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| 干酪乳杆菌arat基因的敲除及其对苯乳酸合成代谢的影响 |
| Knockout of arat Gene in Lactobacillus casei and Its Effect on Phenyllactic Acid Synthesis and Metabolism |
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| DOI：doi:10.3969/j.issn.1005-7021.2022.03.002 |
| 中文关键词: 苯乳酸 苯丙氨酸 芳香族氨基转移酶 干酪乳杆菌 CRISPR/Cas9 |
| 英文关键词: phenyllactic acid phenylalanine aromatic amino acid transferase Lactobacillus casei CRISPR/Cas9 |
| 基金项目:上海市农业科学院青年科技人员“助跑”计划项目（ZP21211）；上海市农业科学院卓越团队项目（沪农科卓〔2022〕016） |
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| 中文摘要: |
| 苯乳酸（PLA）作为一种新型的广谱抑菌物质，在食品行业具有巨大的发展潜力，作为乳酸菌的天然代谢产物之一，对其代谢机理的研究具有非常重要的意义。通过CRISPR/Cas9基因编辑系统对一株干酪乳杆菌（Lactobacillus casei）1.8727的芳香族氨基转移酶基因arat进行敲除，得到一株arat缺失菌株（L. casei）1.8727Δarat。通过HPLC方法分析其代谢产物，发现发酵72 h时，PLA产量比出发菌株提高约66.7%，而苯丙氨酸（Phe）比出发菌株提高约57.8%。首次在L. casei 1.8727中成功应用基因编辑手段敲除了arat基因，研究表明该菌株具有自身合成代谢Phe的能力，arat作为PLA代谢途径的关键酶基因，其缺失并未使PLA产量下调，而是促进了PLA与Phe的合成代谢，证明干酪乳杆菌中可能存在其他的代偿途径，arat基因缺失所造成的代谢流的改变最终造成PLA与Phe产量的提高，同时PLA的合成代谢可能涉及多个基因的参与，是一个复杂的代谢网络。研究结果对于深入研究PLA的合成代谢机制具有重要意义，为后续PLA合成代谢途径提供了新的思路及方法。 |
| 英文摘要: |
| As a new type of broad-spectrum antimicrobial substance, phenyllactic acid (PLA) has great development potential in food industry. It is important to study the metabolic mechanism of PLA as it is also one of the natural metabolites of lactic acid bacteria. In this study, the gene of aromatic amino acid transferase gene arat in Lactobacillus casei 1.8727 was knocked out by CRISPR/Cas9 gene editing system, and an arat deletion strain L. casei 1.8727 Δarat was obtained. The production of PLA was analyzed by HPLC. It was found that 72 h after fermentation, the production of PLA increased by 66.7% and phenylalanine (PPA) 57.8% as compared with original strain. This study firstly knocked out arat gene successfully in L.casei 1.8727 adopting gene editing system. The study showed that the strain had its own ability to synthesize and metabolize Phe. As a key enzyme in PLA metabolic pathway, its deletion did not reduce the PLA yield, but promoted the anabolism of PLA and Phe, and proved that there may be another compensatory pathways in L. casei. The changes in metabolic flow caused by the deletion of arat gene eventually lead to the increase of the production of PLA and Phe. Meanwhile, the anabolism of PLA may involve the participation of multiple genes and is a complex metabolic network. The findings of this study are of great significance for pervasive study of the mechanism of PLA anabolism, and provide a new idea and method for the follow-up PLA anabolism pathway. |
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