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


D-p-Hydroxyphenylglycine (D-HPG) is a critical intermediary in the biosynthesis of lactam antibiotics such as cephalexin, cefadroxil, and amoxicillin produced by using chemical or enzymatic processes. A two-step enzymatic technique has been widely reported to produce D-HPG from DL-hydroxyphenylhydantoin that is chemically produced (DL-HPH). D-hydantoinase converts DL-HPH to N-carbamoyl-D-p-hydroxyphenylglycine (CpHPG), which is subsequently hydrolyzed by N-carbamoyl-Damino acid amidohydrolase to D-HPG (Case). The synthesis of D-HPG is inefficient and wasteful, with a poor decarbamoylation efficiency. Green pathways for the manufacturing of D-HPG have been created in response to increased environmental concerns. The enzymatic approach is difficult to employ in industry due to the poor solubility of DL-HPH. Surprisingly, the DL-HPH substrate is still made via an environmentally harmful chemical procedure. A full fermentation strategy based on renewable feedstocks has a lot of potential as a biomanufacturing option. Despite the recent finding of orthologous genes encoding enzymes involved in L-HPG biosynthesis in several actinomycete strains, no de novo biosynthetic route for D-HPG production has been revealed in nature. Overexpressing hydroxymandelic acid synthase (HmaS), hydroxymandelic acid oxidase (HmO), and D-phenylglycine aminotransferase in Escherichia coli revealed a metabolic route for generating D-phenylglycine from phenylpyruvate in previous investigations (D-HpgAT).

In a new study published in ACS Synthetic Biology, Dr. Yang Liu, Dr. Bo Yu and Dr. Nengzhong Xie from the Chinese Academy of Sciences developed a new de novo D-HPG synthesis method from glucose. This novel synthetic process has three main benefits: (1) it generates D-HPG from L-phenylalanine, a low-cost, biomass-derived substrate; (2) the cofactor/co-substrate can self-regenerate along the pathway, lowering operating costs; and (3) it is a simple process that emits only CO2, making purification easier. To complete the de novo manufacture, the L-phenylalanine and D-HPG synthesis modules were combined in this study. The creation of an artificial microbiome can reduce the metabolic burden on each host strain, resulting in improved performance. By adjusting the initial inoculation ratio, the co-culture approach may also provide alternate conditions for protein expression, reduce unwanted interference from other pathways, and simply balance the biosynthetic modules. Two amino acid intermediates in the synthetic route, L-phenylalanine and L-tyrosine, were employed as auxotroph supplements to ensure that strain growth and product synthesis were firmly coupled, resulting in a stable and interacting synthetic microbiome. The co-culture approach has the benefit of allowing the biosynthetic modules to balance fast by changing the initial inoculation ratio. In this study, various E. coli and P. putida ratios were shown to contribute to diverse D-HPG production capacities. The majority of products remained stuck in the HMA conversion phase, suggesting poor R-MldB activity, despite the fact that mining enzymes from several sources significantly increased production capacity. In addition to mining from natural sources, protein design and rational mutation will be effective approaches for dramatically improving enzyme performance. Finally, the researchers point out that the D-HPG titer generated in this study was small, making it inappropriate for industrial use. As a result, further research is needed to increase performance. The first priority should be to develop a stable R-MldB enzyme with high activity in physiological conditions.

According to the authors, this is the first research to employ a co-culture approach to investigate cofactor-independent de novo D-HPG synthesis from glucose. Finding new enzymes with relatively high activity at neutral pH and boosting the cellular cofactor NAD+ availability increased the limiting step in D-HPG synthesis catalyzed by R-MdlB. A complementary auxotrophic pattern was engineered to sustain synthetic microbial communities for the de novo synthesis of D-HPG. Indeed, the authors successfully developed a viable alternative for producing D-HPG in a green and sustainable manner.

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

About the author

Bo Yu, PhD, Professor and Deputy Director of CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

Dr. Yu obtained his PhD in Microbiology in Shandong University, Jinan, China in 2006 and had his postdoctoral training at German Helmholtz Centre for Infection Research and Hamburg University of Technology, both funded by German Alexander von Humboldt Fellowship. From 2010, he served as a group leader at Institute of Microbiology, Chinese Academy of Sciences. Dr. Yu currently also serves as the executive director of CAS-TWAS Centre of Excellence for Biotechnology (CoEBio), which the center is focusing on strategic intelligence and international collaboration on bioeconomy as well as industrial biotechnology, both for developing and developed countries.

Current research interests in Dr. Yu’s group are in synthetic biotechnology, including 1) reconstruction of novel metabolic pathway for bio-production of bulk and high value-added fine chemicals; 2) metabolic engineering of strains with better performance for traditional bioprocesses; 3) engineering microbes with improved efficiency and biosecurity features for environmental bioremediation. His research has been funded by the key national and international research grants and resulted in high-quality original research articles published in the peer-reviewed journals including Nucleic Acids Research, Green Chemistry, Metabolic Engineering, Journal of Agricultural and Food Chemistry, ACS Synthetic Biology, etc., as well as patent publications.

E-mail: [email protected]


Liu Y, Xie N, Yu B. De Novo Biosynthesis of D‑p‑Hydroxyphenylglycine by a Designed Cofactor Self-Sufficient Route and Co-culture Strategy. ACS Synthetic Biology. 2022 3;11:1361-1372.

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