Fermentation strategies for 1,3-propanediol production from glycerol using a genetically engineered Klebsiella pneumoniae strain to eliminate by-product formation

We generated a genetically engineered Klebsiella pneumoniae strain (AK-VOT) to eliminate by-product formation during production of 1,3-propanediol (1,3-PD) from glycerol. In the present study, the glycerol-metabolizing properties of the recombinant strain were examined during fermentation in a 5 L bioreactor. As expected, by-product formation was completely absent (except for acetate) when the AK-VOT strain fermented glycerol. However, 1,3-PD productivity was severely reduced owing to a delay in cell growth attributable to a low rate of glycerol consumption. This problem was solved by establishing a two-stage process separating cell growth from 1,3-PD production. In addition, nutrient co-supplementation, especially with starch, significantly increased 1,3-PD production from glycerol during fed-batch fermentation by AK-VOT in the absence of by-product formation.     

Identification and utilization of a 1,3-propanediol oxidoreductase isoenzyme for production of 1,3-propanediol from glycerol in Klebsiella pneumoniae

In a previous study, we showed that 1,3-propanediol (1,3-PD) was still produced from glycerol by the Klebsiella pneumoniae mutant strain defective in 1,3-PD oxidoreductase (DhaT), although the production level was lower compared to the parent strain. As a potential candidate for another putative 1,3-PD oxidoreductase, we identified and characterized a homolog of Escherichia coli yqhD (88% homology in amino acid sequence), which encodes an alcohol dehydrogenase and is well known to replace the function of DhaT in E. coli. Introduction of multiple copies of the yqhD homolog restored 1,3-PD production in the mutant K. pneumoniae strain defective in DhaT. In addition, by-product formation was still eliminated in the recombinant strain due to the elimination of the glycerol oxidative pathway. An increase in NADP-dependent 1,3-PD oxidoreductase activity was observed in the recombinant strain harboring multiple copies of the yqhD homolog. The level of 1,3-PD production during batch fermentation in the recombinant strain was comparable to that of the parent strain; further engineering can generate an industrial strain producing 1,3-propanediol.     

克雷伯杆菌甘油脱氢酶和1,3-丙二醇氧化还原酶的动力学机制

本研究主要对克雷伯杆菌甘油转化1,3-丙二醇代谢途径中的2个关键酶甘油脱氢酶(GDH)、1,3-丙二醇氧化还原酶(PDOR)反应机制和动力学进行了研究。首先,通过初速度和产物抑制动力学研究确定了GDH、PDOR双底物酶促反应机制为有序BiBi机制,明确了由反应物消耗到产物生成之间的历程。其次,建立了GDH、PDOR双底物酶促反应动力学模型,由动力学模型可知,在偶合反应中,如果GDH和PDOR酶量相同,GDH氧化反应成为限速反应,而辅酶I将主要以氧化型NAD+形式存在。动力学信息为酶法合成1,3-丙二醇和代谢工程研究提供理论指导。     

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.     

克雷伯肺炎杆菌1,3-丙二醇氧化还原酶基因克隆及表达条件研究

1,3-丙二醇氧化还原酶是甘油歧化为1,3-丙二醇的一种关键酶。本研究从克雷伯肺炎杆菌(Klebsiella pneumoniae)基因组中,用PCR方法克隆了其编码基因dhaT。TA克隆测序正确后,构建胞内表达载体pET-28a-dhaT和分泌表达载体pET-22b-dhaT,然后转化E.coliBL21(DE3)进行原核表达。表达部位确定、SDS-PAGE和酶活分析表明,该酶得到了高水平表达。其中使用pET-22b表达的目的蛋白大都是不溶的包涵体;而使用pET-28a表达的目的蛋白胞内可溶,占胞内可溶总蛋白的45%,占菌体总蛋白的25%。常规(30℃)诱导表达即呈现1,3-丙二醇氧化还原酶活性,但低温(20℃)14 h诱导显示3.7倍的酶活性。     

Production of 1,3-propanediol by Klebsiella pneumoniae from glycerol broth

Broth containing 152 g glycerol l⁻¹ from Candida krusei culture was converted to 1,3-propanediol by Klebsiella pneumoniae. Residual glucose in the broth promoted growth of K. pneumoniae while acetate was inhibitory. After desalination treatment of glycerol broth by electrodialysis, the acetate in the broth was removed. A fed-batch culture with electrodialytically pretreated broth as␣substrate was developed giving 53 g 1,3- propanediol l⁻¹ with a yield of 0.41 g g⁻¹ glycerol and a productivity of 0.94 g l⁻¹ h⁻¹.     

