Mechanism of 2,3-butanediol stereoisomer formation in Klebsiella pneumoniae

Klebsiella pneumoniae is known to produce meso-2,3-butanediol and 2S,3S-butanediol, whereas 2R,3R-butanediol was detected in the culture broth of K. pneumoniae CGMCC 1.6366. The ratio of 2R,3R-butanediol to all isomers obtained using glycerol as the carbon source was higher than that obtained using glucose as the carbon source. Therefore, enzymes involved in glycerol metabolism are likely related to 2R,3R-butanediol formation. In vitro reactions show that glycerol dehydrogenase catalyzes the stereospecific conversion of R-acetoin to 2R,3R-butanediol and S-acetoin to meso-2,3-butanediol. Butanediol dehydrogenase exhibits high (S)-enantioselectivity in ketone reduction. Genes encoding glycerol dehydrogenase, α-acetolactate decarboxylase, and butanediol dehydrogenase were individually disrupted in K. pneumoniae CGMCC 1.6366, and the 2,3-butanediol synthesis characteristics of these mutants were investigated. K. pneumoniae ΔdhaD lost the ability to synthesize 2R,3R-butanediol. K. pneumoniae ΔbudA showed reduced 2R,3R-butanediol synthesis. However, K. pneumoniae ΔbudC produced a high level of 2R,3R-butanediol, and R-acetoin was accumulated in the broth. The metabolic characteristics of these mutants and in vitro experiment results demonstrated the mechanism of the 2,3-butanediol stereoisomer synthesis pathway. Glycerol dehydrogenase, encoded by dhaD, exhibited 2R,3R-butanediol dehydrogenase activity and was responsible for 2R,3R-butanediol synthesis from R-acetoin. This enzyme also contributed to meso-2,3-butanediol synthesis from S-acetoin. Butanediol dehydrogenase, encoded by budC, was the only enzyme that catalyzed the conversion of diacetyl to S-acetoin and further to 2S,3S-butanediol.     

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 Fe²⁺ and for (3S/3R)-acetoin reduction in the presence of Mn²⁺, while several cations inhibited its activity, particularly Fe²⁺ and Fe³⁺ for (3S/3R)-acetoin reduction. The properties provided potential application for single configuration production of acetoin and 2,3-butanediol .     

High-yield production of (R)-acetoin in Saccharomyces cerevisiae by deleting genes for NAD(P)H-dependent ketone reductases producing meso-2,3-butanediol and 2,3-dimethylglycerate

Acetoin is widely used in food and cosmetics industries as a taste and fragrance enhancer. To produce (R)-acetoin in Saccharomyces cerevisiae, acetoin biosynthetic genes encoding α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD) from Bacillus subtilis and water-forming NADH oxidase (NoxE) from Lactococcus lactis were integrated into delta-sequences in JHY605 strain, where the production of ethanol, glycerol, and (R,R)-2,3-butanediol (BDO) was largely eliminated. We further improved acetoin production by increasing acetoin tolerance by adaptive laboratory evolution, and eliminating other byproducts including meso-2,3-BDO and 2,3-dimethylglycerate, a newly identified byproduct. Ara1, Ypr1, and Ymr226c (named Ora1) were identified as (S)-alcohol-forming reductases, which can reduce (R)-acetoin to meso-2,3-BDO in vitro. However, only Ara1 and Ypr1 contributed to meso-2,3-BDO production in vivo. We elucidate that Ora1, having a substrate preference for (S)-acetoin, reduces (S)-α-acetolactate to 2,3-dimethylglycerate, thus competing with AlsD-mediated (R)-acetoin production. By deleting ARA1, YPR1, and ORA1, 101.3 g/L of (R)-acetoin was produced with a high yield (96% of the maximum theoretical yield) and high stereospecificity (98.2%).     

