Glycerol Dehydrogenase Activity Research Articles

Effective multi-biocatalyst system with reusable NADH for transformation of glycerol to value-added dihydroxyacetone

Glycerol-based biorefinery can be a highly profitable process by producing highly value-added products such as dihydroxyacetone via combined catalytic strategies. Here, two-enzyme system is adopted for the transformation of glycerol into highly valuable dihydroxyacetone as well as cofactor regeneration at the same time. Glycerol dehydrogenase (GDH) and alcohol dehydrogenase (ADH) are co-immobilized within magnetically separable and spherical mesocellular silica foam (Mag-S-MCF), to prepare NER-(GDH/ADH). In details, GDH and ADH are adsorbed into the mesopores of Mag-S-MCF, and further crosslinked within the mesopores of Mag-S-MCF. The resulting nanoscale enzyme reactors (NER) of crosslinked GDH and ADH molecules within the bottle-neck structured mesopores can effectively prevent larger sized crosslinked enzyme aggregates from being leached out of smaller mesopores, due to the bottle-neck mesopore structure of Mag-S-MCF, as well as stabilize the activity of GDH and ADH upon chemical crosslinking, effectively preventing the denaturation of enzyme molecules. More importantly, the proximity of GDH and ADH molecules within mesopores of NER improves the efficiency of cofactor-mediated dual-enzymatic reactions by relieving mass-transfer limitations and improving cofactor recycling in an effective way, expediting both glycerol oxidation and dihydroxyacetone generation at the same time. As a result, the DHA concentration of NER-(GDH/ADH) and the simple mixture of NER-GDH and NER-ADH were 410 μM and 336 μM, respectively. To the best of our knowledge, this report is the first demonstration of stabilized nanoscale multi-enzyme reactor system, equipped with efficient cofactor regeneration within confined mesopores, for efficient glycerol transformation to high-valued dihydroxyacetone.Graphical

Characterization of emergent toxigenic M1UK Streptococcus pyogenes and associated sublineages.

Streptococcus pyogenes genotype emm1 is a successful, globally distributed epidemic clone that is regarded as inherently virulent. An emm1 sublineage, M1UK, that produces increased levels of SpeA toxin was associated with increased scarlet fever and invasive infections in England in 2015/2016. Defined by 27 SNPs in the core genome, M1UK is now dominant in England. To more fully characterize M1UK, we undertook comparative transcriptomic and proteomic analyses of M1UK and contemporary non-M1UK emm1 strains (M1global). Just seven genes were differentially expressed by M1UK compared with contemporary M1global strains. In addition to speA, five genes in the operon that includes glycerol dehydrogenase were upregulated in M1UK (gldA, mipB/talC, pflD, and phosphotransferase system IIC and IIB components), while aquaporin (glpF2) was downregulated. M1UK strains have a stop codon in gldA. Deletion of gldA in M1global abrogated glycerol dehydrogenase activity, and recapitulated upregulation of gene expression within the operon that includes gldA, consistent with a feedback effect. Phylogenetic analysis identified two intermediate emm1 sublineages in England comprising 13/27 (M113SNPs) and 23/27 SNPs (M123SNPs), respectively, that had failed to expand in the population. Proteomic analysis of invasive strains from the four phylogenetic emm1 groups highlighted sublineage-specific changes in carbohydrate metabolism, protein synthesis and protein processing; upregulation of SpeA was not observed in chemically defined medium. In rich broth, however, expression of SpeA was upregulated ~10-fold in both M123SNPs and M1UK sublineages, compared with M113SNPs and M1global. We conclude that stepwise accumulation of SNPs led to the emergence of M1UK. While increased expression of SpeA is a key indicator of M1UK and undoubtedly important, M1UK strains have outcompeted M123SNPs and other emm types that produce similar or more superantigen toxin. We speculate that an accumulation of adaptive SNPs has contributed to a wider fitness advantage in M1UK on an inherently successful emm1 streptococcal background.

