Coordination of metabolic pathways: Enhanced carbon conservation in 1,3-propanediol production by coupling with optically pure lactate biosynthesis

Metabolic engineering has emerged as a powerful tool for bioproduction of both fine and bulk chemicals. The natural coordination among different metabolic pathways contributes to the complexity of metabolic modification, which hampers the development of biorefineries. Herein, the coordination between the oxidative and reductive branches of glycerol metabolism was rearranged in Klebsiella oxytoca to improve the 1,3-propanediol production. After deliberating on the product value, carbon conservation, redox balance, biological compatibility and downstream processing, the lactate-producing pathway was chosen for coupling with the 1,3-propanediol-producing pathway. Then, the other pathways of 2,3-butanediol, ethanol, acetate, and succinate were blocked in sequence, leading to improved d-lactate biosynthesis, which as return drove the 1,3-propanediol production. Meanwhile, efficient co-production of 1,3-propanediol and l-lactate was also achieved by replacing ldhD with ldhL from Bacillus coagulans. The engineered strains PDL-5 and PLL co-produced over 70 g/L 1,3-propanediol and over 100 g/L optically pure d-lactate and l-lactate, respectively, with high conversion yields of over 0.95 mol/mol from glycerol.     

Anaerobic Degradation of Glycerol by Desulfovibrio fructosovorans and D. carbinolicus and Evidence for Glycerol-Dependent Utilization of 1,2-Propanediol

The degradation of glycerol by Desulfovibrio carbinolicus and Desulfovibrio fructosovorans was tested in pure culture with sulfate and in coculture with Methanospirillum hungatei. Desulfovibrio carbinolicus degraded glycerol into 3-hydroxypropionate with the formation of sulfide in pure culture and methane in the coculture. The maximum growth rates were 0.063 h⁻¹ in pure culture and 0.014 h⁻¹ in coculture (corresponding growth yields: 8.9 and 6.0 g dry weight/mol glycerol). With D. fructosovorans, the pathway of glycerol degradation depended upon the terminal electron acceptor. Acetate and sulfide were produced in the presence of sulfate, while 3-hydroxypropionate and methane were formed by the syntrophic association with M. hungatei. The maximum growth rates were 0.057 h⁻¹ in pure culture and 0.020 h⁻¹ in coculture (corresponding growth yields: 8.9 and 6.0 g dry weight/mol glycerol). In a medium containing both glycerol and 1,2-propanediol but no sulfate, D. carbinolicus and D. fructosovorans degraded both substrates. A drop in the concentration of 1,3-propanediol was observed, and propionate and n-propanol production was recorded. Putative biochemical pathways of 1,2-propanediol degradation by D. carbinolicus and D. fructosovorans indicated that the enzymes involved in this metabolism are present only when the strains are grown on a mixture of 1,2-propanediol and glycerol without sulfate. Received: 1 October 1997 / Accepted: 3 November 1997     

Production of 1,3-propanediol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> VPI 3266 using a synthetic medium and raw glycerol

Growth inhibition of Clostridium butyricum VPI 3266 by raw glycerol, obtained from the biodiesel production process, was evaluated. C. butyricum presents the same tolerance to raw and to commercial glycerol, when both are of similar grade, i.e. above 87% (w/v). A 39% increase of growth inhibition was observed in the presence of 100 g l^–1 of a lower grade raw glycerol (65% w/v). Furthermore, 1,3-propanediol production from two raw glycerol types (65% w/v and 92% w/v), without any prior purification, was observed in batch and continuous cultures, on a synthetic medium. No significant differences were found in C. butyricum fermentation patterns on raw and commercial glycerol as the sole carbon source. In every case, 1,3-propanediol yield was around 0.60 mol/mol glycerol consumed.     

