RESUMEN
BACKGROUND: (R,R)-2,3-butanediol (BDO) is employed in a variety of applications and is gaining prominence due to its unique physicochemical features. The use of glycerol as a carbon source for 2,3-BDO production in Klebsiella pneumoniae has been limited, since 1,3-propanediol (PDO) is generated during glycerol fermentation. RESULTS: In this study, the inactivation of the budC gene in K. pneumoniae increased the production rate of (R,R)-2,3-BDO from 21.92 ± 2.10 to 92.05 ± 1.20%. The major isomer form of K. pneumoniae (meso-2,3-BDO) was shifted to (R,R)-2,3-BDO. The purity of (R,R)-2,3-BDO was examined by agitation speed, and 98.54% of (R,R)-2,3-BDO was obtained at 500 rpm. However, as the cultivation period got longer, the purity of (R,R)-2,3-BDO declined. For this problem, a two-step agitation speed control strategy (adjusted from 500 to 400 rpm after 24 h) and over-expression of the dhaD gene involved in (R,R)-2,3-BDO biosynthesis were used. Nevertheless, the purity of (R,R)-2,3-BDO still gradually decreased over time. Finally, when pure glycerol was replaced with crude glycerol, the titer of 89.47 g/L of (R,R)-2,3-BDO (1.69 g/L of meso-2,3-BDO), productivity of 1.24 g/L/h, and yield of 0.35 g/g consumed crude glycerol was achieved while maintaining a purity of 98% or higher. CONCLUSIONS: This study is meaningful in that it demonstrated the highest production and productivity among studies in that produced (R,R)-2,3-BDO with a high purity in Klebsiella sp. strains. In addition, to the best of our knowledge, this is the first study to produce (R,R)-2,3-BDO using glycerol as the sole carbon source.
Asunto(s)
Butileno Glicoles , Fermentación , Glicerol , Klebsiella pneumoniae , Klebsiella pneumoniae/metabolismo , Klebsiella pneumoniae/genética , Glicerol/metabolismo , Butileno Glicoles/metabolismo , Ingeniería Metabólica/métodos , Oxidación-Reducción , Estereoisomerismo , Glicoles de Propileno/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genéticaRESUMEN
BACKGROUND: Chemical fertilizers have greatly contributed to the development of agriculture, but alternative fertilizers are needed for the sustainable development of agriculture. 2,3-butanediol (2,3-BDO) is a promising biological plant growth promoter. RESULTS: In this study, we attempted to develop an effective strategy for the biological production of highly pure R,R-2,3-butanediol (R,R-2,3-BDO) by Paenibacillus polymyxa fermentation. First, gamma-ray mutagenesis was performed to obtain P. polymyxa MDBDO, a strain that grew faster than the parent strain and had high production of R,R-2,3-BDO. The activities of R,R-2,3-butanediol dehydrogenase and diacetyl reductase of the mutant strain were increased by 33% and decreased by 60%, respectively. In addition, it was confirmed that the carbon source depletion of the fermentation broth affects the purity of R,R-2,3-BDO through batch fermentation. Fed-batch fermentation using controlled carbon feeding led to production of 77.3 g/L of R,R-2,3-BDO with high optical purity (> 99% of C4 products) at 48 h. Additionally, fed-batch culture using corn steep liquor as an alternative nitrogen source led to production of 70.3 g/L of R,R-2,3-BDO at 60 h. The fed-batch fermentation broth of P. polymyxa MDBDO, which contained highly pure R,R-2,3-BDO, significantly stimulated the growth of soybean and strawberry seedlings. CONCLUSIONS: This study suggests that P. polymyxa MDBDO has potential for use in biological plant growth promoting agent applications. In addition, our fermentation strategy demonstrated that high-purity R,R-2,3-BDO can be produced at high concentrations using P. polymyxa.
