利用CRISPR/Cas9技术提高保加利亚乳杆菌合成L-乳酸的研究

In the process of yogurt fermentation, Lactobacillus bulgaricus is widely used for producing acid, extracellular polysaccharide and characteristic flavor substances. It is well known that L. bulgaricus is a good D-lactic acid producer. Since only the L-lactate dehydrogenase is existed in human body, the excessive intake of D-lactic acid will cause the metabolic burden. Therefore, the CRISPR-Cas9 system is determined in this study for the first time in gene editing of L. bulgaricus to change the synthetic of the D-lactic acid configuration to L-lactic acid configuration. The L. bulgaricus ATCC 11842 is chosen in this study to construct the D-lactate dehydrogenase gene deletion mutants using CRISPR-Cas9 technology to improve its ability in L-lactate production; HPLC and LC/ESI/MS methods are used to study metabolites of the mutants; the methods such as heterologous protein expression and purification, protein activity, RT-PCR, and Western-blot are used to compare the expression differences of the D-LDH gene deficient mutants in transcriptional level and protein level. The result of this study is helpful for understanding the regulatory mechanism of L. bulgaricus in lactic acid production. Furthermore, the high optical purity L-lactate L. bulgaricus is constructed based on the metabolic flux analysis and the metabolic network optimization. This study will provide a theoretical basis of industrial application and will meet the differentiated social demand.

在酸奶发酵过程中，保加利亚乳杆菌因发酵产酸、产胞外多糖及特征性风味物质而被广泛使用。该菌株以合成D-乳酸为主，由于人体只含有L-乳酸脱氢酶，过多D-乳酸的摄入会造成代谢负担。因此，本项目首次利用CRISPR-Cas9技术对保加利亚乳杆菌进行基因编辑以改变其乳酸的合成构型。以Lactobacillus bulgaricus ATCC 11842为研究对象，利用CRISPR-Cas9技术构建D-乳酸脱氢酶基因缺失的突变株；结合HPLC和LC/ESI/MS等方法研究突变株发酵代谢产物的变化规律；采用蛋白异源表达与纯化、蛋白活性染色、RT-PCR和Western-blot等技术研究突变株在转录水平和蛋白水平的表达差异，从而阐明保加利亚乳杆菌乳酸合成的调控机制；结合代谢流分析确定关键节点，优化代谢网络，构建合成高纯度L-乳酸的保加利亚乳杆菌，为高品质、差异化发酵乳的社会需求提供理论依据和研究基础。

保加利亚乳杆菌是乳酸菌中重要的一员，作为发酵剂被广泛应用于食品中。该菌株是厌氧菌，且以合成D-乳酸为主。为深入解析其乳酸合成机理，本项目首次利用CRISPR-Cas9技术对保加利亚乳杆菌进行基因编辑，构建了基于Crispr-Cas9 pLCNICK为载体骨架的D-乳酸脱氢酶基因敲除载体和基于Cre-LoxP同源重组pNZ5319为骨架的D-乳酸脱氢酶基因敲除载体。通过将5种D-乳酸脱氢酶同工酶和2种L-乳酸脱氢酶同工酶基因的异源表达、蛋白纯化、酶活测定和酶学性质的研究，结合厌氧和好氧发酵条件下重组菌D-/L-乳酸的合成，确定了保加利亚乳杆菌中负责D-乳酸合成的关键酶为LDB0101和LDB1010，负责L-乳酸合成的关键酶为LDB0094。进一步采用实时荧光定量PCR方法对L. bulgaricus ATCC11842厌氧发酵过程中生长对数期和稳定期D乳酸脱氢酶和L-乳酸脱氢酶基因转录水平进行了分析，结果显示所测基因均可以正常转录，且LDB0101具有较高的相对转录水平。本研究结合乳酸脱氢酶基因的克隆、酶活性测定、转录组测序和乳酸的合成，从DNA、RNA、蛋白和代谢物四个层面解析了保加利亚乳杆菌合成乳酸的调控机制，为乳酸菌在食品中的应用提供了理论依据。