An enhanced toluene dioxygenase platform for the production of cis-1,2-dihydrocatechol in Escherichia coli BW25113 lacking glycerol dehydrogenase activity

The compound cis-1,2-dihydrocatechol (DHC) is highly valuable since it finds wide application in the production of fine chemicals and bioactive compounds with medical relevance. The biotechnological process to generate DHC involves a dearomatizing dihydroxylation reaction catalyzed by toluene dioxygenase (TDO) from P. putida F1, employing benzene as substrate. We aimed to enhance the biotechnological E. coli BW25113 platform for DHC production by identifying the key operational parameters positively influencing the final isolated yield. Thereby, we observed an unreported downstream reaction, generating catechol from DHC, affecting, in a negative manner, the final titer for the product. Expression temperature for the TDO-system showed to have the highest influence in terms of final isolated yield. A KEIO-collection-based screening approach highlighted glycerol dehydrogenase (GldA) as the main responsible enzyme for the undesired reaction. We transferred the TDO-system to E. coli BW25113 ΔgldA and applied the enhanced operational set-up on it. This enhanced platform enabled the production of 1.41 g L−1 DHC in isolated yield, which represents a two-fold increase compared with the starting working conditions. To our knowledge, this is the highest DHC production accomplished in recombinant E. coli at semi-preparative scale, providing a robust and accessible biotechnological platform for DHC synthesis.

Dihydroxyacetone production from glycerol using Gluconobacter oxydans: Study of medium composition and operational conditions in shaken flasks.

The production of dihydroxyacetone from glycerol employing aerobic cultures of Gluconobacter oxydans is studied. Dihydroxyacetone is one of the most important value-added products obtained from glycerol, a by-product of biodiesel production. The effect of organic nitrogen source and initial substrate concentrations has been studied together with the possibility of product inhibition. Afterward, the influence of the main operating conditions (temperature, shaking speed, and initial biomass concentration) on in vivo glycerol dehydrogenase activity has also been considered. The results show no evidence of glycerol inhibition, but an important product inhibition was detected, which has been taken into account in a kinetic model for enzymatic activity description. In terms of operating conditions, pH was found to exert a great impact on glycerol conversion, being necessary to keep it above 4 to ensure complete glycerol conversion. The minimum temperature that maximized enzymatic activity was found to be 30°C. In addition, a surprising decoupling between biomass concentration and dihydroxyacetone production rate was observed when adding increasing nitrogen source concentrations at a fixed shaking speed. Glycerol dehydrogenase activity remains constant despite the increase in biomass concentration, contrary to what would be expected. This fact revealed the existence of a rate limiting factor, identified subsequently as oxygen transfer rate depending on the biomass concentration.

Investigations in sonication-induced intensification of crude glycerol fermentation to dihydroxyacetone by free and immobilized Gluconobacter oxydans

This study reports crude glycerol fermentation by G. oxydans for dihydroxyacetone (DHA) production, and intensification of fermentation with sonication. Fermentation was carried out using both free and immobilized cells (on polyurethane foam support) for initial glycerol concentrations of 20, 30 and 50 g/L. Sonication at 20% duty cycle enhanced glycerol consumption by 60–84% with no significant change in cell morphology. Lesser DHA yield in crude glycerol fermentation was attributed to possible formation of inhibitory products. Slight reduction in DHA yield for initial glycerol concentration of 50 g/L was attributed to substrate inhibition. Higher DHA productivity was obtained for immobilized cells. Circular dichroism analysis of intracellular proteins obtained from ultrasound-treated G. oxydans revealed significant reduction in α-helix and β-sheet content. These conformational changes in protein structure could augment activity of intracellular glycerol dehydrogenase, which is manifested in terms of enhanced metabolism of glycerol by G. oxydans.