<Emphasis Type="Italic">R</Emphasis>-acetoin accumulation and dissimilation in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Klebsiella pneumoniae is a 2,3-butanediol producer, and R-acetoin is an intermediate of 2,3-butanediol production. R-acetoin accumulation and dissimilation in K. pneumoniae was studied here. A budC mutant, which has lost 2,3-butanediol dehydrogenase activity, accumulated high levels of R-acetoin in culture broth. However, after glucose was exhausted, the accumulated R-acetoin could be reused by the cells as a carbon source. Acetoin dehydrogenase enzyme system, encoded by acoABCD, was responsible for R-acetoin dissimilation. acoABCD mutants lost the ability to grow on acetoin as the sole carbon source, and the acetoin accumulated could not be dissimilated. However, in the presence of another carbon source, the acetoin accumulated in broth of acoABCD mutants was converted to 2,3-butanediol. Parameters of R-acetoin production by budC mutants were optimized in batch culture. Aerobic culture and mildly acidic conditions (pH 6–6.5) favored R-acetoin accumulation. At the optimized conditions, in fed-batch fermentation, 62.3 g/L R-acetoin was produced by budC and acoABCD double mutant in 57 h culture, with an optical purity of 98.0 %, and a substrate conversion ratio of 28.7 %.     

Characterization and functional role of Saccharomyces cerevisiae 2,3-butanediol dehydrogenase

Using a conserved sequence motif, a new gene (YAL060W) of the MDR family has been identified in Saccharomyces cerevisiae. The expressed protein was a stereoespecific (2R,3R)-2,3-butanediol dehydrogenase (BDH). The best substrates were (2R,3R)-2,3-butanediol for the oxidation and (3R/3S)-acetoin and 1-hydroxy-2-propanone for the reduction reactions. The enzyme is extremely specific for NAD(H) as cofactor, probably because the presence of Glu223 in the cofactor binding site, instead of the highly conserved Asp223. BDH is inhibited competitively by 4-methylpyrazole with a K_i of 34 μM. Yeast could grow on 2,3-butanediol or acetoin as a sole energy and carbon sources, and a 3.6-fold increase in BDH activity was observed when cells were grown in 2,3-butanediol, suggesting a role of the enzyme in 2,3-butanediol metabolism. However, the disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions, and the disrupted strain could also grow in 2,3-butanediol and acetoin. This suggests that other enzymes, in addition to BDH, can also metabolize 2,3-butanediol in yeast.     

Enantioselective Synthesis of Vicinal (R,R)-Diols by Saccharomyces cerevisiae Butanediol Dehydrogenase

Butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae belongs to the superfamily of the medium-chain dehydrogenases and reductases and converts reversibly R-acetoin and S-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol, respectively. It is specific for NAD(H) as a coenzyme, and it is the main enzyme involved in the last metabolic step leading to (2R,3R)-2,3-butanediol in yeast. In this study, we have used the activity of Bdh1p in different forms—purified enzyme, yeast extracts, permeabilized yeast cells, and as a fusion protein (with yeast formate dehydrogenase, Fdh1p)—to transform several vicinal diketones to the corresponding diols. We have also developed a new variant of the delitto perfetto methodology to place BDH1 under the control of the GAL1 promoter, resulting in a yeast strain that overexpresses butanediol dehydrogenase and formate dehydrogenase activities in the presence of galactose and regenerates NADH in the presence of formate. While the use of purified Bdh1p allows the synthesis of enantiopure (2R,3R)-2,3-butanediol, (2R,3R)-2,3-pentanediol, (2R,3R)-2,3-hexanediol, and (3R,4R)-3,4-hexanediol, the use of the engineered strain (as an extract or as permeabilized cells) yields mixtures of the diols. The production of pure diol stereoisomers has also been achieved by means of a chimeric fusion protein combining Fdh1p and Bdh1p. Finally, we have determined the selectivity of Bdh1p toward the oxidation/reduction of the hydroxyl/ketone groups from (2R,3R)-2,3-pentanediol/2,3-pentanedione and (2R,3R)-2,3-hexanediol/2,3-hexanedione. In conclusion, Bdh1p is an enzyme with biotechnological interest that can be used to synthesize chiral building blocks. A scheme of the favored pathway with the corresponding intermediates is proposed for the Bdh1p reaction.     

