Deletion of the budBAC operon in Klebsiella pneumoniae to understand the physiological role of 2,3-butanediol biosynthesis

Klebsiella pneumoniae is known to produce 2,3-butanediol (2,3-BDO), a valuable chemical. In K. pneumoniae, the 2,3-BDO operon (budBAC) is involved in the production of 2,3-BDO. To observe the physiological role of the 2,3-BDO operon in a mixed acid fermentation, we constructed a budBAC-deleted strain (SGSB109). The production of extracellular metabolites, CO₂ emission, carbon distribution, and NADH/NAD⁺ balance of SGSB109 were compared with the parent strain (SGSB100). When comparing the carbon distribution at 15 hr, four significant differences were observed: in 2,3-BDO biosynthesis, lactate and acetate production (lactate and acetate production increased 2.3-fold and 4.1-fold in SGSB109 compared to SGSB100), CO₂ emission (higher in SGSB100), and carbon substrate uptake (higher in SGSB100). Previous studies on the inactivation of the 2,3-BDO operon were focused on the increase of 1,3-propanediol production. Few studies have been done observing the role of 2,3-BDO biosynthesis. This study provides a prime insight into the role of 2,3-BDO biosynthesis of K. pneumoniae.     

The regulation of 2,3-butanediol synthesis in Klebsiella pneumoniae as revealed by gene over-expressions and metabolic flux analysis

A variety of microorganism species are able naturally to produce 2,3-butanediol (2,3-BDO), although only a few of them are suitable for consideration as having potential for mass production purposes. Klebsiella pneumoniae (K. pneumoniae) is one such strain which has been widely studied and used industrially to produce 2,3-BDO. In the central carbon metabolism of K. pneumoniae, the 2,3-BDO synthesis pathway is dominated by three essential enzymes, namely acetolactate decarboxylase, acetolactate synthase, and butanediol dehydrogenase, which are encoded by the budA, budB, and budC genes, respectively. The mechanisms of the three enzymes have been characterized with regard to their function and roles in 2,3-BDO synthesis and cell growth (Blomqvist et al. in J Bacteriol 175(5):1392–1404, 1993), while a few studies have focused on the cooperative mechanisms of the three enzymes and their mutual interactions. Therefore, the K. pneumoniae KCTC2242::ΔwabG wild-type strain was utilized to reconstruct seven new mutants by single, double, and triple overexpression of the three enzymes key to this study. Subsequently, continuous cultures were performed to obtain steady-state metabolism in the organisms and experimental data were analyzed by metabolic flux analysis (MFA) to determine the regulation mechanisms. The MFA results showed that the seven overexpressed mutants all exhibited enhanced 2,3-BDO production, and the strain overexpressing the budBA gene produced the highest yield. While the enzyme encoded by the budA gene produced branched-chain amino acids which were favorable for cell growth, the budB gene enzyme rapidly enhanced the conversion of acetolactate to acetoin in an oxygen-dependent manner, and the budC gene enzyme catalyzed the reversible conversion of acetoin to 2,3-BDO and regulated the intracellular NAD⁺/NADH balance.     

Characterization of acetoin production in a budC gene disrupted mutant of Serratia marcescens G12

The 2,3-butanediol (2,3-BD) dehydrogenase gene budC of Serratia marcescens G12 was disrupted to construct the acetoin (AC) producing strain G12M. In shake-flask cultures, AC production was enhanced by increased concentrations of glucose or sodium acetate in G12M. In fed-batch fermentation, G12M produced 47.5 g/L AC along with 9.8 g/L 2,3-BD. The expression of the key enzymes for AC synthesis was further investigated. Alpha-acetolactate synthase gene budB decreased its expression significantly in G12M compared with G12. This probably explained the moderate AC production in G12M cultures. Additionally, overexpression of budB gene and α-acetolactate decarboxylase gene budA was conducted in G12M and no significant increase of AC was observed. The results suggested that intracellular AC accumulation might inhibit the expression of budB and budA gene and induce budC gene expression in G12M. Our analyses offered the bases for further genetic manipulations in improving AC production in microbial fermentations.     

