果汁中脂环酸芽孢杆菌代谢生成愈创木酚分子机制研究

Alicyclobacillus acidoterrestris can cause spoilage in pasteurized fruit juices through the production of guaiacol and has been considered as an important quality control target in the fruit juice industry. Vanillic acid decarboxylase (Vdc) is the key enzyme in the formation pathway of guaiacol in A. acidoterrestris, it plays an important regulatory role in the biosynthesis of guaiacol. In this project, the relationship between guaiacol production and vdc gene expression will be firstly established. Then, in order to understand the function of vdc gene in the production of guaiacol, a vdc-negative mutant of A. acidoterrestris will be constructed by insertional inactivation via a single crossover recombination, and its ability to produce guaiacol will be evaluated. Meanwhile, to identify the differentially expressed genes and proteins in guaiacol producing and non-producing conditions, transcriptomics and proteomics analyses of A. acidoterrestris will be performed when incubated with and without vanillic acid, using the Illumina method of high throughout cDNA sequencing (RNA-seq) and isobaric tags for relative and absolute quantitation (iTRAQ) technique, respectively. An integrative analysis of transcriptomic and proteomic data will be conducted to explore the other key genes involved in guaiacol production, and then systematically reveal the molecular mechanisms for the synthesis of guaiacol in A. acidoterrestris. This work will contribute to the development of effective methods to prevent guaiacol production and could be very useful for a better control of A. acidoterrestris-related spoilage in the fruit juice industry.

脂环酸芽孢杆菌是果汁生产加工中关键的质量安全因子，其可以在果汁中代谢生成愈创木酚，引起果汁口感及风味劣变。香草酸脱羧酶（Vdc）作为脂环酸芽孢杆菌代谢产生愈创木酚的关键作用酶，对于愈创木酚的生成调控具有重要作用。因此，本项目以脂环酸芽孢杆菌代谢产生愈创木酚与vdc基因表达关系构建为基础，通过单交换插入失活基因的方法，构建脂环酸芽孢杆菌vdc基因缺陷株，评价其在果汁中代谢产生愈创木酚的能力，明确vdc基因在脂环酸芽孢杆菌代谢生成愈创木酚中的生物学功能；同时，以基于RNA-seq技术的转录组学和基于iTRAQ技术的蛋白质组学为核心手段，解析脂环酸芽孢杆菌在产生和不产生愈创木酚条件下的差异表达基因和蛋白，通过转录组学和蛋白质组学数据关联分析，挖掘与解析愈创木酚产生相关基因，全面系统地揭示脂环酸芽孢杆菌产愈创木酚的分子机制，为阻断愈创木酚的产生及实现果汁工业中脂环酸芽孢杆菌危害的有效控制奠定基础。

脂环酸芽孢杆菌是果汁工业中关键的质量安全因子，其对果汁的危害主要表现在代谢生成愈创木酚，引起果汁口感及风味劣变。本项目基于RNA-Seq技术的转录组学和基于iTRAQ技术的蛋白质组学，系统解析了脂环酸芽孢杆菌在产生和不产生愈创木酚条件下的差异表达基因和蛋白，通过转录组学和蛋白质组学数据关联分析，挖掘与解析了愈创木酚产生的相关基因，全面系统地揭示了脂环酸芽孢杆菌产愈创木酚的分子机制。研究结果表明：基于RNA-Seq和iTRAQ分析分别鉴定到了225个差异表达基因和77个差异表达蛋白。（1）香草酸脱羧酶的编码基因vdcB、vdcC和vdcD的转录水平分别上调9.27、7.52和5.71倍，它们是上调水平最高的基因；其次是编码吡哆胺5'-磷酸氧化酶家族蛋白的vdcBCD下游基因，表达上调5.56倍。其它与芳香族化合物降解有关的酶，如儿茶酚2,3-加双氧酶、2-酮基-4-戊烯酸酯水合酶、2-羟粘康酸-6-半醛脱氢酶、3,4-二羟基苯乙酸2,3-加双氧酶等，其基因或蛋白表达水平也明显上调。（2）苯甲酸膜转运蛋白、镰刀菌酸抗性蛋白、RND型外排泵家族基因、ABC和MFS转运体的基因或蛋白表达明显上调，表明这些转运蛋白参与了香草酸和愈创木酚的运输。（3）在脂环酸芽孢杆菌代谢香草酸生成愈创木酚的过程中，与三羧酸循环及核糖体蛋白合成相关的差异基因表达水平升高，而与缬氨酸、亮氨酸和异亮氨酸合成相关的基因表达下降。以上研究结果对后期针对性地建立愈创木酚生成阻断方法，继而有效控制果汁工业中脂环酸芽孢杆菌污染问题奠定了理论基础。