Industrializing methanotrophs and other methylotrophic bacteria: from bioengineering to product recovery.

Microbes that use the single-carbon substrates methanol and methane offer great promise to bioindustry along with substantial environmental benefits. Methanotrophs and other methylotrophs can be engineered and optimized to produce a wide range of products, from biopolymers to biofuels and beyond. While significant limitations remain, including delivery of single-carbon feedstock to bioreactors, efficient growth, and scale-up, these challenges are being addressed and notable improvements have been rapid. Development of expression chassis, use of genome-scale and regulatory models based on omics data, improvements in bioreactor design and operation, and development of green product recovery schemes are enabling the rapid development of single-carbon bioconversion in the industrial space.

Elucidating the regulation of glucose tolerance in a ß-glucosidase from Halothermothrix orenii by active site pocket engineering and computational analysis.

ß-Glucosidase catalyzes the hydrolysis of ß-1,4 linkage between two glucose molecules in cello-oligosaccharides and is prone to inhibition by the reaction product glucose. Relieving the glucose inhibition of ß-glucosidase is a significant challenge. Towards the goal of understanding how glucose interacts with ß-glucosidase, we expressed in Escherichia coli, the Hore_15280 gene encoding a ß-glucosidase in Halothermothrix orenii. Our results show that the enzyme is glucose tolerant, and its activity on p-nitrophenyl D-glucopyranoside stimulated in the presence of up to 0.5 M glucose. NMR analyses show the unexpected interactions between glucose and the ß-glucosidase at lower concentrations of glucose that, however, does not lead to enzyme inhibition. We identified non-conserved residues at the aglycone-binding and the gatekeeper site and show that increased hydrophobicity at the pocket entrance and a reduction in steric hindrances are critical towards enhanced substrate accessibility and significant improvement in activity. Analysis of structures and in combination with molecular dynamics simulations show that glucose increases the accessibility of the substrate by enhancing the structural flexibility of the active site pocket and may explain the stimulation in specific activity up to 0.5 M glucose. Such novel regulation of ß-glucosidase activity by its reaction product may offer novel ways of engineering glucose tolerance.

Understanding the role of residues around the active site tunnel towards generating a glucose-tolerant ß-glucosidase from Agrobacterium tumefaciens 5A.

Most ß-glucosidases are subjected to inhibition by the final hydrolysis product glucose resulting in the accumulation of cellobiose and oligosaccharides. This accumulated cellobiose and oligosaccharides further inhibit the activities of endoglucanase and cellobiohydrolases, resulting in the inhibition of cellulose degradation and a more expensive biofuel. To elucidate the mechanism(s) of glucose tolerance, we designed and characterised six mutations of a moderately glucose-tolerant ß-glucosidase (H0HC94) from the mesophilic bacterium Agrobacterium tumefaciens 5A. The hydrophobicity and steric were varied across non-conserved residues in specific regions of the active site tunnel. In contrast to the uncompetitive inhibition of WT enzyme by glucose, C174V and H229S are competitively inhibited pointing towards a possible glucose-binding site in the protein at these positions. Increasing hydrophobicity at the +1 subsite and increasing hydrophobicity and steric at +2 subsites seemed to be critical for glucose tolerance for this BG. Additionally, in L178E, specific activity was 1.8 times higher on the natural substrate cellobiose while both W127F and L178E mutants showed an enhancement in thermostability. The kinetic stability of W127F, V176A, L178A and L178E also increased between 2- and 3-folds compared to WT. Our results indicate that while the structure between subsites +1 and +2 is critical for the glucose tolerance, the specific residues may not be identical across such enzymes.

Exploiting non-conserved residues to improve activity and stability of Halothermothrix orenii ß-glucosidase.

ß-glucosidase (EC 3.2.1.21; BG) cleaves ß-glucosidic linkages in disaccharide or glucose-substituted molecules. In an effort towards designing better BGs, we focused on the role of non-conserved residues across an otherwise homologous BG active site tunnel and designed mutants across the aglycone-binding site (V169C) and the gatekeeper residues (I246A) of the active site tunnel. We expressed in Escherichia coli, the Hore_15280 gene encoding a ß-glucosidase (BG) in Halothermothrix orenii. The overexpressed and purified wild-type (B8CYA8) has a high specific activity of 345 µmol/min/mg on pNPGlc and a half-life of 1.13 h when assayed with pNPGlc at pH 7.1 and 70 °C. The specific activities of V169C and I246A were 1.7 and 1.2 times higher than that of wild-type (WT) enzyme with the model substrate pNPGlc, while the activity on the natural substrate cellobiose was slightly higher to the WT. The two mutants were kinetically stable with 4.4- to 11-fold longer half-life compared to the WT enzyme. When the two mutations were combined to generate the V169C/I246A mutant, the specific activity increased to nearly twofold higher than WT on both substrates and the half-life increased fivefold. The two single mutants also show enhanced saccharification of insoluble natural biomass on supplementation of Trichoderma viride cellulase cocktail. These enhanced properties suggest the need for a closer look at the active site tunnel of these enzymes, especially across residues that are not conserved towards improving catalytic efficiencies.