Reductive dehalogenation of vinyl chloride (VC) to ethene is the key step in complete anaerobic degradation of chlorinated ethenes. VC-reductive dehalogenase was partially purified from a highly enriched culture of the VC-respiring Dehalococcoides sp. strain VS. The enzyme reduced VC and all dichloroethene (DCE) isomers, but not tetrachloroethene (PCE) or trichloroethene (TCE), at high rates. By using reversed genetics, the corresponding gene (vcrA) was isolated and characterized. Based on the predicted amino acid sequence, VC reductase is a novel member of the family of corrinoid/iron-sulfur cluster containing reductive dehalogenases. The vcrA gene was found to be cotranscribed with vcrB, encoding a small hydrophobic protein presumably acting as membrane anchor for VC reductase, and vcrC, encoding a protein with similarity to transcriptional regulators of the NosR/NirI family. The vcrAB genes were subsequently found to be present and expressed in other cultures containing VC-respiring Dehalococcoides organisms and could be detected in water samples from a field site contaminated with chlorinated ethenes. Therefore, the vcrA gene identified here may be a useful molecular target for evaluating, predicting, and monitoring in situ reductive VC dehalogenation.Contamination of groundwater with the chlorinated solvents tetrachloroethene (PCE) and trichloroethene (TCE) threatens numerous drinking water supplies (6, 36). Conventional approaches for groundwater remediation have placed a multibillion-dollar burden on society and have consequently stimulated research in alternative clean-up strategies (22). One such strategy, the removal of these contaminants by naturally occurring, chloroethene-degrading microorganisms (bioremediation), appears to be a viable and cost-effective alternative. The microbial degradation of PCE and TCE has been observed most frequently under anaerobic conditions where the chlorinated ethenes can be reductively dehalogenated via the less chlorinated ethenes cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC) to harmless ethene. However, at many chloroethene-contaminated sites, reductive dehalogenation ceases or is significantly slowed down at the level of VC, resulting in its accumulation. Because VC is a known human carcinogen and is the most toxic compound of all chloroethenes, reduction of VC to ethene is the key step in the complete anaerobic degradation of these compounds.Reductive dehalogenation of VC has been linked to the genus Dehalococcoides (3, 7-9, 18). Dehalococcoides ethenogenes strain 195, the first microorganism isolated in pure culture that dehalogenates VC to ethene (18), catalyzes this reduction only in a slow, cometabolic reaction (14,15,19). Recently, enrichment cultures containing Dehalococcoides-like organisms which couple VC reduction with energy conservation have been reported (3, 7). The isolation of an axenic culture of one of those organisms, strain BAV1, was subsequently described (8).While reductive dehalogenation of higher chlorinated ethenes and of some chlorin...

Polyamides are important industrial polymers. Currently, they are produced exclusively from petrochemical monomers. Herein, we report the production of a novel bio-nylon, PA5.10 through an integration of biological and chemical approaches. First, systems metabolic engineering of Corynebacterium glutamicum was used to create an effective microbial cell factory for the production of diaminopentane as the polymer building block. In this way, a hyper-producer, with a high diaminopentane yield of 41% in shake flask culture, was generated. Subsequent fed-batch production of C. glutamicum DAP-16 allowed a molar yield of 50%, a productivity of 2.2gL(-1)h(-1), and a final titer of 88gL(-1). The streamlined producer accumulated diaminopentane without generating any by-products. Solvent extraction from alkalized broth and two-step distillation provided highly pure diaminopentane (99.8%), which was then directly accessible for poly-condensation. Chemical polymerization with sebacic acid, a ten-carbon dicarboxylic acid derived from castor plant oil, yielded the bio-nylon, PA5.10. In pure form and reinforced with glass fibers, the novel 100% bio-polyamide achieved an excellent melting temperature and the mechanical strength of the well-established petrochemical polymers, PA6 and PA6.6. It even outperformed the oil-based products in terms of having a 6% lower density. It thus holds high promise for applications in energy-friendly transportation. The demonstration of a novel route for generation of bio-based nylon from renewable sources opens the way to production of sustainable bio-polymers with enhanced material properties and represents a milestone in industrial production.

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Basfia succiniciproducens has been recently isolated as novel producer for succinate, an important platform chemical. In batch culture, the wild type exhibited a high natural yield of 0.75 mol succinate (mol glucose)⁻¹. Systems-wide ¹³C metabolic flux analysis identified undesired fluxes through pyruvate-formate lyase (PflD) and lactate dehydrogenase (LdhA). The double deletion strain B. succiniciproducens ΔldhA ΔpflD revealed a 45% improved product yield of 1.08 mol mol⁻¹. In addition, metabolic flux analysis unraveled the parallel in vivo activity of the oxidative and reductive branch of the TCA cycle in B. succiniciproducens, whereby the oxidative part mainly served for anabolism. The wild type re-directed surplus NADH via a cycle involving malic enzyme or via transhydrogenase, respectively, to supply NADPH for anabolism, because the fluxes through the oxidative PPP and isocitrate dehydrogenase, that also provide this cofactor, were not sufficient. This was not observed for the deletion mutants, B. succiniciproducens ΔpflD and ΔldhA ΔpflD, where PPP and isocitrate dehydrogenase flux alone matched with the reduced anabolic NADPH demand. The integration of the production performance into the theoretical flux space, computed by elementary flux mode analysis, revealed that B. succiniciproducens ΔldhA ΔpflD reached 62% of the theoretical maximum yield.