Microbial production of 1,3-propanediol by Klebsiella pneumoniae using crude glycerol from biodiesel preparations

1,3-Propanediol (1,3-PD) was produced by Klebsiella pneumoniae using crude glycerol obtained from biodiesel production. The 1,3-PD concentration of 51.3 g/l⁻¹ on crude glycerol from alkali-catalyzed methanolysis of soybean oil was comparable to that of 53 g/l⁻¹ on crude glycerol derived from a lipase-catalyzed process. The productivities of 1.7 g l⁻¹ h⁻¹ on crude glycerol were comparable to that of 2 g l⁻¹ h⁻¹ on pure glycerol. It could be concluded that the crude glycerol could be directly converted to 1,3-PD without any prior purification.     

利用PDOR同工酶基因yqhD对产1,3-丙二醇克雷伯氏杆菌进行基因工程改造

由于Klebsiella pneumoniae 1,3-丙二醇合成途径中,加强甘油脱水酶基因表达,导致因NADH供应不足使3-羟基丙醛累积,并对菌体生长及1,3-丙二醇合成造成负面影响。为改善Klebsiella pneumoniae 1,3-丙二醇合成途径,本文利用PCR技术从大肠杆菌(Escherichia coli)中扩增出以NADPH 为辅酶的1,3-丙二醇氧化还原酶同工酶编码基因yqhD,从克雷伯氏杆菌中扩增出2.66kb的甘油脱水酶基因（dhaB）,构建了产1,3-丙二醇关键酶基因的串联载体pEtac-dhaB-tac-yqhD,并将其转入到野生克雷伯氏杆菌(Klebsiella pneumoniae)中,重组载体得到了表达。通过初步发酵,重组后的克雷伯氏杆菌产量比原始菌高20%左右,副产物中乙酸和丁二醇分别下降30%左右。     

Pilot-scale production of 1,3-propanediol using Klebsiella pneumoniae

The conversion of glycerol to 1,3-propanediol (PDO) using Klebsiella pneumoniae M5al under anaerobic condition was scaled up from scale 5 to 5000 l in series. A simple strategy for scale-up was to transfer the optimized conditions of a lab scale bioreactor to pilot-scale fermentation. Multistage inocula were developed and their fermentation abilities were assessed in a small-scale fermenter. The experimental results showed that inoculum development in the early steps of a scale-up process could influence the outcomes of a large scale fermentation. Through three-stage liquid inoculum development and a pulse addition of (NH₄)₂SO₄ and yeast extract at 30 h of fermentation, the best results in a 5000 l fermentation were achieved leading to 58.8 g l⁻¹ 1,3-propanediol with a yield of 0.53 mol mol⁻¹ glycerol and productivity of 0.92 g l⁻¹ h⁻¹. This is the first report on pilot-scale 1,3-propanediol production using K. pneumoniae.     

1,3-Propanediol oxidoreductase production with Klebsiella pneumoniae DSM2026

We report a Klebsiella pneumoniae DSM2026 fermentation procedure for the efficient production of a key enzyme of 1,3-propanediol formation: 1,3-propanediol oxidoreductase (E.C. 1.1.1.202). The fermentation process is composed of an aerobic batch phase on glucose and glycerol and an anaerobic phase on glycerol. The role of the aerobic phase is to produce sufficiently high cell mass (12.9–14.6 g/l dry weight) and to activate the aerobic branch of the Klebsiella glycerol pathway, whereas in the anaerobic phase there is a rapid initiation of 1,3-propanediol oxidoreductase formation. A fast change from an aerobic to an anaerobic environment led to a redox imbalance, which resulted in the abrupt activation of the anaerobic branch of glycerol utilization, with the occurrence of a high 1,3-propanediol-oxidoreductase activity. A mathematical model with substrate inhibition showed that the adequate glycerol concentration for enzyme production was 14–16 g/l. The combination of the optimal substrate concentration together with the subsequent use of glucose and glycerol resulted in 90.6 ± 11.6 U enzyme activity referred to 1 l of fermentation broth and 10.3 ± 0.9 U/(1 h) productivity.     

High-level expression of the 1,3-propanediol oxidoreductase from klebsiella pneumoniae in escherichia coli

As one of four key enzymes in glycerol dismutation process, 1,3-propanediol oxidoreductase (EC.1.1.1.202) is important in converting glycerol to 1,3-propanediol in Klebsiella pneumoniae. The dhaT gene encoding 1,3-propanediol oxidoreductase was amplified by polymerase chain reaction (PCR) using the genome DNA of K. pneumoniae as template, and then cloned into cloning vector pMD18-T. After DNA sequence was determined, the dhaT gene was subcloned into Escherichia coli expression vector pET-22b (+) and pET-28a (+). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that both the recombinant E. coli BL21 (DE3) (pET-22b (+)-dhaT) and E. coli BL21(DE3)(pET-28a (+)-dhaT) expressed predicted 42-kDa 1,3-propanediol oxidoreductase after induced by isopropyl-β-d-thiogalactopyranoside (IPTG), and the recombinant enzyme of E. coli BL21 (DE3) (pET-28a (+)-dhaT) was mostly in soluble form, and exhibited high activity (96.8 U/mL culture). The recombinant enzyme was purified and biochemically characterized. The apparent K _m values of the enzyme for 1,3-propanediol and NAD⁺ were 8.5 and 0.21 mM, respectively. The enzyme had maximum activity at pH 9.5 and 30°C.     

