Production of glucaric acid from myo-inositol in engineered Pichia pastoris

• A glucaric acid synthetic pathway was firstly constructed in Pichia pastoris.
• A fusion expression strategy was applied to improve the production of glucaric acid.
• High glucaric acid titer (6.61 ± 0.30 g/L) was achieved from glucose and myo-inositol.

A potential myo-inositol oxygenase (ppMIOX) was identified as a functional enzyme and a glucaric acid synthetic pathway was firstly constructed in Pichia pastoris. Coexpression of the native ppMIOX and the urinate dehydrogenase (Udh) from Pseudomonas putida KT2440 led to obvious accumulation of glucaric acid (90.46 ± 0.04 mg/L) from myo-inositol whereas no glucaric acid was detected from glucose. In comparison, coexpression of the heterologous mouse MIOX (mMIOX) and Udh resulted in higher titers of glucaric acid from glucose and myo-inositol, 107.19 ± 11.91 mg/L and 785.4 ± 1.41 mg/L, respectively. By applying a fusion expression strategy with flexible peptides, the mMIOX specific activity and the glucaric acid concentration were significantly increased. Using glucose and myo-inositol as carbon substrates, the production of glucaric acid was substantially enhanced to 6.61 ± 0.30 g/L in fed-batch cultures. To the best of our knowledge, this is the highest reported value to date.