Functional Verification of the Citrate Transporter Gene in a Wine Lactic Acid Bacterium, .

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 () 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 XJ25, to verify its function in citrate metabolism for the first time. The results showed that the gene knockout mutant XJ25-Δ lost the ability to utilize citric acid, while the gene complement mutant XJ25-Δ-pMG36ek11- fully recovered the ability of citric acid utilization. Meanwhile, knockout and complement barely affected the utilization of l-malic acid. These indicated that in 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 showed distinct transcription efficiency. The transcription efficiency of in XJ25-Δ-pMG36ek11- mutant was 4.01 times higher than that in XJ25-Δ-pMG36ek- 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.