Improved l-phenylglycine synthesis by introducing an engineered cofactor self-sufficient system

Area of Science:

• Biotechnology and Metabolic Engineering
• Synthetic Biology
• Pharmaceutical Chemistry

Background:

• l-phenylglycine (L-phg) is a crucial non-proteinogenic amino acid for pharmaceuticals like antibiotics and anticancer drugs.
• Microbial bioconversion offers a sustainable and enantioselective alternative to traditional organic synthesis for L-phg production.
• Existing microbial synthesis routes face challenges with low yields and cofactor limitations.

Purpose of the Study:

• To engineer a whole-cell biocatalyst in *Escherichia coli* for efficient L-phg synthesis from l-phenylalanine.
• To enhance L-phg production through the introduction of novel enzymes and a cofactor self-sufficient system.
• To improve the overall yield and productivity of L-phg via metabolic engineering strategies.

Main Methods:

• Construction of an L-phg synthesis pathway in *Escherichia coli*.
• Introduction of engineered hydroxymandelate synthases and oxidases to boost L-phg production.
• Implementation of a cofactor self-sufficient system to regenerate essential cofactors (2-oxoglutarate, NH₄⁺, NADH) and a protein scaffold to minimize cofactor loss.

Main Results:

• Initial L-phg production reached 0.23 mM from 10 mM l-phenylalanine.
• Application of new enzymes increased L-phg production fivefold.
• The cofactor self-sufficient system enhanced L-phg to 2.82 mM, and the protein scaffold further improved it to 3.72 mM with a yield of 0.34 g/g L-phe.

Conclusions:

• A robust whole-cell biocatalyst was developed for the efficient, cofactor self-sufficient production of L-phg.
• This engineered system demonstrates a viable strategy for converting amino acids into value-added chiral amines.
• The approach holds significant potential for sustainable and cost-effective pharmaceutical intermediate manufacturing.