Biosynthesis of rare ketoses through constructing a recombination pathway in an engineered Corynebacterium glutamicum

Abstract

Rare sugars have various known biological functions and potential for applications in pharmaceutical, cosmetics, and food industries. Here we designed and constructed a recombination pathway in Corynebacterium glutamicum, in which dihydroxyacetone phosphate (DHAP), an intermediate of the glycolytic pathway, and a variety of aldehydes were condensed to synthesize rare ketoses sequentially by rhamnulose-1-phosphate aldolase (RhaD) and fructose-1-phosphatase (YqaB) obtained from Escherichia coli. A wild-type strain harboring this artificial pathway had the ability to produce D-sorbose and D-psicose using D-glyceraldehyde and glucose as the substrates. The tpi gene, encoding triose phosphate isomerase was further deleted, and the concentration of DHAP increased to nearly 20-fold relative to that of the wild-type. After additional optimization of expression levels from rhaD and yqaB genes and of the fermentation conditions, the engineered strain SY6(pVRTY) exhibited preferable performance for rare ketoses production. Its yield increased to 0.59 mol/mol D-glyceraldehyde from 0.33 mol/mol D-glyceraldehyde and productivity to 2.35 g/L h from 0.58 g/L h. Moreover, this strain accumulated 19.5 g/L of D-sorbose and 13.4 g/L of D-psicose using a fed-batch culture mode under the optimal conditions. In addition, it was verified that the strain SY6(pVRTY) meanwhile had the ability to synthesize C4, C5, C6, and C7 rare ketoses when a range of representative achiral and homochiral aldehydes were applied as the substrates. Therefore, the platform strain exhibited the potential for microbial production of rare ketoses and deoxysugars.

Biosynthesis of l -Sorbose and l -Psicose Based on C—C Bond Formation Catalyzed by Aldolases in an Engineered Corynebacterium glutamicum Strain

“…Although the inactivation of the tpi gene increased intracellular DHAP formation ability and the yield of L-psicose/L-sorbose, the final concentration of total ketoses still was lower than that of our previous study (26). We attribute this to the lower enzyme activity of FruA from E. coli.…”

“…Therefore, further investigations to screen another type of FruA or TagA with higher activity are under way. Large amounts of dihydroxyacetone were produced from DHAP as a by-product during dual-phase fermentation processes not only in this study but also in our previous research (26). Thus, the phosphatase that catalyzes dephosphorylation of DHAP to produce dihydroxyacetone should be identified and deleted to further increase intracellular DHAP concentrations during dual-phase fermentation processes.…”

“…However, the cost and instability property of DHAP limited the large-scale production of target products. We have presented a rare ketose biosynthesis strategy in which DHAP was accumulated from glucose through the glyco- lytic pathway in C. glutamicum and a recombination pathway based on RhaD aldolase, and YqaB was constructed to produce D-sorbose and D-psicose from glucose and D-glyceraldehyde (26). With this in mind, the recombinant pathways based on FruA or TagA aldolase and YqaB phosphatase were constructed in both E. coli BL21(DE3) and C. glutamicum strains to produce L-psicose and L-sorbose from glucose and L-glyceraldehyde.…”

“…2). Moreover, the dual-phase fermentation process, in which cell and enzyme preparations both were performed in one stage and rare ketose production was in another stage (26), was used.…”

“…Generally, compound DHAP scarcely accumulated in vivo because of triose phosphate isomerase (TPI), which catalyzed isomerization of DHAP to glyceraldehyde 3-phosphate (Gap-3P), which can be further metabolized to pyruvate, permitting the generation of ATP and NADH. We have described that gene tpi mutation helped to accumulate intracellular DHAP and increase rare ketose synthetic ability in C. glutamicum (26). Therefore, the recombinant pathway was introduced further into strain SY6, in which gene tpi was deleted, to obtain SY6(pXFTY).…”