Methanol is an ideal feedstock for bio-manufacturing that can be beneficial for global carbon neutrality. However, the toxicity of methanol limits the efficiency of methanol metabolism toward biochemical production, and it is still challenging in engineering this non-conventional yeast due to serious lack of genetic editing tools. In this presentation, we will show our recent progress in establishing CRISPR-Cas9 based genome editing tools and enhancing the homologous recombination in methylotrophic yeast Ogataea polymorpha. With this genetic platform, we tried to engineer cellular metabolism for fatty acid production from methanol. We found that engineering overproduction of free fatty acids (FFA) from sole methanol resulted cell death with a decreased cellular phospholipid in O. polymorpha, and the cell growth was restored by adaptive laboratory evolution (ALE). Whole genome sequencing of the adapted strains reveals that inactivation of LPL1 (encoding a putative lipase) and IZH3 (encoding a membrane protein related to zinc metabolism) preserve cell survival by restoring phospholipid metabolism. Engineering the pentose phosphate pathway and gluconeogenesis enabled high-level production of FFA (15.9 g/L) from sole methanol. Preventing methanol-associated toxicity underscored the link between lipid metabolism and methanol tolerance, which should contribute to enhancing methanol-based bio-manufacturing.
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