Improved methanol-derived lovastatin production through enhancement of the biosynthetic pathway and intracellular lovastatin efflux in methylotrophic yeast

Yiqi Liu,Menghao Cai,Jiahui Yu,Xiangshan Zhou,Chenxiao Bai,Qin Xu,Yuanxing Zhang

doi:10.1186/s40643-018-0202-z

Yiqi Liu, Menghao Cai + Show 5 more

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https://doi.org/10.1186/s40643-018-0202-z

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Abstract

BackgroundMethanol has attracted interest as a substrate for improvement of product titers and yields of bioprocesses, because methanol has a more reduced state than sugars do and can be renewably synthesized from abundant natural gas supplies. Our aim was to engineer methylotrophic Pichia pastoris to convert methanol into value-added lovastatin more productively.ResultsA strengthened biosynthetic pathway of lovastatin was constructed through the assembly of three modules with increasing module-specific antibiotic stress in the methylotrophic yeast P. pastoris. The resulting strain (P. p/LV_V#9) produced 287.5 ± 2.0 mg/L lovastatin in a 5-L bioreactor from methanol. The production was further improved by identification and overexpression of a statin pump protein, TapA, a membrane protein capable of lovastatin efflux out of the cell. A TapA-overexpressing strain, P. p/LV_V#9-TapA, produced 419.0 ± 9.5 mg/L lovastatin from methanol: 46% more than P. p/LV_V#9 did, and 520% more relative to the strain (P. p/LV_SC) with single-copy genes.ConclusionsA methylotrophic yeast strain producing 419.0 ± 9.5 mg/L lovastatin was constructed by optimization of biosynthetic gene dosages and coexpression of a statin pump protein; these results proved that P. pastoris is a promising chassis organism for natural-product biosynthesis. A membrane protein, TapA was found to perform the function of exporting intracellular lovastatin and enhanced lovastatin production.

Highlights

Methanol has attracted interest as a substrate for improvement of product titers and yields of bioprocesses, because methanol has a more reduced state than sugars do and can be renewably synthesized from abundant natural gas supplies
The monacolin J (MJ) titer increased as the G418 concentration was increased; the obtained improvement was somewhat limited, while the intermediate monacolin L (ML) accumulated remarkably during culture (Fig. 2a)
These results revealed that increasing the gene dosages by strengthening antibiotic stress during pathway assembly efficiently enhanced the production of certain compounds in lovastatin biosynthesis

Summary

Introduction

Methanol has attracted interest as a substrate for improvement of product titers and yields of bioprocesses, because methanol has a more reduced state than sugars do and can be renewably synthesized from abundant natural gas supplies. Our aim was to engineer methylotrophic Pichia pastoris to convert methanol into value-added lovastatin more productively. Methanol can be inexpensively manufactured from natural gas (which is abundant in the deep sea of our planet) and can be renewably obtained through reduction of carbon dioxide (Räuchle et al 2016). We assembled the biosynthetic pathway of lovastatin in the methylotrophic yeast P. pastoris (Fig. 1) and successfully produced lovastatin from methanol (Liu et al 2018). Gene dosages in the lovastatinproducing strains were all maintained at single-copy levels, and a titer of only 250.8 mg/L lovastatin was obtained via an optimal coculture strategy (Liu et al 2018); this titer is still much lower than that of a native lovastatinproducing strain (Singh and Pandey 2013; Huang et al 2017)

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