High-Level Squalene Production from Methane Using a Metabolically Engineered Methylomonas sp. DH-1 Strain

Methane is an inexpensive and sustainable raw material with high potential bioconversion into high-value-added compounds. We report the development of a metabolically engineered type I methanotroph, the Methylomonas sp. DH-1 strain, capable of producing squalene from methane. First, a base strain equipped with a Cre-lox-based automated genetic engineering system was constructed in the genome for efficient strain development. Second, the pds-ald-crtN2 gene cluster, which encodes phytoene desaturase, aldehyde dehydrogenase, and diapolycopene oxygenase, was deleted to increase the pool of presqualene diphosphate, an immediate precursor of squalene. Third, the activity of squalene-hopene cyclase was prohibited using ferulenol to concentrate the squalene. Finally, the squalene synthase regulated by a strong promoter was further overexpressed to carry strong flux toward squalene. The final engineered strain produced 8.4 +/- 1.2 mg/L of squalene with the content and productivity of 12.0 +/- 1.0 mg/g DCW and 58.7 +/- 8.5 mu g/(L/h), respectively. The fed-batch fermentation of the final engineered strain produced 31.3 mg/L of squalene with the content and productivity of 39.3 mg/g DCW and 746.2 mu g/(L/h), respectively, by supplementing KNO3. The final engineered strain exhibited remarkable performance in the bioconversion of methane into squalene with the CH4 consumption rate, yield, and CH4 conversion rate values of 3.09 g/L, 10.2 mg of squalene per gram of methane, and 1.19%, respectively.