Regulation of 3-hydroxypropionaldehyde accumulation in Klebsiella pneumoniae by overexpression of dhaT and dhaD genes

3-Hydroxypropionaldehyde (3-HPA), an important intermediary metabolite of 1,3-propanediol (PDO) production, would be toxic to the cell growth and led to the abnormal cessation of the fermentation process. In this study, the dhaD gene encoding glycerol dehydrogenase (GDH) and dhaT gene encoding 1,3-propanediol oxidoreductase (PDOR) were overexpressed in Klebsiella pneumoniae ACCC 10082 to decrease the 3-HPA accumulation and increase the coenzyme NADH supply. By the construction of pTD plasmid, GDH and PDOR were both overexpressed and their enzyme activities were increased by 2.6- and 3.2-fold, respectively. The enzyme activity ratio of PDOR/GDHt (glycerol dehydratase) also was increased. On the other hand, NADH production was enhanced and the ratio of NADH/NAD⁺ exceeded 1 after the inducement of IPTG for the constructed strain. The two factors enhanced the transformation of 3-HPA to PDO. In the batch and fed-batch fermentation by the constructed strain, the peak of 3-HPA accumulation reduced by 52.2% and 33.3%, respectively, compared with the control. The PDO concentration and yield reached 59.2 g/L and 0.48 mol/mol, respectively. Furthermore, the fed-batch fermentation process appeared easier to be regulated. This work is considered helpful for the further understanding on the PDO metabolic mechanism of K. pneumoniae and also useful for the PDO fermentation in a large-scale bioreactor.     

3-Hydroxypropionaldehyde guided glycerol feeding strategy in aerobic 1,3-propanediol production by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

3-Hydroxypropionaldehyde (3-HPA) is a toxic intermediary metabolite in the biological route of 1,3-propanediol biosynthesis from glycerol. 3-HPA accumulated in culture medium would arouse an irreversible cessation of the fermentation process. The role of substrate (glycerol) on 3-HPA accumulation in aerobic fermentation was investigated in this paper. 1,3-Propanediol oxidoreductase and glycerol dehydratase, two key enzyme catalyzing reactions of 3-HPA formation and consumption, were sensitive to high concentration of 3-HPA. When the concentration of 3-HPA increased to a higher level in medium (ac 10 mmol/L), the activity of 1,3-propanediol oxidoreductase in cell decreased correspondingly, which led to decrease of the 3-HPA conversion rate, then the 3-HPA concentration increasing was accelerated furthermore. 3-HPA accumulation in culture medium was triggered by this positive feedback mechanism. In the cell exponential growth phase, the reaction catalyzed by 1,3-propanediol oxidoreductase was the rate limiting step in 1,3-propanediol production. The level of 3-HPA in culture medium could be controlled by the substrate (glycerol) concentration, and lower level of glycerol could avoid 3-HPA accumulating to a high, lethal concentration. In fed batch fermentation, under the condition of initial glycerol concentration 30 g/L, and keeping glycerol concentration lower than 7–8 g/L in cell exponential growth phase, 3-HPA accumulation could not be incurred. Based on this result, a glycerol feeding strategy was set up in fed batch fermentation. Under the optimized condition, 50.1 g/L of 1,3-propanediol was produced in 24 h, and 73.1 g/L of final 1,3-propanediol concentration was obtained in 54 h.     

Microbial conversion of glycerol to 1,3-propanediol

Abstract: Glycerol produced by cleavage of natural fats can microbially be converted to 1,3-propanediol (PD) by Citrobacter, Klebsiella and Clostridia strains. The fermentation by C. butyricum , product recovery and purification has been investigated in detail up to the 2 m³ scale. Estimation of product costs for a 10,000 t/a plant indicates that the microbial process is obviously more attractive than the chemical route. Presently, 1,3-propanediol has only a low market volume; however, its use for special polycondensates, in particular polyesters, could reduce glycerol surpluses and make plastics a easily biodegradable part of natural cycles.     

