Methyl-coenzyme M reductase (MCR) is the key enzyme of methanogenesis and anaerobic methane oxidation. The activity of MCR is dependent on the unique nickel-containing tetrapyrrole known as coenzyme F430. We used comparative genomics to identify the coenzyme F430 biosynthesis (cfb) genes and characterized the encoded enzymes from Methanosarcina acetivorans C2A. The pathway involves nickelochelation by a nickel-specific chelatase, followed by amidation to form Ni-sirohydrochlorin a,c-diamide. Next, a primitive homolog of nitrogenase mediates a six-electron reduction and γ-lactamization reaction before a Mur ligase homolog forms the six-membered carbocyclic ring in the final step of the pathway. These data show that coenzyme F430 can be synthesized from sirohydrochlorin using Cfb enzymes produced heterologously in a nonmethanogen host and identify several targets for inhibitors of biological methane formation.

pH control has been essential for butanol production with Clostridium acetobutylicum. However, it is not very clear at what pH level the acid crash will occur, at what pH level butanol production will be dominant, and at what pH level butyric acid production will be prevailing. Furthermore, contradictory results have been reported about required acidic conditions for initiation of solventogenesis. In this study, with the aim of further understanding the role of undissociated butyric acid in butanol production, we investigated the correlation between undissociated butyric acid concentration and specific butanol production rate in batch fermentation of Clostridium acetobutylicum by comparing three pH control approaches: NaOH neutralization (at 12, 24 or 36 h), CaCO3 supplementation (2, 5, or 8 g/l) and NaOAc buffering (pH 4.6, 5.0 or 5.6). By neutralizing the fermentation pH to ~5.0 at different time, we observed that neutralization should take place at the beginning of exponential phase (12 h), and otherwise resulting in lower concentrations of undissociated butyric acid, cell biomass and final butanol. CaCO3 supplementation extended cell growth to 36 h and resulted in higher butyrate yield under 8 g/L of CaCO3. In the NaOAc buffering, the highest specific butanol rate (0.58 h−1) was associated with the highest undissociated butyric acid (1.92 g/L). The linear correlation of the undissociated butyric acid with the specific butanol production rates suggested the undissociated butyric acid could be the major driving force for butanol production.

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Gluconobacter oxydans has a lower biomass yield. Uniform design (UD) was applied to determine the optimum composition of the critical media and their mutual interactions for increased biomass yield of Gluconobacter oxydans DSM 2003 in shake flasks. Fed-batch fermentation process for biomass was optimized in a 3.7-l fermentor. By undertaking a preliminary and improved fed-batch fermentation-process strategy, a cell density of 6.0 g/l (DCW) was achieved in 22 h and 14.1 g/l (DCW) in 35 h, which is the highest cell density of G. oxydans produced thus far in a 3.7-l bioreactor. The biomass production was increased by 135% compared with that using the original cultivation strategy. Bioconversion of ethylene glycol to glycolic acid was catalyzed by the resting cells of G. oxydans DSM 2003, and conversion rate reached 86.7% in 48 h. In summary, the approach including high-density fermentation of G. oxydans DSM 2003 and bioconversion process was established and proved to be an effective method for glycolic acid production.

A membrane-bound protein purified from Gluconobacter oxydans M5 was confirmed to be a pyrroloquinoline quinone-dependent D-sorbitol dehydrogenase. Gene disruption and complementation experiments demonstrated that this enzyme is responsible for the oxidation of 1-(2-hydroxyethyl) amino-1-deoxy-D-sorbitol (1NSL) to 6-(2-hydroxyethyl) amino-6-deoxy-L-sorbose (6NSE), which is the precursor of an antidiabetic drug, miglitol.Gluconobacter strains are able to oxidize many sugar alcohols incompletely to produce the corresponding aldehydes, ketones, and organic acids, e.g., L-ascorbic acid, D-gluconic acid, ketogluconic acids, and dihydroxyacetone. Their responsible polyol dehydrogenases have been confirmed (1,4,15). It was reported that Gluconobacter oxydans strain M5 can catalyze the oxidation of 1-(2-hydroxyethyl) amino-1-deoxy-D-sorbitol (1NSL) to 6-(2-hydroxyethyl) amino-6-deoxy-L-sorbose (6NSE), a precursor of the antidiabetic drug miglitol (11,20). However, which dehydrogenase is involved has not been revealed yet. The aim of this work was to find out the responsible enzyme.The strains and plasmids used in this study are listed in Table 1. G. oxydans M5 was grown in a medium containing 20 g of D-sorbitol, 3 g of yeast extract, 10 g of polypeptone, 1 g of KH 2 PO 4 , and 0.2 g of MgSO 4 -7H 2 O in 1 liter of deionized water. The different fractions of the cells were prepared by supercentrifugation at 100,000 ϫ g for 60 min. The activities of 1NSL oxidation were measured in the presence of 200 mM substrate, 1NSL (8). The substrate and product were analyzed by silica gel thin-layer chromatography (TLC), with ethanolmethanol-ammonia (1.5:1:1 [vol/vol/vol]) as its eluant, and then dyed with iodine. The major part of the activities was found in the membrane fraction, but little activity could be found in the cytoplasmic fraction (Fig. 1). A second experiment showed phenazine methosulfate (PMS)-dependent 1NSL dehydrogenase activity but not NAD(P)-dependent 1NSL dehydrogenase activity in the membrane fraction. On the contrary, the cytoplasmic fraction had only NAD(P)-dependent 1NSL dehydrogenase activity. In cell extracts, the total activity of PMS-dependent 1NSL dehydrogenase was some 28-fold higher than that of the NAD(P)-dependent 1NSL dehydrogenase (data not shown). These results suggested that the proteins responsible for 1NSL oxidation in G. oxydans are located mainly in the cytoplasmic membrane.Gluconobacter strains contain a large number of dehydrogenases, which can be classified into two major groups (15). The first group is formed by cytoplasmic-soluble NAD(P)-dependent polyol dehydrogenases, which were believed to participate in the synthesis of precursors and are obviously involved in the maintenance of cells in the stationary growth phase (12, 15). The second group possesses various membranebound dehydrogenases, which are shown to be responsible for the rapid oxidation of some important substrates (16,17). According to our previous work, the oxidation of 1NSL to 6NSE by G. oxydans M5 was fast, with almost 95%...