Enzyme and metabolic engineering offer the potential to develop biocatalysts for converting natural resources into a wide range of chemicals. To broaden the scope of potential products beyond natural metabolites, methods of engineering enzymes to accept alternative substrates and/or perform novel chemistries must be developed. DNA synthesis can create large libraries of enzyme-coding sequences, but most biochemistries lack a simple assay to screen for promising enzyme variants. Our solution to this challenge is structure-guided mutagenesis in which optimization algorithms select the best sequences from libraries based on specified criteria (i.e. binding selectivity). Here, we demonstrate this approach by identifying medium-chain (C6-C12) acyl-ACP thioesterases through structure-guided mutagenesis. Medium-chain fatty acids, products of thioesterase-catalyzed hydrolysis, are limited in natural abundance compared to long-chain fatty acids; the limited supply leads to high costs of C6-C10 oleochemicals such as fatty alcohols, amines, and esters. Here, we applied computational tools to tune substrate binding to the highly-active ‘TesA thioesterase in Escherichia coli. We used the IPRO algorithm to design thioesterase variants with enhanced C12- or C8-specificity while maintaining high activity. After four rounds of structure-guided mutagenesis, we identified three thioesterases with enhanced production of dodecanoic acid (C12) and twenty-seven thioesterases with enhanced production of octanoic acid (C8). The top variants reached up to 49% C12 and 50% C8 while exceeding native levels of total free fatty acids. A comparably sized library created by random mutagenesis failed to identify promising mutants. The chain length-preference of ‘TesA and the best mutant were confirmed in vitro using acyl-CoA substrates. Molecular dynamics simulations, confirmed by resolved crystal structures, of ‘TesA variants suggest that hydrophobic forces govern ‘TesA substrate specificity. We expect that the design rules we uncovered and the thioesterase variants identified will be useful to metabolic engineering projects aimed at sustainable production of medium-chain oleochemicals.

Microbial metabolism is an attractive route for producing medium chain length fatty acids, e.g., octanoic acid, used in the oleochemical industry. One challenge to this strategy is the lack of enzymes that are both highly active in a microbial host and selective toward substrates with desired chain length. Of the many steps in fatty acid biosynthesis, the thioesterase is the most widely used enzyme for controlling chain length. Thioesterases hydrolyze the thioester bond between fatty acids and the acyl-carrier protein (ACP) or coenzyme A (CoA) cofactor. The functional role of thioesterases varies between organisms ( i.e., bacteria vs plant) and therefore so do the substrate specificities. As a result, microbial biocatalysts that utilize a heterologous thioesterase either produce high titers of fatty acids with mixed chain lengths or low titers of products with a narrow chain length distribution. To search for highly active enzymes that selectively hydrolyze octanoyl-ACP, we developed a genetic selection based on the lipoic acid requirement of Escherichia coli. We used the selection to identify variants in a randomly mutagenized library of the C-specific Cuphea palustris FatB1 thioesterase. After optimizing expression of the thioesterase, E. coli cultures produced 1.7 g/L of octanoic acid with >90% specificity from a single chromosomal copy of this thioesterase. In vitro studies confirmed the mutant thioesterase possessed a 15-fold increase in k compared to its native sequence. The high level of specific activity allowed for low levels of expression while maintaining fatty acid titer. The low expression requirement will allow metabolic engineers to use more cellular resources to address other limitations in the pathway and maximize overall productivity.

In S. cerevisiae the thiamin pyrimidine is formed from histidine and pyridoxal phosphate. The origin of all of the pyrimidine atoms has been determined using labeling studies and suggests that the pyrimidine is formed using remarkable chemistry that is without chemical or biochemical precedent. Here we report the overexpression of the closely related Candida albicans pyrimidine synthase (THI5p) and the reconstitution and preliminary characterization of the enzymatic activity. A structure of the C. albicans THI5p shows PLP bound at the active site via an imine with Lys62 and His66 in close proximity to the PLP. Our data suggest that His66 of the THI5 protein is the histidine source for pyrimidine formation and that the pyrimidine synthase is a single turnover enzyme.

Reaction of o-(diphenylphosphino)(N-benzylidene)aniline (P∼N) with [Ir(COD)Cl]2 affords the substitution product [(P∼N)Ir(COD)Cl] (1). Treatment of 1 with AgBF4 yields the cyclometalated iridium hydride complex [P,N,C-(P∼N)Ir(COD)H]BF4 (2). On the other hand, under atmospheric pressure of CO, carrying out the substitution of [Ir(COD)Cl]2 with P∼N results in the formation of [P,N,C-(P∼N)Ir(CO)HCl] (5). Conversion of 4 into 5 can be achieved by the reaction of 4 with CO in the presence of tetraethylammonium chloride. Both 4 and 5 are characterized by spectroscopic and X-ray structural analyses. All iridium complexes are not good catalysts for hydroamination. However, the combination of 5 with NaB[3,5-C6H3(CF3)2]4 (denoted as NaBArF 4) provides a potent catalytic system for both intra- and intermolecular hydroamination of alkynes. Intramolecular reaction of o-(2-phenylethynyl)anilines produces the corresponding indoles in good yields. Furthermore, intermolecular hydroamination takes place smoothly to generate the imine intermediates, which could be subsequently reduced by triethylsilane using the same catalyst, giving N-silylated amines. However, the N-silylated amines readily hydrolyze to produce secondary amines.