Role of Saccharomyces cerevisiae Oxidoreductases Bdh1p and Ara1p in the Metabolism of Acetoin and 2,3-Butanediol

NAD-dependent butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae reversibly transforms acetoin to 2,3-butanediol in a stereospecific manner. Deletion of BDH1 resulted in an accumulation of acetoin and a diminution of 2,3-butanediol in two S. cerevisiae strains under two different growth conditions. The concentrations of (2R,3R)-2,3-butanediol are mostly dependent on Bdh1p activity, while those of (meso)-2,3-butanediol are also influenced by the activity of NADP(H)-dependent oxidoreductases. One of them has been purified and shown to be d-arabinose dehydrogenase (Ara1p), which converts (R/S)-acetoin to meso-2,3-butanediol and (2S,3S)-2,3-butanediol. Deletion of BDH2, a gene adjacent to BDH1, whose encoded protein is 51% identical to Bdh1p, does not significantly alter the levels of acetoin or 2,3-butanediol in comparison to the wild-type strain. Furthermore, we have expressed Bdh2p with a histidine tag and have shown it to be inactive toward 2,3-butanediol. A whole-genome expression analysis with microarrays demonstrates that BDH1 and BDH2 are reciprocally regulated.Acetoin and 2,3-butanediol are minor products generated by Saccharomyces cerevisiae during alcohol fermentation. Their sensory impacts on wine are poorly documented. Acetoin may affect the wine bouquet, although its perception threshold in wine is relatively high, around 150 mg/liter (21, 31). On the other hand, 2,3-butanediol is odorless (33) and cannot be expected to appreciably affect the sensory quality of wine. However, the compound may contribute to the wine body (28).Acetaldehyde, pyruvate, and α-acetolactate are the main precursors of acetoin in S. cerevisiae. Acetoin can be formed from acetaldehyde and/or pyruvate through an anomalous reaction of pyruvate decarboxylase. Thus, although its main activity is to irreversibly decarboxylate pyruvate to acetaldehyde, it can also catalyze carbon-carbon bond formation, yielding acetoin from pyruvate and/or acetaldehyde (2, 4). In addition, α-acetolactate would produce acetoin through its nonenzymatic decarboxylation to diacetyl and subsequent reduction to acetoin through the action of several NADH- and NADPH-dependent oxidoreductases (12). However, the situation is more complex in wine fermentation, where other yeasts and bacteria display supplementary enzymatic activities capable of producing both acetoin and 2,3-butanediol (1, 27).We have previously characterized a butanediol dehydrogenase (Bdh1p) as a medium-chain dehydrogenase/reductase (MDR) that can reversibly transform R-acetoin and S-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol, respectively, in a NAD(H)-dependent reaction (10). BDH2 is a gene adjacent to BDH1 whose uncharacterized protein product (Bdh2p) shares 51% sequence identity with Bdh1p. To evaluate the in vivo roles of Bdh1p and Bdh2p, we compared the levels of several extracellular metabolites in cultures of wild-type and deficient strains. The results show that, although Bdh1p is the main enzyme in 2,3-butanediol production [essentially the (2R,3R)-2,3-butanediol stereoisomer], some meso-2,3-butanediol is still produced by the bdh1Δ strains. We have characterized Ara1p as an oxidoreductase that can reduce racemic acetoin to meso-2,3-butanediol and (2S,3S)-2,3-butanediol in the presence of NADPH.Furthermore, we have overexpressed Bdh2p with a histidine tag at its carboxyl terminus and have shown it to be inactive toward acetoin and 2,3-butanediol. A microarray study indicated that BDH1 and BDH2 are reciprocally regulated under the conditions studied.     

Potential and limitations of Klebsiella pneumoniae as a microbial cell factory utilizing glycerol as the carbon source

Klebsiella pneumoniae is a Gram-negative facultative anaerobe that metabolizes glycerol efficiently under both aerobic and anaerobic conditions. This microbe is considered an outstanding biocatalyst for transforming glycerol into a variety of value-added products. Crude glycerol is a cheap carbon source and can be converted by K. pneumoniae into useful compounds such as lactic acid, 3-hydroxypropionic acid, ethanol, 1,3-propanediol, 2,3-butanediol, and succinic acid. This review summarizes glycerol metabolism in K. pneumoniae and its potential as a microbial cell factory for the production of commercially important acids and alcohols. Although many challenges remain, K. pneumoniae is a promising workhorse when glycerol is used as the carbon source.     

