Functional Verification of the Citrate Transporter Gene in a Wine Lactic Acid Bacterium, Lactiplantibacillus plantarum
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 verifi...
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Frontiers Media S.A.
2022-05-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2022.894870/full |
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| author | Xiangke Yang Xiangke Yang Lili Zhao Qiling Chen Nan Wang Kan Shi Kan Shi Kan Shi Kan Shi Kan Shi Shuwen Liu Shuwen Liu Shuwen Liu Shuwen Liu Shuwen Liu |
| author_facet | Xiangke Yang Xiangke Yang Lili Zhao Qiling Chen Nan Wang Kan Shi Kan Shi Kan Shi Kan Shi Kan Shi Shuwen Liu Shuwen Liu Shuwen Liu Shuwen Liu Shuwen Liu |
| author_sort | Xiangke Yang |
| collection | DOAJ |
| description | 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 (citP) 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, Lactiplantibacillus plantarum XJ25, to verify its function in citrate metabolism for the first time. The results showed that the gene knockout mutant XJ25-ΔcitP lost the ability to utilize citric acid, while the gene complement mutant XJ25-ΔcitP-pMG36ek11-citP fully recovered the ability of citric acid utilization. Meanwhile, citP knockout and complement barely affected the utilization of l-malic acid. These indicated that citP in L. plantarum 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 L. plantarum showed distinct transcription efficiency. The transcription efficiency of citP in XJ25-ΔcitP-pMG36ek11-citP mutant was 4.01 times higher than that in XJ25-ΔcitP-pMG36ek-citP 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. |
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| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-12-12T20:56:59Z |
| publishDate | 2022-05-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-98da307ccadf4363a05f03d95309f88f2022-12-22T00:12:16ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852022-05-011010.3389/fbioe.2022.894870894870Functional Verification of the Citrate Transporter Gene in a Wine Lactic Acid Bacterium, Lactiplantibacillus plantarumXiangke Yang0Xiangke Yang1Lili Zhao2Qiling Chen3Nan Wang4Kan Shi5Kan Shi6Kan Shi7Kan Shi8Kan Shi9Shuwen Liu10Shuwen Liu11Shuwen Liu12Shuwen Liu13Shuwen Liu14College of Enology, Northwest A&F University, Yangling, ChinaCollege of Food and Bioengineering, Henan University of Animal Husbandry and Economy, Zhengzhou, ChinaCollege of Enology, Northwest A&F University, Yangling, ChinaCollege of Enology, Northwest A&F University, Yangling, ChinaCollege of Enology, Northwest A&F University, Yangling, ChinaCollege of Enology, Northwest A&F University, Yangling, ChinaShaanxi Engineering Research Center for Viti-Viniculture, Yangling, ChinaEngineering Research Center for Viti-Viniculture, National Forestry and Grassland Administration, Yangling, ChinaHeyang Experimental and Demonstrational Stations for Grape, Northwest A&F University, Weinan, ChinaNingxia Helan Mountain’s East Foothill Wine Experiment and Demonstration Station, Northwest A&F University, Yongning, ChinaCollege of Enology, Northwest A&F University, Yangling, ChinaShaanxi Engineering Research Center for Viti-Viniculture, Yangling, ChinaEngineering Research Center for Viti-Viniculture, National Forestry and Grassland Administration, Yangling, ChinaHeyang Experimental and Demonstrational Stations for Grape, Northwest A&F University, Weinan, ChinaNingxia Helan Mountain’s East Foothill Wine Experiment and Demonstration Station, Northwest A&F University, Yongning, ChinaOrganic 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 (citP) 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, Lactiplantibacillus plantarum XJ25, to verify its function in citrate metabolism for the first time. The results showed that the gene knockout mutant XJ25-ΔcitP lost the ability to utilize citric acid, while the gene complement mutant XJ25-ΔcitP-pMG36ek11-citP fully recovered the ability of citric acid utilization. Meanwhile, citP knockout and complement barely affected the utilization of l-malic acid. These indicated that citP in L. plantarum 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 L. plantarum showed distinct transcription efficiency. The transcription efficiency of citP in XJ25-ΔcitP-pMG36ek11-citP mutant was 4.01 times higher than that in XJ25-ΔcitP-pMG36ek-citP 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.https://www.frontiersin.org/articles/10.3389/fbioe.2022.894870/fullcitrate transporter genefunctional verificationlactiplantibacillus plantarumorganic acidslactic acid bacteriagene editing |
| spellingShingle | Xiangke Yang Xiangke Yang Lili Zhao Qiling Chen Nan Wang Kan Shi Kan Shi Kan Shi Kan Shi Kan Shi Shuwen Liu Shuwen Liu Shuwen Liu Shuwen Liu Shuwen Liu Functional Verification of the Citrate Transporter Gene in a Wine Lactic Acid Bacterium, Lactiplantibacillus plantarum Frontiers in Bioengineering and Biotechnology citrate transporter gene functional verification lactiplantibacillus plantarum organic acids lactic acid bacteria gene editing |
| title | Functional Verification of the Citrate Transporter Gene in a Wine Lactic Acid Bacterium, Lactiplantibacillus plantarum |
| title_full | Functional Verification of the Citrate Transporter Gene in a Wine Lactic Acid Bacterium, Lactiplantibacillus plantarum |
| title_fullStr | Functional Verification of the Citrate Transporter Gene in a Wine Lactic Acid Bacterium, Lactiplantibacillus plantarum |
| title_full_unstemmed | Functional Verification of the Citrate Transporter Gene in a Wine Lactic Acid Bacterium, Lactiplantibacillus plantarum |
| title_short | Functional Verification of the Citrate Transporter Gene in a Wine Lactic Acid Bacterium, Lactiplantibacillus plantarum |
| title_sort | functional verification of the citrate transporter gene in a wine lactic acid bacterium lactiplantibacillus plantarum |
| topic | citrate transporter gene functional verification lactiplantibacillus plantarum organic acids lactic acid bacteria gene editing |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2022.894870/full |
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