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

Abstract

Background: 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. Results:A 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. Conclusions:A 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.

Fungal statin pump protein improves monacolin J efflux and regulates its production in Komagataella phaffii

“…Also, heterologous expression of a homolog of MlcE did not trigger increased resistance to LV in a LV-sensitive strain Aspergillus nidulans (Hutchinson et al 2000). Differently, our previous work revealed that a homolog (TapA) of MlcE from A. terreus promoted LV release and improved LV production by a methanol-utilizing yeast Komagataella phaffii (syn Pichia pastoris) (Liu et al 2018a).…”

“…K. phaffii GS115 was purchased from Invitrogen and used for construction of expression strains. J#9 and GS-P GAP -TapA strains were constructed and stored in our laboratory (Liu et al 2018a). The linearized vector was assembled with different fragments by ClonExpress ™ II One Step Cloning Kit (Vazyme Biotech Co., Ltd., China), generating various target plasmids.…”

“…Chemical synthesis of SV requires a key intermediate of monacolin J (MJ). Currently, industrial production of MJ is realized through hydrolysis of LV with large amounts of concentrated alkali, acids, and organic reagents (Jaeheon et al 2005); while industrial production of LV is usually implemented by large-scale fermentation of Aspergillus terreus strains (Liu et al 2018a). This manufacturing process faces serious problems such as various byproducts, complex process, high requirements for operators, and costly wastewater treatment (Liu et al 2018a).…”

“…Currently, industrial production of MJ is realized through hydrolysis of LV with large amounts of concentrated alkali, acids, and organic reagents (Jaeheon et al 2005); while industrial production of LV is usually implemented by large-scale fermentation of Aspergillus terreus strains (Liu et al 2018a). This manufacturing process faces serious problems such as various byproducts, complex process, high requirements for operators, and costly wastewater treatment (Liu et al 2018a). With the development of synthetic biology, heterologous productions of MJ have been achieved in yeast cells recently (Xie and Tang 2007;Gao et al 2010;Xu et al 2013;Liu et al 2018a, b;Bond and Tang 2019).…”

Chemical Production from Methanol Using Natural and Synthetic Methylotrophs

“…In addition, through enhancement of intracellular lovastatin efflux by overexpression of TapA , a putative efflux pump protein from the native lovastatin‐producing strain A. terreus , increment of lovastatin production was achieved with 419 mg L −1 of lovastatin produced using a monoculture fermentation. [ 46 ] In another study, citrinin production pathway was expressed in P. pastoris . Citrinin is regarded as a model compound to explore the potential for synthesis polyketides.…”