肠杆菌中以葡萄糖为原料利用苯丙酮酸途径合成苯乙醇的代谢调控研究

2-Phenylethanl (2-PE) is one of the most important flavour and fragrance in the present market. However, most fermentations used phenylalanine as feedstock, the prices of which barred the industrial scale production of 2-PE. To address this issue, new methods with cheaper feedstock to produce 2-PE were needed. In our previous study, a new strain Enterobacter sp. CGMCC 5087 was isolated and identified. The strain was verified to produce 2-PE using de-novo synthetic pathway with monosaccharide as a carbon source and NH4Cl as a nitrogen source. In this study, we would like to regulate the phenylpyruvate pathway at the transcription levels based on the regulations in prokaryote and the character of phenylpyruvate pathway, according to the principle of economy and balance. Two genes of rate-limiting enzymes, chorismate mutase p-prephenate dehydratase (PheA) and 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase (DAHP), were tried to be controlled by promoters at different strength to produce the two enzymes at fixed quantities and ratios. A constructed temperature-sensitive promoter was applied to control the expression of 2-keto-acid decarboxylases (KDCs) at the proper time and quantities to balance the phenylpyruvate in phenylpyruvate pathway. Combined with the results in feedstock fermentation, we try to reveal the metabolic regulation mechanism of the phenylpyruvate pathway in E. sp. CGMCC 5087. This study is expected to lay the foundation for microbial synthesis of 2-PE using renewable sugar as feedstock. It also provides data for the metabolic regulation of other chemicals using shikimate pathway.

苯乙醇是市场份额居第二位的香料。目前微生物法合成苯乙醇的主要原料为苯丙氨酸。由于该化合物价格过高，极大限制了苯乙醇的生物合成。因此，挖掘新的合成途径，建立以廉价原料合成苯乙醇的新方法，成为解决该问题的有效手段。本工作前期鉴定了一株以葡萄糖为原料通过苯丙酮酸途径合成苯乙醇的菌株Enterobacter. sp. CGMCC 5087，并初步确定了两个参与该代谢路径的关键酶。基于上述基础，本研究依据微生物代谢中体现出的"生物平衡性与经济性原则"为指导，结合原核微生物的调控和苯丙酮酸代谢途径关键酶的特点，从转录水平上进行调控，利用启动子工程和温控启动子等技术，实现苯丙酮酸代谢途径关键酶DAHP、PheA和苯丙酮酸脱羧酶在宿主中的最经济性表达，在提高转化效率的同时保持菌株代谢的稳定性；结合发酵控制，对改菌株苯丙酮酸代谢途径的调控进行研究。研究有望为以可再生糖为原料，微生物法生产苯乙醇奠定基础。

苯乙醇是市场份额居第二位的香料。目前微生物法合成苯乙醇的主要原料为苯丙氨酸。由于该化合物价格过高，极大限制了苯乙醇的生物合成。因此，挖掘新的合成途径，建立以廉价原料合成苯乙醇的新方法，成为解决该问题的有效手段。本工作前期鉴定了一株以葡萄糖为原料通过苯丙酮酸途径合成苯乙醇的菌株Enterobacter. sp. CGMCC 5087，并初步确定了两个参与该代谢路径的关键酶。本研究实现了利用质粒表达强度和启动子强度对关键酶NAHP和Phe A从代谢经济性和平衡性方面进行控制；利用温控启动子对苯丙酮酸脱羧酶进行表达量和表达时序性的优化，使关键酶以最为经济的量进行表达；获得了三个关键酶的调控方案，进行了发酵罐发酵并发现苯乙醇和乙偶姻联产及Enterobacter sp. CGMCC 5087可降解苯乙醇的现象。在此基础上进而进行以下计划外研究：过对E. sp. CGMCC 5087进行全基因组测序挖掘苯乙醇合成途径的关键基因；并成功鉴定了苯乙醇合成途径的限速酶-苯丙酮酸脱羧酶KDC4427和ADH4428；对苯乙醇生物合成代谢途径进行调控，过表达aroF、pheA 、tktA和ppsA，苯乙醇产量可达到320 mg/L；还开展了KDC4427 及ADH4428的转录调控相关的工作，发现二者以头对头方式位于同一启动子的两侧，其关键区域为1-145bp，其转录表达受到L-Phe的诱导，受到2-PE的抑制；进行了苯乙烯合成的相关工作，经过系统优化，苯乙烯的产量由最初的55 mg/L提高到350 mg/L；对金属离子在调整有机溶剂的耐受性方面进行了研究，提高了宿主的产物耐受性。这是目前在摇瓶发酵水平上最高的产量，这为以后苯乙烯和苯乙醇的工业化生产奠定了基础。同时研究过程中发现的细菌来源的苯乙醇双向启动子和苯丙酮酸脱羧酶具有重要的理论研究价值。