Effects of Acetate and Butyrate During Glycerol Fermentation by Clostridium butyricum

The effects of acetate and butyrate during glycerol fermentation to 1,3-propanediol at pH 7.0 by Clostridium butyricum CNCM 1211 were studied. At pH 7.0, the calculated quantities of undissociated acetic and butyric acids were insufficient to inhibit bacterial growth. The initial addition of acetate or butyrate at concentrations of 2.5 to 15 gL⁻¹ had distinct effects on the metabolism and growth of Clostridium butyricum. Acetate increased the biomass and butyrate production, reducing the lag time and 1,3-propanediol production. In contrast, the addition of butyrate induced an increase in 1,3-propanediol production (yield: 0.75 mol/mol glycerol, versus 0.68 mol/mol in the butyrate-free culture), and reduced the biomass and butyrate production. It was calculated that reduction of butyrate production could provide sufficient NADH to increase 1,3-propanediol production. The effects of acetate and butyrate highlight the metabolic flexibility of Cl. butyricum CNCM 1211 during glycerol fermentation. Received: 2 January 2001 / Accepted: 6 February 2001     

Inactivation of aldehyde dehydrogenase: a key factor for engineering 1,3-propanediol production by Klebsiella pneumoniae

Production of 1,3-propanediol (1,3-PD) from glycerol by Klebsiella pneumoniae is restrained by ethanol formation. The first step in the formation of ethanol from acetyl-CoA is catalyzed by aldehyde dehydrogenase (ALDH), an enzyme that competes with 1,3-PD oxidoreductase for the cofactor NADH. This study aimed to improve the production of 1,3-PD by engineering the ethanol formation pathway. An inactivation mutation of the aldA gene encoding ALDH in K. pneumoniae YMU2 was generated by insertion of a tetracycline resistance marker. Inactivation of ALDH resulted in a nearly abolished ethanol formation but a significantly improved 1,3-PD production. Metabolic flux analysis revealed that a pronounced redistribution of intracellular metabolic flux occurred. The final titer, the productivity of 1,3-PD and the yield of 1,3-PD relative to glycerol of the mutant strain reached 927.6 mmol L(-1), 14.05 mmol L(-1)h(-1) and 0.699 mol mol(-1), respectively, which were much higher than those of the parent strain. In addition, the specific 1,3-PD-producing capability (1,3-PD produced per gram of cells) of the mutant strain was 2-fold that of the parent strain due to a lower growth yield of the mutant. By increasing NADH availability, this study demonstrates an important metabolic engineering approach to improve the efficiency of oxidoreduction-coupled bioprocesses.     

1,3-Propanediol formation with product-tolerant mutants of Clostridium butyricum DSM 5431 in continuous culture: productivity, carbon and electron flow

Glycerol fermentation and product formation of two product-tolerant mutants of Clostridium butyricum DSM 5431 were investigated in continuous culture at increasing glycerol feed concentrations. Under conditions of glycerol excess (above 55 g l⁻¹ at D = 0·15 h⁻¹), the mutants maintained a constant level of glycerol consumption and product formation, whereas the parent strain exhibited a substantial decrease in substrate conversion, 1,3-propanediol and butyrate formation, and an increase in acetate formation. The activities of the glycerol dehydrogenase, the glycerol dehydratase and the 1,3-propanediol dehydrogenase showed only slight changes with glycerol concentrations in the mutants, but dropped markedly at high concentrations in the wild type. Intracellular concentrations of NADH, NAD ⁺ and acetyl-CoA remained at a relatively constant level in the mutants, but increased sharply with the wild type strain. The NADH content was always higher than the NAD ⁺ content in the mutants as well as in the wild type.     

Improved 1,3-propanediol production by engineering the 2,3-butanediol and formic acid pathways in integrative recombinant Klebsiella pneumoniae

In the biotechnological process, insufficient cofactor NADH and multiple by-products restrain the final titer of 1,3-propanediol (1,3-PD). In this study, 1,3-PD production was improved by engineering the 2,3-butanediol (2,3-BD) and formic acid pathways in integrative recombinant Klebsiella pneumoniae. The formation of 2,3-BD is catalysed by acetoin reductase (AR). An inactivation mutation of the AR in K. pneumoniae CF was generated by insertion of a formate dehydrogenase gene. Inactivation of AR and expression of formate dehydrogenase reduced 2,3-BD formation and improved 1,3-PD production. Fermentation results revealed that intracellular metabolic flux was redistributed pronouncedly. The yield of 1,3-PD reached 0.74 mol/mol glycerol in flask fermentation, which is higher than the theoretical yield. In 5 L fed-batch fermentation, the final titer and 1,3-PD yield of the K. pneumoniae CF strain reached 72.2 g/L and 0.569 mol/mol, respectively, which were 15.9% and 21.7% higher than those of the wild-type strain. The titers of 2,3-BD and formic acid decreased by 52.2% and 73.4%, respectively. By decreasing the concentration of all nonvolatile by-products and by increasing the availability of NADH, this study demonstrates an important strategy in the metabolic engineering of 1,3-PD production by integrative recombinant hosts.     

