Construction Of Biosynthetic Pathway And Heterologous Production Of Citrinin In Pichia Pastoris

With the development of synthetic biology and combinatorial biochemistry, production of rare native compounds and bulk pharmaceutical chemical products in metabolically engineered cells has caused widespread concern. Fungal polyketides show great potential in clinical treatment and commercial value. However, polyketides production by fermentation in native strains may cause many difficulties such as long period, multiple byproducts, complex process and low yield. Heterologous production of polyketides in generally used and efficient hosts is not only beneficial for basic research on biosynthesis mechanism, but also in keeping with the need of industrial production. In this study, we selected a model compound citrinin with a relatively intricate biosynthetic pathway and constructed a foreign biosynthetic pathway involving multiple functional genes in Pichia pastoris. We aimed to explore the potential of P. pastoris for heterologous synthesis of polyketides.In previous reports, citrinin biosynthetic gene cluster consisted of pksCT (encoding polyketide synthase), orfl (encoding the putative dehydrogenase), orf2 (encoding a positive regulator), orf1 (encoding a plausible oxygenase), orf4 (encoding a plausible oxidoreductase), orf5 (encoding a plausible transporter). As for heterologous synthesis, transporter is not necessary and the promoters and transcription factors in heterologous hosts are usually utilized. Thus in this study, pksCT, orf1, orf3 and orf4 were combinationally expressed and the products were detected.Firstly, we correctly removed the intron in pksCT from Monascus purpureus. The ACP domain in PKS requires posttranslational 4-phosphopantetheinylation catalyzed by 4-phosphopantetheinyl transferases (PPTase). Coexpresseion of pksCT and npgA encoding PPTase from Aspergillus nidulans generated citrinin intermediate, trimethylated pentaketide aldehyde. Then, according to the relationship between the functions of genes and the synthesis of products, orf4 encoding oxidoreductase was added. Proteins were expressed normally but the intermediate remained unchanged. Orf3 encoding oxygenase and orfl encoding dehydrogenase were further added. Proteins were expressed normally but no citrinin was detected, neither were other newly produced compounds.Citrinin has a poor stability and is sensitive to pH. Thus we analyzed the effects of different pH on the synthesis of products. The results showed that citrinin tended to degrade under acidic conditions (pH=2). Extraction efficiency was also influenced by pH and could be quite low under neutral conditions (pH=6). Maintaining pH>4 during fermentation and pH=4 before extraction can remit the degradation. Cultivation, induction, and extraction were rearranged under this condition but still no citrinin or other new compounds were detected. Therefore, we suspected that the deduced gene cluster of citrinin may be incomplete.At the end of 2015, German scientists reported the biosynthetic pathway of citrinin and the completed gene cluster in Monascus ruber. Based on their results, we obtained two other genes (mpl6 and mpl7) participating in the synthesis of citrinin by genome walking in M. purpureus and co-transformed mpl6, mpl7 into the P. Pastoris strain carrying pksCT, npgA, orf1 (mpl4), orf3 (mpl2) and orf4 (mpll). All genes involved in the synthesis of citrinin were activated by PAOX1 and functional enzymes were expressed successfully. We achieved construction of a complete pathway and synthesis of citrinin. In addition, the citrinin gene cluster in M. purpureus shows high homology with M. ruber, and the catalytic mechanisms of synthesis are very similar.