期刊
BIOTECHNOLOGY FOR BIOFUELS
卷 12, 期 -, 页码 -出版社
BMC
DOI: 10.1186/s13068-019-1490-z
关键词
Methane; Methanotroph; Methylomicrobium alcaliphilum 20Z; Putrescine; Metabolic engineering
资金
- C1 Gas Refinery Program through the National Research Foundation of Korea (NRF) - Ministry of Science and ICT [2015M3D3A1A01064882]
- National Research Foundation of Korea [2015M3D3A1A01064882] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
BackgroundMethane is the primary component of natural gas and biogas. The huge abundance of methane makes it a promising alternative carbon source for industrial biotechnology. Herein, we report diamine compound, putrescine, production from methane by an industrially promising methanotroph Methylomicrobium alcaliphilum 20Z.ResultsWe conducted adaptive evolution to improve putrescine tolerance of M. alcaliphilum 20Z because putrescine highly inhibits the cell growth. The evolved strain 20ZE was able to grow in the presence of 400mM of putrescine dihydrochloride. The expression of linear pathway ornithine decarboxylase genes from Escherichia coli and Methylosinus trichosporium OB3b allowed the engineered strain to produce putrescine. A higher putrescine titer of 12.44mg/L was obtained in the strain 20ZE-pACO with ornithine decarboxylase from M. trichosporium OB3b. For elimination of the putrescine utilization pathway, spermidine synthase (MEALZ_3408) was knocked out, resulting in no spermidine formation in the strain 20ZES1-pACO with a putrescine titer of 18.43mg/L. Next, a genome-scale metabolic model was applied to identify gene knockout strategies. Acetate kinase (MEALZ_2853) and subsequently lactate dehydrogenase (MEALZ_0534) were selected as knockout targets, and the deletion of these genes resulted in an improvement of the putrescine titer to 26.69mg/L. Furthermore, the putrescine titer was improved to 39.04mg/L by overexpression of key genes in the ornithine biosynthesis pathway under control of the pTac promoter. Finally, suitable nitrogen sources for growth of M. alcaliphilum 20Z and putrescine production were optimized with the supplement of 2mM ammonium chloride to nitrate mineral salt medium, and this led to the production of 98.08mg/L putrescine, almost eightfold higher than that from the initial strain. Transcriptome analysis of the engineered strains showed upregulation of most genes involved in methane assimilation, citric acid cycle, and ammonia assimilation in ammonia nitrate mineral salt medium, compared to nitrate mineral salt medium.ConclusionsThe engineered M. alcaliphilum 20ZE4-pACO strain was able to produce putrescine up to 98.08mg/L, almost eightfold higher than the initial strain. This study represents the bioconversion of methane to putrescinea high value-added diamine compound.
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