Engineering microorganisms based on molecular evolutionary analysis: a succinate production case study.

Evolution has resulted in thousands of species possessing similar metabolic enzymes with identical functions that are, however, regulated by different mechanisms. It is thus difficult to select optimal gene to engineer novel or manipulated metabolic pathways. Here, we tested the ability of molecular evolutionary analysis to identify appropriate genes from various species. We calculated the fraction of synonymous substitution and the effective number of codons (ENC) for nine genes stemming from glycolysis. Our research indicated that an enzyme gene with a stronger selective constraint in synonymous sites would mainly regulate corresponding reaction flux through altering the concentration of the protein, whereas those with a more relaxed selective constraint would primarily affect corresponding reaction flux by changing kinetic properties of the enzyme. Further, molecular evolutionary analysis was investigated for three types of genes involved in succinate precursor supply by catalysis of pyruvate. In this model, overexpression of Corynebacterium glutamicum pyc should result in greater conversion of pyruvate. Succinate yields in two Escherichia coli strains that overexpressed each of the three types of genes supported the molecular evolutionary analysis. This approach may thus provide an alternative strategy for selecting genes from different species for metabolic engineering and synthetic biology.