Intrinsic and Extrinsic Programming of Product Chain Length and Release Mode in Fungal Collaborating Iterative Polyketide Synthases

Abstract

Combinatorial biosynthesis with fungal polyketide synthases (PKSs) promises to produce unprecedented bioactive "unnatural" natural products (uNPs) for drug discovery. Genome mining of the dothideomycete Rhytidhysteron rufulum uncovered a collaborating highly reducing PKS (hrPKS) -nonreducing PKS (nrPKS) pair. These enzymes produce trace amounts of rare S-type benzenediol macrolactone congeners with a phenylacetate core in a heterologous host. However, subunit shuffling and domain swaps with voucher enzymes demonstrated that all PKS domains are highly productive. This contradiction led us to reveal novel programming layers exerted by the starter unit acyltransferase (SAT) and the thioesterase (TE) domains on the PKS system. First, macrocyclic vs. linear product formation is dictated by the intrinsic biosynthetic program of the TE domain. Next, the chain length of the hrPKS product is strongly influenced in trans by the offloading preferences of the nrPKS SAT domain. Last, TE domains are size-selective filters that facilitate or obstruct product formation from certain priming units. Thus, the intrinsic programs of the SAT and TE domains are both part of the extrinsic program of the hrPKS subunit, and modulate the observable metaprogram of the whole PKS system. Reconstruction of SAT and TE *

Cloning and Functional Characterization of the Polyketide Synthases Based on Genome Mining of Preussia isomera XL-1326

“…The PCR products were purified with a GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Vilnius, Lithuania). The three amplicons and the Nde I/ Pme I fragment of YEpADH2p-FLAG-URA ( S. cerevisiae empty expression vector; Wang et al, 2019 , 2020 ) were linked together end-to-end with the SE Seamless Cloning and Assembly Kit (ZOMANBIO, Beijing, China) according to homologous recombination principle. The recombination reaction mix (10.0 μl) was prepared with 2.0 μl of SE cloning buffer (5 × ), 2.0 μl of fragments 1, 2, and 3, respectively, 1.0 μl of Nde I/ Pme I fragment of YEpADH2p-FLAG-URA, and 1.0 μl of SE recombinase, and then proceeded to the ligation step at 37°C for 0.5 h to generate plasmid YEpPreu3.…”

“…Fungal polyketides (PKs) are one of the largest classes of small-molecule natural products with diverse structures and various interesting biological activities. They can typically be classified into naphthopyrones, isocoumarins, acetyl tetrahydroxynaphthalene (ATHN), anthraquinones, benzenediol lactones (BDLs), and orsellinic acid (OA) derivatives (Lackner et al, 2013 ; Hussain et al, 2017 ; Wang et al, 2020 ). Importantly, they also provide lead compounds and inspiration for pharmaceutical drug discovery, which is evidenced by the cholesterol-lowering agents/statins, the antibiotic griseofulvin, and the immunosuppressant mycophenolic acid (Keller, 2019 ).…”

One Polyketide Synthase, Two Distinct Products: Trans‐Acting Enzyme‐Controlled Product Divergence in Calbistrin Biosynthesis

“…On the other hand, no enoyl reduction is required at this point for the synthesis of 3 , and therefore, the production of 3 could be hampered in the presence of CalK. Chain length of fungal type I PKSs is known to be controlled by several factors, such as in cis ‐control by KR domains [28] and in trans ‐control by starter unit acyltransferase (SAT) [29] or TE domains, [30] and CalK thus adds another example of a chain length control mechanism in fungal PKS systems.…”

One Polyketide Synthase, Two Distinct Products: Trans‐Acting Enzyme‐Controlled Product Divergence in Calbistrin Biosynthesis

“…However,inthe third round of chain processing,the carbonyl reduction occurs completely in the presence of the trans-ER CalK, leading to the production of 1.O nt he other hand, no enoyl reduction is required at this point for the synthesis of 3,and therefore,the production of 3 could be hampered in the presence of CalK. Chain length of fungal type IPKSs is known to be controlled by several factors,such as in cis-control by KR domains [28] and in trans-control by starter unit acyltransferase (SAT) [29] or TE domains, [30] and CalK thus adds another example of ac hain length control mechanism in fungal PKS systems.…”