Efficient L-valine production using systematically metabolic engineered Klebsiella oxytoca.

L-Valine, a branched-chain amino acid with diversified applications, is biosynthesized with α-acetolactate as the key precursor. In this study, the metabolic flux in Klebsiella oxytoca PDL-K5, a Risk Group 1 organism producing 2,3-butanediol as the major fermentation product, was rearranged to L-valine production by introducing exogenous L-valine biosynthesis pathway and blocking endogenous 2,3-butanediol generation at the metabolic branch point α-acetolactate. After further enhancing L-valine efflux, strengthening pyruvate polymerization and selecting of key enzymes for L-valine synthesis, a plasmid-free K. oxytoca strain VKO-9 was obtained. Fed-batch fermentation with K. oxytoca VKO-9 in a 7.5 L fermenter generated 122 g/L L-valine with a yield of 0.587 g/g in 56 h. In addition, repeated fed-batch fermentation was conducted to prevent precipitation of L-valine due to oversaturation. The average concentration, yield, and productivity of produced L-valine in three cycles of repeated fed-batch fermentation were 81.3 g/L, 0.599 g/g, and 3.39 g/L/h, respectively.

Non-Sterilized Fermentation of 2,3-Butanediol with Seawater by Metabolic Engineered Fast-Growing Vibrio natriegens.

Sustainable and environment-friendly microbial fermentation processes have been developed to produce numerous chemicals. However, the high energy input required for sterilization and substantial fresh water consumption restrict the economic feasibility of traditional fermentation processes. To address these problems, Vibrio natriegens, a promising microbial chassis with low nutritional requirements, high salt tolerance and rapid growth rate can be selected as the host for chemical production. In this study, V. natriegens was metabolic engineered to produce 2,3-butanediol (2,3-BD), an important platform chemical, through non-sterilized fermentation with seawater-based minimal medium after expressing a 2,3-BD synthesis cluster and deleting two byproduct encoding genes. Under optimized fermentative conditions, 41.27 g/L 2,3-BD was produced with a productivity of 3.44 g/L/h and a yield of 0.39 g/g glucose by recombinant strain V. natriegensΔfrdAΔldhA-pETRABC. This study confirmed the feasibility of non-sterilized fermentation using seawater to replace freshwater and other valuable chemicals may also be produced through metabolic engineering of the emerging synthetic biology chassis V. natriegens.

Biotechnological production of chiral acetoin.

Acetoin (AC) is an important platform bulk chemical with versatile applications. It exists in two stereoisomeric forms: (3R)-AC and (3S)-AC. Both stereoisomers could be potentially applied in the pharmaceutical industry, agriculture, and in optically active α-hydroxyketone derivative synthesis. Chiral AC production has recently become a new research focus in biotechnology. Fermentative and biocatalytic routes that can produce (3R)-AC or (3S)-AC with high optical purity have been developed over the past several years. In this review we summarize recent advances in strain screening, metabolic engineering, and biocatalytic system construction aimed at improving the production of chiral AC. Limiting factors and possible solutions for chiral AC production are discussed.

2,3-Butanediol synthesis from glucose supplies NADH for elimination of toxic acetate produced during overflow metabolism.

Overflow metabolism-caused acetate accumulation is a major problem that restricts industrial applications of various bacteria. 2,3-Butanediol (2,3-BD) synthesis in microorganisms is an ancient metabolic process with unidentified functions. We demonstrate here that acetate increases and then decreases during the growth of a bacterium Enterobacter cloacae subsp. dissolvens SDM. Both bifunctional acetaldehyde/ethanol dehydrogenase AdhE-catalyzed ethanol production and acetate-induced 2,3-BD biosynthesis are indispensable for the elimination of acetate generated during overflow metabolism. 2,3-BD biosynthesis from glucose supplies NADH required for acetate elimination via AdhE-catalyzed ethanol production. The coupling strategy involving 2,3-BD biosynthesis and ethanol production is widely distributed in bacteria and is important for toxic acetate elimination. Finally, we realized the co-production of ethanol and acetoin from chitin, the second most abundant natural biopolymer whose catabolism involves inevitable acetate production through the coupling acetate elimination strategy. The synthesis of a non-toxic chemical such as 2,3-BD may be viewed as a unique overflow metabolism with desirable metabolic functions.

