Biochemical and molecular characterization of the isocitrate dehydrogenase with dual coenzyme specificity from the obligate methylotroph Methylobacillus Flagellatus.

The isocitrate dehydrogenase (MfIDH) with unique double coenzyme specificity from Methylobacillus flagellatus was purified and characterized, and its gene was cloned and overexpressed in E. coli as a fused protein. This enzyme is homodimeric,-with a subunit molecular mass of 45 kDa and a specific activity of 182 U mg -1 with NAD+ and 63 U mg -1 with NADP+.

Genome tailoring powered production of isobutanol in continuous CO2/H2 blend fermentation using engineered acetogen biocatalyst.

The cell energy fraction that powered maintenance and expression of genes encoding pro-phage elements, pta-ack cluster, early sporulation, sugar ABC transporter periplasmic proteins, 6-phosphofructokinase, pyruvate kinase, and fructose-1,6-disphosphatase in acetogen Clostridium sp. MT871 was re-directed to power synthetic operon encoding isobutanol biosynthesis at the expense of these genes achieved via their elimination. Genome tailoring decreased cell duplication time by 7.

UVC-mutagenesis in acetogens: resistance to methanol, ethanol, acetone, or n-butanol in recombinants with tailored genomes as the step in engineering of commercial biocatalysts for continuous CO₂/H₂ blend fermentations.

Time- and cost-efficient six-step UVC-mutagenesis was developed and validated to generate acetogen mutants with preliminary reduced genomes to prevent product inhibition in the to-be-engineered commercial biocatalysts. Genome reduction was performed via elimination of pta, ack, spo0A, spo0J and some pro-phage genes. UVC-mutants such as Clostridium sp.

Mevalonate production by engineered acetogen biocatalyst during continuous fermentation of syngas or CO₂/H₂ blend.

Naturally mevalonate-resistant acetogen Clostridium sp. MT1243 produced only 425 mM acetate during syngas fermentation. Using Clostridium sp.

Synthetic 2,3-butanediol pathway integrated using Tn7-tool and powered via elimination of sporulation and acetate production in acetogen biocatalyst.

Acetogen Clostridium sp. MT1802 originally producing 336-mM acetate from inorganic carbon of CO₂/CO was engineered to eliminate acetate production and sporulation using Cre-lox66/lox71-approach. The recombinant started producing 105-mM formate expressing synthetic formate dehydrogenase integrated in two copies.

Selective methanol or formate production during continuous CO₂ fermentation by the acetogen biocatalysts engineered via integration of synthetic pathways using Tn7-tool.

Methanol-resistant mutant acetogen Clostridium sp. MT1424 originally producing only 365 mM acetate from CO₂/CO was engineered to eliminate acetate production and spore formation using Cre-lox66/lox71-system to power subsequent methanol production via expressing synthetic methanol dehydrogenase, formaldehyde dehydrogenase and formate dehydrogenase, three copies of each, assembled in cluster and integrated to chromosome using Tn7-based approach. Production of 2.

Selective n-butanol production by Clostridium sp. MTButOH1365 during continuous synthesis gas fermentation due to expression of synthetic thiolase, 3-hydroxy butyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, and NAD-dependent butanol dehydrogenase.

Acetogen Clostridum sp. MT1962 produced 287 mM acetate (p < 0.005) and 293 mM ethanol (p < 0.

"Curing" of plasmid DNA in acetogen using microwave or applying an electric pulse improves cell growth and metabolite production as compared to the plasmid-harboring strain.

Plasmid-free acetogen Clostridium sp. MT962 electrotransformed with a small cryptic plasmid pMT351 was used to develop time- and cost-effective methods for plasmid elimination. Elimination of pMT351 restored production of acetate and ethanol to the levels of the plasmid-free strain with no dry cell weight changes.

Cre-lox66/lox71-based elimination of phosphotransacetylase or acetaldehyde dehydrogenase shifted carbon flux in acetogen rendering selective overproduction of ethanol or acetate.

Acetogen strain Clostridium sp. MT1121 produced 300 mM acetate (p<0.005) and 321 mM ethanol (p<0.

Elimination of acetate production to improve ethanol yield during continuous synthesis gas fermentation by engineered biocatalyst Clostridium sp. MTEtOH550.

Acetogen strain Clostridum sp. MT653 produced acetate 273 mM (p < 0.005) and ethanol 250 mM (p < 0.

Dynamic hydration numbers for biologically important ions.

The role of ionized groups in biological systems is determined by their affinity for water [Biophys. J. 72 (1997) 65-76].