Development of an industrial yeast strain for efficient production of 2,3-butanediol.

As part of the transition from a fossil resources-based economy to a bio-based economy, the production of platform chemicals by microbial cell factories has gained strong interest. 2,3-butanediol (2,3-BDO) has various industrial applications, but its production by microbial fermentation poses multiple challenges. We have engineered the bacterial 2,3-BDO synthesis pathway, composed of AlsS, AlsD and BdhA, in a pdc-negative version of an industrial Saccharomyces cerevisiae yeast strain. The high concentration of glycerol caused by the excess NADH produced in the pathway from glucose to 2,3-BDO was eliminated by overexpression of NoxE and also in a novel way by combined overexpression of NDE1, encoding mitochondrial external NADH dehydrogenase, and AOX1, encoding a heterologous alternative oxidase expressed inside the mitochondria. This was combined with strong downregulation of GPD1 and deletion of GPD2, to minimize glycerol production while maintaining osmotolerance. The HGS50 strain produced a 2,3-BDO titer of 121.04 g/L from 250 g/L glucose, the highest ever reported in batch fermentation, with a productivity of 1.57 g/L.h (0.08 g/L.h per gCDW) and a yield of 0.48 g/g glucose or with 96% the closest to the maximum theoretical yield ever reported. Expression of Lactococcus lactis NoxE, encoding a water-forming NADH oxidase, combined with similar genetic modifications, as well as expression of Candida albicans STL1, also minimized glycerol production while maintaining high osmotolerance. The HGS37 strain produced 130.64 g/L 2,3-BDO from 280 g/L glucose, with productivity of 1.58 g/L.h (0.11 g/L.h per gCDW). Both strains reach combined performance criteria adequate for industrial implementation.

Metabolic engineering of Bacillus subtilis for chiral pure meso-2,3-butanediol production.

BACKGROUND: 2,3-Butanediol (2,3-BD) with low toxicity to microbes, could be a promising alternative for biofuel production. However, most of the 2,3-BD producers are opportunistic pathogens that are not suitable for industrial-scale fermentation. In our previous study, wild-type Bacillus subtilis 168, as a class I microorganism, was first found to generate only d-(-)-2,3-BD (purity >99 %) under low oxygen conditions. RESULTS: In this work, B. subtilis was engineered to produce chiral pure meso-2,3-BD. First, d-(-)-2,3-BD production was abolished by deleting d-(-)-2,3-BD dehydrogenase coding gene bdhA, and acoA gene was knocked out to prevent the degradation of acetoin (AC), the immediate precursor of 2,3-BD. Next, both pta and ldh gene were deleted to decrease the accumulation of the byproducts, acetate and l-lactate. We further introduced the meso-2,3-BD dehydrogenase coding gene budC from Klebsiella pneumoniae CICC10011, as well as overexpressed alsSD in the tetra-mutant (ΔacoAΔbdhAΔptaΔldh) to achieve the efficient production of chiral meso-2,3-BD. Finally, the pool of NADH availability was further increased to facilitate the conversion of meso-2,3-BD from AC by overexpressing udhA gene (coding a soluble transhydrogenase) and low dissolved oxygen control during the cultivation. Under microaerobic oxygen conditions, the best strain BSF9 produced 103.7 g/L meso-2,3-BD with a yield of 0.487 g/g glucose in the 5-L batch fermenter, and the titer of the main byproduct AC was no more than 1.1 g/L. CONCLUSION: This work offered a novel strategy for the production of chiral pure meso-2,3-BD in B. subtilis. To our knowledge, this is the first report indicating that metabolic engineered B. subtilis could produce chiral meso-2,3-BD with high purity under limited oxygen conditions. These results further demonstrated that B. subtilis as a class I microorganism is a competitive industrial-level meso-2,3-BD producer.

Enhanced biodegradation of alkane hydrocarbons and crude oil by mixed strains and bacterial community analysis.

In this study, two strains, Acinetobacter sp. XM-02 and Pseudomonas sp. XM-01, were isolated from soil samples polluted by crude oil at Bohai offshore. The former one could degrade alkane hydrocarbons (crude oil and diesel, 1:4 (v/v)) and crude oil efficiently; the latter one failed to grow on alkane hydrocarbons but could produce rhamnolipid (a biosurfactant) with glycerol as sole carbon source. Compared with pure culture, mixed culture of the two strains showed higher capability in degrading alkane hydrocarbons and crude oil of which degradation rate were increased from 89.35 and 74.32 ± 4.09 to 97.41 and 87.29 ± 2.41 %, respectively. In the mixed culture, Acinetobacter sp. XM-02 grew fast with sufficient carbon source and produced intermediates which were subsequently utilized for the growth of Pseudomonas sp. XM-01 and then, rhamnolipid was produced by Pseudomonas sp. XM-01. Till the end of the process, Acinetobacter sp. XM-02 was inhibited by the rapid growth of Pseudomonas sp. XM-01. In addition, alkane hydrocarbon degradation rate of the mixed culture increased by 8.06 to 97.41 % compared with 87.29 % of the pure culture. The surface tension of medium dropping from 73.2 × 10(-3) to 28.6 × 10(-3) N/m. Based on newly found cooperation between the degrader and the coworking strain, rational investigations and optimal strategies to alkane hydrocarbons biodegradation were utilized for enhancing crude oil biodegradation.