An L-2-hydroxyglutarate biosensor based on specific transcriptional regulator LhgR.

L-2-Hydroxyglutarate (L-2-HG) plays important roles in diverse physiological processes, such as carbon starvation response, tumorigenesis, and hypoxic adaptation. Despite its importance and intensively studied metabolism, regulation of L-2-HG metabolism remains poorly understood and none of regulator specifically responded to L-2-HG has been identified. Based on bacterial genomic neighborhood analysis of the gene encoding L-2-HG oxidase (LhgO), LhgR, which represses the transcription of lhgO in Pseudomonas putida W619, is identified in this study. LhgR is demonstrated to recognize L-2-HG as its specific effector molecule, and this allosteric transcription factor is then used as a biorecognition element to construct an L-2-HG-sensing FRET sensor. The L-2-HG sensor is able to conveniently monitor the concentrations of L-2-HG in various biological samples. In addition to bacterial L-2-HG generation during carbon starvation, biological function of the L-2-HG dehydrogenase and hypoxia induced L-2-HG accumulation are also revealed by using the L-2-HG sensor in human cells.

Pyruvate Production from Whey Powder by Metabolic Engineered Klebsiella oxytoca.

Pyruvate is an important platform material widely used in food, pharmaceutical, and chemical industries. Pyruvate-tolerant Klebsiella oxytoca PDL-0 was chosen as a chassis for pyruvate production via metabolic engineering. Genes related to by-product generation were knocked out to decrease the production of 2,3-butantediol, acetate, ethanol, and succinate. The NADH oxidase encoding gene nox was inserted into the locus of the lactate dehydrogenase encoding gene ldhD in the genome of K. oxytoca to simultaneously block lactate production and regenerate NAD+. The pyruvate importers CstA and YjiY were identified, and their encoding genes were deleted to increase pyruvate accumulation. The engineered strain K. oxytoca PDL-YC produced 71.0 g/L pyruvate from glucose. Furthermore, K. oxytoca PDL-YC can use whey powder, an abundant by-product of the cheese making process, as substrate for pyruvate production. Pyruvate production with a concentration of 62.3 g/L and a productivity of 1.60 g/[L·h] was realized using whey powder as substrate.

Efficient 2,3-butanediol production from whey powder using metabolically engineered Klebsiella oxytoca.

BACKGROUND: Whey is a major pollutant generated by the dairy industry. To decrease environmental pollution caused by the industrial release of whey, new prospects for its utilization need to be urgently explored. Here, we investigated the possibility of using whey powder to produce 2,3-butanediol (BDO), an important platform chemical. RESULTS: Klebsiella oxytoca strain PDL-0 was selected because of its ability to efficiently produce BDO from lactose, the major fermentable sugar in whey. After deleting genes pox, pta, frdA, ldhD, and pflB responding for the production of by-products acetate, succinate, lactate, and formate, a recombinant strain K. oxytoca PDL-K5 was constructed. Fed-batch fermentation using K. oxytoca PDL-K5 produced 74.9 g/L BDO with a productivity of 2.27 g/L/h and a yield of 0.43 g/g from lactose. In addition, when whey powder was used as the substrate, 65.5 g/L BDO was produced within 24 h with a productivity of 2.73 g/L/h and a yield of 0.44 g/g. CONCLUSION: This study demonstrated the efficiency of K. oxytoca PDL-0 for BDO production from whey. Due to its non-pathogenicity and efficient lactose utilization, K. oxytoca PDL-0 might also be used in the production of other important chemicals using whey as the substrate.