A Novel Whole-Cell Biocatalyst with NAD+ Regeneration for Production of Chiral Chemicals

Background

The high costs of pyridine nucleotide cofactors have limited the applications of NAD(P)-dependent oxidoreductases on an industrial scale. Although NAD(P)H regeneration systems have been widely studied, NAD(P)⁺ regeneration, which is required in reactions where the oxidized form of the cofactor is used, has been less well explored, particularly in whole-cell biocatalytic processes.

Methodology/Principal Findings

Simultaneous overexpression of an NAD⁺ dependent enzyme and an NAD⁺ regenerating enzyme (H₂O producing NADH oxidase from Lactobacillus brevis) in a whole-cell biocatalyst was studied for application in the NAD⁺-dependent oxidation system. The whole-cell biocatalyst with (2R,3R)-2,3-butanediol dehydrogenase as the catalyzing enzyme was used to produce (3R)-acetoin, (3S)-acetoin and (2S,3S)-2,3-butanediol.

Conclusions/Significance

A recombinant strain, in which an NAD⁺ regeneration enzyme was coexpressed, displayed significantly higher biocatalytic efficiency in terms of the production of chiral acetoin and (2S,3S)-2,3-butanediol. The application of this coexpression system to the production of other chiral chemicals could be extended by using different NAD(P)-dependent dehydrogenases that require NAD(P)⁺ for catalysis.     

Characterization of a stereospecific acetoin(diacetyl) reductase from Rhodococcus erythropolis WZ010 and its application for the synthesis of (2S,3S)-2,3-butanediol

Rhodococcus erythropolis WZ010 was capable of producing optically pure (2S,3S)-2,3-butanediol in alcoholic fermentation. The gene encoding an acetoin(diacetyl) reductase from R. erythropolis WZ010 (ReADR) was cloned, overexpressed in Escherichia coli, and subsequently purified by Ni-affinity chromatography. ReADR in the native form appeared to be a homodimer with a calculated subunit size of 26,864, belonging to the family of the short-chain dehydrogenase/reductases. The enzyme accepted a broad range of substrates including aliphatic and aryl alcohols, aldehydes, and ketones. It exhibited remarkable tolerance to dimethyl sulfoxide (DMSO) and retained 53.6 % of the initial activity after 4 h incubation with 30 % (v/v) DMSO. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2S,3S)-2,3-butanediol via (S)-acetoin. The optimal pH and temperature for diacetyl reduction were pH 7.0 and 30 °C, whereas those for (2S,3S)-2,3-butanediol oxidation were pH 9.5 and 25 °C. Under the optimized conditions, the activity of diacetyl reduction was 11.9-fold higher than that of (2S,3S)-2,3-butanediol oxidation. Kinetic parameters of the enzyme showed lower K _m values and higher catalytic efficiency for diacetyl and NADH in comparison to those for (2S,3S)-2,3-butanediol and NAD⁺, suggesting its physiological role in favor of (2S,3S)-2,3-butanediol formation. Interestingly, the enzyme showed higher catalytic efficiency for (S)-1-phenylethanol oxidation than that for acetophenone reduction. ReADR-catalyzed asymmetric reduction of diacetyl was coupled with stereoselective oxidation of 1-phenylethanol, which simultaneously formed both (2S,3S)-2,3-butanediol and (R)-1-phenylethanol in great conversions and enantiomeric excess values.     

Inhibition of acetate accumulation leads to enhanced production of (R,R)-2,3-butanediol from glycerol in Escherichia coli

This work describes the production of (R,R)-2,3-butanediol in Escherichia coli using glycerol by metabolic engineering approaches. The introduction of a synthetic pathway converting pyruvate to (R,R)-2,3-butanediol into wild-type E. coli strain BW25113 led to the production of (R,R)-2,3-butanediol at a titer of 3.54?g/l and a yield of 0.131?g product/g glycerol (26.7?% of theoretical maximum) with acetate (around 3.00?g/l) as the dominant by-product. We therefore evaluated the impacts of deleting the genes ackA or/and poxB that are responsible for the major by-product, acetate. This increased production of (R,R)-2,3-butanediol to 9.54?g/l with a yield of 0.333?g product/g glycerol (68.0?% of theoretical maximum) in shake flask studies. The utilization of low-priced crude glycerol to produce value-added chemicals is of great significance to the economic viability of the biodiesel industry.     

