Interrogating the Role of the Two Distinct Fructose-Bisphosphate Aldolases of Bacillus methanolicus by Site-Directed Mutagenesis of Key Amino Acids and Gene Repression by CRISPR Interference

The Gram-positive Bacillus methanolicus shows plasmid-dependent methylotrophy. This facultative ribulose monophosphate (RuMP) cycle methylotroph possesses two fructose bisphosphate aldolases (FBA) with distinct kinetic properties. The chromosomally encoded FBA(C) is the major glycolytic aldolase. The gene for the major gluconeogenic aldolase FBA(P) is found on the natural plasmid pBM19 and is induced during methylotrophic growth. The crystal structures of both enzymes were solved at 2.2 Å and 2.0 Å, respectively, and they suggested amino acid residue 51 to be crucial for binding fructose-1,6-bisphosphate (FBP) as substrate and amino acid residue 140 for active site zinc atom coordination. As FBA(C) and FBA(P) differed at these positions, site-directed mutagenesis (SDM) was performed to exchange one or both amino acid residues of the respective proteins. The aldol cleavage reaction was negatively affected by the amino acid exchanges that led to a complete loss of glycolytic activity of FBA(P). However, both FBA(C) and FBA(P) maintained gluconeogenic aldol condensation activity, and the amino acid exchanges improved the catalytic efficiency of the major glycolytic aldolase FBA(C) in gluconeogenic direction at least 3-fold. These results confirmed the importance of the structural differences between FBA(C) and FBA(P) concerning their distinct enzymatic properties. In order to investigate the physiological roles of both aldolases, the expression of their genes was repressed individually by CRISPR interference (CRISPRi). The fba(C) RNA levels were reduced by CRISPRi, but concomitantly the fba(P) RNA levels were increased. Vice versa, a similar compensatory increase of the fba(C) RNA levels was observed when fba(P) was repressed by CRISPRi. In addition, targeting fba(P) decreased tkt(P) RNA levels since both genes are cotranscribed in a bicistronic operon. However, reduced tkt(P) RNA levels were not compensated for by increased RNA levels of the chromosomal transketolase gene tkt(C).