In addition to studies on ethanol production using Z. mobilis, research on this bacterium has also focused on the production of higher value metabolites via the engineering of its central metabolic pathways. As examples, the present study aims to overproduce gluconolactone and pyruvate from Z. mobilis ZM4 using a gluconolactonase deficient mutant (gnl-) and a thiamine-synthesis pathway deficient mutant (thiD-) respectively. Studies have shown that wild type ZM4 can grow on synthetic medium in the absence of thiamine, thereby confirming the presence of a thiamine producing pathway in Z. mobilis. These mutant strains were created via homologous recombination with the transfer of cat gene encoding chloramphenicol acetyl transferase into the gnl and thiD sites of its genome using the vectors pGEM::gnl::cat and pGEM::thiD::cat respectively. Enzymatic assays on both mutants confirmed the loss of gluconolactonase and hydroxymethylpyrimidine/ phosphomethylpyrimidine kinase activities. The analyses showed that the both gnl- and thiD- mutants resulted in no detrimental effect on the cell growth or ethanol production in complex media. Following the fermentation studies, the biomass of gnl- was collected and permeabilised to investigate the biotransformation of glucose into gluconolactone. Preliminary results of the biotransformation studies using gnl- mutant showed some gluconolactone accumulation within the system. Studies on the thiamine dependent mutant (thiD-) strain showed that the expression of pyruvate decarboxylase, the thiamine-dependent enzyme in the ethanol metabolism, could be regulated with low level concentrations of thiamine supplementation in synthetic media. This presentation will further discuss characteristics of both mutant strains for various biotransformation/ fermentation conditions.