Regulation of glycerol metabolism in <Emphasis Type="Italic">Enterobacter aerogenes</Emphasis> NBRC12010 under electrochemical conditions

Enterobacter aerogenes NBRC12010 was able to ferment glycerol to ethanol and hydrogen gas. Fermentation of glycerol ceased in the stationary phase of growth, and it was activated by electrochemical reactions using thionine as an electron transfer mediator from bacterial cells to an electrode. Using resting cells of E. aerogenes NBRC12010 in only citrate buffer solution, the cells did not consume glycerol at all, but they could metabolize glucose. These results suggest that the regulation of glycerol metabolism occurred at enzymatic steps before glycolysis. In E. aerogenes NBRC12010, glycerol was metabolized via glycerol dehydrogenase (GDH) and then dehydroxyacetone kinase. The GDH-catalyzed reaction mainly depended on the ratio of NAD⁺/NADH. At a NAD⁺/NADH ratio of nearly 1 or less, it was substantially suppressed and glycerol metabolism stopped. When the ratio was higher than 1, GDH was activated and glycerol was metabolized. Thus, the reaction of glycerol metabolism depended on the balance of cellular NAD⁺/NADH. Exogenous NADH was oxidized to NAD⁺ by electrochemical reactions with thionine. We proposed the activation mechanism of glycerol metabolism under electrochemical conditions.     

Enhancement of 1,3-propanediol production by expression of pyruvate decarboxylase and aldehyde dehydrogenase from Zymomonas mobilis in the acetolactate-synthase-deficient mutant of Klebsiella pneumoniae

The acetolactate synthase (als)-deficient mutant of Klebsiella pneumoniae fails to produce 1,3-propanediol (1,3-PD) or 2,3-butanediol (2,3-BD), and is defective in glycerol metabolism. In an effort to recover production of the industrially valuable 1,3-PD, we introduced the Zymomonas mobilis pyruvate decarboxylase (pdc) and aldehyde dehydrogenase (aldB) genes into the als-deficient mutant to activate the conversion of pyruvate to ethanol. Heterologous expression of pdc and aldB efficiently recovered glycerol metabolism in the 2,3-BD synthesis-defective mutant, enhancing the production of 1,3-PD by preventing the accumulation of pyruvate. Production of 1,3-PD in the pdc- and aldB-expressing als-deficient mutant was further enhanced by increasing the aeration rate. This system uses metabolic engineering to produce 1,3-PD while minimizing the generation of 2,3-BD, offering a breakthrough for the industrial production of 1,3-PD from crude glycerol.     

Efficient production of ethanol from crude glycerol by a Klebsiella pneumoniae mutant strain

A mutant strain of Klebsiella pneumoniae, termed GEM167, was obtained by γ irradiation, in which glycerol metabolism was dramatically affected on exposure to γ rays. Levels of metabolites of the glycerol reductive pathway, 1,3-propanediol (1,3-PD) and 3-hydroxypropionic acid (3-HP), were decreased in the GEM167 strain compared to a control strain, whereas the levels of metabolites derived from the oxidative pathway, 2,3-butanediol (2,3-BD), ethanol, lactate, and succinate, were increased. Notably, ethanol production from glycerol was greatly enhanced upon fermentation by the mutant strain, to a maximum production level of 21.5 g/l, with a productivity of 0.93 g/l/h. Ethanol production level was further improved to 25.0 g/l upon overexpression of Zymomonas mobilispdc and adhII genes encoding pyruvate decarboxylase (Pdc) and aldehyde dehydrogenase (Adh), respectively in the mutant strain GEM167.     

