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. 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. 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.

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系统工程能够在大肠杆菌中利用廉价的芳香族前体高效生物合成 L-苯丙氨酸

L-苯丙氨酸是一种必需氨基酸，具有多种有前景的应用。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，限制了产量。寻找其他候选物很有吸引力，它们可用于建立简洁且具有成本效益的 L-苯丙氨酸生产途径。在这里，我们开发了一种人工生物转化工艺，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，这项工作开启了在辅因子自给自足的系统中，无需添加任何还原剂，从苯甲醇生产 L-苯丙氨酸的可能性。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，芳香族前体和甘氨酸被转化为苯丙酮酸，这是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，可以产生效价为4.3 g/L的苯丙酮酸。基因ridA的过表达可以使苯丙酮酸产量进一步增加16.3%，达到5 g/L。第二个模块催化苯丙酮酸生成L-苯丙氨酸，通过共表达PheDH和FDHV120S，苯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生产L-苯丙氨酸，转化率为69%。最后，我们扩展了芳香族前体，以苯甲醇为原料生产L-苯丙氨酸，并首次构建了辅因子自给自足的生物合成途径，无需任何额外的还原剂（例如甲酸盐）即可合成L-苯丙氨酸。系统的生物转化工艺已经被设计和构建，这可以为从低成本的起始原料芳香族前体生产高价值的L-苯丙氨酸提供潜在的生物基策略。