Multi-gene metabolic engineering of Pichia pastoris to synthesize ectoine

As a promising osmolyte, ectoine has been widely applied in cosmetics, food, and pharmaceutical industries in recent years, therefore its biomanufacturer has attracted increasing interest. Ectoine-producing isolates were previously screened from halophilic microorganisms. After ectoine synthetase was identified, genetic engineering of Escherichia coli, Corynebacterium glutamicum, and Hansenula polymorpha were employed to produce ectoine. However, Pichia pastoris, another successful host capable of high-density cell culture, had not yet been exploited as an ectoine-synthesizing host. In this study, therefore, P. pastoris was employed for the first time to produce ectoine through multi-gene metabolic engineering. Firstly, Chromohalobacter salexigens HZS/E, a halophilic isolate producing 46.96 mg/mL ectoine, was identified, while ectABC encoding ectoine synthetase was cloned. Later, ectABC was introduced into P. pastoris GS115 under the control of two different promoters. The results showed that P_GAP-based HZS02 accumulated 8.03 g/L, 12.62 % higher than 7.13 g/L produced by P_AOX-based HZS01. Finally, to enhance the supply of the precursor l-aspartate-β-semialdehyde, three genes (aspC, aK, and asD) were individually and collectively overexpressed. The highest ectoine yield was achieved at 10.88 g/L by GS115/pGAPZ A-ectABC-aspC-aK-asD. This study demonstrated that P. pastoris was a highly effective host for ectoine biosynthesis.