Effects of over-expression of glycerol dehydrogenase and 1,3-propanediol oxidoreductase on bioconversion of glycerol into 1,3-propandediol by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> under micro-aerobic conditions

Glycerol dehydrogenase (GDH) and 1,3-propanediol (1,3-PD) oxidoreductase had been proved two key enzymes for 1,3-PD production by Klebsiella pneumoniae. Fed-batch fermentations of the recombinant K. pneumoniae strains, over-expressing the two enzymes individually, were carried out under micro-aerobic conditions, and the behaviors of the recombinants were investigated. Results showed that over-expression of 1,3-PD oxidoreductase did not affect the concentration of 1,3-PD. However, it enhanced the molar yield from 50.6 to 64.0% and reduced the concentration of by-products. Among them, the concentrations of lactic acid, ethanol and succinic acid were decreased by 51.8, 50.6 and 47.4%, respectively. Moreover, in the recombinant the maximal concentration of 3-hydroxypropionaldehyde decreased by 73.6%. Over-expression of GDH decreased the yield of ethanol and 2,3-butanediol, meanwhile it increased the concentration of acetic acid. No significant changes were observed both in 1,3-PD yield and glycerol flux distributed to oxidative branch.     

Codon-Optimized NADH Oxidase Gene Expression and Gene Fusion with Glycerol Dehydrogenase for Bienzyme System with Cofactor Regeneration

NADH oxidases (NOXs) play an important role in maintaining balance of NAD⁺/NADH by catalyzing cofactors regeneration. The expression of nox gene from Lactobacillus brevis in Escherichia coli BL21 (BL21 (DE3)) was studied. Two strategies, the high AT-content in the region adjacent to the initiation codon and codon usage of the whole gene sequence consistent with the host, obtained the NOX activity of 59.9 U/mg and 73.3 U/mg (crude enzyme), with enhanced expression level of 2.0 and 2.5-folds, respectively. Purified NOX activity was 213.8 U/mg. Gene fusion of glycerol dehydrogenase (GDH) and NOX formed bifuctional multi-enzymes for bioconversion of glycerol coupled with coenzyme regeneration. Kinetic parameters of the GDH-NOX for each substrate, glycerol and NADH, were calculated as V _{max(Glycerol)} 20 μM/min, K _m(Glycerol) 19.4 mM, V _{max (NADH)} 12.5 μM/min and K _{m (NADH)} 51.3 μM, respectively, which indicated the potential application of GDH-NOX for quick glycerol analysis and dioxyacetone biosynthesis.     

Erratum to: Production of isobutanol from crude glycerol by a genetically-engineered Klebsiella pneumoniae strain

Klebsiella pneumoniae was engineered to produce isobutanol from crude glycerol as a sole carbon source by expressing acetolactate synthase (ilvIH), keto-acid reducto-isomerase (ilvC) and dihydroxy-acid dehydratase (ilvD) from K. pneumoniae, and α-ketoisovalerate decarboxylase (kivd) and alcohol dehydrogenase (adhA) from Lactococcus lactis. Engineered K. pneumonia, ?ldhA/pBR-iBO (ilvIH–ilvC–ilvD–kivd–adhA), produced isobutanol (160 mg l^?1) from crude glycerol. To increase the yield of isobutanol, we eliminated the 2,3-butanediol pathway from the recombinant strain by inactivating α-acetolactate decarboxylase (adc). This further engineering step improved the yield of isobutanol from 160 to 320 mg l^?1. This represents the first successful attempt to produce isobutanol from crude glycerol.     

