AUTHOR=Yang Xiangke , Zhao Lili , Chen Qiling , Wang Nan , Shi Kan , Liu Shuwen 

TITLE=Functional Verification of the Citrate Transporter Gene in a Wine Lactic Acid Bacterium, Lactiplantibacillus plantarum

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=10

YEAR=2022

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2022.894870

DOI=10.3389/fbioe.2022.894870

ISSN=2296-4185

ABSTRACT=<p>Organic acid metabolism by lactic acid bacteria plays a significant role in improving wine quality. During this process, the uptake of extracellular organic acids by the transporters is the first rate-limiting step. However, up to now, there is very little published research on the functional verification of organic acid transporter genes in wine lactic acid bacteria. In this study, a predicted citrate transporter gene JKL54_04345 (<italic>citP</italic>) by protein homology analysis was knocked out using a CRISPR/Cas9-based gene-editing system, and then complemented using the modified pMG36e vectors in a major wine lactic acid bacterium<italic>, Lactiplantibacillus plantarum</italic> XJ25, to verify its function in citrate metabolism for the first time. The results showed that the gene knockout mutant XJ25-Δ<italic>citP</italic> lost the ability to utilize citric acid, while the gene complement mutant XJ25-Δ<italic>citP</italic>-pMG36ek11-<italic>citP</italic> fully recovered the ability of citric acid utilization. Meanwhile, <italic>citP</italic> knockout and complement barely affected the utilization of <sc>l</sc>-malic acid. These indicated that <italic>citP</italic> in <italic>L. plantarum</italic> functioned as a citrate transporter and was the only gene responsible for citrate transporter. In addition, two modified plasmid vectors used for gene supplement in <italic>L. plantarum</italic> showed distinct transcription efficiency. The transcription efficiency of <italic>citP</italic> in XJ25-Δ<italic>citP</italic>-pMG36ek11-<italic>citP</italic> mutant was 4.01 times higher than that in XJ25-Δ<italic>citP</italic>-pMG36ek-<italic>citP</italic> mutant, and the utilization rate of citric acid in the former was 3.95 times higher than that in the latter, indicating that pMG36ek11 can be used as a high-level expression vector in lactic acid bacteria.</p>