Enhanced production of ectoine from methane using metabolically engineered Methylomicrobium alcaliphilum 20Z

Abstract

Background Ectoine (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. Results We 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. Conclusions Ectoine 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 Abstract

Genome-scale flux balance analysis reveals redox trade-offs in the metabolism of the thermoacidophile Methylacidiphilum fumariolicum under auto-, hetero-and methanotrophic conditions

“…Moreover, extremophile bacteria are sources of novel and robust industrially relevant compounds (Tao et al, 2016) and proteins (Aulitto et al, 2017). Acidophile methanotrophs have been used for the co-degradation of organochlorine compounds (Choi et al, 2021), whereas halotolerant methanotrophs have been successfully used to produce ectoine (Cantera et al, 2017;Cho et al, 2022).…”

High production of ectoine from methane in genetically engineered Methylomicrobium alcaliphilum 20Z by preventing ectoine degradation

“…Halophilic, methanotrophic Methylomicrobium alcaliphilum 20Z was used for ectoine production. Specifically, the ectD and ectR gene-deficient 20ZDP2 strain was constructed in our previous study [22] and 20ZDP3, 20ZDP4, and 20ZDP5 defective in doeA, doeD, ectB genes, respectively, from 20ZDP2 were constructed in this study (Table 1). The bacteria were cultured in a modified Methylomicrobium medium described in the previous study comprising 30% (v/v) methane or 9.93 g/L methanol [22].…”

“…Specifically, the ectD and ectR gene-deficient 20ZDP2 strain was constructed in our previous study [22] and 20ZDP3, 20ZDP4, and 20ZDP5 defective in doeA, doeD, ectB genes, respectively, from 20ZDP2 were constructed in this study (Table 1). The bacteria were cultured in a modified Methylomicrobium medium described in the previous study comprising 30% (v/v) methane or 9.93 g/L methanol [22]. The strains were cryopreserved with 10% (v/v) dimethylsulfoxide added in the early exponential phase.…”

Transcription regulation strategies in methylotrophs: progress and challenges

“…RTK17.1 nifA Nitrogen-fixing factor Methylacidiphilum sp . RTK17.1 M. buryatense 5GB1 (Guo et al 2022 ) phoB Phosphate transport regulatory M. buryatense 5GB1 (Hu et al 2020 ) phoU Phosphate transport regulatory M. buryatense 5GB1 rpoN Sigma-54 factor M. trichosporium OB3b (Stafford et al 2003 ) ectR Ectoine biosynthesis gene repressor M. alcaliphilum 20Z (Cho et al 2022 ) …”

“…Cho et al recognized a MarR-like TF ectR in M. alcaliphilum 20Z, which was found to suppress the expression of the ectoine biosynthesis gene ectD by binding to the putative -10 sequence. Knocking out ectR could strengthen the transcription of ectD and ectoine production was enhanced 1.6-fold comparing the mutants to the original strains, thus solving the problem of transcription rate limiting in the conversion from methane to ectoine (Cho et al 2022 ).…”