The rumen represents the first section of a ruminant animal's stomach, where feed is collected and mixed with microorganisms for initial digestion. The major gas produced in the rumen is CO(2) (65.5 mol%), yet the metabolic characteristics of capnophilic (CO(2)-loving) microorganisms are not well understood. Here we report the 2,314,078 base pair genome sequence of Mannheimia succiniciproducens MBEL55E, a recently isolated capnophilic Gram-negative bacterium from bovine rumen, and analyze its genome contents and metabolic characteristics. The metabolism of M. succiniciproducens was found to be well adapted to the oxygen-free rumen by using fumarate as a major electron acceptor. Genome-scale metabolic flux analysis indicated that CO(2) is important for the carboxylation of phosphoenolpyruvate to oxaloacetate, which is converted to succinic acid by the reductive tricarboxylic acid cycle and menaquinone systems. This characteristic metabolism allows highly efficient production of succinic acid, an important four-carbon industrial chemical.

In this study, we developed recombinant Escherichia coli strains expressing Lactococcus lactis subsp. lactis Il1403 glutamate decarboxylase (GadB) for the production of GABA from glutamate monosodium salt (MSG). Syntheses of GABA from MSG were examined by employing recombinant E. coli XL1-Blue as a whole cell biocatalyst in buffer solution. By increasing the concentration of E. coli XL1-Blue expressing GadB from the OD₆₀₀ of 2-10, the concentration and conversion yield of GABA produced from 10 g/L of MSG could be increased from 4.3 to 4.8 g/L and from 70 to 78 %, respectively. Furthermore, E. coli XL1-Blue expressing GadB highly concentrated to the OD₆₀₀ of 100 produced 76.2 g/L of GABA from 200 g/L of MSG with 62.4 % of GABA yield. Finally, nylon 4 could be synthesized by the bulk polymerization using 2-pyrrolidone that was prepared from microbially synthesized GABA by the reaction with Al₂O₃ as catalyst in toluene with the yield of 96 %.

SummaryBacterial pathogenesis requires the precise spatial and temporal control of gene expression, the dynamics of which are controlled by regulatory networks. A network encoded within Salmonella Pathogenicity Island 1 controls the expression of a type III protein secretion system involved in the invasion of host cells. The dynamics of this network are measured in single cells using promotergreen fluorescent protein (gfp) reporters and flow cytometry. During induction, there is a temporal order of gene expression, with transcriptional inputs turning on first, followed by structure, and effector genes. The promoters show varying stochastic properties, where graded inputs are converted into all-or-none and hybrid responses. The relaxation dynamics are measured by shifting cells from inducing into non-inducing conditions and measuring the fluorescence decay. The gfp expressed from promoters controlling the transcriptional inputs (hilC and hilD) and structural genes (prgH) decay exponentially with a characteristic time of 50-55 minutes. In contrast, the gfp expressed from a promoter controlling the expression of effectors (sicA) persists for 110 ± 9 minutes. This promoter is controlled by a genetic circuit formed by a transcription factor (InvF), chaperone (SicA) and secreted protein (SipC) that regulates effector expression in response to the secretion capacity of the cell. A mathematical model of this circuit demonstrates that the delay is due to a split positive feedback loop. This model is tested in a ΔsicA knockout where sicA is complemented with and without the feedback loop. The delay is eliminated when the feedback loop is deleted. Further, a robustness analysis of the model predicts that the delay time can be tuned by changing the affinity of SicA:InvF multimers to an operator in the sicA promoter. This prediction is used to construct a targeted library, which contains mutants with both longer and shorter delays. This combination of theory and experiments provides a platform to predict how genetic perturbations lead to changes in the global dynamics of a regulatory network.

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A biosynthetic pathway for the production of (S)-3-hydroxybutyric acid (S3HB) from glucose was established in recombinant Escherichia coli by introducing the beta-ketothiolase gene from Ralstonia eutropha H16, the (S)-3-hydroxybutyryl-CoA dehydrogenase gene from R. eutropha H16, or Clostridium acetobutylicum ATCC824, and the 3-hydroxyisobutyryl-CoA hydrolase gene from Bacillus cereus ATCC14579. Artificial operon consisting of these genes was constructed and was expressed in E. coli BL21 (DE3) codon plus under T7 promoter by isopropyl beta-D: -thiogalactoside (IPTG) induction. Recombinant E. coli BL21 (DE3) codon plus expressing the beta-ketothiolase gene, the (S)-3-hydroxybutyryl-CoA dehydrogenase gene, and the 3-hydroxyisobutyryl-CoA hydrolase gene could synthesize enantiomerically pure S3HB to the concentration of 0.61 g l(-1) from 20 g l(-1) of glucose in Luria-Bertani medium. Fed-batch cultures of recombinant E. coli BL21 (DE3) codon plus were carried out to achieve higher titer of S3HB with varying induction time and glucose concentration during fermentation. Protein expression was induced by addition of 1 mM IPTG when cell concentration reached 10 and 20 g l(-1) (OD(600) = 30 and 60), respectively. When protein expression was induced at 60 of OD(600) and glucose was fed to the concentration of 15 g l(-1), 10.3 g l(-1) of S3HB was obtained in 38 h with the S3HB productivity of 0.21 g l(-1)h(-1). Lowering glucose concentration to 5 g l(-1) and induction of protein expression at 30 of OD(600) significantly reduced final S3HB concentration to 3.7 g l(-1), which also resulted in the decrease of the S3HB productivity to 0.05 g l(-1)h(-1).