Development of a novel methanotrophic platform to produce ectoine from methane and lignocellulose-derived sugars

Methane is considered a promising carbon source in the field of biotechnology. Using methane as the carbon source for producing chemicals reduces manufacturing costs and tackles the issue of global warming. Like methane, lignocellulose biomass is also an inexpensive and ample carbon source, which could be used for in-dustrial biomanufacturing. In this study, Methylotuvimicrobium alcaliphilum 20Z was genetically modified to simultaneously consume methane and lignocellulose-derived sugars (glucose and xylose) to produce ectoine, a native osmoprotectant. The xylose utilization pathway, comprising xylA (xylose isomerase) and xylB (xyluloki-nase) genes from Escherichia coli, was first integrated into the chromosome of M. alcaliphilum 20Z. Further expression of the glucose utilization, consisting of glf (glucose facilitator) from Zymmonas mobilis, glk (endoge-nous glucokinase), and pgi (glucose 6-phosphate isomerase) from E. coli, made the engineered strain 20ZXG consume methane, xylose, and glucose concurrently. Cultivation on three substrates upregulated the Emb-den-Meyerhof-Parnas and ectoine biosynthesis pathways of 20Z. As a result, the ectoine content produced by 20ZXG grown on the three substrates was 1.7-fold enhanced compared with that obtained on methane solely. Furthermore, the knock-out of ectoine biosynthesis repressor (ectR1) led to an output of 37.93 +/- 3.27 mg/g-DCW, which was the highest quantity of ectoine produced after 48 h of cultivation in the presence of three substrates and was 2.3-fold higher than the initial output.