Enzymatic Conversion of Glycerol to 2,3-Butanediol and Acetoin by Serratia proteamaculans SRWQ1

Biodiesel, a renewable and environment friendly biofuel is produced by transesterification process using animal fats and vegetable oils. However, the flourishing of biodiesel industries has led to produce a huge amount (10% v/v) of crude glycerol as a core by-product, created an overflow problem. Therefore, biotransformation of glycerol into biofuel and other value-added products is one of the promising applications of glycerol due to its high availability at low cost. In this study, we report the capability of converting glycerol as a sole carbon source to 2,3-butanediol (2,3-BDO) and acetoin by using a newly isolated Serratia proteamaculans SRWQ1 strain in batch biotransformation process under aerobic condition. Strain SRWQ1 displayed a maximum up to 18.43 ± 1.55 g/L of 2,3-BDO, yielding 0.4 g/g using 49.0 g/L glycerol which was 98.0% of glycerol utilization. The strain SRWQ1 also successfully produced a significant amount of acetoin 8.38 ± 0.76 g/L with yields 0.06 g/g. Moreover, the maximum activity of glycerol dehydrogenase (GDH) which is a key enzyme of glycerol metabolisms was 408.69 ± 0.069 units/mg protein. The newly isolated strain S. proteamaculans SRWQ1 displayed the best ability to synthesize 2,3-BDO and acetoin using glycerol as the sole substrate, and it is the first report on biotransformation of glycerol by S. proteamaculans. Therefore, this aerobic conversion of glycerol to value-added green products 2,3-BDO and acetoin with potential industrial applications would represent a noteworthy alternative to add value for biodiesel production helping biodiesel industries development.

Utilization of a waste glycerol fraction using and reusing immobilized Gluconobacter oxydans ATCC 621 cell extract

Abstract Background Depletion of petroleum resources has enforced the search for alternative sources of renewable energy. Introduction of biofuels into the market was expected to become a solution to this disadvantageous situation. Attempts to cover fuel demand have, however, caused another severe problem—the waste glycerol generated during biodiesel production at a concentration of approximately 10% w/w. This, in turn, prompted a global search for effective methods of valorization of the waste fraction of glycerol. Results Utilization of the waste fraction at 48 h with an initial glycerol concentration of 30 g·L -1 and proceeding with 62% efficiency enabled the production of 9 g·L -1 dihydroxyacetone at 50% substrate consumption. The re-use of the immobilized biocatalyst resulted in a similar concentration of dihydroxyacetone (8.7 g·L -1 ) in two-fold shorter time, with an efficiency of 85% and lower substrate consumption (35%). Conclusions The method proposed in this work is based on the conversion of waste glycerol to dihydroxyacetone in a reaction catalyzed by immobilized Gluconobacter oxydans cell extract with glycerol dehydrogenase activity, and it could be an effective way to convert waste glycerol into a valuable product.

Metabolic networks to generate pyruvate, PEP and ATP from glycerol in Pseudomonas fluorescens

Glycerol is a major by-product of the biodiesel industry. In this study we report on the metabolic networks involved in its transformation into pyruvate, phosphoenolpyruvate (PEP) and ATP. When the nutritionally-versatile Pseudomonas fluorescens was exposed to hydrogen peroxide (H2O2) in a mineral medium with glycerol as the sole carbon source, the microbe reconfigured its metabolism to generate adenosine triphosphate (ATP) primarily via substrate-level phosphorylation (SLP). This alternative ATP-producing stratagem resulted in the synthesis of copious amounts of PEP and pyruvate. The production of these metabolites was mediated via the enhanced activities of such enzymes as pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC). The high energy PEP was subsequently converted into ATP with the aid of pyruvate phosphate dikinase (PPDK), phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK) with the concomitant formation of pyruvate. The participation of the phospho-transfer enzymes like adenylate kinase (AK) and acetate kinase (ACK) ensured the efficiency of this O2-independent energy-generating machinery. The increased activity of glycerol dehydrogenase (GDH) in the stressed bacteria provided the necessary precursors to fuel this process. This H2O2-induced anaerobic life-style fortuitously evokes metabolic networks to an effective pathway that can be harnessed into the synthesis of ATP, PEP and pyruvate. The bioconversion of glycerol to pyruvate will offer interesting economic benefit.

