植物乳植杆菌类胡萝卜素生物合成关键基因LpispA的功能鉴定  

Functional Characterization of LpispA,a Key Gene Involved in Carotenoid Biosynthesis in Lactiplantibacillus plantarum

作　　者：李舒丽 谢卓婷 沈润泽 郭丽琼[1,2] 林俊芳 陈谋通[3] 叶志伟 LI Shuli;XIE Zhuoting;SHEN Runze;GUO Liqiong;LIN Junfang;CHEN Moutong;YE Zhiwei(College of Food Science,South China Agricultural University,Guangzhou 510642,China;Research Center for Micro-ecological Agent Engineering and Technology of Guangdong Province,Guangzhou 510642,China;Guangdong Institute of Microbiology,State Key Laboratory of Applied Microbiology Southern China,Guangdong Provincial Key Laboratory of Microbial Safety and Health,Key Laboratory of Agricultural Microbiome and Precision Application,Ministry of Agriculture and Rural Affairs,Guangzhou 510070,China)

机构地区：[1]华南农业大学食品学院,广东广州510642 [2]广东省微生态制剂工程技术研究中心,广东广州510642 [3]广东省科学院微生物研究所,华南应用微生物国家重点实验室,广东省微生物安全与健康重点实验室,农业农村部农业微生物组学与精准应用重点实验室,广东广州510070

出　　处：《现代食品科技》2024年第2期131-138,共8页Modern Food Science and Technology

基　　金：国家自然科学基金项目(32272298,31801918);广东省自然科学基金项目(2022A1515010057,2015A030310225);广东省重点领域研发计划重点项目(2022B0202050002);广东省农业科研项目和农业技术推广项目(2022KJ103,2023KJ103)。

摘　　要：类胡萝卜素具有抗氧化和抗肿瘤等多种重要生理活性。与化学合成法和植物提取法相比较,利用微生物法合成新型的、高价值的类胡萝卜素,具有食用安全和经济可行等优势。植物乳植杆菌作为天然C_(30)类胡萝卜素来源的几种细菌之一,其类胡萝卜素生物合成途径却尚未被阐明清楚。该论文基于转录组数据对一株海洋源植物乳植杆菌中的LpispA基因进行克隆、绘制系统进化树,并构建了类胡萝卜素合成功能互补的大肠杆菌表达系统pAC-BETA∆E-ispA,再采用紫外可见光谱法和反相高效液相色谱法验证产物成分。结果表明,植物乳植杆菌LpispA与桃色欧文氏菌的亲缘关系最近;构建的大肠杆菌工程菌株pAC-BETA∆E-ispA的β-胡萝卜素(C_(40))产量为0.05 mg/g DCW。因此,LpispA在大肠杆菌功能互补表达系统中成功表达了Geranylgeranyl Pyrophosphate(GGPP)合成酶的活性,也即说明LpispA在细菌类胡萝卜素合成途径中可作为类胡萝卜素合成多功能酶,共同参与C_(30)和C_(40)类胡萝卜素的合成。该研究为植物乳植杆菌类胡萝卜素生物合成机制研究和利用植物乳植杆菌作为类胡萝卜素补充剂的应用奠定了实验基础。Carotenoids exhibit various important physiological activities,including anti-oxidation and anti-tumor properties.Compared with plant extracts and chemically synthesized compounds,carotenoids are considered safe for human consumption.Moreover,microbial biosynthesis offers an economically viable approach to the synthesis of carotenoids.As a novel source of the carotenoid C_(30),the carotenoid biosynthetic pathway in Lactiplantibacillus plantarum has not been fully elucidated.Based on transcriptome analyses performed previously,in this study,the LpispA gene was cloned from a marine L.plantarum strain,and phylogenetic analysis was performed.Subsequently,the cloned open reading frame sequence was expressed in the reconstructed plasmid pAC-BETA∆E-ispA within a functional complementation system using Escherichia coli for carotenoid biosynthesis.In addition,ultraviolet-visible light spectroscopy and reversed-phase high-performance liquid chromatography were employed for component detection of the pigment productions.The results indicated that the L.plantarum LpispA gene exhibited the closest genetic relationship with the corresponding gene from Erwinia persicina.Aβ-carotene(C_(40))yield of 0.05 mg/g(dry cell weight)was achieved in the engineered E.coli strain with pAC-BETA∆E-ispA.In conclusion,the bioactivity of geranylgeranyl pyrophosphate synthase expressed by LpispA in the functional complementation E.coli system resulted in the biosynthesis ofβ-carotene.In other words,LpispA,isolated from L.plantarum,is a multifunctional enzyme in carotenoid biosynthesis,contributing to the biosynthesis of both C_(30) and C_(40) carotenoids in bacteria.These results may establish an experimental foundation for investigating carotenoid biosynthesis in L.plantarum and pave the way for potential applications of L.plantarum in the production of carotenoid supplements.

关 键 词：类胡萝卜素 植物乳植杆菌 LpispA基因 互补表达 

分 类 号：TS201.2[轻工技术与工程—食品科学]