2-Ketogluconic acid production by Klebsiella pneumoniae CGMCC 1.6366

Klebsiella pneumoniae CGMCC 1.6366 is a bacterium isolated for 1,3-propanediol or 2,3-butanediol production previously. K. pneumoniae ΔbudA, a 2,3-butanediol synthesis pathway truncated mutant with the gene deletion of budA which encodes alpha-acetolactate decarboxylase, was found to execrate an unknown chemical at a high titer when grown in the broth using glucose as carbon source. Later this chemical was identified to be 2-ketogluconic acid, which was formed through the glucose oxidation pathway in K. pneumoniae. It was found that 2-ketogluconic can also be produced by the wild strain. The fermentation studies showed that the production of this metabolite is strictly pH dependent, when the fermenting broth was maintained at pH 6–7, the main metabolite produced by K. pneumoniae CGMCC 1.6366 was 2,3-butanediol, or some organic acids in the budA mutated strain. However, if the cells were fermented at pH 4.7, 2-ketogluconic acid was formed, and the secretion of all other organic acids or 2,3-butanediol were limited. In the 5L bioreactors, a final level of 38.2 and 30.2 g/L 2-ketogluconic acid were accumulated by the wild type and the budA mutant K. pneumoniae, respectively, in 26 and 56 h; and the conversion ratios of glucose to 2-ketogluconic acid reached 0.86 and 0.91 mol/mol for the wild and the budA mutant, respectively.     

Removal of pathogenic factors from 2,3-butanediol-producing Klebsiella species by inactivating virulence-related wabG gene

Klebsiella species are the most extensively studied among a number of 2,3-butanediol (2,3-BDO)-producing microorganisms. The ability to metabolize a wide variety of substrates together with the ease of cultivation made this microorganisms particularly promising for the application in industrial-scale production of 2,3-BDO. However, the pathogenic characteristics of encapsulated Klebsiella species are considered to be an obstacle hindering their industrial applications. Here, we removed the virulence factors from three 2,3-BDO-producing strains, Klebsiella pneumoniae KCTC 2242, Klebsiella oxytoca KCTC1686, and K. oxytoca ATCC 43863 through site-specific recombination technique. We generated deletion mutation in wabG gene encoding glucosyltransferase which plays a key role in the synthesis of outer core lipopolysaccharides (LPS) by attaching the first outer core residue d-GalAp to the O-3 position of the l,d-HeppII residue. The morphologies and adhesion properties against epithelial cells were investigated, and the results indicated that the wabG mutant strains were devoid of the outer core LPS and lost the ability to retain capsular structure. The time profile of growth and 2,3-BDO production from K. pneumoniae KCTC 2242 and K. pneumoniae KCTC 2242 ΔwabG were analyzed in batch culture with initial glucose concentration of 70 g/l. The growth was not affected by disrupting wabG gene, but the production of 2,3-BDO decreased from 31.27 to 22.44 g/l in mutant compared with that of parental strain. However, the productions of acetoin and lactate from wabG mutant strain were negligible, whereas that from parental strain reached to ~5 g/l.     

A shortened,two-enzyme pathway for 2,3-butanediol production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The platform chemical 2,3-butanediol (2,3-BDO) is produced by a number of microorganisms via a three-enzyme pathway starting from pyruvate. Here, we report production of 2,3-BDO via a shortened, two-enzyme pathway in Escherichia coli. A synthetic operon consisting of the acetolactate synthase (ALS) and acetoin reductase (AR) genes from Enterobacter under control of the T7 promoter was cloned in an episomal plasmid. E. coli transformed with this plasmid produced 2,3-BDO and the pathway intermediate acetoin, demonstrating that the shortened pathway was functional. To assemble a synthetic operon for inducer- and plasmid-free production of 2,3-BDO, ALS and AR genes were integrated in the E. coli genome under control of the constitutive ackA promoter. Shake flask-level cultivation led to accumulation of ~1 g/L acetoin and ~0.66 g/L 2,3-BDO in the medium. The novel biosynthetic route for 2,3-BDO biosynthesis described herein provides a simple and cost-effective approach for production of this important chemical.     