Asunto(s)
Paenibacillus polymyxa , Paenibacillus , Paenibacillus polymyxa/genética , Carbono , Fertilizantes , Butileno Glicoles , Fermentación , Paenibacillus/genéticaRESUMEN
This study investigated the effects of different amino acids on the fermentation of pure inulin and glucose using Paenibacillus polymyxa ZJ-9 in order to improve the production of R,R-2,3-butanediol (R,R-2,3-BD) respectively. The inulin extract from Jerusalem artichoke tubers contained 19 common amino acids, which were detected by HPLC. Arg featured the highest content (1290 mg l-1 ). A single add-back experiment of 20 common amino acids indicated that Asn, Ser, His and Arg are key amino acids in R,R-2,3-BD synthesis during inulin fermentation using P. polymyxa ZJ-9. The corresponding yields of R,R-2,3-BD reached 24·32, 22·32, 22·03 and 21·04 g l-1 after the four key amino acids (1·5 g l-1 each) and glucose were evaluated in this fermentation. The yields were considerably higher than that of the control group (12·11 g l-1 ). With the addition of the mixture of four amino acids (1·5 g l-1 each), the highest yields of R,R-2,3-BD (25·07 and 17·47 g l-1 ) were obtained with the increase of 107·0 and 89·1% during the fermentation of glucose and pure inulin respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Paenibacillus polymyxa is a micro-organism with a reported potential for industrialized production of R,R-2,3-butanediol. The nitrogen sources have a significant effect on R,R-2,3-butanediol formation using P. polymyxa. This study demonstrated a highly efficient new method to improve the yield of R,R-2,3-butanediol without adding other nitrogen sources except amino acids during the fermentation. This will therefore decrease the production cost of R,R-2,3-butanediol and provide a new strategy for promoting synthesis of amino acid-dependent products.
Asunto(s)
Aminoácidos/metabolismo , Butileno Glicoles/metabolismo , Glucosa/metabolismo , Inulina/metabolismo , Paenibacillus polymyxa/metabolismo , Fermentación , Helianthus/químicaRESUMEN
The present study describes the use of metabolic engineering to achieve the production of R,R-2,3-butanediol (R,R-2,3-BD) of ultra-high optical purity (>99.99%). To this end, the diacetyl reductase (DAR) gene (dud A) of Paenibacillus polymyxa ZJ-9 was knocked out via homologous recombination between the genome and the previously constructed targeting vector pRN5101-L'C in a process based on homologous single-crossover. PCR verification confirmed the successful isolation of the dud A gene disruption mutant P. polymyxa ZJ-9-â³dud A. Moreover, fermentation results indicated that the optical purity of R,R-2,3-BD increased from about 98% to over 99.99%, with a titer of 21.62â¯g/L in Erlenmeyer flasks. The latter was further increased to 25.88â¯g/L by fed-batch fermentation in a 5-L bioreactor.
Asunto(s)
Butileno Glicoles/química , Paenibacillus , Fermentación , Recombinación Homóloga , Paenibacillus polymyxa/genética , Paenibacillus polymyxa/metabolismoRESUMEN
When modifying the metabolism of living organisms with the aim of achieving biosynthesis of useful compounds, it is essential to ensure that it is possible to achieve overall redox balance. We propose a generalized strategy for this, based on fine-tuning of respiration. The strategy was applied on metabolically engineered Lactococcus lactis strains to optimize the production of acetoin and (R,R)-2,3-butanediol (R-BDO). In the absence of an external electron acceptor, a surplus of two NADH per acetoin molecule is produced. We found that a fully activated respiration was able to efficiently regenerate NAD+, and a high titer of 371mM (32g/L) of acetoin was obtained with a yield of 82% of the theoretical maximum. Subsequently, we extended the metabolic pathway from acetoin to R-BDO by introducing the butanediol dehydrogenase gene from Bacillus subtilis. Since one mole of NADH is consumed when acetoin is converted into R-BDO per mole, only the excess of NADH needs to be oxidized via respiration. Either by fine-tuning the respiration capacity or by using a dual-phase fermentation approach involving a switch from fully respiratory to non-respiratory conditions, we obtained 361mM (32g/L) R-BDO with a yield of 81% or 365mM (33g/L) with a yield of 82%, respectively. These results demonstrate the great potential in using finely-tuned respiration machineries for bio-production.