Glycerol fermentation by a new 1,3-propanediol-producing microorganism:Enterobacter agglomerans

 According to their ability to synthesize 1,3-propanediol from glycerol, two species were isolated from the anoxic mud of a distillery waste-water digestor: Clostridium butyricum and Enterobacter agglomerans. The latter, a facultatively anaerobic gram-negative bacterium, is described for the first time as a microorganism producing 1,3-propanediol from glycerol. The products of glycerol conversion by E. agglomerans were identified using nuclear magnetic resonance. A 20-g/l glycerol solution was fermented mainly to 1,3-propanediol (0.51 mol/mol) and acetate (0.18 mol/mol). Ethanol, formate, lactate and succinate were formed as by-products. Gas production was very low; 1,3-propanediol production perfectly balanced the oxido-reduction state of the microorganism. Acetate was the predominant metabolite generating energy for growth. High-glycerol-concentration fermentations (71 g/l and 100 g/l) resulted in an increase of the 1,3-propanediol yield (0.61 mol/mol) at the expense of lactate and ethanol production. Specific rates of glycerol consumption and 1, 3-propanediol and acetate production increased whereas the growth rate decreased. The decrease in ATP yield was linearly correlated with the specific rate of 1,3-propanediol production. Incomplete glycerol consumption (about 40 g/l) was systematically observed when high glycerol concentrations were used. The unbalanced oxido-reduction state, the low carbon recovery and the detection of an unknown compound by HPLC observed in these cases indicate the formation of another metabolite, which is possibly an inhibitory factor. Received: 17 November 1994 / Accepted: 15 December 1994     

克雷伯氏肺炎杆菌HR526快速合成1,3-丙二醇发酵特性研究

研究了实验室筛选的一株高产1,3-丙二醇(PDO)菌株克雷伯氏肺炎杆菌HR526(Klebsiella pneumoniae HR526), 在5 L B. Braun发酵罐进行甘油补料流加发酵30 h, PDO达到91.47 g/L, 胞外代谢通量分析显示, PDO在对数中期通量达到最大, 而乳酸在稳定期通量达到最大。结合酶学检测分析了PDO合成关键酶PDO氧化还原酶(PDOR)、甘油脱水酶(GDHt)和甘油脱氢酶(GDH)酶活的变化, PDO氧化还原酶活性在对数中期达到最高, 甘油脱水酶/甘油脱氢酶在对数期远大于稳定期、衰退期, 与代谢通量变化一致甘油脱水酶/甘油脱氢酶活性比例不均衡是3-HPA对数期积累的原因, PDO合成主要集中在对数期, 是生长偶联的代谢产物。     

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.     

异源表达木糖异构酶基因对克雷伯氏菌合成1,3-丙二醇的影响

【目的】提高克雷伯氏菌胞内还原力以强化1,3-丙二醇合成。【方法】将来源于大肠杆菌的木糖异构酶基因在克雷伯氏菌中异源表达,构建重组菌。研究重组菌添加不同浓度木糖为辅底物与甘油共发酵过程中代谢产物和NADH的变化规律。【结果】与对照菌相比,重组菌细胞内还原力NADH提高了0.1?0.3倍,1,3-丙二醇产量达到23.31 g/L,提高20%,1,3-丙二醇转化率从0.60 mol/mol提高到0.73 mol/mol。【结论】木糖异构酶基因的表达强化了木糖代谢途径,经磷酸戊糖途径积累大量还原力,促进了1,3-丙二醇的生成。     

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.     