Engineering Cupriavidus necator H16 for the autotrophic production of (R)-1,3-butanediol

Butanediols are widely used in the synthesis of polymers, specialty chemicals and important chemical intermediates. Optically pure R-form of 1,3-butanediol (1,3-BDO) is required for the synthesis of several industrial compounds and as a key intermediate of β-lactam antibiotic production. The (R)-1,3-BDO can only be produced by application of a biocatalytic process. Cupriavidus necator H16 is an established production host for biosynthesis of biodegradable polymer poly-3-hydroxybutryate (PHB) via acetyl-CoA intermediate. Therefore, the utilisation of acetyl-CoA or its upstream precursors offers a promising strategy for engineering biosynthesis of value-added products such as (R)-1,3-BDO in this bacterium. Notably, C. necator H16 is known for its natural capacity to fix carbon dioxide (CO₂) using hydrogen as an electron donor. Here, we report engineering of this facultative lithoautotrophic bacterium for heterotrophic and autotrophic production of (R)-1,3-BDO. Implementation of (R)-3-hydroxybutyraldehyde-CoA- and pyruvate-dependent biosynthetic pathways in combination with abolishing PHB biosynthesis and reducing flux through the tricarboxylic acid cycle enabled to engineer strain, which produced 2.97 g/L of (R)-1,3-BDO and achieved production rate of nearly 0.4 Cmol Cmol⁻¹ h⁻¹ autotrophically. This is first report of (R)-1,3-BDO production from CO₂.     

Effect of pH on the metabolic flux of Klebsiella oxytoca producing 2,3-butanediol in continuous cultures at different dilution rates

The efficiency of the bioconversion process and the achievable end-product concentration decides the economic feasibility of microbial 2,3-butanediol (2,3-BDO) production. In 2,3-BDO production, optimization of culture condition is required for cell growth and metabolism. Also, the pH is an important factor that influences microbial performance. For different microorganisms and substrates, it has been shown that the distribution of the metabolites in 2,3-BDO fermentation is greatly affected by pH, and the optimum pH for 2,3-BDO production seems dependently linked to the particular strain and the substrate employed. Quantification analysis of intracellular metabolites and metabolic flux analysis (MFA) were used to investigate the effect of pH on the Klebsiella oxytoca producing 2,3-BDO and other organic acids. The main objectives of MFA are the estimation of intracellular metabolic fluxes and the identification of rate-limiting step and the key enzymes. This study was conducted under continuous aerobic conditions at different dilution rates (0.1, 0.2, and 0.3 h^?1) and different pH values (pH 5.5 and 7.0) for the steady-state experimental data. In order to obtain the flux distribution, the extracellular specific rates were calculated from the experimental data using the metabolic network model of K. oxytoca. Intracellular metabolite concentration profiles were generated using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.     

Enhanced 2,3-Butanediol Production by Optimizing Fermentation Conditions and Engineering Klebsiella oxytoca M1 through Overexpression of Acetoin Reductase

Microbial production of 2,3-butanediol (2,3-BDO) has been attracting increasing interest because of its high value and various industrial applications. In this study, high production of 2,3-BDO using a previously isolated bacterium Klebsiella oxytoca M1 was carried out by optimizing fermentation conditions and overexpressing acetoin reductase (AR). Supplying complex nitrogen sources and using NaOH as a neutralizing agent were found to enhance specific production and yield of 2,3-BDO. In fed-batch fermentations, 2,3-BDO production increased with the agitation speed (109.6 g/L at 300 rpm vs. 118.5 g/L at 400 rpm) along with significantly reduced formation of by-product, but the yield at 400 rpm was lower than that at 300 rpm (0.40 g/g vs. 0.34 g/g) due to acetoin accumulation at 400 rpm. Because AR catalyzing both acetoin reduction and 2,3-BDO oxidation in K. oxytoca M1 revealed more than 8-fold higher reduction activity than oxidation activity, the engineered K. oxytoca M1 overexpressing the budC encoding AR was used in fed-batch fermentation. Finally, acetoin accumulation was significantly reduced by 43% and enhancement of 2,3-BDO concentration (142.5 g/L), yield (0.42 g/g) and productivity (1.47 g/L/h) was achieved compared to performance with the parent strain. This is by far the highest titer of 2,3-BDO achieved by K. oxytoca strains. This notable result could be obtained by finding favorable fermentation conditions for 2,3-BDO production as well as by utilizing the distinct characteristic of AR in K. oxytoca M1 revealing the nature of reductase.     

