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

摘要

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.