De Novo Biosynthesis of D-p-Hydroxyphenylglycine by a Designed Cofactor Self-Sufficient Route and Co-culture Strategy,ACS Synthetic Biology

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De Novo Biosynthesis of D-p-Hydroxyphenylglycine by a Designed Cofactor Self-Sufficient Route and Co-culture Strategy
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2022-03-04 , DOI: 10.1021/acssynbio.2c00007
Yang Liu _{1,

2} , Nengzhong Xie ₃ , Bo Yu ₁

Affiliation

d-p-Hydroxyphenylglycine (D-HPG) is an important intermediate for the synthesis of β-lactam antibiotics with an annual market demand of thousands of tons. Currently, the main production processes are via chemical approaches. Although enzymatic conversion has been investigated for D-HPG production, synthesis of the substrate DL-hydroxyphenylhydantoin is still chemically based, which suffers from high pollution and harsh reaction conditions. In this study, one cofactor self-sufficient route for D-HPG production from l-phenylalanine was newly designed and the artificial pathway was functionalized by selecting suitable enzymes and adjusting their expressions in strain Pseudomonas putida KT2440. Notably, a new R-mandelate dehydrogenase from Lactococcus lactis with relatively high activity under pH neutral conditions was successfully mined to demonstrate the biosynthetic pathway in vivo. The performance of the engineered P. putida strain was further increased by enhancing cellular NAD availability and blocking l-phenylalanine consumption. Coupled with the l-phenylalanine producer, Escherichia coli strain ATCC 31884, a stable and interactive co-culture process was also developed by engineering a “cross-link auxotrophic” system to produce D-HPG directly from glucose. Thus, this study is the first approach for the de novo biosynthesis of D-HPG by engineering a non-natural pathway and lays the foundation for further improving the efficiency of D-HPG production via a green and sustainable route.

中文翻译：

通过设计的辅因子自给自足途径和共培养策略从头生物合成 Dp-羟基苯基甘氨酸

d - p-羟基苯基甘氨酸（D-HPG）是合成β-内酰胺类抗生素的重要中间体，年市场需求量达数千吨。目前，主要的生产过程是通过化学方法。尽管已经研究了用于 D-HPG 生产的酶促转化，但底物 DL-羟基苯基乙内酰脲的合成仍以化学为基础，具有高污染和苛刻的反应条件。在这项研究中，新设计了一条从L-苯丙氨酸生产D-HPG的辅助因子自给自足途径，并通过选择合适的酶并调节其在恶臭假单胞菌KT2440菌株中的表达来对人工途径进行功能化。值得注意的是，新的R从乳酸乳球菌中提取的-扁桃酸脱氢酶在pH中性条件下具有较高的活性，被成功地挖掘出来，以证明体内的生物合成途径。通过增强细胞 NAD 可用性和阻断l-苯丙氨酸消耗进一步提高了工程化恶臭假单胞菌菌株的性能。与l-苯丙氨酸生产者大肠杆菌菌株 ATCC 31884 相结合，还通过设计“交联营养缺陷型”系统以直接从葡萄糖生产 D-HPG，开发了一种稳定且互动的共培养过程。因此，这项研究是从头开始的第一种方法通过工程非自然途径生物合成D-HPG，为通过绿色和可持续途径进一步提高D-HPG生产效率奠定基础。

更新日期：2022-03-04

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