Aerobic Conversion of Glycerol to 2,3-Butanediol by a Novel Klebsiella variicola SRP3 Strain

The manufacture of biodiesel generates about 10% crude glycerol as a core by- product. This research is to identify novel bacterial strains which are capable of efficiently converting glycerol aerobically, and improve the strains for large scale production of value-added products. The production of a major product 2,3-butanediol (2,3- BD) from glycerol as a sole carbon source by the newly isolated Klebsiella variicola SRP3 are reported in a series of batch processes under aerobic condition. This study also compares the bacterial cell biomass, bio-products and glycerol dehydrogenase (GDH) enzyme production of K. variicola SRP3 isolated from paper mill waste when grown in aerobic condition. The incubation temperature, pH, glycerol concentration and nitrogen sources are the most important factors ruling the glycerol dehydrogenase. Acidic initial pH (pH 5.0) led to enhanced GDH activity (558.2 μmol/min/mg protein), yielding 25.33 g/L 2,3-BD, 7.6 g/L 1,3-PDO and 2.2 g/L acetoin from 50.0 g/L glycerol. In our aerobic batch culture the mutated strain K. variicola SRM3 exhibited 1.3 fold increased GDH activity of 721.5 units/mg protein from 558.2 units/mg protein, yielding 29.87 g/L 2,3-BD, 7.08 g/L 1,3-PDO and 2.02 g/L acetoin from 50.0 g/L glycerol. In our report, the optimal conditions for maximal GDH enzyme activity were defined, and 0.79 g/g product yield was achieved by the muted strain K. variicola SRM3, which is the highest amount obtained from glycerol as a sole carbon source until now. The research has for the first time proved that this K. variicola species can efficiently convert glycerol.

Cloning, expression and characterization of glycerol dehydrogenase involved in 2,3-butanediol formation in Serratia marcescens H30.

The meso-2,3-butanediol dehydrogenase (meso-BDH) from S. marcescens H30 is responsible for converting acetoin into 2,3-butanediol during sugar fermentation. Inactivation of the meso-BDH encoded by budC gene does not completely abolish 2,3-butanediol production, which suggests that another similar enzyme involved in 2,3-butanediol formation exists in S. marcescens H30. In the present study, a glycerol dehydrogenase (GDH) encoded by gldA gene from S. marcescens H30 was expressed in Escherichia coli BL21(DE3), purified and characterized for its properties. In vitro conversion indicated that the purified GDH could catalyze the interconversion of (3S)-acetoin/meso-2,3-butanediol and (3R)-acetoin/(2R,3R)-2,3-butanediol. (2S,3S)-2,3-Butanediol was not a substrate for the GDH at all. Kinetic parameters of the GDH enzyme showed lower K m value and higher catalytic efficiency for (3S/3R)-acetoin in comparison to those for (2R,3R)-2,3-butanediol and meso-2,3-butanediol, implying its physiological role in favor of 2,3-butanediol formation. Maximum activity for reduction of (3S/3R)-acetoin and oxidations of meso-2,3-butanediol and glycerol was observed at pH 8.0, while it was pH 7.0 for diacetyl reduction. The enzyme exhibited relative high thermotolerance with optimum temperature of 60°C in the oxidation-reduction reactions. Over 60% of maximum activity was retained at 70°C. Additionally, the GDH activity was significantly enhanced for meso-2,3-BD oxidation in the presence of Fe(2+) and for (3S/3R)-acetoin reduction in the presence of Mn(2+), while several cations inhibited its activity, particularly Fe(2+) and Fe(3+) for (3S/3R)-acetoin reduction. The properties provided potential application for single configuration production of acetoin and 2,3-butanediol .

The improvement of stability, activity, and substrate promiscuity of glycerol dehydrogenase substituted by divalent metal ions

The substitution of the catalytic zinc ion of glycerol dehydrogenase (GDH) from Klebsiella pneumonia sp. by divalent metal ions, Mn2+ and Mg2+, enabled improvements of activity, substrate promiscuity and stability. The activity of Mn-GDH and Mg-GDH improved several folds in comparison to the native GDH. The activity of substituted GDH towards non-natural substrates, 4-chloroacetoacetate, 3-chloroacetylpyridine, p-chloroacetophenone, and acetophenone was 30 folds higher than native GDH. Manganese substitution increased the half-life of GDH by 6 folds at 60 and 70°C. The two-fraction first order inactivation models fitted the nonlinear thermal inactivation curves well. Combined with the kinetic and thermodynamic analysis, further mechanistic insights to the metal ion roles in thermostability enhancements were studied. The thermodynamic parameters of inactivation, enthalpy, entropy and the Gibbs free energy indicated that Mn-GDH was stabilized entropically and elucidated the mechanisms of enzyme inactivation.

