In the first and second year, we used Escherichia coli, the model microorganism for biorefinery, as the basis for our initial characterization of indigo and indirubin biosynthetic pathway. Upon protein engineering of the essential oxygenase, we have successfully demonstrated significant accumulation of indigo and indirubin production in E. coli, reaching about 1.2 and 0.3 g/L respectively. It is by far the highest indirubin titer reported using microorganism in literature. Additionally, we identified the stable method of bacterial waste pretreatment using E. coli for the preparation of protein hydrolysates. The treated cell lysates could provide a protein concentration between 5 to 11 g/L, which could support the growth of wild type Bacillus marmarensis to an OD between 4 to 8. Using the tryptophan analog 7-azatryptophan, we were able to mutate and select strains of Bacillus marmarensis that are capable of producing tryptophan to a level around 75 mg/L. Combining with the optimized oxygenase, microbial conversion of protein waste into high valued chemicals should be greatly enhanced.
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