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Abstract
Microorganisms are widely used to synthesize chemicals. One set of microbial conversions involves the transfer of electron from a starting material to a microbial electron carrier (such as NAD+): A + NAD+ to B + NADH. L-xylulose formation is a good example of this microbial conversion, which is a rare sugar generated from xylitol by an NAD+-dependent xylitol 4-dehydrogenase (XDH) from Pantoea ananatis. Since the general conversion of A to B (or the specific example of xylitol to L-xylulose) involves the utilization of NAD+ and the formation of NADH, the continued presence of NAD+ is necessary to drive the process. Two fundamental challenges exist, however. First, to drive the reaction forward at a fast rate toward the product, a relatively high ratio of NAD+/NADH is required. Second, microbes continually degrade both NAD+ and NADH as part of biochemical recycling that occurs in living systems, which will lead essentially to a loss of NAD(H) availability. Using the conversion of xylitol to L-xylulose as a model system, we co-expressed water-forming NADH oxidase (NOX) from Streptococcus pneumoniae with XDH to regenerate NAD+, and also modified the biochemical pathways involved in NAD(H) degradation. Controlled batch process demonstrated that the final equilibrium L-xylulose/xylitol ratio was correlated to an elevated ratio of NAD+/NADH. The E. coli strain (MEC697) with deletions of the nadR nudC mazG genes in the degradation pathway of NAD(H) increased the total amount of NAD(H) and delayed the degradation of this cofactor, further improving the conversion of xylitol to L-xylulose. As a potential platform for enhancing formation of the oxidized biochemical product, MEC697 also successfully improved the conversion of galactitol to L-tagatose, with almost a 60% increase in the final product.In addition, MEC697 reduced the acetate formation when grown on glucose and improved galactosidase production. Using steady-state cultures, we demonstrated the enhanced total NAD(H) in MEC697 delayed the threshold growth rate for acetate formation to beyond 0.27 h-1. At the 1 liter scale, a batch process with 10 g/L glucose, MEC697 as the host strain for recombinant protein production reduced acetate by about 50% and doubled the formation of galactosidase.