English

Glycerol can be converted into more valuable compound dihydroxyacetone by the nicotinamide adenine dinucleotide (NAD+)-dependent glycerol dehydrogenase. However, it is economically prohibitive to produce dihydroxyacetone using purified glycerol dehydrogenase at the expense of a stoichiometric amount of the cofactor NAD+. In this study, Escherichia coli was engineered for dihydroxyacetone production by enhancing its glycerol dehydrogenase activity and introducing NADH oxidase activity. Under optimized conditions, dihydroxyacetone productivity reached 0.13 g/h/g wet cell mass by recombinant E. coli D4 (pET-24b-gldA+nox) cells co-expressinggldA gene from E. coli and nox gene from Enterococcus faecalis. It was interesting to note that exogenous NAD+ greatly improved dihydroxyacetone production for the whole-cell biotransformation process. These results should be useful for the development of advanced bioprocess in terms of glycerol utilization. Key words: Dihydroxyacetone, Glycerol dehydrogenase, NAD+, whole-cell biotransformation, Escherichia coli.

Metabolism of biodiesel-derived glycerol in probiotic Lactobacillus strains

Three probiotic Lactobacillus strains, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus delbrueckii, were tested for their ability to assimilate and metabolize glycerol. Biodiesel-derived glycerol was used as the main carbon and energy source in batch microaerobic growth. Here, we show that the tested strains were able to assimilate glycerol, consuming between 38 and 48 % in approximately 24 h. L. acidophilus and L. delbrueckii showed a similar growth, higher than L. plantarum. The highest biomass reached was 2.11 g L⁻¹ for L. acidophilus, with a cell mass yield (Y (X/S)) of 0.37 g g⁻¹. L. delbrueckii and L. plantarum reached a biomass of 2.06 and 1.36 g L⁻¹. All strains catabolize glycerol mainly through glycerol kinase (EC 2.7.1.30). For these lactobacillus species, kinetic parameters for glycerol kinase showed Michaelis-Menten constant (K(m)) ranging from 1.2 to 3.8 mM. The specific activities for glycerol kinase in these strains were in the range of 0.18 to 0.58 U mg protein⁻¹, with L. acidophilus ATCC 4356 showing the maximum specific activity after 24 h of cultivation. Glycerol dehydrogenase activity was also detected in all strains studied but only for the reduction of glyceraldehyde with NADPH (K(m) for DL-glyceraldehyde ranging from 12.8 to 32.3 mM). This enzyme shows a very low oxidative activity with glycerol and NADP+ and, most likely, under physiological conditions, the oxidative reaction does not occur, supporting the assumption that the main metabolic flux concerning glycerol metabolism is through the glycerol kinase pathway.

High-temperature sorbose fermentation with thermotolerant Gluconobacter frateurii CHM43 and its mutant strain adapted to higher temperature

We succeeded in obtaining a strain adapted to higher temperature from a thermotolerant strain, Gluconobacter frateurii CHM43, for sorbose fermentation. The adapted strain showed higher growth and L-sorbose production than original CHM43 strain at higher temperature around 38.5-40 °C. It was also shown to be useful even with the fermentation without temperature control. To understand the sorbose fermentation ability of the adapted strain at higher temperature, D-sorbitol-oxidizing respiratory chain was compared with the CHM43 strain and the adapted strain. We found that the activity of pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase (GLDH), which is a primary dehydrogenase of the respiratory chain and responsible for L-sorbose production, was decreased when the temperature increased, but the decreased activity of GLDH was recovered by the addition of PQQ. Since the adapted strain was found to produce more PQQ than the CHM43 strain, it was suggested that the adapted strain keeps GLDH as holoenzyme with the increased PQQ production, and thus produces more L-sorbose and grows better under higher temperature.