Identification of factors regulating Escherichia coli 2,3-butanediol production by continuous culture and metabolic flux analysis

2,3-Butanediol (2,3-BDO) is an organic compound with a wide range of industrial applications. Although Escherichia coli is often used for the production of organic compounds, the wild-type E. coli does not contain two essential genes in the 2,3-BDO biosynthesis pathway, and cannot ferment 2,3-BDO. Therefore, a 2,3-BDO biosynthesis mutant strain of Escherichia coli was constructed and cultured. To determine the optimum culture factors for 2,3-BDO production, experiments were conducted under different culture environments ranging from strongly acidic to neutral pH. The extracellular metabolite profiles were obtained using high-performance liquid chromatography (HPLC), and the intracellular metabolite profiles were analyzed by ultra-performance liquid chromatography and quadruple time-of-flight mass spectrometry (UPLC/ Q-TOF-MS). Metabolic flux analysis (MFA) was used to integrate these profiles. The metabolite profiles showed that 2,3-BDO production favors an acidic environment (pH 5), whereas cell mass favors a neutral environment. Furthermore, when the pH of the culture fell below 5, both the cell growth and 2,3-BDO production were inhibited.     

Rapid and stable production of 2,3-butanediol by an engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain in a continuous airlift bioreactor

Utilization of renewable feedstocks for the production of bio-based bulk chemicals, such as 2,3-butanediol (2,3-BDO), by engineered strains of the non-pathogenic yeast, Saccharomyces cerevisiae, has recently become an attractive option. In this study, to realize rapid production of 2,3-BDO, a flocculent, 2,3-BDO-producing S. cerevisiae strain YPH499/dPdAdG/BDN6-10/FLO1 was constructed from a previously developed 2,3-BDO-producing strain. Continuous 2,3-BDO fermentation was carried out by the flocculent strain in an airlift bioreactor. The strain consumed more than 90 g/L of glucose, which corresponded to 90% of the input, and stably produced more than 30 g/L of 2,3-BDO over 380 h. The maximum 2,3-BDO productivity was 7.64 g/L/h at a dilution rate of 0.200/h, which was higher than the values achieved by continuous fermentation using pathogenic bacteria in the previous reports. These results demonstrate that continuous 2,3-BDO fermentation with flocculent 2,3-BDO-producing S. cerevisiae is a promising strategy for practical 2,3-BDO production.     

Metabolic engineering of a Saccharomyces cerevisiae strain capable of simultaneously utilizing glucose and galactose to produce enantiopure (2R,3R)-butanediol

2,3-Butanediol (BDO) is an important chemical with broad industrial applications and can be naturally produced by many bacteria at high levels. However, the pathogenicity of these native producers is a major obstacle for large scale production. Here we report the engineering of an industrially friendly host, Saccharomyces cerevisiae, to produce BDO at high titer and yield. By inactivation of pyruvate decarboxylases (PDCs) followed by overexpression of MTH1 and adaptive evolution, the resultant yeast grew on glucose as the sole carbon source with ethanol production completely eliminated. Moreover, the pdc- strain consumed glucose and galactose simultaneously, which to our knowledge is unprecedented in S. cerevisiae strains. Subsequent introduction of a BDO biosynthetic pathway consisting of the cytosolic acetolactate synthase (cytoILV2), Bacillus subtilis acetolactate decarboxylase (BsAlsD), and the endogenous butanediol dehydrogenase (BDH1) resulted in the production of enantiopure (2R,3R)-butanediol (R-BDO). In shake flask fermentation, a yield over 70% of the theoretical value was achieved. Using fed-batch fermentation, more than 100 g/L R-BDO (1100 mM) was synthesized from a mixture of glucose and galactose, two major carbohydrate components in red algae. The high titer and yield of the enantiopure R-BDO produced as well as the ability to co-ferment glucose and galactose make our engineered yeast strain a superior host for cost-effective production of bio-based BDO from renewable resources.     

2,3-Butanediol production by the non-pathogenic bacterium Paenibacillus brasilensis

