Expression of 1,3-propanediol oxidoreductase and its isoenzyme in Klebsiella pneumoniae for bioconversion of glycerol into 1,3-propanediol

In the Klebsiella pneumoniae reduction pathway for 1,3-propanediol (1,3-PD) synthesis, glycerol is first dehydrated to 3-hydroxypropionaldehyde (3-HPA) and then reduced to 1,3-PD with NADH consumption. Rapid conversion of 3-HPA to 1,3-PD is one of the ways to improve the yield of 1,3-PD from glycerol and to avoid 3-HPA accumulation, which depends on enzyme activity of the reaction and the amount of reducing equivalents available from the oxidative pathway of glycerol. In the present study, the yqhD gene, encoding 3-propanediol oxidoreductase isoenzyme from Escherichia coli and the dhaT gene, encoding 3-propanediol oxidoreductase from K. pneumoniae were expressed individually and co-expressed in K. pneumoniae using the double tac promoter expression plasmid pEtac-dhaT-tac-yqhD. The three resultant recombinant strains (K. pneumoniae/pEtac-yqhD, K. pneumoniae/pEtac-dhaT, and K. pneumoniae/pEtac-dhaT-tac-yqhD) were used for fermentation studies. Experimental results showed that the peak values for 3-HPA production in broth of the three recombinant strains were less than 25% of that of the parent strain. Expression of dhaT reduced formation of by-products (ethanol and lactic acid) and increased molar yield of 1,3-PD slightly, while expression of yqhD did not enhance molar yield of 1,3-PD, but increased ethanol concentration in broth as NADPH participation in transforming 3-HPA to 1,3-PD allowed more cellular NADH to be used to produce ethanol. Co-expression of both genes therefore decreased by-products and increased the molar yield of 1,3-PD by 11.8%, by catalyzing 3-HPA conversion to 1,3-propanediol using two cofactors (NADH and NADPH). These results have important implications for further studies involving use of YqhD and DhaT for bioconversion of glycerol into 1,3-PD.     

Expression of dha Operon Required for 1,3-PD Formation in Escherichia coli and Saccharomyces cerevisiae

The 1,3-propanediol (1,3-PD) synthesis operon (dha operon) was mainly composed of four genes: dhaB, dhaT, gdrA, and gdrB, which encoded glycerol dehydratase, 1,3-PD oxidoreductase and reactivating factor for glycerol dehydratase, respectively. In the present study, dha operon was cloned from 1,3-PD producing strain Klebsiella pneumoniae. Heterologous expression of cloned dha operon was carried out in Escherichia coli and Saccharomyces cerevisiae W303-1A, respectively. The results indicated that recombinant E. coli harboring the dha operon can produce 8–9 g/l 1,3-PD from glycerol while the 1,3-PD yield of recombinant strain W303-1A-dha could not be detected. In order to complete the 1,3-PD production from glucose, further, we also constructed the recombinant S. cerevisiae W303-1A-BT harboring plasmid pZ-BT. The 1,3-PD production and enzymatic activities of DhaB and DhaT were found in the engineered strain W303-1A-BT. Our results demonstrated that the recombinant S. cerevisiae strain W303-1A-BT that can produce 1,3-PD at low cost was constructed. This study might open a novel way to a safe and cost-efficient method for microbial production of 1,3-PD.     

Characterization of 1,3-propanediol oxidoreductase (DhaT) from <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> J2B

1,3-propanediol oxidoreductase (DhaT) of Klebsiella pneumoniae converts 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD) during microbial production of 1,3-PD from glycerol. In this study, DhaT from newly isolated K. pneumoniae J2B was cloned, expressed, purified, and studied for its kinetic properties. It showed, on its physiological substrate 3-HPA, higher activity than similar aldehydes such as acetaldehyde, propionaldehyde and butyraldehyde. The turnover numbers (k _cat , 1/s) were estimated as 59.4 for the forward reaction (3-HPA to 1,3-PD at pH 7.0) and 10.0 for the reverse reaction (1,3-PD to 3-HPA at pH 9.0). The Michaelis constants (K _m , mM) were 0.77 (for 3-HPA) and 0.03 (for NADH) for the forward reaction (at pH 7.0), and 7.44 (for 1,3-PD) and 0.23 (for NAD⁺) for the reverse reaction (at pH 9.0). Between these forward and reverse reactions, the optimum temperature and pH were significantly different (37°C and 7.0 vs. 55°C and 9.0, respectively). These results indicate that, under physiological conditions, DhaT mostly catalyzes the forward reaction. The enzyme was seriously inhibited by heavy metal ions such as Ag⁺ and Hg²⁺. DhaT was highly unstable when incubated with its own substrate 3-HPA, indicating the necessity of enhancing its stability for improved 1,3-PD production from glycerol.     