Use of glycerol for producing 1,3-dihydroxyacetone by Gluconobacter oxydans in an airlift bioreactor

1,3-Dihydroxyacetone can be produced by biotransformation of glycerol with glycerol dehydrogenase from Gluconobacter oxydans cells. Firstly, improvement the activity of glycerol dehydrogenase was carried out by medium optimization. The optimal medium for cell cultivation was composed of 5.6g/l yeast extract, 4.7g/l glycerol, 42.1g/l mannitol, 0.5g/l K2HPO4, 0.5g/l KH2PO4, 0.1g/l MgSO4·7H2O, and 2.0g/l CaCO3 with the initial pH of 4.9. Secondly, an internal loop airlift bioreactor was applied for DHA production from glycerol by resting cells of G. oxydans ZJB09113. Furthermore, the effects of pH, aeration rate and cell content on DHA production and glycerol feeding strategy were investigated. 156.3±7.8g/l of maximal DHA concentration with 89.8±2.4% of conversion rate of glycerol to DHA was achieved after 72h of biotransformation using 10g/l resting cells at 30°C, pH 5.0 and 1.5vvm of aeration rate.

Molecular Characterization of the Glycerol-Oxidative Pathway of Clostridium butyricum VPI 1718

The glycerol oxidative pathway of Clostridium butyricum VPI 1718 plays an important role in glycerol dissimilation. We isolated, sequenced, and characterized the region coding for the glycerol oxidation pathway. Five open reading frames (ORFs) were identified: dhaR, encoding a putative transcriptional regulator; dhaD (1,142 bp), encoding a glycerol dehydrogenase; and dhaK (995 bp), dhaL (629 bp), and dhaM (386 bp), encoding a phosphoenolpyruvate (PEP)-dependent dihydroxyacetone (DHA) kinase enzyme complex. Northern blot analysis demonstrated that the last four genes are transcribed as a 3.2-kb polycistronic operon only in glycerol-metabolizing cultures, indicating that the expression of this operon is regulated at the transcriptional level. The transcriptional start site of the operon was determined by primer extension, and the promoter region was deduced. The glycerol dehydrogenase activity of DhaD and the PEP-dependent DHA kinase activity of DhaKLM were demonstrated by heterologous expression in different Escherichia coli mutants. Based on our complementation experiments, we proposed that the HPr phosphoryl carrier protein and His9 residue of the DhaM subunit are involved in the phosphoryl transfer to dihydroxyacetone-phosphate. DhaR, a potential regulator of this operon, was found to contain conserved transmitter and receiver domains that are characteristic of two-component systems present in the AraC family. To the best of our knowledge, this is the first molecular characterization of a glycerol oxidation pathway in a Gram-positive bacterium.

Influence of dhaT mutation of K. pneumoniae on 1,3-propanediol fermentation.

Metabolic role of 1,3-propanediol oxidoreductase (PDOR) in the production of 1,3-propanediol (1,3-PDO) with K. pneumonia was investigated by knocking out the coded gene dhaT. Fermentation with both the wide-type and mutant were studied in 5l fermentor. A PDOR-deficient mutant K. pneumonia T1.9131 with 19% PDOR activity of the wild type was constructed. The cultures of the mutant indicated that PDOR inactivation had great effect on the other dha regulon enzymes: activity of glycerol dehydratase decreased by 70% while activity of glycerol dehydrogenase increased by 68%. Fed-batch fermentation showed that more metabolic flux of glycerol was directed to lactate and ethanol in the mutant. Lactate was identified as major metabolite and received an increase in the final concentration from 45 to 91gl(-1), while the concentration of 1,3-PDO production dropped from 94 to 36gl(-1). The results demonstrated PDOR was not indispensable in glycerol metabolism but was crucial in high 1,3-PDO productivity. It is postulated that a hypothetical oxidoreductase was expressed and replaced the function of PDOR. Blocking the pathway towards lactate and ethanol could be a plausible scheme to enhance 1,3-PDO productivity.

