Metabolic Engineering of Low-pH-Tolerant Non-Model Yeast, Issatchenkia orientalis, for Production of Citramalate

Themes: Conversion

Keywords: Metabolomics

Citation

Wu, Z.Sun, W.Shen, Y.Suthers, P.Hsieh, P.H.Dwaraknath, S.Rabinowitz, J.D.Maranas, C.D.Shao, Z.Yoshikuni, Y. Feb. 16, 2023. “Metabolic Engineering of Low-pH-Tolerant Non-Model Yeast, Issatchenkia orientalis, for Production of Citramalate.” Metabolic Engineering Communications 16:e00220. DOI: 10.1016/j.mec.2023.e00220.

Overview

(A) I. orientalis could tolerate 80 g/L of citramalate at pH 3.0. (B) The genome-integrated-cimA strains, I. orientalis SB814 and SB816, produced the most citramalate, with a titer of 2.0 g/L, which is 6-fold higher than their plasmid counterpart.

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. 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.

Data

Download (28.6KB) includes:

  • Citramalate production by strain
  • Strains and plasmids
  • Sequence data