(R)-1,3-butanediol ((R)-1,3-BD) is an important substrate for the synthesis of industrial chemicals. Despite its large demand, a bioprocess for the efficient production of 1,3-BD from renewable resources has not been developed. We previously reported the construction of recombinant Escherichia coli that could efficiently produce (R)-1,3-BD from glucose. In this study, the fermentation conditions were optimized to further improve 1,3-BD production by the recombinant strain. A batch fermentation was performed with an optimized overall oxygen transfer coefficient (82.3 h(-1)) and pH (5.5); the 1,3-BD concentration reached 98.5 mM after 36 h with high-yield (0.444 mol (mol glucose)(-1)) and a high maximum production rate (3.63 mM h(-1)). In addition, a fed-batch fermentation enabled the recombinant strain to produce 174.8 mM 1,3-BD after 96 h cultivation with a yield of 0.372 mol (mol glucose)(-1), a maximum production rate of 3.90 mM h(-1), and a 98.6% enantiomeric excess (% ee) of (R)-1,3-BD.
Acetic acid bacteria catalyze the two-step oxidation of ethanol to acetic acid using the membrane-bound enzymes pyrroloquinoline quinone-dependent alcohol dehydrogenase and molybdopterin-dependent aldehyde dehydrogenase (ALDH). Although the reducing equivalents from the substrate are transferred to ubiquinone in the membrane, intramolecular electron transport in ALDH is not understood. Here, we purified the AldFGH complex, the membrane-bound ALDH that is physiologically relevant to acetic acid fermentation in Gluconacetobacter diazotrophicus strain PAL5. The purified AldFGH complex showed acetaldehyde:ubiquinone (Q2) oxidoreductase activity. c-type cytochromes of the AldFGH complex (in the AldF subunit) were reduced by acetaldehyde. Next, we genetically dissected the AldFGH complex into AldGH and AldF units and reconstituted them. The AldGH subcomplex showed acetaldehyde:ferricyanide oxidoreductase activity but not Q2 reductase activity. The ALDH activity of AldGH was not found in membranes but was found in the soluble fraction of the recombinant strain, suggesting that the AldF subunit is responsible for membrane binding of the AldFGH complex. The absorption spectra of the purified AldGH subcomplex suggested the presence of an [Fe-S] cluster, which can be reduced by acetaldehyde. The AldFGH complex reconstituted from the AldGH subcomplex and AldF showed Q2 reductase activity. We propose a model in which electrons from the substrate are abstracted by a molybdopterin in the AldH subunit and transferred to the [Fe-S] cluster(s) in the AldG subunit, followed by electron transport to c-type cytochrome centers in the AldF subunit, which is the site of ubiquinone reduction in the membrane. IMPORTANCE Two membrane-bound enzymes of acetic acid bacteria, pyrroloquinoline quinone-dependent alcohol dehydrogenase and molybdopterin-dependent aldehyde dehydrogenase (ALDH), are responsible for vinegar production. Upon the oxidation of acetaldehyde, ALDH reduces ubiquinone in the cytoplasmic membrane. ALDH is an enzyme complex of three subunits. Here, we tried to understand how ALDH works by using a classical biochemical approach and genetic engineering to dissect the enzyme complex into soluble and membrane-bound parts. The soluble part had limited activity in vitro and did not reduce ubiquinone. However, the enzyme complex reconstituted from the soluble and membrane-bound parts showed ubiquinone reduction activity. The proposed working model of ALDH provides a better understanding of how the enzyme works in the vinegar fermentation process.
ABSTRACT Incomplete oxidation of glucose by Gluconobacter sp. strain CHM43 produces gluconic acid and then 2- or 5-ketogluconic acid. Although 2-keto-D-gluconate (2KG) is a valuable compound, it is sometimes consumed by Gluconobacter itself via an unknown metabolic pathway. We anticipated that 2KG reductase (2KGR) would be a key enzyme in 2KG metabolism. GLF_0478 and GLF_1777 were identified in the genome of strain CHM43, which encode proteins with 70% and 48% amino acid sequence identity, respectively, to the 2KGR of Gluconobacter oxydans strain 621H. Constructed mutant derivatives of strain CHM43 lacking GLF_0478 , GLF_1777 , or both were examined for their 2KG consumption ability. Strains ∆ GLF_0478 and ∆ GLF_1777 consumed 2KG like the parental strain. However, the double-deletion (∆∆) strain did not consume 2KG at all, although it produced 2KG like the parental strain. Strains ∆ GLF_0478 and ∆ GLF_1777 each showed decreased 2KGR activity compared with the parental strain, and strain ΔΔ lost 2KGR activity. These results suggest that reduction of 2KG catalyzed by GLF_0478 and GLF_1777 is the committed step in 2KG metabolism in Gluconobacter sp. strain CHM43. The two 2KGRs showed high activity at neutral pH and lower K M values for NADPH than NADH. Results of induction experiments suggest that GLF_0478 is constitutively expressed at a low level but induced by 2KG, and GLF_1777 is also inducible by 2KG but repressed in the absence of an inducer. Our study that characterizes the key genes for 2KG consumption in Gluconobacter gives insights for improvement of biological 2KG production systems. IMPORTANCE 2-Keto-D-gluconate (2KG), a product of incomplete oxidation of glucose by acetic acid bacteria including Gluconobacter spp., is used for various purposes, including in the food industry. Gluconobacter also consumes 2KG via an unclear metabolic pathway. It is reported that Pseudomonas spp. and Cupriavidus necator phosphorylate 2KG in the first step of 2KG metabolism, but some enteric bacteria including Escherichia coli reduce 2KG. This study evaluated the 2KG consumption ability of a mutant derivative of a strain of Gluconobacter that lacks two putative 2KGR-encoding genes. The mutant strain did not consume 2KG at all; the two 2KGRs were each found to catalyze 2KG reduction. It is concluded that reduction of 2KG is the committed step in 2KG metabolism in Gluconobacter . The results presented here give insights that might facilitate improvement of 2KG production systems that use Gluconobacter .
The nematode Caenorhabditis elegans yields a substance(s) inducing the larval diapause, called dauer-inducing pheromone. We discovered that the crude pheromone extract extends the adult lifespan in the animal. This extension does not occur in the mutant animal, in which expansion of the lifespan caused by other mutations reducing insulin signaling is suppressed. This is the first description concerning the relevancy of the pheromone to the longevity in the animal.
A new architecture for OCDMA-based PON systems is proposed, that allows contentionless, asynchronous access at 10 Gb/s data rate, without using any laser source at the users' premises.
