sec4001"><title>Discussion</title><p>Consequently, the recombinant strains <italic>L. lactis-cbh</italic>, <italic>L. lactis-eg</italic>, and <italic>L. lactis-eg-cbh</italic> were successfully constructed, which could successfully expressed EG, CBH, and EG-CBH. <italic>L. lactis-eg</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.</p></sec>