基于芽孢杆菌关键生理调控策略的苯基乳酸生物合成研究

Phenyllactic acid (PLA) biosynthesis is a research focus for its good security and broad-spectrum antimicrobial activity. Previous research reported Bacillus spp. synthesized PLA with much more higher productivity than the most studied lactic acid bacteria did. Not merely high productivity but also high growth rate and low nutrition requirment, Bacillus spp. are becoming the potential powerful PLA-producing strains. However, the specific physiological process of Bacillus spp., such as sporulation and cannibalism impeded PLA biosynthesis. For further improving PLA productivity of Bacillu spp.,the project will carry out the following research items: (1)Knocking out the key regulator encoding gene spo0A associated closely with sporulation and cannibalism and figure out the difference in physiological process and PLA synthesis between the wild and mutant strains;(2)Elucidating the mechanism on cell dencity inhibiting PLA synthesis based on the integrated RNA-seq and iTRAQ technologies; (3)Purification and characterization of the key enzyme that converting phenylpyruvic acid to PLA. The results of these researches will provide the fundermental data for improving PLA productivity and PLA biosynthesis with high cell dencity.

3-苯基乳酸（PLA）因其安全性高、抗菌谱广而受到广泛关注。前期研究发现一些芽孢杆菌属细菌具有较乳酸菌相比更强的PLA合成能力，并且因其营养要求低、生长迅速、安全性好而具有良好的开发潜力。但由于芽孢杆菌属特有的芽孢形成和同类相食效应，抑制了PLA合成能力的进一步提高。本课题以巨大芽孢杆菌为研究对象，对以下几个问题展开研究，便于开发出基于芽孢杆菌关键生理过程调控策略的PLA合成过程优化方法。主要内容为：敲除紧密调控芽孢生成与同类相食过程的主调控子编码基因spo0A，分析突变株与野生菌生理过程差异及由此导致的PLA合成过程差异。

项目背景：.研究发现乳酸菌具备合成苯基乳酸的能力，Lavermicocca（2000年）在乳酸菌Lactobacillus plantarum 21B的检测到抗真菌性能的苯基乳酸，Jiang Bo等从自然发酵的泡菜中筛选到高产苯基乳酸的乳酸菌Lactobacillus sp. SK007，转化率为51%。.主要研究内容、结果和关键数据：.（1）重组ldhL大肠杆菌全细胞合成L-苯基乳酸体系的构建与优化.来源于Bacillus megaterium Z2013513的ldhL在大肠杆菌中表达，对底物苯丙酮酸的活力增强。全细胞合成结果表明该基因的过量表达能使重组大肠杆菌具有高效合成L-PLA的能力，底物转化率达到71.9%，光学纯度达到96.9%。.（2）基于Bacillus megaterium Z2013513来源的葡萄糖脱氢酶NADH再生体系构建.葡萄糖脱氢酶过量表达强化了细胞内NADH的再生，产量和底物转化率均提高。在流加条件下，PLA产量达到103 mM/h。.（3）重组大肠杆菌辅因子再生循环系统的构建.为克服辅酶再生瓶颈构建了辅因子再生循环系统，再生系统强化了全细胞转化合成L-PLA。E. coli BL21(DE3)/pETduet-Lpldh-gdh合成L-PLA产量始终高于E. coli BL21(DE3)/pETduet-Lpldh。经60 min转化后，L-PLA产量为28.03 mM，PPA转化率达70.08%，转化率提高约10个百分点。.（4）共表达重组大肠杆菌全细胞转化L-苯基乳酸条件优化.共表达菌株E. coli BL21(DE3)/pETduet-Lpldh-gdh全细胞转化的最优转化组合：底物PPA浓度80 mM，缓冲液pH 7.5，菌体浓度OD600为30，反应温度42℃，葡萄糖浓度100 mM。90 min分批补料，L-PLA 产量为108.51 mM，生产速率72.34 mM/h。实现高效不对称还原PPA合成高光学纯度L-PLA。.（5）重组大肠杆菌双相体系合成D-苯基乳酸.水/正己烷双相中，重组大肠杆菌BL21(DE3)/pET-28a-ldhY52V全细胞催化合成D-PLA的最优转化条件为：菌体浓度为15 g/L；正己烷40%；底物浓度15 g/L；转化温度40℃；转速300 rpm；分批补加底物，产率达8.77 g/L/h.