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

Abstract

Background: Polyketides 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 pharmaceutical 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.

Results: A 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 identified 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 specific 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.

Conclusions: An 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.

Figure 1

The HPLC chromatogram of organic extracts from fermentation broth of strain GS115, GS115-ATX and GS115-NpgA-ATX. Samples preparation were described in the Section 6-MSA extraction and identification in Methods.

Figure 2

The EI-MS identification of product 6-MSA by GS115-NpgA-ATX and transcription analysis of npgA and atX. (A) EI-MS analysis of extract from GS115-NpgA-ATX. m/z, mass-to-charge ratio; (B) The ¹HNMR analysis of extract from GS115-NpgA-ATX. Samples preparation were in the Section 6-MSA extraction and identification in Methods; (C) Lane 1–3: PCR using cDNA of wild type GS115 and primers 5AOX1/3AOX1, NpgA-F/R1 and BstpF/AtxR, respectively; Lane 4–6: PCR using GS115-NpgA-ATX cDNA and primers 5AOX1/3AOX1, NpgAF/ NpgAR1 and BstpF/AtxR, respectively; M: DNA marker Hind III. Gene transcription induced by 0.5% methanol for 24 h.

Figure 3

Time profiles of GS115-NpgA-ATX in 5-L stirred-tank bioreactor fermentation. The first arrow (32 h) indicated the starting point of glycerol feeding; The second arrow (47 h) indicated the starting point of methanol induction. Culture conditions were shown in Methods.

Figure 4

Transcription of npgA and pksCT and western blot assay of PPtase induced by 0.5% methanol. (A) PCR with primers NpgA-HIS6-F/ NpgA-HIS6-R; (B) PCR with primers 1977 F/4088R. Lane 1: GS115-3.5 K-CT-SKL; Lane 2: GS115-NpgA-CT; Lane 3: GS115-NpgA-SKL-CT-SKL. (C) Western blot of PPtase. Lane1: GS115; Lane2: GS115-3.5 K-CT-SKL; Lane 3: GS115-NpgA-CT; Lane 4: GS115-NpgA-SKL-CT-SKL. The arrows indicates the positive bands of nucleic acids and proteins. Samples preparation and experimenal procedure were shown in the Section Transcription and western blot analysis in Methods.

Figure 5

Nucleotide and amino acid sequences of ACP amd SDS-PAGE of PPTase and ACP and ACPm. (A) nucleotide and amino acid sequences ACP domain of citrinin polyketide synthase. The conserved serine at site 56 which marked by square frame was mutated by overlap PCR using primers mutant5/mutant3 to generate ACPm. (B) SDS-PAGE of PPTase expressed by GS115-NpgA-HIS₆; (C) SDS-PAGE of ACP and ACPm expressed by E. coli BL21. Lane 1: Lysate supernatant of GS115-NpgA-HIS₆; Lane 2: Flow-through fraction of GS115-NpgA-HIS₆; Lane 3: Eluted protein of GS115-NpgA-HIS₆; Lane 4: Lysate supernatant of wild type E. coli BL21 strain as negative control; Lane 5: Lysate supernatant of BL21-ACP; Lane 6: Lysate supernatant of BL21-ACPm. M: Protein marker. Protein purification were described in the Section Protein expression and purification in Methods. The arrows indicates the objective proteins.

Figure 6

Fluorescent assay of ACP and ACPm that were fluorescently labelled. The KODAK In-vivo multispectral imaging systerm F was used for scanning. (A) Image parameter of 480 nm laser and 535 nm emission filter; (B) Image parameter of 580 nm laser and 670 nm emission filter; (C) Image of the same SDS-PAGE gel stained with Coomassie brilliant blue R-250. Lane 1: In vitro reaction containing Bodipy FL-CoA, PPtase and ACPm; Lane 2: In vitro reaction containing Bodipy-CoA, PPtase and ACP; Lane 3: In vitro reaction containing Alexa Fluor 647-CoA, PPtase and ACPm; Lane 4: In vitro reaction containing Alexa Fluor 647-CoA, PPtase and ACP. Protein preparation were described in Methods.