Metabolic engineering of Pichia pastoris for production of isopentanol (3-Methyl-1-butanol)

Abstract

In recent years, the increasingly serious and clear effects of climate change have increased interest in renewable fuels and platform chemicals. Microbial platforms that can produce these compounds in an economically efficient way have emerged as an attractive alternative to the traditional production approaches. Here, we engineered the industrially-relevant yeast Pichia pastoris to produce the platform chemical 3-methyl-1-butanol (3M1B, isopentanol) directly from the renewable carbon source glucose. Specifically, we overexpressed the endogenous valine and leucine biosynthetic pathways to increase the production of the key pathway intermediate, 2-ketoisocaproate (2-KIC). Overexpression of the artificial keto-acid degradation pathway converted 2-KIC into 3M1B. Down-regulation of the side-product ethanol production using the CRISPR/Cas9 system led to a strain that is able to produce 3M1B at a titer of 191.0 ± 9.6 mg/L, the highest titer reported to date in a non-conventional yeast. We envision that our yeast system will pave the way for an efficient production system for this important class of platform compounds.

Keywords: 3-methyl-1-butanol; Biofuel; Isopentanol; Metabolic engineering; Pichia pastoris; Yeast.