Construction and Characterization of a 1,3-Propanediol Operon

The genes for the production of 1,3-propanediol (1,3-PD) in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, are naturally under the control of two different promoters and are transcribed in different directions. These genes were reconfigured into an operon containing dhaB followed by dhaT under the control of a single promoter. The operon contains unique restriction sites to facilitate replacement of the promoter and other modifications. In a fed-batch cofermentation of glycerol and glucose, Escherichia coli containing the operon consumed 9.3 g of glycerol per liter and produced 6.3 g of 1,3-PD per liter. The fermentation had two distinct phases. In the first phase, significant cell growth occurred and the products were mainly 1,3-PD and acetate. In the second phase, very little growth occurred and the main products were 1,3-PD and pyruvate. The first enzyme in the 1,3-PD pathway, glycerol dehydratase, requires coenzyme B₁₂, which must be provided in E. coli fermentations. However, the amount of coenzyme B₁₂ needed was quite small, with 10 nM sufficient for good 1,3-PD production in batch cofermentations. 1,3-PD is a useful intermediate in the production of polyesters. The 1,3-PD operon was designed so that it can be readily modified for expression in other prokaryotic hosts; therefore, it is useful for metabolic engineering of 1,3-PD pathways from glycerol and other substrates such as glucose.     

Improvement of 1,3-propanediol oxidoreductase (DhaT) stability against 3-hydroxypropionaldehyde by substitution of cysteine residues

1,3-propanediol oxidoreductase (DhaT), which catalyzes the conversion of 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD) with the oxidation of NADH to NAD⁺, is a key enzyme in the production of 1,3-PD from glycerol. DhaT is known to be severely inactivated by its physiological substrate, 3-HPA, due to the reaction of 3-HPA with the thiol group of the cysteine residues. In this study, using site-directed mutagenesis, four cysteine residues in Klebsiella pneumoniae J2B DhaT were substituted to alanine, the amino acid commonly found in cysteine’s positions in other DhaT, individually and in combination. Among the total of 15 mutants developed, a double mutant (C28A_C107A) and a triple mutant (C28A_C93A_C107A) exhibited approximately 50 and 16% higher activity than the wild-type counterpart, respectively, after 1 h incubation with 10 mM 3-HPA. According to detailed kinetic studies, the double mutant had slightly better kinetic properties (V _max , K _cat , and K _m for both 3-HPA and NADH) than wild-type DhaT. This study shows that DhaT stability against 3-HPA can be increased by cysteine-residue removal, albeit to a limited extent.     

Molecular Cloning, Co-expression, and Characterization of Glycerol Dehydratase and 1,3-Propanediol Dehydrogenase from Citrobacter freundii

1,3-Propanediol (1,3-PD), an important material for chemical industry, is biologically synthesized by glycerol dehydratase (GDHt) and 1,3-propanediol dehydrogenase (PDOR). In present study, the dhaBCE and dhaT genes encoding glycerol dehydratase and 1,3-propanediol dehydrogenase respectively were cloned from Citrobacter freundii and co-expressed in E. coli. Sequence analysis revealed that the cloned genes were 85 and 77 % identical to corresponding gene of C. freundii DSM 30040 (GenBank No. U09771), respectively. The over-expressed recombinant enzymes were purified by nickel-chelate chromatography combined with gel filtration, and recombinant GDHt and PDOR were characterized by activity assay, kinetic analysis, pH, and temperature optimization. This research may form a basis for the future work on biological synthesis of 1,3-PD.     

1,3-Propanediol production by Escherichia coli expressing genes from the Klebsiella pneumoniae dha regulon.

