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Short-chain fatty acid (SCFA) biosynthesis is pertinent to production of biofuels, industrial compounds, and pharmaceuticals from renewable resources. To expand on Escherichia coli SCFA products, we previously implemented a coenzyme A (CoA)-dependent pathway that condenses acetyl-CoA to a diverse group of short-chain fatty acyl-CoAs. To increase product titers and reduce premature pathway termination products, we conducted in vivo and in vitro analyses to understand and improve the specificity of the acyl-CoA thioesterase enzyme, which releases fatty acids from CoA. A total of 62 putative bacterial thioesterases, including 23 from the cow rumen microbiome, were inserted into a pathway that condenses acetyl-CoA to an acyl-CoA molecule derived from exogenously provided propionic or isobutyric acid. Functional screening revealed thioesterases that increase production of saturated (valerate), unsaturated (trans-2-pentenoate), and branched (4-methylvalerate) SCFAs compared to overexpression of E. coli thioesterase tesB or native expression of endogenous thioesterases. To determine if altered thioesterase acyl-CoA substrate specificity caused the increase in product titers, six of the most promising enzymes were analyzed in vitro. Biochemical assays revealed that the most productive thioesterases rely on promiscuous activity but have greater specificity for product-associated acyl-CoAs than for precursor acyl-CoAs. In this study, we introduce novel thioesterases with improved specificity for saturated, branched, and unsaturated short-chain acyl-CoAs, thereby expanding the diversity of potential fatty acid products while increasing titers of current products. The growing uncertainty associated with protein database annotations denotes this study as a model for isolating functional biochemical pathway enzymes in situations where experimental evidence of enzyme function is absent.

The production of mycobactin (MBT) by Mycobacterium tuberculosis is essential for this bacterium to access iron when it is in an infected host. Due to this essential function, there is considerable interest in deciphering the mechanism of MBT assembly, with the goal of targeting select biosynthetic steps for antituberculosis drug development. The proposed scheme for MBT biosynthesis involves assembly of the MBT backbone by a hybrid nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) megasynthase followed by the tailoring of this backbone by N(6) acylation of the central l-Lys residue and subsequent N(6)-hydroxylation of the central N(6)-acyl-l-Lys and the terminal caprolactam. A complete testing of this hypothesis has been hindered by the inability to heterologously produce soluble megasynthase components. Here we show that soluble forms of the NRPS components MbtB, MbtE, and MbtF are obtained when these enzymes are coproduced with MbtH. Using these soluble enzymes we determined the amino acid specificity of each adenylation (A) domain. These results suggest that the proposed tailoring enzymes are actually involved in precursor biosynthesis since the A domains of MbtE and MbtF are specific for N(6)-acyl-N(6)-hydroxy-l-Lys and N(6)-hydroxy-l-Lys, respectively. Furthermore, the preference of the A domain of MbtB for l-Thr over l-Ser suggests that the megasynthase produces MBT derivatives with β-methyl oxazoline rings. Since the most prominent form of MBT produced by M. tuberculosis lacks this β-methyl group, a mechanism for demethylation remains to be discovered. These results suggest revisions to the MBT biosynthesis pathway while also identifying new targets for antituberculosis drug development.

Most functional metagenomic studies have been limited by the poor expression of many genes derived from metagenomic DNA in Escherichia coli, which has been the predominant surrogate host to date. To expand the range of expressed genes, we developed tools for construction and functional screening of metagenomic libraries in Streptomyces lividans. We expanded on previously published protocols by constructing a system that enables retrieval and characterization of the metagenomic DNA from biologically active clones. To test the functionality of these methods, we constructed and screened two metagenomic libraries in S. lividans. One was constructed with pooled DNA from 14 bacterial isolates cultured from Alaskan soil and the second with DNA directly extracted from the same soil. Functional screening of these libraries identified numerous clones with hemolytic activity, one clone that produces melanin by a previously unknown mechanism, and one that induces the overproduction of a secondary metabolite native to S. lividans. All bioactive clones were functional in S. lividans but not in E. coli, demonstrating the advantages of screening metagenomic libraries in more than one host.

We designed a week-long laboratory workshop in metagenomics for a cohort of undergraduate student researchers. During this course, students learned and utilized molecular biology and microbiology techniques to construct a metagenomic library from Puerto Rican soil. Pre-and postworkshop assessments indicated student learning gains in technical knowledge, skills, and confidence in a research environment. Postworkshop construction of additional libraries demonstrated retention of research techniques by the students.