背景:L-苯丙氨酸是一种具有多种有前途的应用的必需氨基酸。在野生菌株中从葡萄糖合成L-苯丙氨酸的微生物途径涉及冗长的步骤和严格的反馈调节,这限制了产量。寻找其他候选人很有吸引力,该方法可用于建立一个简洁且经济有效的L-苯丙氨酸生产途径。这里,我们开发了一种人工生物转化方法,从廉价的芳香族前体(苯甲醛或苯甲醇)合成L-苯丙氨酸。特别是,这项工作打开了在辅因子自给自足系统中从苯甲醇生产L-苯丙氨酸的可能性,而无需添加任何还原剂。
结果:工程化的L-苯丙氨酸生物合成途径包括两个模块:在第一个模块中,芳香前体和甘氨酸转化为苯丙酮酸,L-苯丙氨酸的关键前体.高活性酶组合是天然苏氨酸醛缩酶LtaEP。p和苏氨酸脱水酶A8HB。t,可以产生滴度为4.3g/L的丙酮酸苯酯。过表达基因ridA可使丙酮酸产量进一步提高16.3%,达到5g/L第二个模块催化苯丙酮酸合成L-苯丙氨酸,通过共表达PheDH和FDHV120S,丙酮酸的转化率高达93%。然后,含有这两个模块的工程大肠杆菌可以从苯甲醛中生产L-苯丙氨酸,转化率为69%。最后,我们扩展了芳香前体,从苯甲醇生产L-苯丙氨酸,首先构建了辅因子自给自足的生物合成途径,在没有任何额外还原剂如甲酸盐的情况下合成L-苯丙氨酸。
结论:已经设计和构建了系统的生物转化过程,这可以为从低成本起始材料芳香族前体生产高价值L-苯丙氨酸提供潜在的生物基策略。
BACKGROUND: L-phenylalanine is an essential amino acid with various promising applications. The microbial pathway for L-phenylalanine synthesis from glucose in wild strains involves lengthy steps and stringent feedback regulation that limits the production yield. It is attractive to find other candidates, which could be used to establish a succinct and cost-effective pathway for L-phenylalanine production. Here, we developed an artificial bioconversion process to synthesize L-phenylalanine from inexpensive aromatic precursors (benzaldehyde or benzyl alcohol). In particular, this work opens the possibility of L-phenylalanine production from benzyl alcohol in a cofactor self-sufficient system without any addition of reductant.
RESULTS: The engineered L-phenylalanine biosynthesis pathway comprises two modules: in the first module, aromatic precursors and glycine were converted into phenylpyruvate, the key precursor for L-phenylalanine. The highly active enzyme combination was natural threonine aldolase LtaEP.p and threonine dehydratase A8HB.t, which could produce phenylpyruvate in a titer of 4.3 g/L. Overexpression of gene ridA could further increase phenylpyruvate production by 16.3%, reaching up to 5 g/L. The second module catalyzed phenylpyruvate to L-phenylalanine, and the conversion rate of phenylpyruvate was up to 93% by co-expressing PheDH and FDHV120S. Then, the engineered E. coli containing these two modules could produce L-phenylalanine from benzaldehyde with a conversion rate of 69%. Finally, we expanded the aromatic precursors to produce L-phenylalanine from benzyl alcohol, and firstly constructed the cofactor self-sufficient biosynthetic pathway to synthesize L-phenylalanine without any additional reductant such as formate.
CONCLUSIONS: Systematical bioconversion processes have been designed and constructed, which could provide a potential bio-based strategy for the production of high-value L-phenylalanine from low-cost starting materials aromatic precursors.