De Novo Biosynthesis of D‑p‑Hydroxyphenylglycine by a Designed Cofactor Self-Sufficient Route and Co-culture Strategy

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.