ABSTRACT
The economic viability of the biomass-based biorefinery is readily acknowledged by implementation of a cascade process that produces value-added products such as enzymes prior to biofuels. Proteins from the waste stream of biorefinery processes generally contain glutamate (Glu) in abundance. Accordingly, this study was initiated to explore the potential of Glu for production of recombinant proteins in Escherichia coli. The approach was first adopted by expression of D-hydantoinase (HDT) in commercially-available BL21(DE3) strain. Equipped with the mutant gltS (gltS*), the strain grown on Glu produced the maximum HDT as compared to the counterpart on glucose, glycerol, or acetate. The Glu-based production scheme was subsequently reprogrammed based on the L-arabinose-regulated T7 expression system. The strain with gltS* was further engineered by rewiring metabolic pathways. With low ammonium, the resulting strain produced 1.63-fold more HDT. The result indicates that Glu can serve as a carbon and nitrogen source. Overall, our proposed approach may open up a new avenue for the enzyme biorefinery platform based on Glu.
ABSTRACT
BACKGROUND: The economic viability of a protein-production process relies highly on the production titer and the price of raw materials. Crude glycerol coming from the production of biodiesel is a renewable and cost-effective resource. However, glycerol is inefficiently utilized by Escherichia coli. RESULTS: This issue was addressed by rewiring glycerol metabolism for redistribution of the metabolic flux. Key steps in central metabolism involving the glycerol dissimilation pathway, the pentose phosphate pathway, and the tricarboxylic acid cycle were pinpointed and manipulated to provide precursor metabolites and energy. As a result, the engineered E. coli strain displayed a 9- and 30-fold increase in utilization of crude glycerol and production of the target protein, respectively. CONCLUSIONS: The result indicates that the present method of metabolic engineering is useful and straightforward for efficient adjustment of the flux distribution in glycerol metabolism. The practical application of this methodology in biorefinery and the related field would be acknowledged.
ABSTRACT
Enzymes have a wide range of applications in many sectors of the industry, and the market value has skyrocketed in recent years. Glucose and glycerol are two renewable carbon sources of importance. Therefore, it is appealing to produce recombinant enzymes with these carbon substrates on the basis of economic viability. In this study, glycerol metabolism and glucose metabolism in Escherichia coli (E. coli) were manipulated in a systematic way. In addition, glutamate (Glu) was used for replacement of yeast extract to reduce the cost and the quality-variation problem. A strategy was further developed to incorporate Glu into the central metabolism. The engineered E. coli strain finally enabled efficient co-utilization of glucose and glycerol and improved biomass and protein production by 4.3 and 8.2-folds, respectively. The result illustrates that this proposed approach is promising for effective production of recombinant proteins.