Asunto(s)
Acetoína/metabolismo , Butileno Glicoles/metabolismo , Lactococcus lactis/enzimología , Ingeniería Metabólica , Consumo de Oxígeno , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Lactococcus lactis/genéticaRESUMEN
R,R-2,3-butanediol (R,R-2,3-BD) was produced by Paenibacillus polymyxa ZJ-9, which was capable of utilizing inulin without previous hydrolysis. The Jerusalem artichoke pomace (JAP) derived from the conversion of Jerusalem artichoke powder into inulin extract, which was usually used for biorefinery by submerged fermentation (SMF), was utilized in solid state fermentation (SSF) to produce R,R-2,3-BD. In this study, the fermentation parameters of SSF were optimized and determined in flasks. A novel bioreactor was designed and assembled for the laboratory scale-up of SSF, with a maximum yield of R,R-2,3-BD (67.90 g/kg (JAP)). This result is a 36.3% improvement compared with the flasks. Based on the same bath of Jerusalem artichoke powder, the total output of R,R-2,3-BD increased by 38.8% for the SSF of JAP combined with the SMF of inulin extraction. Overall, the utilization of JAP for R,R-2,3-BD production was beneficial to the comprehensive utilization of Jerusalem artichoke tuber.
Asunto(s)
Reactores Biológicos , Butileno Glicoles/metabolismo , Helianthus/química , Paenibacillus polymyxa/crecimiento & desarrollo , Tubérculos de la Planta/químicaRESUMEN
Many microorganisms could naturally produce (R, R)-2,3-butanediol ((R, R)-2,3-BD), which has unique applications due to its special chiral group and spatial configuration. But the low enantio-purity of the product hindered the development of large-scale production. In this work, a synthetic constitutive metabolic pathway for enantiomerically pure (R, R)-2,3-BD biosynthesis was constructed in Escherichia coli with vector pUC6S, which does not contain any lac sequences. The expression of this artificial constructed gene cluster was optimized by using two different strength of promoters (AlperPLTet01 (P01) and AlperBB (PBB)). The strength of P01 is twice stronger than PBB. The fermentation results suggested that the yield of (R, R)-2,3-BD was higher when using the stronger promoter. Compared with the wild type, the recombinant strain E. coli YJ2 produced a small amount of acetic acid and showed higher glucose consumption rate and higher cell density, which indicated a protection against acetic acid inhibition. In order to further increase the (R, R)-2,3-BD production by reducing the accumulation of its precursor acetoin, the synthetic operon was reconstructed by adding the strong promoter P01 in front of the gene ydjL coding for the enzyme of (R, R)-2,3-BD dehydrogenase which catalyzes the conversion of acetoin to (R, R)-2,3-BD. The engineered strain E. coli YJ3 showed a 20 % decrease in acetoin production compared with that of E. coli YJ2. After optimization the fermentation conditions, 30.5 g/L of (R, R)-2,3-BD and 3.2 g/L of acetoin were produced from 80 g/L of glucose within 18 h, with an enantio-purity over 99 %.
Asunto(s)
Butileno Glicoles/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Redes y Vías Metabólicas , Acetoína/metabolismo , Técnicas de Cultivo Celular por Lotes , Fermentación , Glucosa/metabolismo , Operón Lac , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas/genética , Familia de Multigenes , Regiones Promotoras Genéticas , Biología Sintética/métodosRESUMEN
The NAD(+)-dependent formate dehydrogenase (FDH) gene from Candida boidinii was introduced into Paenibacillus polymyxa ZJ-9. The effects of this exogenous gene on the growth of the recombinant strain P. polymyxa XG-1, FDH activity, intracellular NADH and NAD(+) level and the synthesis of R,R-2,3-butanediol (R,R-2,3-BD) were determined. Results from the fermentation in the 7.5L bioreactor showed that the exogenous FDH was highly expressed in the recombinant strain. The titers of NADH, lactic acid, ethanol, NADH/NAD(+), and CO2 excretion rate (CER) of the recombinant strain increased considerably, while acetoin and formic acid decreased significantly. The highest titers of R,R-2,3-BD by the recombinant strain in batch and fed-batch fermentation were 36.8g/L and 51.3g/L, increased 10.2% and 8.0% compared with the parent strain, respectively. This study confirmed that coenzyme regeneration system can manipulate substance metabolism in bacteria, and is an efficient way for promoting the synthesis of NADH-dependent products.
