Spangler, A. P. | bioRxiv Science

bioRxiv2024

Combined biosynthesis and site-specific incorporation of phenylalanine derivatives from aryl aldehydes or carboxylic acids in engineered bacteria

Applications of genetic code expansion in live cells are widespread and continually emerging, yet they have been limited by their reliance on the supplementation of non-standard amino acids (nsAAs) to cell culturing media. While advances in cell-free biocatalysis are improving nsAA synthesis cost and sustainability, such processes remain reliant on multi-step processes of product isolation followed by supplementation to engineered cells. Here, we report the design of a modular and genetically encoded system that combines the steps of biosynthesis of diverse phenylalanine derivatives, which are the most frequently used family of nsAAs for genetic code expansion, and their site-specific incorporation within target proteins using a single engineered bacterial host. Unlike previous demonstrations in which individual nsAAs were targeted for biosynthesis and site-specific incorporation, our system serves as a platform that exhibits broad substrate specificity towards commercially ubiquitous, achiral building blocks of aryl aldehydes or carboxylic acids. We demonstrate that this modular system enables high fidelity biosynthesis and incorporation of nsAAs for multiple industrially relevant nsAAs, such as the incorporation of 4,4-L-biphenylalanine within proteins after supplementation with biphenylaldehyde and the incorporation of 4-azido-L-phenylalanine within proteins after supplementation with 4-azido-benzoic acid. Finally, we show that the combination of nsAA biosynthesis and incorporation steps can extend the chemical reach of the intrinsic biological containment strategy of synthetic auxotrophy from nsAAs to low-cost and achiral building blocks. We anticipate that our system will aid industrial-scale manufacturing of proteins that contain nsAAs and democratize access to expensive or commercially unavailable chemistries for labs that lack separations or synthesis expertise.

synthetic biology↗

bioRxiv2024

A One-Pot Biocatalytic Cascade to Access Diverse L-Phenylalanine Derivatives from Aldehydes or Carboxylic Acids

Non-standard amino acids (nsAAs) that are L-phenylalanine derivatives with aryl ring functionalization have long been harnessed in natural product synthesis, therapeutic peptide synthesis, and diverse applications of genetic code expansion. Yet, to date these chiral molecules have often been the products of poorly enantioselective and environmentally harsh organic synthesis routes. Here, we reveal the broad specificity of multiple natural pyridoxal 5-phosphate (PLP)-dependent enzymes, specifically an L-threonine transaldolase, a phenylserine dehydratase, and an aminotransferase, towards substrates that contain aryl side chains with diverse substitutions. We exploit this tolerance to construct a one-pot biocatalytic cascade that achieves high-yield synthesis of 18 diverse L-phenylalanine derivatives from aldehydes under mild aqueous reaction conditions. We demonstrate addition of a carboxylic acid reductase module to this cascade to enable the biosynthesis of L-phenylalanine derivatives from carboxylic acids that may be less expensive or less reactive than the corresponding aldehydes. Finally, we investigate the scalability of the cascade by developing a lysate-based route for preparative-scale synthesis of 4-formyl-L-phenylalanine, a nsAA with a bio-orthogonal handle that is not readily market-accessible. Overall, this work offers an efficient, versatile, and scalable route with the potential to lower manufacturing cost and democratize synthesis for many valuable nsAAs.

synthetic biology↗