Abstract
BACKGROUND: Dopaxanthin is a natural pigment betaxanthins family member with the highest antioxidant and free radical scavenging activities. However, its relatively low content in plants limited the wide range of applications. Cost-efficient microbial production, therefore, showed an attractive alternative. RESULTS: Here, an Escherichia coli strain equipped with the de novo biosynthetic pathway for hyperproducing dopaxanthin was constructed by combining metabolic engineering and protein engineering. Firstly, a high-performance rate-limiting levodopa 4,5-dioxygenase (DODA) was mined and characterized based on sequence similarity searching followed by whole-cell catalysis and de novo synthesis strategy. Then, the catalytic efficiency of DODA was increased 34 times with directed evolution. The mutated DODA significantly facilitated the production of dopaxanthin, with an increase of 40.17% in plasmid expression and 64.11% in genome expression, respectively. Finally, through connecting the blocked pathway from 3-dehydroshikimate to levodopa (L-DOPAOPA) and enhancing the expression level of DODA, a titer of dopaxanthin of 22.87 g/L was achieved from glucose as feedstock, which is 286 times higher than that in the previous report. CONCLUSION: This work not only established a promising platform for the environmentally friendly production of dopaxanthin but also laid a foundation for the commercialization of other betalain.