Methylotrophic yeast Pichia pastoris as a chassis organism for polyketide synthesis via the full citrinin biosynthetic pathway

毕赤酵母 聚酮 桔霉素 聚酮合酶 生物 基因簇 生物化学 基因 毕赤酵母 酵母 遗传学 生物合成 重组DNA 真菌毒素 植物
作者
Ying Xue,Chuixing Kong,Wei Shen,Chenxiao Bai,Yanna Ren,Xiangshan Zhou,Yuanxing Zhang,Menghao Cai
出处
期刊:Journal of Biotechnology [Elsevier]
卷期号:242: 64-72 被引量:32
标识
DOI:10.1016/j.jbiotec.2016.11.031
摘要

With the rapid development of synthetic biology, exploring various chassis organisms has become necessary to improve the heterologous biosynthesis of natural products and pharmaceuticals. In this study, we tested the potential of the industrial methylotrophic yeast strain Pichia pastoris for the heterologous synthesis of polyketides. A recombinant P. pastoris GS-pksCT-npgA carrying the Monascus purpureus citrinin polyketide synthase gene pksCT and the Aspergillus nidulans phosphopantetheinyl transferase gene npgA was constructed. Subsequently, a specific compound was isolated and identified as citrinin intermediate trimethylated pentaketide aldehyde. On account of the hypothetic functions of the genes in the citrinin gene cluster, mpl1 encoding serine hydrolase, mpl2 encoding oxygenase, and mpl4 encoding dehydrogenase were gradually expressed. Proteins were also normally expressed, but a new compound was undetected. Basing on the recently reported citrinin gene cluster in Monascus ruber, we obtained two other genes (mpl6 and mpl7) participating in citrinin biosynthesis by genome walking in M. purpureus. Then, we co-transformed intron-removed mpl6 and mpl7 into the P. pastoris strain carrying pksCT, npgA, mpl1, mpl2, and mpl4. All genes were activated by the methanol-induced AOX1 promoter, and a complete biosynthetic pathway of citrinin was assembled. Finally, citrinin was successfully produced under methanol induction in P. pastoris. These results prove that P. pastoris is a promising chassis organism for polyketide production.
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