Glycerol Metabolism Is Important for Cytotoxicity of Mycoplasma pneumoniae

Glycerol is one of the few carbon sources that can be utilized by Mycoplasma pneumoniae. Glycerol metabolism involves uptake by facilitated diffusion, phosphorylation, and the oxidation of glycerol 3-phosphate to dihydroxyacetone phosphate, a glycolytic intermediate. We have analyzed the expression of the genes involved in glycerol metabolism and observed constitutive expression irrespective of the presence of glycerol or preferred carbon sources. Similarly, the enzymatic activity of glycerol kinase is not modulated by HPr-dependent phosphorylation. This lack of regulation is unique among the bacteria for which glycerol metabolism has been studied so far. Two types of enzymes catalyze the oxidation of glycerol 3-phosphate: oxidases and dehydrogenases. Here, we demonstrate that the enzyme encoded by the M. pneumoniae glpD gene is a glycerol 3-phosphate oxidase that forms hydrogen peroxide rather than NADH₂. The formation of hydrogen peroxide by GlpD is crucial for cytotoxic effects of M. pneumoniae. A glpD mutant exhibited a significantly reduced formation of hydrogen peroxide and a severely reduced cytotoxicity. Attempts to isolate mutants affected in the genes of glycerol metabolism revealed that only the glpD gene, encoding the glycerol 3-phosphate oxidase, is dispensable. In contrast, the glpF and glpK genes, encoding the glycerol facilitator and the glycerol kinase, respectively, are essential in M. pneumoniae. Thus, the enzymes of glycerol metabolism are crucial for the pathogenicity of M. pneumoniae but also for other essential, yet-to-be-identified functions in the M. pneumoniae cell.Mycoplasma pneumoniae causes infections of the upper and lower respiratory tracts. These bacteria are responsible for a large fraction of community-acquired pneumonias. Although usually harmless for adult patients, M. pneumoniae may cause severe disease in children or elderly people. In addition, M. pneumoniae is involved in extrapulmonary complications such as pediatric encephalitis and erythema multiforme (for reviews, see references 15, 21, and 34).M. pneumoniae and its relatives, the Mollicutes, are all characterized by the lack of a cell wall and a very close adaptation to a life within a eukaryotic host. This close adaptation is reflected by degenerative genome evolution that resulted in an extreme genome reduction. As a result, the Mollicutes are the organisms that are capable of independent life with the smallest known genome. M. pneumoniae has a genome of 816 kb and encodes only 688 proteins (18). This genome reduction is taken even further in the close relative Mycoplasma genitalium, which has only 482 protein-coding genes (18). Thus, the analysis of the Mollicutes allows us to study a minimal form of natural life. This question has recently attracted much interest and resulted in the determination of the essential gene sets of M. pneumoniae, M. genitalium, and, more recently, Mycoplasma pulmonis (6, 20). In M. genitalium, with the most reduced genomes, only 100 out of the 482 protein-coding genes are dispensable, suggesting that the remaining 382 genes form the essential gene set (7).Reductive genome evolution in M. pneumoniae is still under way: the genes for the utilization of mannitol as a carbon source seem to be present in M. pneumoniae; however, this substrate cannot be used by the bacteria. M. genitalium, which is further advanced in genome reduction, has lost the genes for mannitol transport and oxidation. It was therefore suggested that the genes for mannitol utilization in M. pneumoniae either are not expressed or encode inactive proteins (12).In M. pneumoniae as well as in other Mollicutes, pathogenicity is closely linked to carbon metabolism (13). M. pneumoniae can use glucose, fructose, and glycerol as the only carbon sources (12). Studies with Mycoplasma mycoides revealed that glycerol metabolism has a major impact on the pathogenicity of these bacteria. Oxidation of glycerol involves the glycerol 3-phosphate oxidase, which produces hydrogen peroxide rather than NADH₂, which is generated by the glycerol 3-phosphate dehydrogenase in most other bacteria (28). In addition to the induction of autoimmune responses, the formation of hydrogen peroxide is the only established mechanism by which mycoplasmas cause damage to their hosts (31, 34). Pathogenic strains of M. mycoides possess a highly active ABC transport system for glycerol in addition to the ubiquitous glycerol facilitator (33). The efficient formation of hydrogen peroxide by the membrane-bound glycerol 3-phosphate oxidase is the major virulence factor of the highly pathogenic strains of M. mycoides (28).M. pneumoniae possesses the complete set of genes for glycerol utilization, and the bacteria do indeed use this carbon source (12). The first component in glycerol metabolism is the glycerol facilitator encoded by the glpF gene. The transported glycerol is then phosphorylated by the glycerol kinase (product of glpK), and glycerol 3-phosphate is subsequently oxidized to dihydroxyacetone phosphate, a glycolytic intermediate. The relevant enzyme is annotated as glycerol 3-phosphate dehydrogenase (encoded by the gene glpD) in M. pneumoniae (17).In all organisms studied so far, glycerol metabolism is under dual control: the genes involved in glycerol utilization are expressed only if glycerol or glycerol 3-phosphate is present in the medium, and they are not expressed in the presence of glucose, the preferred carbon source (3, 4). This second mode of regulation, carbon catabolite repression, involves two distinct mechanisms in the Firmicutes, from which the Mollicutes evolved. In the presence of preferred sugars, the CcpA repressor protein binds in the promoter regions of glycerol utilization genes and prevents their expression. Moreover, the molecular inducer of the system, glycerol 3-phosphate, is formed only in the absence of glucose. This results from the low activity of the glycerol kinase. This enzyme is activated upon phosphorylation by HPr, a protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). HPr can phosphorylate other proteins only in the absence of glucose, thus providing a link between glucose availability, the activity of the glycerol kinase, and the induction of the glycerol utilization genes (3). Nothing is known about the regulation of glycerol utilization in any member of the Mollicutes; however, regulatory events seem to be rare in these organisms due to the lack of regulatory proteins, among them CcpA.In this work, we studied the mechanisms of glycerol utilization in M. pneumoniae, its regulation, and its contribution to cytotoxicity. We demonstrate constitutive expression of the genes for glycerol utilization in M. pneumoniae. As observed in M. mycoides, glycerol 3-phosphate oxidation involves the formation of hydrogen peroxide and is important for damaging the host cells.     