Engineering of glycerol dehydrogenase for improved activity towards 1, 3-butanediol

The objective of this study was to use protein engineering techniques to enhance the catalytic activity of glycerol dehydrogenase (GlyDH) on racemic 1, 3-butanediol (1, 3-BDO) for the bioproduction of the important pharmaceutical intermediate 4-hydroxy-2-butanone. Three GlyDH genes (gldA) from Escherichia coli K-12, Salmonella enterica, and Klebsiella pneumoniae MGH78578 were shuffled to generate a random mutagenesis library. The nitroblue tetrazolium/phenazine methosulfate high throughput screening protocol was used to select four chimeric enzymes with up to a 2.6-fold improved activity towards 1, 3-BDO. A rational design method was also employed to further improve the enzyme activity after DNA shuffling. Based on the homology model of GlyDH (Escherichia coli), Asp121 was predicted to influence 1, 3-BDO binding and replaced with Ala by site-directed mutagenesis. Combination of the mutations from both DNA shuffling and rational design produced the best mutant with a V (max) value of 126.6 U/mg, a 26-fold activity increase compared with that of the wild type GlyDH from E. coli.

Enhanced production of dihydroxyacetone from glycerol by overexpression of glycerol dehydrogenase in an alcohol dehydrogenase-deficient mutant of Gluconobacter oxydans

Gluconobacter oxydans can rapidly and incompletely oxidize glycerol to dihydroxyacetone (DHA), a versatile product extensively used in cosmetic, chemical and pharmaceutical industries. To improve DHA production, the glycerol dehydrogenase (GDH) responsible for DHA formation was overexpressed in G. oxydans M5AM, in which the gene coding for the membrane-bound alcohol dehydrogenase (ADH) was interrupted. Real-time PCR and enzyme activity assay revealed that the absence of ADH together with the overexpression of GDH gene resulted in an increased GDH activity in the resulting strain M5AM/GDH, which led to a substantially enhanced production of DHA in a resting cell system. In a batch biotransformation process, M5AM/GDH exhibited a 2.4-fold increased DHA productivity of 2.4 g/g CDW/h from 1.0 g/g CDW/h, yielding 96 g/L DHA from 100 g/L glycerol. When 140 g/L glycerol was supplied, a final DHA concentration of 134 g/L was accumulated within 14 h. In four repeated batch runs, 385 g DHA over a time period of 34 h was achieved from 400 g glycerol with an average productivity of 2.2 g/g CDW/h. These results indicated that this newly developed strain G. oxydans M5AM/GDH with high productivity and increased tolerance against product inhibition has potential for DHA production in an industrial bioconversion process.

Metabolic engineering to improve ethanol production in Thermoanaerobacter mathranii

Thermoanaerobacter mathranii can produce ethanol from lignocellulosic biomass at high temperatures, but its biotechnological exploitation will require metabolic engineering to increase its ethanol yield. With a cofactor-dependent ethanol production pathway in T. mathranii, it may become crucial to regenerate cofactor to increase the ethanol yield. Feeding the cells with a more reduced carbon source, such as mannitol, was shown to increase ethanol yield beyond that obtained with glucose and xylose. The ldh gene coding for lactate dehydrogenase was previously deleted from T. mathranii to eliminate an NADH oxidation pathway. To further facilitate NADH regeneration used for ethanol formation, a heterologous gene gldA encoding an NAD(+)-dependent glycerol dehydrogenase was expressed in T. mathranii. One of the resulting recombinant strains, T. mathranii BG1G1 (Deltaldh, P(xyl)GldA), showed increased ethanol yield in the presence of glycerol using xylose as a substrate. With an inactivated lactate pathway and expressed glycerol dehydrogenase activity, the metabolism of the cells was shifted toward the production of ethanol over acetate, hence restoring the redox balance. It was also shown that strain BG1G1 acquired the capability to utilize glycerol as an extra carbon source in the presence of xylose, and utilization of the more reduced substrate glycerol resulted in a higher ethanol yield.