Photosynthetic Reduction of Xylose to Xylitol Using Cyanobacteria.

Photosynthetic generation of reducing power makes cyanobacteria an attractive host for biochemical reduction compared to cell-free and heterotrophic systems, which require burning of additional resources for the supply of reducing equivalent. Here, using xylitol synthesis as an example, efficient uptake and reduction of xylose photoautotrophically in Synechococcus elongatus PCC7942 are demonstrated upon introduction of an effective xylose transporter from Escherichia coli (Ec-XylE) and the NADPH-dependent xylose reductase from Candida boidinii (Cb-XR). Simultaneous activation of xylose uptake and matching of cofactor specificity enabled an average xylitol yield of 0.9 g g-1 xylose and a maximum productivity of about 0.15 g L-1 day-1 OD-1 with increased level of xylose supply. While long-term cellular maintenance still appears challenging, high-density conversion of xylose to xylitol using concentrated resting cell further pushes the titer of xylitol formation to 33 g L-1 in six days with 85% of maximum theoretical yield. While the results show that the unknown dissipation of xylose can be minimized when coupled to a strong reaction outlet, it remains to be the major hurdle hampering the yield despite the reported inability of cyanobacteria to metabolize xylose.

Biotransformation of 5-Hydroxymethylfurfural to 2,5-Furandicarboxylic Acid by a Syntrophic Consortium of Engineered Synechococcus elongatus and Pseudomonas putida.

2,5-furandicarboxylic acid (FDCA) is one of the top platform chemicals that can be produced from biomass feedstock. To make the cost of industrial FDCA production compatible with plastics made from fossils, the price of substrates and process complexity should be reduced. The aim of this research is to create a CO2 -driven syntrophic consortium for the catalytic conversion of renewable biomass-derived 5-hydroxymethylfurfural (HMF) to FDCA. Sucrose produced from carbon fixation by the engineered Synechococcus elongatus serves as the sole carbon source for the engineered Pseudomonas putida to catalyze the reaction of HMF to FDCA. The yield of FDCA by the consortium reaches around 70% while the conversion of HMF is close to 100%. With further surface engineering to clump the two strains, the FDCA yield is elevated to almost 100% via the specific association between an Src homology 3 (SH3) domain and its ligand. The syntrophic consortium successfully demonstrates its green and cost-effective characteristics for the conversion of CO2 and biomass into platform chemicals.

Self-regulated 1-butanol production in Escherichia coli based on the endogenous fermentative control.

BACKGROUND: As a natural fermentation product secreted by Clostridium species, bio-based 1-butanol has attracted great attention for its potential as alternative fuel and chemical feedstock. Feasibility of microbial 1-butanol production has also been demonstrated in various recombinant hosts. RESULTS: In this work, we constructed a self-regulated 1-butanol production system in Escherichia coli by borrowing its endogenous fermentation regulatory elements (FRE) to automatically drive the 1-butanol biosynthetic genes in response to its natural fermentation need. Four different cassette of 5' upstream transcription and translation regulatory regions controlling the expression of the major fermentative genes ldhA, frdABCD, adhE, and ackA were cloned individually to drive the 1-butanol pathway genes distributed among three plasmids, resulting in 64 combinations that were tested for 1-butanol production efficiency. Fermentation of 1-butanol was triggered by anaerobicity in all cases. In the growth-decoupled production screening, only combinations with formate dehydrogenase (Fdh) overexpressed under FRE adhE demonstrated higher titer of 1-butanol anaerobically. In vitro assay revealed that 1-butanol productivity was directly correlated with Fdh activity under such condition. Switching cells to oxygen-limiting condition prior to significant accumulation of biomass appeared to be crucial for the induction of enzyme synthesis and the efficiency of 1-butanol fermentation. With the selection pressure of anaerobic NADH balance, the engineered strain demonstrated stable production of 1-butanol anaerobically without the addition of inducer or antibiotics, reaching a titer of 10 g/L in 24 h and a yield of 0.25 g/g glucose under high-density fermentation. CONCLUSIONS: Here, we successfully engineered a self-regulated 1-butanol fermentation system in E. coli based on the natural regulation of fermentation reactions. This work also demonstrated the effectiveness of selection pressure based on redox balance anaerobically. Results obtained from this study may help enhance the industrial relevance of 1-butanol synthesis using E. coli and solidifies the possibility of strain improvement by directed evolution.