Thioesterase-Catalyzed Aminoacylation and Thiolation of Polyketides in Fungi

Abstract

Fungal highly reducing polyketide synthases (HRPKSs) biosynthesize polyketides using a single set of domains iteratively. Product release is a critical step in HRPKS function to ensure timely termination and enzyme turnover. Nearly all of the HRPKSs characterized to date employ a separate thioesterase (TE) or acyltransferase enzyme for product release. In this study, we characterized two fungal HRPKSs that have fused C-terminal TE domains, a new domain architecture for fungal HRPKSs. We showed that both HRPKS-TEs synthesize aminoacylated polyketides in an ATP-independent fashion. The KU42 TE domain selects cysteine and homocysteine, and catalyzes transthioesterification using the side chain thiol group as the nucleophile. In contrast, the KU43 TE domain selects leucine methyl ester and performs a direct amidation of the polyketide, a reaction typically catalyzed by nonribosomal peptide synthetase (NRPS) domains. The characterization of these HRPKS-TE enzymes showcase the functional diversity of HRPKS enzymes, and provides potential TE domains as biocatalytic tools to diversify HRPKS structures.

A CRISPR Interference Screen of Essential Genes Reveals that Proteasome Regulation Dictates Acetic Acid Tolerance in Saccharomyces cerevisiae

“…In this strain collection of roughly 9,000 strains, nearly 99% of the essential and 98% of the respiratory growth-essential genes have been targeted with up to 17 gRNAs per target gene ( 12 ). Recently, the construction and phenotypic screening of CRISPR technology-based S. cerevisiae libraries have been demonstrated to be very efficient to identify bioengineering genetic candidates to increase the production of β-carotene or endoglucanase ( 15 ), regulate polyketide synthesis ( 16 ), or improve tolerance to furfural ( 11 ) or lignocellulose hydrolysates ( 13 ).…”

Chain release mechanisms in polyketide and non-ribosomal peptide biosynthesis

“…Another thioesterase domain capable of catalysing intermolecular trans-esterication, specically a transthioesterication, was identi-ed in the biosynthesis pathway for an aminoacylated polyketide (11) in basidiomycete Punctularia strigosozonata. 33 Here, the TE domain of the PKS KU42 was shown to catalyse release of the sorbyl unit using the thiol group of L-cysteine or Lhomocysteine as an intermolecular nucleophile (Fig. 4B).…”

“…4B). 33 3.1.4 Amines (amidation). TE domains that catalyse chain release via an intermolecular amidation reaction are also uncommon.…”

A CRISPRi screen of essential genes reveals that proteasome regulation dictates acetic acid tolerance inSaccharomyces cerevisiae

“…The deletions of genes encoding the vacuolar membrane ATPase complex (VMA2-8, 13,16,21,22) has been shown to decrease the tolerance to acetic acid (22,36), presumably as cells struggle to maintain a neutral cytosolic pH (42). Similarly, single gene deletions of VPS genes (encoding a GTPases required for vacuolar sorting) have been shown to result in a drastically enhanced sensitivity to acetic acid and a drop in intracellular pH (43).…”

“…Recently, the construction and phenotypic screening of CRISPR technology-based S. cerevisiae libraries have been demonstrated to be very efficient to identify bioengineering genetic candidates to increase production of β-carotene or endoglucanase (15), regulate polyketide synthesis (16) or improve tolerance to furfural (11) or lignocellulose hydrolysate (13).…”