The dha regulon in Klebsiella pneumoniae enables the organism to grow anaerobically on glycerol and produce 1,3-propanediol (1,3-PD). Escherichia coli, which does not have a dha system, is unable to grow anaerobically on glycerol without an exogenous electron acceptor and does not produce 1,3-PD. A genomic library of K. pneumoniae ATCC 25955 constructed in E. coli AG1 was enriched for the ability to grow anaerobically on glycerol and dihydroxyacetone and was screened for the production of 1,3-PD. The cosmid pTC1 (42.5 kb total with an 18.2-kb major insert) was isolated from a 1,3-PD-producing strain of E. coli and found to possess enzymatic activities associated with four genes of the dha regulon: glycerol dehydratase (dhaB), 1,3-PD oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK). All four activities were inducible by the presence of glycerol. When E. coli AG1/pTC1 was grown on complex medium plus glycerol, the yield of 1,3-PD from glycerol was 0.46 mol/mol. The major fermentation by-products were formate, acetate, and D-lactate. 1,3-PD is an intermediate in organic synthesis and polymer production. The 1,3-PD fermentation provides a useful model system for studying the interaction of a biochemical pathway in a foreign host and for developing strategies for metabolic pathway engineering.     

1,3-Propanediol production by Escherichia coli expressing genes from the Klebsiella pneumoniae dha regulon.

The dha regulon in Klebsiella pneumoniae enables the organism to grow anaerobically on glycerol and produce 1,3-propanediol (1,3-PD). Escherichia coli, which does not have a dha system, is unable to grow anaerobically on glycerol without an exogenous electron acceptor and does not produce 1,3-PD. A genomic library of K. pneumoniae ATCC 25955 constructed in E. coli AG1 was enriched for the ability to grow anaerobically on glycerol and dihydroxyacetone and was screened for the production of 1,3-PD. The cosmid pTC1 (42.5 kb total with an 18.2-kb major insert) was isolated from a 1,3-PD-producing strain of E. coli and found to possess enzymatic activities associated with four genes of the dha regulon: glycerol dehydratase (dhaB), 1,3-PD oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK). All four activities were inducible by the presence of glycerol. When E. coli AG1/pTC1 was grown on complex medium plus glycerol, the yield of 1,3-PD from glycerol was 0.46 mol/mol. The major fermentation by-products were formate, acetate, and D-lactate. 1,3-PD is an intermediate in organic synthesis and polymer production. The 1,3-PD fermentation provides a useful model system for studying the interaction of a biochemical pathway in a foreign host and for developing strategies for metabolic pathway engineering.     

Physiological predisposition of various Clostridium species to synthetize 1,3-propanediol from glycerol

1,3-Propanediol (1,3-PD) production by fermentation of glycerol was first described in 1881 but little attention was paid to this biosynthesis for over a century. An increasing interest in microbial 1,3-PD production is observed since late 1980s. The high growth rate of the biofuel market and the perspective of glycerol becoming abundant attract even more attention to this valuable chemical.Glycerol conversion to 1,3-PD is known to occur in Clostridia, Enterobacteriaceae and Lactobacillaceae. Some clostridial species are among the best 1,3-PD producers.This work is a review of the current state of research on Clostridium spp. strains that ferment glycerol to 1,3-PD. It focuses on the metabolic pathways and factors that influence the production of this diol. The effects of different environmental stresses on the process of 1,3-PD synthesis are also covered. Moreover, various genetic engineering methods utilized in order to improve the capabilities of bacteria used in this process are presented.     

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.     

利用甘油发酵耦联生产3-羟基丙酸及1,3-丙二醇重组菌的构建及筛选

在肺炎克雷伯杆菌(Klebsiella pneumoniae)代谢甘油生产1,3-丙二醇(1,3-PD)的过程中,为了减少有毒中间产物3-羟基丙醛(3-HPA)的积累,可将其转化为3-羟基丙酸(3-HP),从而实现1,3-丙二醇和3-羟基丙酸的联产。克隆来自于酿酒酵母的NAD+依赖型的乙醛脱氢酶(ALDH)的基因aldh4,构建了表达载体pKP-aldh,转化K.pneumoniae,得到了有效表达乙醛脱氢酶的重组肺炎克雷伯杆菌(K.pneumoniae A+)。在此基础上,使用紫外诱变联合菌种驯化的方法对K.pneumoniae A+进行筛选,获得了可耐受较高3-HP浓度(≥35 g/L)的重组肺炎克雷伯杆菌K.pneumoniae A+5-3。发酵实验结果表明,K.pneumoniae A+5-3可将3-HPA转化为3-HP,能够同时利用甘油耦联生产3-HP和1,3-PD,产量分别达到5.0 g/L和74.5 g/L。     