来源于克雷伯氏菌的1,3-丙二醇氧化还原酶的同源建模

用Swiss—Model和Modeller对来源于Klebsiella pneumonide的1,3-丙二醇氧化还原酶（PDOR）进行三级结构建模,并对所得的6个目标模型进行评价和比较,从中选择最好的一个模型,预测了辅酶NADP＋和Fe2＋在PDOR结构空间的近似位置,并定位了与NADP^＋和Fe^2＋作用的相关残基。     

Simultaneous production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol by a recombinant strain of Klebsiella pneumoniae

In this study, an aldehyde dehydrogenase (ALDH) was over-expressed in Klebsiella pneumoniae for simultaneous production of 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO). Various genes encoding ALDH were cloned and expressed in K. pneumoniae, and expression of Escherichia colialdH resulted in the highest 3-HP titer in anaerobic cultures in shake flasks. Anaerobic fed-batch culture of this recombinant strain was further performed in a 5-L reactor. The 3-HP concentration and yield reached 24.4 g/L and 0.18 mol/mol glycerol, respectively, and at the same time 1,3-PDO achieved 49.3 g/L with a yield of 0.43 mol/mol in 24 h. The overall yield of 3-HP plus 1,3-PDO was 0.61 mol/mol. Over-expression of the E. coli AldH also reduced the yields of by-products except for lactate. This study demonstrated the possibility of simultaneous production of 3-HP and 1,3-PDO by K. pneumoniae under anaerobic conditions without supply of vitamin B₁₂.     

Fermentation of glycerol to 1,3-propanediol and 2,3-butanediol by Klebsiella pneumoniae

Klebsiella pneumoniae was shown to convert glycerol to 1,3-propanediol, 2,3-butanediol and ethanol under conditions of uncontrolled pH. Formation of 2,3-butanediol starts with some hours' delay and is accompanied by a reuse of the acetate that was formed in the first period. The fermentation was demonstrated in the type strain of K. pneumoniae, but growth was better with the more acid-tolerant strain GT1, which was isolated from nature. In continuous cultures in which the pH was lowered stepwise from 7.3 to 5.4, 2,3-butanediol formation started at pH 6.6 and reached a maximum yield at pH 5.5, whereas formation of acetate and ethanol declined in this pH range. 2,3-Butanediol and acetoin were also found among the products in chemostat cultures grown at pH 7 under conditions of glycerol excess but only with low yields. At any of the pH values tested, excess glycerol in the culture enhanced the butanediol yield. Both effects are seen as a consequence of product inhibition, the undissociated acid being a stronger trigger than the less toxic diols and acid anions. The possibilities for using the fermentation type described to produce 1,3-propanediol and 2,3-butanediol almost without by-products are discussed. Received: 4 February 1998 / Received revision: 30 March 1998 / Accepted: 13 April 1998     

Microbial production of 1,3-propanediol from glycerol by Klebsiella pneumoniae under micro-aerobic conditions up to a pilot scale

1,3-Propanediol (1,3-PD) can be produced from glycerol by Klebsiella pneumoniae under micro-aerobic conditions. Recently, this fed-batch fermentation process has been successfully scaled up to 1 m³. The final 1,3-PD concentration, molar yield and volumetric productivity of 72 g l⁻¹, 57% and 2.1 g l⁻¹ h⁻¹, respectively, are close to those of 75 g l⁻¹, 61%, and 2.2 g l⁻¹ h⁻¹ under anaerobic conditions. This process would be suitable for the production of 1,3-PD on a large scale.     

