维生素B6是重要的水溶性维生素之一,在生物医药、饲料及食品美容等方面具有广泛的应用。目前工业上通常采用噁唑法生产维生素B6,该方法存在安全隐患,易造成环境污染。该研究以大肠杆菌为底盘细胞构建了具有潜力的环境友好型维生素B6细胞工厂。为了降低中间代谢物4-磷酸羟基-L-苏氨酸(4-hydroxy-L-threonine,4HTP)对大肠杆菌的生物毒性,提升维生素B6在大肠杆菌中的发酵产量,通过构建整合型蛋白支架,并结合随机突变技术对关键酶PdxA3进行了改造。结果显示,4种不同来源的4HTP脱氢酶(PdxA)中,Sinorhizobium meliloti来源的PdxA3活性最高,使产量达到2.24 mg/L;针对PdxA3进行的随机突变使维生素B6产量提升了60%,产量达到3.92 mg/L;整合型蛋白支架优化比例为1∶1∶2时,产量有了大幅提升,与出发菌株相比,产量提升约20倍,最终达到了21.23 mg/L。通过实验发现,最适来源和最优表达比例的途径酶能使产量大幅提升。该研究基于蛋白支架技术,避免了代谢过程有毒中间产物过度积累,对于大肠杆菌维生素B6工程菌株的构建具有重要作用。
Vitamin B6 is one of the important water-soluble vitamins, which has been widely applied in biomedicine, feed, food ,and beauty industries. At present, oxazole method is usually used for the production of vitamin B6 in industry, which has safety risks and causes environmental pollution. In this study, we constructed a potential and environmental vitamin B6 cell factory using Escherichia coli as chassis cells. In order to enhance the fermentation yield of vitamin B6 in E. coli and reduce the biotoxicity of the intermediate metabolite 4-hydroxy-L-threonine (4HTP) to E. coli, the key enzymes were modified by constructing an integrated protein scaffold and combining with random mutation technology. The results showed that among four different sources of 4HTP dehydrogenase (PdxA), PdxA3 from Sinorhizobium meliloti had the highest activity, reaching a yield of 2.24 mg/L; random mutation targeting PdxA3 resulted in a 60% increase in vitamin B6 titer, with a yield of 3.92 mg/L; the protein scaffold optimized at a ratio of 1∶1∶2 resulted in a greatly improved, with an approximately 20-fold increase in yield, compared to the starting strain, and eventually reached 21.23 mg/L. It was found that the pathway enzyme with optimal source and optimal expression ratio could significantly increase titer. This study is based on protein scaffolding technology, which avoids excessive accumulation of toxic intermediates in the metabolic process and is important for the construction of E. coli vitamin B6 engineered strains.
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