Effect of aeration strategy on the metabolic flux of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> producing 1,3-propanediol in continuous cultures at different glycerol concentrations

The microbial production of 1,3-propaneidol (1,3-PD) by Klebsiella pneumoniae in continuous fermentation was investigated under low, medium and high glycerol concentrations in the absence and presence of oxygen. The production of 1,3-PD increased with increasing glycerol concentrations, reaching a maximum (266 mmol l⁻¹) under high glycerol concentration (760 mmol l⁻¹) with air sparging at 0.04 vvm. The yield of 1,3-PD, however, decreased gradually with increasing glycerol concentrations, with the highest yield (0.52 mol mol⁻¹) obtained for low glycerol concentration (270 mmol l⁻¹) under anaerobic condition. Enzyme activity assays showed that the specific activity of glycerol dehydratase was highest (0.04 U mg⁻¹) for culture sparged with 0.04 vvm air under high glycerol concentration. The specific activities of glycerol dehydrogenase and 1,3-propanediol oxidoreductase were also improved for all glycerol concentrations and in the presence of oxygen, implying that the dha operon was not repressed under microaerobic conditions. Analysis of metabolic fluxes showed that more carbon flux was shifted to the oxidative pathway with increasing glycerol concentrations, resulting in a reduced flux to 1,3-PD formation. However, the increases in carbon fluxes were not evenly distributed among the oxidative branches of the pathway. Furthermore, ethanol and acetic acid levels were slightly increased whereas 2,3-butanediol and lactic levels were greatly enhanced.     

Complex metabolic network of 1,3-propanediol transport mechanisms and its system identification via biological robustness

The bioconversion of glycerol to 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae (K. pneumoniae) can be characterized by an intricate metabolic network of interactions among biochemical fluxes, metabolic compounds, key enzymes and genetic regulation. Since there are some uncertain factors in the fermentation, especially the transport mechanisms of 1,3-PD across cell membrane, the metabolic network contains multiple possible metabolic systems. Considering the genetic regulation of dha regulon and inhibition of 3-hydroxypropionaldehyde to the growth of cells, we establish a 14-dimensional nonlinear hybrid dynamical system aiming to determine the most possible metabolic system and the corresponding optimal parameter. The existence, uniqueness and continuity of solutions are discussed. Taking the robustness index of the intracellular substances together as a performance index, a system identification model is proposed, in which 1,395 continuous variables and 90 discrete variables are involved. The identification problem is decomposed into two subproblems and a parallel particle swarm optimization procedure is constructed to solve them. Numerical results show that it is most possible that 1,3-PD passes the cell membrane by active transport coupled with passive diffusion.     

Improving 1,3-propanediol production from glycerol in a metabolically engineered Escherichia coli by reducing accumulation of sn-glycerol-3-phosphate

High levels of glycerol significantly inhibit cell growth and 1,3-propanediol (1,3-PD) production in anaerobic glycerol fermentation by genetically engineered Escherichia coli (E. coli) strains expressing genes from the Klebsiella pneumoniae dha (K.pneumoniae) regulon. We have previously demonstrated that 1,3-PD production by the engineered E. coli can be improved by reducing the accumulation of methylglyoxal. This study focuses on investigation of another lesser-known metabolite in the pathways related to 1,3-PD production-glycerol-3-phosphate (G3P). When grown anaerobically on glycerol in the absence of an exogenous acceptor, the engineered E. coli strains have intracellular G3P levels that are significantly higher than those in K. pneumoniae, a natural 1,3-PD producer. Furthermore, in the engineered E. coli strains, the G3P levels increase with increasing glycerol concentrations, whereas, in K. pneumoniae, the concentrations of G3P remain relatively constant. Addition of fumarate, which can stimulate activity of anaerobic G3P dehydrogenase, into the fermentation medium led to a greater than 30-fold increase in the specific activity of anaerobic G3P dehydrogenase and a significant decrease in concentrations of intracellular G3P and resulted in better cell growth and an improved production of 1,3-PD. This indicates that the low activity of G3P dehydrogenase in the absence of an exogenous electron acceptor is one of the reasons for G3P accumulation. In addition, spent media from E.coli Lin61, a glycerol kinase (responsible for conversion of glycerol to G3P) mutant, contains greatly decreased concentrations of G3P and shows improved production of 1,3-PD (by 2.5-fold), when compared to media from its parent strain E. coli K10. This further suggests that G3P accumulation is one of the reasons for the inhibition of 1,3-PD production during anaerobic fermentation.     

