Coculture of Gluconobacter oxydans and Escherichia coli for 3,4-Dihydroxybutyric Acid Production from Xylose

Abstract

3,4-Dihydroxybutyric acid (3,4-DHBA) is an important platform chemical with versatile applications. Traditional chemical approaches for 3,4-DHBA production involving hazardous materials and harsh reaction conditions are environmentally unfriendly. The reported biotechnological routes for 3,4-DHBA production rely on microbial monocultures with low productivity and yield. In this study, a coculture system to synthesize 3,4-DHBA from xylose was established using Gluconobacter oxydans and Escherichia coli. First, it was confirmed that G. oxydans 621H can oxidize xylose and 3,4-dihydroxybutanal (3,4-DHB) into xylonate and 3,4-DHBA, respectively. Second, xylonate dehydratase and α-ketoisovalerate decarboxylase were overexpressed and the competing pathways in the host strain were knocked out for 3,4-DHB biosynthesis from xylonate in E. coli. Third, the conditions for the coculture of G. oxydans 621H and engineered E. coli were optimized to enhance 3,4-DHBA biosynthesis from xylose. Finally, the coculture system produced 3.26 g/L 3,4-DHBA from 7.0 g/L xylose with a high yield of 0.47 g/g, achieving the highest titer and yield of 3,4-DHBA reported so far. Besides the biotechnological production of 3,4-DHBA, the coculture of G. oxydans 621H with engineered E. coli would be a useful engineering strategy in the production of other important biochemicals.

An in vitro cascade with four enzymes for the production of d-3,4-dihydroxybutyric acid from d-xylose

“…14,44,45 LlKivD from L. lactis has been reported to catalyze the decarboxylation of d -2-KDX into d -3,4-DHB. 14,21 Thus, CcXylD and LlKivD were separately overexpressed in E. coli BL21 (DE3) and purified to homogeneity (Fig. S3B and S3C†).…”

“…Recently, we used a complex modular co-culture system composed of Gluconobacter oxydans 621H and engineered E. coli to produce d -3,4-DHBA from d -xylose. 21 Membrane-bound glucose dehydrogenase (mGDH) and membrane-bound aldehyde dehydrogenase (mAcDH) of G. oxydans were utilized to avoid the intracellular redox pressure in engineered E. coli . 21…”

“…21 Membrane-bound glucose dehydrogenase (mGDH) and membrane-bound aldehyde dehydrogenase (mAcDH) of G. oxydans were utilized to avoid the intracellular redox pressure in engineered E. coli . 21…”

“…Recently, we used a complex modular co-culture system composed of Gluconobacter oxydans 621H and engineered E. coli to produce D-3,4-DHBA from D-xylose. 21 Membrane-bound glucose dehydrogenase (mGDH) and membrane-bound aldehyde dehydrogenase (mAcDH) of G. oxydans were utilized to avoid the intracellular redox pressure in engineered E. coli. 21 The in vitro enzymatic cascade, which assembles individual enzymes according to the designed metabolic pathway, is emerging as a promising biosynthetic platform for chemical production.…”

“…21 Membrane-bound glucose dehydrogenase (mGDH) and membrane-bound aldehyde dehydrogenase (mAcDH) of G. oxydans were utilized to avoid the intracellular redox pressure in engineered E. coli. 21 The in vitro enzymatic cascade, which assembles individual enzymes according to the designed metabolic pathway, is emerging as a promising biosynthetic platform for chemical production. [22][23][24][25] Compared to the cell-based system, this strategy has several advantages such as fast reaction rates, high selectivity, and easy manipulation, but also suffers from the high cost of biocatalyst preparation and exogenous cofactors.…”