Abstract
BackgroundPolyketides are one of the most important classes of secondary metabolites and usually make good drugs. Currently, heterologous production of fungal polyketides for developing a high potential industrial application system with high production capacity and pharmacutical feasibility was still at its infancy. Pichia pastoris is a highly successful system for the high production of a variety of heterologous proteins. In this work, we aim to develop a P. pastoris based in vivo fungal polyketide production system for first time and evaluate its feasibility for future industrial application.ResultsA recombinant P. pastoris GS115-NpgA-ATX with Aspergillus nidulans phosphopantetheinyl transferase (PPtase) gene npgA and Aspergillus terrus 6-methylsalicylic acid (6-MSA) synthase (6-MSAS) gene atX was constructed. A specific compound was isolated and idenified as 6-MSA by HPLC, LC-MS and NMR. Transcription of both genes were detected. In 5-L bioreactor, the GS115-NpgA-ATX grew well and produced 6-MSA quickly until reached a high value of 2.2 g/L by methanol induction for 20 hours. Thereafter, the cells turned to death ascribing to high concentration of antimicrobial 6-MSA. The distribution of 6-MSA changed that during early and late induction phase it existed more in supernatant while during intermediate stage it mainly located intracellular. Different from 6-MSA production strain, recombinant M. purpureus pksCT expression strains for citrinin intermediate production, no matter PksCT located in cytoplasm or in peroxisomes, did not produce any specfic compound. However, both npgA and pksCT transcripted effectively in cells and western blot analysis proved the expression of PPtase. Then the PPTase was expressed and purified, marked by fluorescent probes, and reacted with purified ACP domain and its mutant ACPm of PksCT. Fluoresence was only observed in ACP but not ACPm, indicating that the PPTase worked well with ACP to make it bioactive holo-ACP. Thus, some other factors may affect polyketide synthesis that include activities of the individual catalytic domains and release of the product from the synthase of PksCT.ConclusionsAn efficient P. pastoris expression system of fungal polyketides was successfully constructed. It produced a high production of 6-MSA and holds potential for future industrial application of 6-MSA and other fungal polyketides.
Highlights
Polyketides are one of the most important classes of secondary metabolites and usually make good drugs
A. nidulans npgA (GenBank: AAF12814) encoding the phosphopantetheine transferase (PPTase) consisted of 344 amino acids was selected
Commercial vector pPIC3.5 K (Invitrogen) with AOX1 promoter and selective marker HIS4 was used for plasmid pPIC3.5 K-NpgA construction and transformed auxotrophic his4 wild type P. pastoris GS115 by a single homologous recombination event to integrate at HIS4 locus
Summary
Polyketides are one of the most important classes of secondary metabolites and usually make good drugs. We aim to develop a P. pastoris based in vivo fungal polyketide production system for first time and evaluate its feasibility for future industrial application. Polyketides are one of the most important classes of secondary metabolites They occur in bacteria, fungi, plants, marine organisms, etc., showing flexible structural diversity and a variety of bioactivities [1]. Polyketides and their derivatives make good drugs. The recycling mechanism of fungal type I PKS makes the biosynthesis process efficiently with relatively small genes
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