Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities.

Enzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales. Using a continuous directed evolution platform called OrthoRep, we rapidly evolve the Thermotoga maritima tryptophan synthase ß-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only on TmTrpB's primary activity of synthesizing L-tryptophan from indole and L-serine. We find that the resulting sequence-diverse TmTrpB variants span a range of substrate profiles useful in industrial biocatalysis and suggest that the depth and scale of evolution that OrthoRep affords will be generally valuable in enzyme engineering and the evolution of biomolecular functions.

Engineering enzymes for noncanonical amino acid synthesis.

The standard proteinogenic amino acids grant access to a myriad of chemistries that harmonize to create life. Outside of these twenty canonical protein building blocks are countless noncanonical amino acids (ncAAs), either found in nature or created by man. Interest in ncAAs has grown as research has unveiled their importance as precursors to natural products and pharmaceuticals, biological probes, and more. Despite their broad applications, synthesis of ncAAs remains a challenge, as poor stereoselectivity and low functional-group compatibility stymie effective preparative routes. The use of enzymes has emerged as a versatile approach to prepare ncAAs, and nature's enzymes can be engineered to synthesize ncAAs more efficiently and expand the amino acid alphabet. In this tutorial review, we briefly outline different enzyme engineering strategies and then discuss examples where engineering has generated new 'ncAA synthases' for efficient, environmentally benign production of a wide and growing collection of valuable ncAAs.

Engineered Biosynthesis of ß-Alkyl Tryptophan Analogues.

Noncanonical amino acids (ncAAs) with dual stereocenters at the α and ß positions are valuable precursors to natural products and therapeutics. Despite the potential applications of such bioactive ß-branched ncAAs, their availability is limited due to the inefficiency of the multistep methods used to prepare them. Herein we report a stereoselective biocatalytic synthesis of ß-branched tryptophan analogues using an engineered variant of Pyrococcus furiosus tryptophan synthase (PfTrpB), PfTrpB7E6 . PfTrpB7E6 is the first biocatalyst to synthesize bulky ß-branched tryptophan analogues in a single step, with demonstrated access to 27 ncAAs. The molecular basis for the efficient catalysis and broad substrate tolerance of PfTrpB7E6 was explored through X-ray crystallography and UV/Vis spectroscopy, which revealed that a combination of active-site and remote mutations increase the abundance and persistence of a key reactive intermediate. PfTrpB7E6 provides an operationally simple and environmentally benign platform for the preparation of ß-branched tryptophan building blocks.

Improved Synthesis of 4-Cyanotryptophan and Other Tryptophan Analogues in Aqueous Solvent Using Variants of TrpB from Thermotoga maritima.

The use of enzymes has become increasingly widespread in synthesis as chemists strive to reduce their reliance on organic solvents in favor of more environmentally benign aqueous media. With this in mind, we previously endeavored to engineer the tryptophan synthase ß-subunit (TrpB) for production of noncanonical amino acids that had previously been synthesized through multistep routes involving water-sensitive reagents. This enzymatic platform proved effective for the synthesis of analogues of the amino acid tryptophan (Trp), which are frequently used in pharmaceutical synthesis as well as chemical biology. However, certain valuable compounds, such as the blue fluorescent amino acid 4-cyanotryptophan (4-CN-Trp), could only be made in low yield, even at elevated temperature (75 °C). Here, we describe the engineering of TrpB from Thermotoga maritima that improved synthesis of 4-CN-Trp from 24% to 78% yield. Remarkably, although the final enzyme maintains high thermostability ( T50 = 93 °C), its temperature profile is shifted such that high reactivity is observed at ∼37 °C (76% yield), creating the possibility for in vivo 4-CN-Trp production. The improvements are not specific to 4-CN-Trp; a boost in activity at lower temperature is also demonstrated for other Trp analogues.