Asunto(s)
Coenzimas/metabolismo , Espacio Intracelular/metabolismo , Análisis de Flujos Metabólicos , NAD/metabolismo , Paenibacillus/metabolismo , Técnicas de Cultivo Celular por Lotes , Vías Biosintéticas , Butileno Glicoles/metabolismo , Fermentación , Formiato Deshidrogenasas/genética , Formiato Deshidrogenasas/metabolismo , Genes Bacterianos , Vectores Genéticos/metabolismo , Paenibacillus/enzimología , Paenibacillus/genética , Paenibacillus/crecimiento & desarrolloRESUMEN
Microbial fermentation produces a racemic mixture of 2,3-butanediol ((R,R)-BD, (S,S)-BD, and meso-BD), and the compositions and physiochemical properties vary from microorganism to microorganism. Although the meso form is much more difficult to transport and store because of its higher freezing point than those of the optically active forms, most microorganisms capable of producing 2,3-BD mainly yield meso-2,3-BD. Thus, we developed a metabolically engineered (R,R)-2,3-BD overproducing strain using a Klebsiella oxytoca ΔldhA ΔpflB strain, which shows an outstanding 2,3-BD production performance with more than 90 % of the meso form. A budC gene encoding 2,3-BD dehydrogenase in the K. oxytoca ΔldhA ΔpflB strain was replaced with an exogenous gene encoding (R,R)-2,3-BD dehydrogenase from Paenibacillus polymyxa (K. oxytoca ΔldhA ΔpflB ΔbudC::PBDH strain), and then its expression level was further amplified with using a pBBR1MCS plasmid. The fed-batch fermentation of the K. oxytoca ΔldhA ΔpflB ΔbudC::PBDH (pBBR-PBDH) strain with intermittent glucose feeding allowed the production of 106.7 g/L of (R,R)-2,3-BD [meso-2,3-BD, 9.3 g/L], with a yield of 0.40 g/g and a productivity of 3.1 g/L/h, which should be useful for the industrial application of 2,3-BD.
Asunto(s)
Butileno Glicoles/metabolismo , Klebsiella oxytoca/metabolismo , Ingeniería Metabólica , Bacillus/enzimología , Bacillus/genética , Técnicas de Cultivo Celular por Lotes , Reactores Biológicos , Fermentación , Glucosa/metabolismo , Glucosa/farmacología , Klebsiella oxytoca/efectos de los fármacos , Klebsiella oxytoca/enzimología , Klebsiella oxytoca/genéticaRESUMEN
The objective of this work was to construct a non-pathogenic Klebsiella pneumonia strain that can produce optically high concentrated (R,R)-2,3-BDO. A K. pneumonia mutant lacking the pathogenic factor was used as the host strain. In order to construct a K. pneumonia strain that would biosynthesize high concentrated (R,R)-2,3-BDO, gene deletion and over-expression methods were combined; firstly, the 2,3-BDO dehydrogenase (budC) gene was deleted to re-direct utilization of the carbon source to (R,R)-2,3-BDO biosynthesis; secondly, the two glycerol dehydrogenase (GDH) enzymes in K. pneumonia (DhaD and GldA) were over-expressed to maximize (R,R)-2,3-BDO biosynthesis; and thirdly, the lactate dehydrogenase (ldhA) gene was deleted to minimize the accumulation of lactate. SGSB112, a non-pathogenic strain of K. pneumonia that can produce optically high concentrated (R,R)-2,3-BDO, was constructed as above. Approximately 36% of the carbon source was converted to (R,R)-2,3-BDO by SGSB112, achieving a production of 61gL(-1) (R,R)-2,3-BDO in a fed-batch fermentation. On the other hand, meso-2,3-BDO was produced 1.4gL(-1) and (S,S)-2,3-BDO was not detected. This study provides an insight into 2,3-BDO biosynthesis in K. pneumonia and demonstrates the achievement of high-yield production of optically high concentrated (R,R)-2,3-BDO through constructing a strain by genetic modification and metabolic engineering.