Use of ceramic-based cell immobilization to produce 1,3-propanediol from biodiesel-derived waste glycerol with Klebsiella pneumoniae

Aims: The feasibility of the continuous production of a valuable bioplastic raw material, namely 1,3‐propanediol (1,3‐PDO) from biodiesel by‐product glycerol, using immobilized cells was investigated. In addition, the effect of hydraulic retention time (HRT) was also analysed. Methods and Results: Ceramic balls and ceramic rings were used for the immobilization of a locally isolated strain; Klebsiella pneumoniae (GenBank no. 27F HM063413 ). HRT of 1 h is the best one in terms of volumetric production rate (g 1,3‐PDO l^?1 h^?1). The results indicated that ceramic‐based cell immobilization achieved a 2‐fold higher production rate (10 g 1,3‐PDO l^?1 h^?1) in comparison with suspended cell system (4·9 g 1,3‐PDO l^?1 h^?1). Conclusions: Continuous cultures with immobilized cells revealed that 1,3‐PDO production was more effective and more stable than suspended culture systems. Furthermore, cell immobilization had also obvious benefits especially for resistance of the production for extreme conditions (high organic loading rates, cell washouts). The results were important for understanding the significance of continuous immobilization process among other well‐known 1,3‐PDO fermentation processes. Significance and Impact of the Study: This work is a promising process for further studies, as the immobilized micro‐organism was able to reach high volumetric production rates at short HRT, it has an important role in tolerating and converting glycerol during fermentation. Therefore, HRT is a very significant operational parameter (P value <0·05) directly affecting the bioreactor performance and production rate.     

Fast conversion of glycerol to 1,3-propanediol by a new strain of Klebsiella pneumoniae

Five bacterial strains screened from a batch of 39 samples could convert glycerol anaerobically to 1,3-propanediol (1,3-PD). One of the strains, XJ-Li, which could synthesize 1,3-PD with a higher concentration, was identified and characterized. Phylogenetic analysis of the strain XJ-Li included the study of morphology, physiological and biochemical characteristics. In addition, 16SrDNA sequences were created. The results indicated that this strain is a member of Klebsiella pneumoniae. The optimal cultivation parameters for pH and temperature were determined as 8.0 and 40 °C, respectively. The optimized nitrogen source and carbon source were 6.0 g/L of (NH₄)₂SO₄ and 20 g/L of glycerol, respectively. After 8 h in batch fermentation, both the 1,3-PD concentration and glycerol consumption reached the maximum, with 12.2 g/L of 1,3-PD and 1.53 g/L h of productivity, and a molar yield of 1,3-PD to glycerol of 0.75. Fed-batch fermentation also indicated a higher molar yield of 0.70, and the concentration of 1,3-PD reached 38.1 g/L after 66.4 g/L of glycerol consumption. The results of batch and fed-batch fermentations demonstrated that K. pneumoniae XJ-Li would be an excellent 1,3-PD producer.     

Microbial fed-batch production of 1,3-propanediol by Klebsiella pneumoniae under micro-aerobic conditions

The microbial production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae under micro-aerobic conditions was investigated in this study. The experimental results of batch fermentation showed that the final concentration and yield of 1,3-PD on glycerol under micro-aerobic conditions approached values achieved under anaerobic conditions. However, less ethanol was produced under microaerobic than anaerobic conditions at the end of fermentation. The batch micro-aerobic fermentation time was markedly shorter than that of anaerobic fermentation. This led to an increment of productivity of 1,3-PD. For instance, the concentration, molar yield, and productivity of 1,3-PD of batch micro-aerobic fermentation by K. pneumoniae DSM 2026 were 17.65 g/l, 56.13%, and 2.94 g l^–1 h^–1, respectively, with a fermentation time of 6 h and an initial glycerol concentration of 40 g/l. Compared with DSM 2026, the microbial growth of K. pneumoniae AS 1.1736 was slow and the concentration of 1,3-PD was low under the same conditions. Furthermore, the microbial growth in fed-batch fermentation by K. pneumoniae DSM 2026 was faster under micro-aerobic than anaerobic conditions. The concentration, molar yield, and productivity of 1,3-PD in fed-batch fermentation under micro-aerobic conditions were 59.50 g/l, 51.75%, and 1.57 g l^–1 h^–1, respectively. The volumetric productivity of 1,3-PD under microaerobic conditions was almost twice that of anaerobic fed-batch fermentation, at 1.57 and 0.80 g l^–1 h^–1, respectively.     

1,3-Propandiol production by engineered Hansenula polymorpha expressing dha genes from Klebsiella pneumoniae

Currently, 1,3-propanediol (1,3-PD) is an important chemical widely used in polymer production, but its availability is being restricted owing to its expensive chemical synthesis. A methylotrophic yeast Hansenula polymorpha was engineered by expression of dhaB1, dhaB2, dhaB3, dhaB _RA1 and dhaB _RA2 encoding glycerol dehydratase complex and dhaT encoding 1,3-PD oxidoreductase from Klebsiella pneumoniae under direction of promoter of glyceraldehyde-3 phosphate dehydrogenase (GAPDH). The engineered recombinant yeast strain can produce 1,3-PD from glucose (2.4 g L⁻¹) as well as glycerol (0.8 g L⁻¹), which might lead to a safe and cost-effective method for industrial production of 1,3-PD from various biomass resources.