Metabolic engineering of low-pH-tolerant non-model yeast, , for production of citramalate. Author Zong-Yen Wu, Wan Sun, Yihui Shen, Jimmy Pratas, Patrick Suthers, Ping-Hung Hsieh, Sudharsan Dwaraknath, Joshua Rabinowitz, Costas Maranas, Zengyi Shao, Yasuo Yoshikuni Publication Year 2023 Type Journal Article Abstract Methyl methacrylate (MMA) is an important petrochemical with many applications. However, its manufacture has a large environmental footprint. Combined biological and chemical synthesis (semisynthesis) may be a promising alternative to reduce both cost and environmental impact, but strains that can produce the MMA precursor (citramalate) at low pH are required. A non-conventional yeast, , may prove ideal, as it can survive extremely low pH. Here, we demonstrate the engineering of for citramalate production. Using sequence similarity network analysis and subsequent DNA synthesis, we selected a more active citramalate synthase gene () variant for expression in . We then adapted a piggyBac transposon system for that allowed us to simultaneously explore the effects of different gene copy numbers and integration locations. A batch fermentation showed the genome-integrated- strains produced 2.0 g/L citramalate in 48 h and a yield of up to 7% mol citramalate/mol consumed glucose. These results demonstrate the potential of as a chassis for citramalate production. Journal Metab Eng Commun Volume 16 Pages e00220 Date Published 2023/06/30 ISSN Number 2214-0301 DOI 10.1016/j.mec.2023.e00220 Alternate Journal Metab Eng Commun PMCID PMC9969067 PMID 36860699 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML