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Glycerol dehydrogenase plays a dual role in glycerol metabolism and 2,3-butanediol formation in Klebsiella pneumoniae.

ABSTRACT: Glycerol dehydrogenase (GDH) is an important polyol dehydrogenase for glycerol metabolism in diverse microorganisms and for value-added utilization of glycerol in the industry. Two GDHs from Klebsiella pneumoniae, DhaD and GldA, were expressed in Escherichia coli, purified and characterized for substrate specificity and kinetic parameters. Both DhaD and GldA could catalyze the interconversion of (3R)-acetoin/(2R,3R)-2,3-butanediol or (3S)-acetoin/meso-2,3-butanediol, in addition to glycerol oxidation. Although purified GldA appeared more active than DhaD, in vivo inactivation and quantitation of their respective mRNAs indicate that dhaD is highly induced by glycerol and plays a dual role in glycerol metabolism and 2,3-butanediol formation. Complementation in K. pneumoniae further confirmed the dual role of DhaD. Promiscuity of DhaD may have vital physiological consequences for K. pneumoniae growing on glycerol, which include balancing the intracellular NADH/NAD(+) ratio, preventing acidification, and storing carbon and energy. According to the kinetic response of DhaD to modified NADH concentrations, DhaD appears to show positive homotropic interaction with NADH, suggesting that the physiological role could be regulated by intracellular NADH levels. The co-existence of two functional GDH enzymes might be due to a gene duplication event. We propose that whereas DhaD is specialized for glycerol utilization, GldA plays a role in backup compensation and can turn into a more proficient catalyst to promote a survival advantage to the organism. Revelation of the dual role of DhaD could further the understanding of mechanisms responsible for enzyme evolution through promiscuity, and guide metabolic engineering methods of glycerol metabolism.

SUBMITTER: Wang Y

PROVIDER: S-EPMC3937674 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Glycerol dehydrogenase plays a dual role in glycerol metabolism and 2,3-butanediol formation in Klebsiella pneumoniae.

Wang Yu Y Tao Fei F Xu Ping P

The Journal of biological chemistry 20140115 9

Glycerol dehydrogenase (GDH) is an important polyol dehydrogenase for glycerol metabolism in diverse microorganisms and for value-added utilization of glycerol in the industry. Two GDHs from Klebsiella pneumoniae, DhaD and GldA, were expressed in Escherichia coli, purified and characterized for substrate specificity and kinetic parameters. Both DhaD and GldA could catalyze the interconversion of (3R)-acetoin/(2R,3R)-2,3-butanediol or (3S)-acetoin/meso-2,3-butanediol, in addition to glycerol oxid ...[more]

PMID: 24429283

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Project description:Biodiesel, a renewable fuel produced by transesterification of animal fats and vegetable oils, generates about 10% (v/v) of crude glycerol as a core byproduct. The high volume of this non bio-degradable glycerol is becoming of a great environmental and economical concern due to its worldwide ever-growing surplus. Herein we report a high production of 2,3-butanediol (2,3-BD) from pure and biodiesel derived crude glycerol using a mutant K. pneumoniae SRM2 obtained from a newly isolated strain Klebsiella pneumoniae SRP2. The mutant strain SRM2 with standing high glycerol concentration (220 g L-1 of medium) could rapidly convert glycerol aerobically to 2,3-BD, a versatile product extensively used in chemical, pharmaceutical and fuel industries Our study revealed that an increased GDH activity led to a substantially enhanced production of 2,3-BD. The mutant strain exhibited 1.3-fold higher activity of GDH than that of parent strain (500.08 vs. 638.6 µmol min -1 mg -1 protein), yielding of 32.3 g L-1 and 77.5 g L-1 2,3-BD with glycerol in batch and fed-batch process respectively. However, in batch culture with crude glycerol, cell growth and glycerol consumption were expressively boosted, and 2,3-BD production was 27.7 g L-1 from 75.0 g/L crude glycerol. In this report, the optimal conditions for high production of 2,3-BD were defined in a completely aerobic process, and 0.59 g g-1 product yield of 2,3-BD was attained by the mutated strain K. pneumoniae SRM2, which is the highest amount obtained from batch biotransformation process of glycerol metabolism till today. These results indicated that our newly developed mutant can tolerate high concentration of glycerol, have a high glycerol utilization rate, and high product yield of 2,3-BD. It is demonstrated that the mutant strain K. pneumoniae SRM2 has an ability to produce fewer co-products at trace concentrations at higher glycerol concentrations, and could be a potential candidate for 2,3-DB production in an industrial bioconversion process.

Project description:BACKGROUND:2,3-Butanediol (2,3-BDO) is a promising bio-based chemical because of its wide industrial applications. Previous studies on microbial production of 2,3-BDO has focused on sugar fermentation. Alternatively, biodiesel-derived crude glycerol can be used as a cheap resource for 2,3-BDO production; however, a considerable formation of 1,3-propanediol (1,3-PDO) and low concentration, productivity, and yield of 2,3-BDO from glycerol fermentation are limitations. RESULTS:Here, we report a high production of 2,3-BDO from crude glycerol using the engineered Klebsiella oxytoca M3 in which pduC (encoding glycerol dehydratase large subunit) and ldhA (encoding lactate dehydrogenase) were deleted to reduce the formation of 1,3-PDO and lactic acid. In fed-batch fermentation with the parent strain K. oxytoca M1, crude glycerol was more effective than pure glycerol as a carbon source in 2,3-BDO production (59.4 vs. 73.8 g/L) and by-product reduction (1,3-PDO, 8.9 vs. 3.7 g/L; lactic acid, 18.6 vs. 9.8 g/L). When the double mutant was used in fed-batch fermentation with pure glycerol, cell growth and glycerol consumption were significantly enhanced and 2,3-BDO production was 1.9-fold higher than that of the parent strain (59.4 vs. 115.0 g/L) with 6.9 g/L of 1,3-PDO and a small amount of lactic acid (0.7 g/L). Notably, when crude glycerol was supplied, the double mutant showed 1,3-PDO-free 2,3-BDO production with high concentration (131.5 g/L), productivity (0.84 g/L/h), and yield (0.44 g/g crude glycerol). This result is the highest 2,3-BDO production from glycerol fermentation to date. CONCLUSIONS:2,3-BDO production from glycerol was dramatically enhanced by disruption of the pduC and ldhA genes in K. oxytoca M1 and 1,3-PDO-free 2,3-BDO production was achieved by using the double mutant and crude glycerol. 2,3-BDO production obtained in this study is comparable to 2,3-BDO production from sugar fermentation, demonstrating the feasibility of economic industrial 2,3-BDO production using crude glycerol.

Project description:BackgroundPreviously, a safe strain, Bacillus amyloliquefaciens B10-127 was identified as an excellent candidate for industrial-scale microbial fermentation of 2,3-butanediol (2,3-BD). However, B. amyloliquefaciens fermentation yields large quantities of acetoin, lactate and succinate as by-products, and the 2,3-BD yield remains prohibitively low for commercial production.Methodology/principal findingsIn the 2,3-butanediol metabolic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of 3-phosphate glyceraldehyde to 1,3-bisphosphoglycerate, with concomitant reduction of NAD(+) to NADH. In the same pathway, 2,3-BD dehydrogenase (BDH) catalyzes the conversion of acetoin to 2,3-BD with concomitant oxidation of NADH to NAD(+). In this study, to improve 2,3-BD production, we first over-produced NAD(+)-dependent GAPDH and NADH-dependent BDH in B. amyloliquefaciens. Excess GAPDH reduced the fermentation time, increased the 2,3-BD yield by 12.7%, and decreased the acetoin titer by 44.3%. However, the process also enhanced lactate and succinate production. Excess BDH increased the 2,3-BD yield by 16.6% while decreasing acetoin, lactate and succinate production, but prolonged the fermentation time. When BDH and GAPDH were co-overproduced in B. amyloliquefaciens, the fermentation time was reduced. Furthermore, in the NADH-dependent pathways, the molar yield of 2,3-BD was increased by 22.7%, while those of acetoin, lactate and succinate were reduced by 80.8%, 33.3% and 39.5%, relative to the parent strain. In fed-batch fermentations, the 2,3-BD concentration was maximized at 132.9 g/l after 45 h, with a productivity of 2.95 g/l·h.Conclusions/significanceCo-overexpression of bdh and gapA genes proved an effective method for enhancing 2,3-BD production and inhibiting the accumulation of unwanted by-products (acetoin, lactate and succinate). To our knowledge, we have attained the highest 2,3-BD fermentation yield thus far reported for safe microorganisms.

Dataset Information

Glycerol dehydrogenase plays a dual role in glycerol metabolism and 2,3-butanediol formation in Klebsiella pneumoniae.

Publications

Glycerol dehydrogenase plays a dual role in glycerol metabolism and 2,3-butanediol formation in Klebsiella pneumoniae.

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