Inactivation of <Emphasis Type="Italic">dhaD</Emphasis> and <Emphasis Type="Italic">dhaK</Emphasis> abolishes by-product accumulation during 1,3-propanediol production in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

1,3-Propanediol (1,3-PD) can be used for the industrial synthesis of a variety of compounds, including polyesters, polyethers, and polyurethanes. 1,3-PD is generated from petrochemical and microbial sources. 1,3-Propanediol is a typical product of glycerol fermentation, while acetate, lactate, 2,3-butanediol, and ethanol also accumulate during the process. Substrate and product inhibition limit the final concentration of 1,3-propanediol in the fermentation broth. It is impossible to increase the yield of 1,3-propanediol by using the traditional whole-cell fermentation process. In this study, dhaD and dhaK, the genes for glycerol dehydrogenase and dihydroxyacetone kinase, respectively, were inactivated by homologous recombination in Klebsiella pneumoniae. The dhaD/dhaK double mutant (designated TC100), selected from 5,000 single or double cross homologous recombination mutants, was confirmed as a double cross by using polymerase chain reaction. Analysis of the cell-free supernatant with high-performance liquid chromatography revealed elimination of lactate and 2,3-butanediol, as well as ethanol accumulation in TC100, compared with the wild-type strain. Furthermore, 1,3-propanediol productivity was increased in the TC100 strain expressing glycerol dehydratase and 1,3-PDO dehydrogenase regulated by the arabinose P_BAD promoter. The genetic engineering and medium formulation approaches used here should aid in the separation of 1,3-propanediol from lactate, 2,3-butanediol, and ethanol and lead to increased production of 1,3-propanediol in Klebsiella pneumoniae.     

Production of 2-butanol from crude glycerol by a genetically-engineered Klebsiella pneumoniae strain

Klebsiella pneumoniae was engineered to produce 2-butanol from crude glycerol as a sole carbon source by expressing acetolactate synthase (ilvIH), keto-acid reducto-isomerase (ilvC) and dihydroxy-acid dehydratase (ilvD) from K. pneumoniae, and α-ketoisovalerate decarboxylase (kivd) and alcohol dehydrogenase (adhA) from Lactococcus lactis. Engineered K. pneumonia, ?ldhA/pBR-iBO (ilvIH–ilvC–ilvD–kivd–adhA), produced 2-butanol (160 mg l^?1) from crude glycerol. To increase the yield of 2-butanol, we eliminated the 2,3-butanediol pathway from the recombinant strain by inactivating α-acetolactate decarboxylase (adc). This further engineering step improved the yield of 2-butanol from 160 to 320 mg l^?1. This represents the first successful attempt to produce 2-butanol from crude glycerol.