Coenzyme B-12 can be produced by engineered Escherichia coli under both anaerobic and aerobic conditions

Coenzyme B-12 (Vitamin B-12) is one of the most complex biomolecules and an essential cofactor required for the catalytic activity of many enzymes. Pseudomonas denitrificans synthesizes coenzyme B-12 in an oxygen-dependent manner using a pathway encoded by more than 25 genes that are located in six different operons. Escherichia coli, a robust and suitable host for metabolic engineering was used to produce coenzyme B-12. These genes were cloned into three compatible plasmids and expressed heterologously in E. coli BL21 (DE3). Real-time PCR, SDS-PAGE analysis and bioassay showed that the recombinant E. coli expressed the coenzyme B-12 synthetic genes and successfully produced coenzyme B-12. However, according to the quantitative determination by inductively coupled plasma-mass spectrometry, the amount of coenzyme B-12 produced by the recombinant E. coli (0.21 +/- 0.02 mu g/g cdw) was approximately 13-fold lower than that by P. denitrificans (2.75 +/- 0.22 mu g/g cdw). Optimization of the culture conditions to improve the production of coenzyme B-12 by the recombinant E. coli was successful, and the highest titer (0.65 +/- 0.03 mu g/g cdw) of coenzyme B-12 was obtained. Interestingly, although the synthesis of coenzyme B-12 in P. denitrificans is strictly oxygen-dependent, the recombinant E. coli could produce coenzyme B-12 under anaerobic conditions.X111312sciescopu

Inducible gene expression system by 3-hydroxypropionic acid

Background: 3-Hydroxypropionic acid (3-HP) is an important platform chemical that boasts a variety of industrial applications. Gene expression systems inducible by 3-HP, if available, are of great utility for optimization of the pathways of 3-HP production and excretion. Results: Here we report the presence of unique inducible gene expression systems in Pseudomonas denitrificans and other microorganisms. In P. denitrificans, transcription of three genes (hpdH, mmsA and hbdH-4) involved in 3-HP degradation was upregulated by 3-HP by the action of a transcriptional regulator protein, LysR, and a cis-acting regulatory site for LysR binding. Similar inducible systems having an LysR transcriptional regulator were identified in other microorganisms that also could degrade 3-HP. A docking study showed that the 3-HP binding pocket is located between the N-terminal helix-turn-helix motif and the C-terminal cofactor-binding domain. Conclusions: This LysR-regulated 3-HP-inducible system should prove useful for control of the level of gene expression in response to 3-HP

Biological Production of Hydrogen

The production of H2 from renewable sources, such as water or biomass, is a sustainable strategy for energy supply. Hydrogenases are the only enzymes that specifically catalyze the reversible reaction of H2 production/uptake with almost no overpotential. In this chapter, we review the advances produced in the last decade in the biocatalytic production of H2, including systems based on isolated hydrogenases as well as those using microorganisms through dark fermentation processes