Asunto(s)
Proteínas Bacterianas/genética , Butileno Glicoles/metabolismo , Mejoramiento Genético/métodos , Klebsiella/metabolismo , Técnicas de Cultivo Celular por Lotes/métodos , Eliminación de Gen , Klebsiella/genética , Ingeniería Metabólica/métodosRESUMEN
Enantiomerically pure (R, R)-2,3-butanediol has unique applications due to its special chiral group and spatial configuration. Currently, its chemical production route has many limitations. In addition, no native microorganisms can accumulate (R, R)-2,3-butanediol with an enantio-purity over 99%. Herein, we constructed a synthetic metabolic pathway for enantiomerically pure (R, R)-2,3-butanediol biosynthesis in Escherichia coli. The fermentation results suggested that introduction of the synthetic metabolic pathway redistributed the carbon fluxes to the neutral (R, R)-2,3-butanediol, and thus protected the strain against the acetic acid inhibition. Additionally, it showed that the traditionally used isopropyl beta-D-thiogalactoside (IPTG) induction displayed negative effect on (R, R)-2,3-butanediol biosynthesis in the recombinant E. coli, which was probably due to the protein burden. With no IPTG addition, the (R, R)-2,3-butanediol concentration reached 115 g/L by fed-batch culturing of the recombinant E. coli, with an enantio-purity over 99%, which is suitable for the pilot-scale production.
Asunto(s)
Butileno Glicoles/metabolismo , Escherichia coli/metabolismo , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas , Técnicas de Cultivo Celular por Lotes/métodos , Escherichia coli/genética , Fermentación , Microbiología Industrial/métodos , Isopropil Tiogalactósido/metabolismoRESUMEN
Paenibacillus polymyxa DSM 365, an efficient producer of (R,R)-2,3-butanediol, is known to show the highest production titer and productivity reported to date. Here, the first draft genome sequence of this promising strain may provide the genetic basis for further insights into the molecular mechanisms underlying the production of (R,R)-2,3-butanediol with high optical purity and at a high titer. It will also facilitate the design of rational strategies for further strain improvements, as well as construction of artificial biosynthetic pathways through synthetic biology for asymmetric synthesis of chiral 2,3-butanediol or acetoin in common microbial hosts.
Asunto(s)
Butileno Glicoles/metabolismo , Genoma Bacteriano/genética , Paenibacillus/genética , Paenibacillus/metabolismo , Butileno Glicoles/química , Butileno Glicoles/aislamiento & purificación , Datos de Secuencia Molecular , EstereoisomerismoRESUMEN
Cellular redox status and oxygen availability influence the product formation. Herein, decreasing agitation speed or adding vitamin C (Vc) achieved the 2,3-BDL yield of 0.40 g g(-1) or 0.39 g g(-1)glucose under batch fermentation, respectively. To our knowledge, this is the highest 2,3-BDL yield reported so far for Paenibacillus polymyxa without adding acetic acid. The NADH/NAD(+) ratio and 2,3-BDL titer could be increased significantly by reducing the agitation speed or adding Vc, indicating that the enhancement of 2,3-BDL is closely associated with the adjustment of NADH/NAD(+) ratio. Especially, Vc addition elevated the 2,3-BDL titer from 43.66 g L(-1) to 71.71 g L(-1) within 54 h under fed-batch fermentation. This is the highest titer of 2,3-BDL so far reported for P. polymyxa from glucose fermentation. This work provides a new strategy to improve 2,3-BDL production and helps us to understand the responses of P. polymyxa to extracellular oxidoreduction potential.
Asunto(s)
Butileno Glicoles/metabolismo , Espacio Extracelular/metabolismo , Paenibacillus/metabolismo , Ácido Ascórbico/farmacología , Técnicas de Cultivo Celular por Lotes , Borohidruros/farmacología , Fermentación/efectos de los fármacos , Espacio Intracelular/metabolismo , Datos de Secuencia Molecular , NAD/metabolismo , Oxidación-Reducción/efectos de los fármacos , Oxígeno/farmacología , Paenibacillus/efectos de los fármacos , TemperaturaRESUMEN
A 2,3-butanediol dehydrogenase (BDH) from Paenibacillus polymyxa ZJ-9 was purified to homogeneity via fractional ammonium sulfate precipitation, followed by two steps of anion-exchange chromatography using DEAE-Sepharose and Source 15Q, obtaining a 35-fold increase in specific activity and 34.9% yield. The molecular weights of the purified BDH subunit and holoenzyme were 44.5 and 90.0 kDa, respectively, as detected via SDS-PAGE and gel filtration chromatography. These results were significantly different from those of other reported BDHs. Substrate specificity experiments showed that the enzyme could function preferentially as a reductase rather than as a dehydrogenase, and was mainly responsible for the reduction of R-acetoin to R,R-2,3-butanediol. Gene cloning, sequencing, and expression experiments further demonstrate that this enzyme was a new type of BDH.