Metabolic engineering of low-pH-tolerant non-model yeast, , for production of citramalate.

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.