Constructing a synthetic constitutive metabolic pathway in Escherichia coli for (R, R)-2,3-butanediol production.

Many microorganisms could naturally produce (R, R)-2,3-butanediol ((R, R)-2,3-BD), which has unique applications due to its special chiral group and spatial configuration. But the low enantio-purity of the product hindered the development of large-scale production. In this work, a synthetic constitutive metabolic pathway for enantiomerically pure (R, R)-2,3-BD biosynthesis was constructed in Escherichia coli with vector pUC6S, which does not contain any lac sequences. The expression of this artificial constructed gene cluster was optimized by using two different strength of promoters (AlperPLTet01 (P01) and AlperBB (PBB)). The strength of P01 is twice stronger than PBB. The fermentation results suggested that the yield of (R, R)-2,3-BD was higher when using the stronger promoter. Compared with the wild type, the recombinant strain E. coli YJ2 produced a small amount of acetic acid and showed higher glucose consumption rate and higher cell density, which indicated a protection against acetic acid inhibition. In order to further increase the (R, R)-2,3-BD production by reducing the accumulation of its precursor acetoin, the synthetic operon was reconstructed by adding the strong promoter P01 in front of the gene ydjL coding for the enzyme of (R, R)-2,3-BD dehydrogenase which catalyzes the conversion of acetoin to (R, R)-2,3-BD. The engineered strain E. coli YJ3 showed a 20 % decrease in acetoin production compared with that of E. coli YJ2. After optimization the fermentation conditions, 30.5 g/L of (R, R)-2,3-BD and 3.2 g/L of acetoin were produced from 80 g/L of glucose within 18 h, with an enantio-purity over 99 %.

Genome Sequence of Klebsiella pneumoniae CICC10011, a Promising Strain for High 2,3-Butanediol Production.

Klebsiella pneumoniae CICC10011, a promising 2,3-butanediol producer, has received much attention because of its high productivity. Here, the first draft genome sequence of this efficient strain may provide the genetic basis for further insights into the metabolic and regulatory mechanisms underlying the production of 2,3-butanediol at a high titer.

Constructing a synthetic metabolic pathway in Escherichia coli to produce the enantiomerically pure (R, R)-2,3-butanediol.

Enantiomerically pure (R, R)-2,3-butanediol has unique applications due to its special chiral group and spatial configuration. Currently, its chemical production route has many limitations. In addition, no native microorganisms can accumulate (R, R)-2,3-butanediol with an enantio-purity over 99%. Herein, we constructed a synthetic metabolic pathway for enantiomerically pure (R, R)-2,3-butanediol biosynthesis in Escherichia coli. The fermentation results suggested that introduction of the synthetic metabolic pathway redistributed the carbon fluxes to the neutral (R, R)-2,3-butanediol, and thus protected the strain against the acetic acid inhibition. Additionally, it showed that the traditionally used isopropyl beta-D-thiogalactoside (IPTG) induction displayed negative effect on (R, R)-2,3-butanediol biosynthesis in the recombinant E. coli, which was probably due to the protein burden. With no IPTG addition, the (R, R)-2,3-butanediol concentration reached 115 g/L by fed-batch culturing of the recombinant E. coli, with an enantio-purity over 99%, which is suitable for the pilot-scale production.

Genome Sequence of Paenibacillus polymyxa ATCC 12321, a Promising Strain for Optically Active (R,R)-2,3-Butanediol Production.

Paenibacillus polymyxa is a potential strain for (R,R)-2,3-butanediol production. Here, we report an annotated draft genome sequence of P. polymyxa strain ATCC 12321, which contains 4,429 protein-coding genes and 49 structural RNAs. This genome sequence provides a genetic basis for a better understanding of the mechanism for the accumulation of highly optically active (R,R)-2,3-butanediol.