Bacterial Genome Mining of Enzymatic Tools for Alkyne Biosynthesis

Abstract

The alkyne is an important functionality widely used in material science, pharmaceutical science, and chemical biology, but the importance of this functionality is contrasted by the very limited number of enzymes known to be involved in alkyne biosynthesis. We recently reported the first known carrier protein-dependent pathway for terminal alkyne formation, and in silico analysis suggested that this mechanism could be widespread in bacteria. In this paper, we screened additional homologous gene cassettes presumed to be involved in alkyne biosynthesis using both in vitro biochemical study and an E. coli-polyketide synthase (PKS) reporting system for in vivo analysis. We discovered and characterized a new terminal alkyne biosynthetic pathway comprised of TtuA, B, and C from Teredinibacter turnerae T7901. While the acyl-CoA ligase homolog (TtuA) demonstrated promiscuity in the activation and loading of medium-chain fatty acids onto the carrier protein (TtuC), the desaturase homolog (TtuB) showed stringent substrate specificity towards C10 fatty acyl moieties. In addition, TtuB was demonstrated to be a bifunctional desaturase/acetylenase that efficiently catalyzed two sequential O2-dependent dehydrogenation reactions. A novel terminal-alkyne bearing polyketide was further produced upon co-expression of ttuABC and a PKS gene in E. coli. The discovery and characterization of TtuA, B, and C provides us with a new bifunctional desaturase/acetylenase for mechanistic and structural study and expands the scarce enzyme inventory for the biosynthesis of the alkyne functionality, which has important applications in synthetic and chemical biology.

Probing the Acyl Carrier Protein-Enzyme Interactions within Terminal Alkyne Biosynthetic Machinery

“…We previously reported that a promiscuous type III polyketide synthase (PKS), HsPKS1, could incorporate a CP-bound fatty acyl moiety into the PK backbone, and consequently generate a terminal alkyne-tagged PK 1 in an engineered E. coli BAP1 strain that co-expressed jamABC and hspks1 (named as strain XZ1; Figure 3; Supporting Information Figure S3). 5 As HsPKS1 shows no preference toward different CP-bound acyl moieties, 6,15,31 this E.coli-hspks1 platform could be a feasible reporting system for the study of JamB-ACP interactions. We introduced ACP-encoding gene into the engineered E. coli BAP1 strain that co-expressed hspks1, jamA, and jamB.…”

A fluorogenic screening platform enables directed evolution of an alkyne biosynthetic tool

“…The resulting structure showed that I23 is located at the interface of the two proteins, and thus I23M likely benefits the interaction between JamB and JamC (Figure 3b). M5 is located near the N -terminus of JamB, and interestingly, our recent characterization of JamB and its homologs CamB 14, 27 (identity/similarity = 95%/98%), and TtuB 15 (identity/similarity = 44%/60%) demonstrated that their activities in E. coli were affected by the amino acids at the non-homologous N -terminal region (Figure S7). This was further confirmed by the construction of chimeric JamB mutants with the N -terminal region replaced by the one from the more efficient desaturase/acetylenase TtuB or the less efficient CamB.…”

“…For example, triazole-forming azide-alkyne [3+2] cycloaddition reaction (often referred to as click reaction) and Raman scattering microscopy of alkynes have enabled the imaging and mode of action study of alkyne-tagged glycans, proteins, nucleic acids, lipids, and natural products 3–9 . However, the prevalence of the alkyne functionality in synthetic molecules and its significance in chemical biology are contrasted by the very limited number of known alkyne biosynthetic tools 10–15 .…”

Probing the acyl carrier protein–Enzyme interactions within terminal alkyne biosynthetic machinery

“…In particular, two homologous pathways, JamABC from Moorea producens JHB and TtuABC from Teredinibacter turnerae T7901, have been identified which activate hexanoic acid and decanoic acid, respectively. We further showed that omitting the ACP, either JamC or TtuC in the respective pathway, completely abolished or significantly impaired alkyne synthesis, demonstrating the necessity of ACP in terminal alkyne biosynthesis.…”