BACKGROUND: 2,3-Butanediol (2,3-BD) has a wide range of applications in chiral molecular synthesis, biofuel additives, and in food flavor additive manufacturing. Fermentation is a favorable method for 2,3-BD production. However, it requires much time and produces several NADH related byproducts which compete with 2,3-BD production. Bacillus subtilis has an excellent ability for 2,3-BD production by biocatalysis. However, its production is limited by low intracellular NADH and the reversible property of acetoin reductase (AR/2,3-BDH). The whole cell biocatalyst process with two different NADH regeneration systems was designed for efficient production of 2,3-BD in B. subtilis 168. RESULTS: Formate dehydrogenase and glucose dehydrogenase for NADH regeneration were successfully co-expressed with acetoin reductase in B. subtilis 168. After optimization of biocatalyst bioconversion conditions, B. subtilis 168/pMA5-bdhA-HpaII-fdh yielded 74.5 g L − 1 of 2, 3-BD with 9.3 g L −1 h −1 productivity by fed batch and 115.4 g of 2,3-BD was achieved using same batch bacterium by three repeated batch bioconversions. On the other hand, 63.7 g L −1 of 2, 3-BD was produced with 7.92 g L −1 h −1 productivity by B. subtilis 168/pMA5-bdhA-HpaII-gdh. To our knowledge, the volume productivity obtained here is the highest ever reported for biocatalysis. CONCLUSION: A higher productivity of 2,3-BD from acetoin was achieved by whole cell biocatalysis with NADH regeneration systems in B. subtilis 168. This approach can be applied for NADH related bio-based chemicals production to improve titer, yield and productivity.

Reactive oxygen species (ROS) is an inherent consequence to all aerobically living organisms that might lead to the cells being lethal and susceptible to oxidative stress. Bacillus pumilus is characterized by high-resistance oxidative stress that stimulated our interest to investigate the heterologous expression and characterization of heme-catalase as potential biocatalyst. Results indicated that recombinant enzyme significantly exhibited the high catalytic activity of 55,784 U/mg expressed in Bacillus subtilis 168 and 98.097 µmol/min/mg peroxidatic activity, the apparent K m of catalytic activity was 59.6 ± 13 mM with higher turnover rate (K cat = 322.651 × 10(3) s(-1)). The pH dependence of catalatic and peroxidatic activity was pH 7.0 and pH 4.5 respectively with temperature dependence of 40 °C and the recombinant heme-catalase exhibited a strong Fe(2+) preference. It was further revealed that catalase KatX2 improved the resistance oxidative stress of B. subtilis. These findings suggest that this B. pumilus heme-catalase can be considered among the industrially relevant biocatalysts due to its exceptional catalytic rate and high stability and it can be a potential candidate for the improvement of oxidative resistance of industrially produced strains.

Background Biomass formation and product synthesis decoupling have been proven to be promising to increase the titer of desired value add products. Optogenetics provides a potential strategy to develop light-induced circuits that conditionally control metabolic flux redistribution for enhanced microbial production. However, the limited number of light-sensitive proteins available to date hinders the progress of light-controlled tools. Results To address these issues, two optogenetic systems (TPRS and TPAS) were constructed by reprogramming the widely used repressor TetR and protease TEVp to expand the current optogenetic toolkit. By merging the two systems, a bifunctional optogenetic switch was constructed to enable orthogonally regulated gene transcription and protein accumulation. Application of this bifunctional switch to decouple biomass formation and shikimic acid biosynthesis allowed 35 g/L of shikimic acid production in a minimal medium from glucose, representing the highest titer reported to date by E. coli without the addition of any chemical inducers and expensive aromatic amino acids. This titer was further boosted to 76 g/L when using rich medium fermentation. Conclusion The cost effective and light-controlled switch reported here provides important insights into environmentally friendly tools for metabolic pathway regulation and should be applicable to the production of other value-add chemicals.

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