Identification and characterization of Klebsiella pneumoniae aldehyde dehydrogenases increasing production of 3-hydroxypropionic acid from glycerol

Klebsiella pneumoniae produces 3-hydroxypropionic acid (3-HP) from glycerol with oxidation of 3-hydroxypropionaldehyde (3-HPA) to 3-HP in a reaction catalyzed by aldehyde dehydrogenase (ALDH). In the present study, two putative ALDHs of K. pneumoniae, YneI and YdcW were identified and characterized. Recombinant YneI was specifically active on 3-HPA and preferred NAD⁺ as a cofactor, whereas YdcW exhibited broad substrate specificity and preferred NADP⁺ as a cofactor. Overexpression of ALDHs in the glycerol oxidative pathway-deficient mutant K. pneumoniae AK resulted in a significant increase in 3-HP production upon shake-flask culture. The final titers of 3-HP were 2.4 and 1.8 g L^?1 by recombinants overexpressing YneI and YdcW, respectively. Deletion of the ALDH gene from K. pneumoniae did not affect the extent of 3-HP synthesis, implying non-specific activity of ALDHs on 3-HPA. The ALDHs might play major roles in detoxifying the aldehyde generated in glycerol metabolism.     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Decrease of 3-hydroxypropionaldehyde accumulation in 1,3-propanediol production by over-expressing <Emphasis Type="Italic">dha</Emphasis>T gene in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> TUAC01

Glycerol can be biologically converted to 1,3-propanediol, a key raw material required for the synthesis of polytrimethylene terephthalate and other polyester fibers. In 1,3-propanediol synthesis pathway, 3-hydroxypropionaldehyde (3-HPA) was an inhibitory intermediary metabolite. The accumulation of 3-HPA in broth would cause an irreversible cessation of the fermentation process. With the object of reducing 3-HPA level in the fermentation broth, dhaT gene which encodes 1,3-propanediol oxidoreductase (PDOR) was cloned and over expressed in 1,3-propanediol producing bacterium Klebsiella pneumoniae TUAC01. dhaT gene was linked downstream of the ptac promoter in an expressing vector pDK6 to form plasmid pDK-dhaT. The newly formed pDK-dhaT was transformed to K. pneumoniae TUAC01. Under the inducement of IPTG, PDOR was over-expressed when the constructed strain was cultured on an LB medium or a fermentation medium. A 5 L scale-up fermentation experiment was done to test the 3-HPA accumulation in broth, with the initial substrate glycerol 30 g/L; the peak levels of 3-HPA in broth were 7.55 and 1.49 mmol/L for control host strain and the constructed strain, respectively. In 50 g/L initial glycerol experiment, the peak level of 3-HPA in broth was 12.57 and 2.02 mmol/l for the control host strain and the constructed strain, respectively. Thus the fermentation cessation caused by the toxicity of 3-HPA was alleviated in the constructed strain.     

Enzymatic synthesis of 1,3-dihydroxyphenylacetoyl-sn-glycerol: Optimization by response surface methodology and evaluation of its antioxidant and antibacterial activities

In this study, the enzymatic synthesis of phenylacetoyl glycerol ester was carried out as a response to the increasing consumer demand for natural compounds. 1,3-dihydroxyphenylacetoyl-sn-Glycerol (1,3-di-HPA-Gly), labeled as “natural” compound with interesting biological properties, has been successfully synthesized for the first time in good yield by a direct esterification of glycerol (Gly) with p-hydroxyphenylacetic acid (p-HPA) using immobilized Candida antarctica lipase as a biocatalyst. Spectroscopic analyses of purified esters showed that the glycerol was mono- or di-esterified on the primary hydroxyl group. These compounds were evaluated for their antioxidant activity using two different tests. The glycerol di-esters (1,3-di-HPA-Gly) showed a higher antiradical capacity than that of the butyl hydroxytoluene. Furthermore, compared to the p-HPA, synthesized ester (1,3-di-HPA-Gly) exhibited the most antibacterial effect mainly against Gram + bacteria. Among synthesized esters the 1,3-di-HPA-Gly was most effective as antioxidant and antibacterial compound. These findings could be the basis for a further exploitation of the new compound, 1,3-di-HPA-Gly, as antioxidant and antibacterial active ingredient in the cosmetic and pharmaceutical fields.