2,3-Butanediol (2,3-BDO) is of considerable importance in the chemical, plastic, pharmaceutical, cosmetic, and food industries. The main bacterial species producing this compound are considered pathogenic, hindering large-scale productivity. The species Paenibacillus brasilensis is generally recognized as safe (GRAS) and is phylogenetically similar to P. polymyxa, a species widely used for 2,3-BDO production. Here, we demonstrate, for the first time, that P. brasilensis strains produce 2,3-BDO. Total 2,3-BDO concentrations for 15 P. brasilensis strains varied from 5.5 to 7.6 g/l after 8 h incubation at 32 °C in modified YEPD medium containing 20 g/l glucose. Strain PB24 produced 8.2 g/l of 2,3-BDO within a 12-h growth period, representing a yield of 0.43 g/g and a productivity of 0.68 g/l/h. An increase in 2,3-BDO production by strain PB24 was observed using higher concentrations of glucose, reaching 27 g/l of total 2,3-BDO in YEPD containing about 80 g/l glucose within a 72-h growth period. We sequenced the genome of P. brasilensis PB24 and uncovered at least six genes related to the 2,3-BDO pathway at four distinct loci. We also compared gene sequences related to the 2,3-BDO pathway in P. brasilensis PB24 with those of other spore-forming bacteria, and found strong similarity to P. polymyxa, P. terrae, and P. peoriae 2,3-BDO-related genes. Regulatory regions upstream of these genes indicated that they are probably co-regulated. Finally, we propose a production pathway from glucose to 2,3-BDO in P. brasilensis PB24. Although the gene encoding S-2,3-butanediol dehydrogenase (butA) was found in the genome of P. brasilensis PB24, only R,R-2,3- and meso-2,3-butanediol were detected by gas chromatography under the growth conditions tested here. Our findings can serve as a basis for further improvements to the metabolic capabilities of this little-studied Paenibacillus species in relation to production of the high-value chemical 2,3-butanediol.

     

Screening of novel bacteria for the 2,3-butanediol production

Biotechnologically produced 2,3-butanediol (2,3-BDO) is a potential starting material for industrial bulk chemicals such as butadiene or methyl ethyl ketone which are currently produced from fossil feedstocks. So far, the highest 2,3-BDO concentrations have been obtained with risk group 2 microorganisms. In this study, three risk group 1 microorganisms are presented that are so far unknown for an efficient production of 2,3-BDO. The strains Bacillus atrophaeus NRS-213, Bacillus mojavensis B-14698, and Bacillus vallismortis B-14891 were evaluated regarding their ability to produce high 2,3-BDO concentrations with a broad range of different carbon sources. A maximum 2,3-BDO concentration of 60.4 g/L was reached with the strain B. vallismortis B-14891 with an initial glucose concentration of 200 g/L within 55 h in a batch cultivation. Besides glucose, B. vallismortis B-14891 converts 14 different substrates that can be obtained from residual biomass sources to 2,3-BDO. Therefore B. vallismortis B-14891 is a promising candidate for the large-scale production of 2,3-BDO with low-cost substrates.

     

Engineering of Ralstonia eutropha H16 for Autotrophic and Heterotrophic Production of Methyl Ketones

Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H₂ and CO₂ under aerobic conditions. Under conditions of nutrient imbalance, R. eutropha produces copious amounts of poly[(R)-3-hydroxybutyrate] (PHB). Its ability to utilize CO₂ as a sole carbon source renders it an interesting new candidate host for the production of renewable liquid transportation fuels. We engineered R. eutropha for the production of fatty acid-derived, diesel-range methyl ketones. Modifications engineered in R. eutropha included overexpression of a cytoplasmic version of the TesA thioesterase, which led to a substantial (>150-fold) increase in fatty acid titer under certain conditions. In addition, deletion of two putative β-oxidation operons and heterologous expression of three genes (the acyl coenzyme A oxidase gene from Micrococcus luteus and fadB and fadM from Escherichia coli) led to the production of 50 to 65 mg/liter of diesel-range methyl ketones under heterotrophic growth conditions and 50 to 180 mg/liter under chemolithoautotrophic growth conditions (with CO₂ and H₂ as the sole carbon source and electron donor, respectively). Induction of the methyl ketone pathway diverted substantial carbon flux away from PHB biosynthesis and appeared to enhance carbon flux through the pathway for biosynthesis of fatty acids, which are the precursors of methyl ketones.     

Identification and characterization of a short-chain acyl dehydrogenase from Klebsiella pneumoniae and its application for high-level production of l-2,3-butanediol

Klebsiella pneumoniae synthesize large amounts of l-2,3-butanediol (l-2,3-BD), but the underlying mechanism has been unknown. In this study, we provide the first identification and characterization of an l-2,3-BD dehydrogenase from K. pneumoniae, demonstrating its reductive activities toward diacetyl and acetoin, and oxidative activity toward l-2,3-BD. Optimum pH, temperature, and kinetics determined for reductive and oxidative reactions support the preferential production of 2,3-BD during cell growth. Synthesis of l-2,3-BD was remarkably enhanced by increasing gene dosage, reaching levels that, to the best of our knowledge, are the highest achieved to date.