Abstract
BackgroundEctoine (1,3,4,5-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is an attractive compatible solute because of its wide industrial applications. Previous studies on the microbial production of ectoine have focused on sugar fermentation. Alternatively, methane can be used as an inexpensive and abundant resource for ectoine production by using the halophilic methanotroph, Methylomicrobium alcaliphilum 20Z. However, there are some limitations, including the low production of ectoine from methane and the limited tools for the genetic manipulation of methanotrophs to facilitate their use as industrial strains.ResultsWe constructed M. alcaliphilum 20ZDP with a high conjugation efficiency and stability of the episomal plasmid by the removal of its native plasmid. To improve the ectoine production in M. alcaliphilum 20Z from methane, the ectD (encoding ectoine hydroxylase) and ectR (transcription repressor of the ectABC-ask operon) were deleted to reduce the formation of by-products (such as hydroxyectoine) and induce ectoine production. When the double mutant was batch cultured with methane, ectoine production was enhanced 1.6-fold compared to that obtained with M. alcaliphilum 20ZDP (45.58 mg/L vs. 27.26 mg/L) without growth inhibition. Notably, a maximum titer of 142.32 mg/L was reached by the use of an optimized medium for ectoine production containing 6% NaCl and 0.05 μM of tungsten without hydroxyectoine production. This result demonstrates the highest ectoine production from methane to date.ConclusionsEctoine production was significantly enhanced by the disruption of the ectD and ectR genes in M. alcaliphilum 20Z under optimized conditions favoring ectoine accumulation. We demonstrated effective genetic engineering in a methanotrophic bacterium, with enhanced production of ectoine from methane as the sole carbon source. This study suggests a potentially transformational path to commercial sugar-based ectoine production.Graphical
Highlights
Ectoine (1,3,4,5-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is an attractive compatible solute because of its wide industrial applications
Development of M. alcaliphilum 20Z mutant for efficient genetic engineering by the removal of the native plasmid The process of the genetic manipulation of methanotrophs has not yet been fully established, and this serves as a critical limitation for bioconversion technologies
And 6, ectoine production increased up to 1.3-fold compared to that of M. alcaliphilum 20ZDP1 (45.58 mg/L vs. 34.53 mg/L). These results clearly indicate that the deletion of ectD and ectR had a beneficial effect on ectoine production without the inhibition of cell growth
Summary
Ectoine (1,3,4,5-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is an attractive compatible solute because of its wide industrial applications. There are some limitations, including the low production of ectoine from methane and the limited tools for the genetic manipulation of methanotrophs to facilitate their use as industrial strains. New methanotrophs have been isolated, and current biological engineering approaches have provided new opportunities for the development of industrial methanotroph strains [10,11,12]. Despite these efforts, it is necessary to overcome shortcomings such as their low growth rate, the limited genetic tools for their manipulation, and the insufficient fundamental knowledge for using methanotrophs as industrial strains [13]
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