Wu et al. (2023)

Metabolic engineering of low-pH-tolerant non-model yeast, Issatchenkia orientalis, 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, Issatchenkia orientalis, may prove ideal, as it can survive extremely low pH. Here, we demonstrate the engineering of I. orientalis for citramalate production. Using sequence similarity network analysis and subsequent DNA synthesis, we selected a more active citramalate synthase gene (cimA) variant for expression in I. orientalis. We then adapted a piggyBac transposon system for I. orientalis that allowed us to simultaneously explore the effects of different cimA gene copy numbers and integration locations.


The SSN analysis was used for target gene selection. The cimA variants that have been previously characterized are denoted with blue circles. The cimA variants of eukaryotic origin are denoted with green circles. The orange circles indicate the cimAvariants randomly selected from different clades. The number in each dot represents the synthetic cimA gene ID. The sequences of those genes are listed in Supplementary Tables S3 and S4

A batch fermentation showed the genome-integrated-cimA 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 I. orientalis as a chassis for citramalate production.

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