Engineered fungal polyketide biosynthesis in Pichia pastoris: a potential excellent host for polyketide production

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.

Identification and characterization of N-glycosylation site on a Mucor circinelloides aspartic protease expressed in Pichia pastoris: effect on secretion, activity and thermo-stability

“…Additionally, when the original MCAP gene was adapted to the optimal codon usage of P. pastoris, the production of heterologous MCAP was increased three folds. The synthetic gene is 78% identical (nucleotide sequence level) to the original and the observed increase in production of several other recombinant proteins in P. pastoris using codon optimization has also been reported in the literature (Sinclair and Choy 2002 ; Xiong et al 2005 ; Gao et al 2013 ).…”

Characterization of a thermostable raw-starch hydrolyzing α-amylase from deep-sea thermophile Geobacillus sp.

“…The precipitate was dissolved in 0.05 mol/L sodium phosphate buffer (pH 5.5) and dialyzed overnight against the same buffer. Afterwards, the crude extract was further purified by Ni 2+ -NTA resin affinity chromatography under non-denaturing conditions according to Gao et al [14]. The recombinant a-amylase was eluted with elution buffer (50 mM NaH 2 PO 4 , 250 mM NaCl, and 500 mM imidazole at pH 5.5) and identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).…”

“…Positive cloned vector was then transformed into E. coli BL21 (DE3). Enzyme expression and the intracellular proteins collection were performed according to Gao et al [14]. Gs4j-amyA in the cell free supernatant was precipitated using ammonium sulphate with a 60% of saturation degree.…”

Synthetic biology of fungal natural products

“…One of the most promising studies to date a non filamentous fungal host was done in the yeast Pichia pastoris , which is also known for its high production of heterologous proteins ( Cereghino and Cregg, 2000 ). In this particular study the A. terreus 6-methylsalicylic acid (6-MSA) synthase, which is a PKS, was coexpressed with the A. nidulans phosphopantetheinyl transferase (PPTase) gene and resulted in yields of 6-MSA as high as 2.2 g/L ( Gao et al, 2013 ). This was the highest yield reported thus far from all the studies listed above, which also expressed the PKS for 6-MSA.…”