Systems metabolic engineering of Corynebacterium glutamicum for high-level production of 1,3-propanediol from glucose and xylose

Corynebacterium glutamicum is a versatile chassis which has been widely used to produce various amino acids and organic acids. In this study, we report the development of an efficient C. glutamicum strain to produce 1,3-propanediol (1,3-PDO) from glucose and xylose by systems metabolic engineering approaches, including (1) construction and optimization of two different glycerol synthesis modules; (2) combining glycerol and 1,3-PDO synthesis modules; (3) reducing 3-hydroxypropionate accumulation by clarifying a mechanism involving 1,3-PDO re-consumption; (4) reducing the accumulation of toxic 3-hydroxypropionaldehyde by pathway engineering; (5) engineering NADPH generation pathway and anaplerotic pathway. The final engineered strain can efficiently produce 1,3-PDO from glucose with a titer of 110.4 g/L, a yield of 0.42 g/g glucose, and a productivity of 2.30 g/L/h in fed-batch fermentation. By further introducing an optimized xylose metabolism module, the engineered strain can simultaneously utilize glucose and xylose to produce 1,3-PDO with a titer of 98.2 g/L and a yield of 0.38 g/g sugars. This result demonstrates that C. glutamicum is a potential chassis for the industrial production of 1,3-PDO from abundant lignocellulosic feedstocks.     

一株1,3-丙二醇生产菌的分离鉴定及其发酵特性

从活性污泥中分离筛选得到一株能代谢甘油生产1,3-丙二醇(1,3-PD)的菌株2-1,通过形态学鉴定、生理生化试验、16S rRNA序列分析对菌株分类学地位进行鉴定,用MEGA 4.1软件构建的系统发育树显示菌株2-1与Klebsiella pneumoniae(CP001891)的亲缘关系最近。16S rDNA序列同源性比较发现,菌株2-1与模式菌株同源率为95.4%,疑似为新种。对菌株2-1在5 L发酵罐中进行发酵特性研究,分批补料发酵时得到较高的1,3-PD终浓度,达到63.5 g/L,此时生产强度为2.19 g/(L.h),底物转化率0.64 mol/mol。     

一步法产1,3-丙二醇酿酒酵母基因工程菌的构建

1,3-丙二醇(1,3-PD)是一种重要的化工原料,发酵法生产1,3-PD是一条新颖且具有潜在竞争力的生产途径。本研究在前期工作的基础上,将分别来源于大肠杆菌和肺炎克雷伯氏菌的基因片段yqhD和dhaB串联表达,构建重组表达载体pYX212-zeocin-pGAP-yqhD-pGAP-dhaB;并得到重组酿酒酵母(Saccharomyces cerevisiae)W303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB。该重组菌和对照S.cerevisiae分别以葡萄糖为底物摇瓶发酵72h后,重组酿酒酵母发酵液中1,3-PD含量约为1.5g/L;而对照菌株不产1,3-PD。以上结果表明本研究在国内首次成功构建了直接以葡萄糖为底物发酵生产1,3-PD的酿酒酵母基因工程菌。为进一步将dhaB、yqhD基因导入其他以葡萄糖为底物高产甘油的酵母宿主中表达,获得以葡萄糖为底物一步法发酵高产1,3-丙二醇工程菌打下了坚实的基础。     

Improved 1,3-propanediol production with hemicellulosic hydrolysates (corn straw) as cosubstrate: Impact of degradation products on Klebsiella pneumoniae growth and 1,3-propanediol fermentation

To improve 1,3-propanediol (1,3-PD) production by an economic and efficient approach, hemicellulosic hydrolysates (HH) used as cosubstrate resulted in more biomass and higher reducing power for 1,3-PD production. The effects of primary degradation products such as individual sugars (xylose, glucose, mannose, arabinose and galactose) and major inhibitors (furfural, acetate and formate) on the Klebsiella pneumoiae growth and 1,3-PD production were investigated in this study. Xylose and mannose could efficiently promote the 1,3-PD production and cell growth. Furfural (0.28 g/l) and sodium acetate (1.46 g/l) in low concentration were not inhibitory to Klebsiella pneumoniae, rather they have stimulatory effect on the growth and 1,3-PD biosynthesis, especially the acetate. In fed-batch fermentation with HH as cosubstrate, the final 1,3-PD production, conversion from glycerol and productivity were 71.58 g/l, 0.65 mol/mol and 1.93 g/l/h, respectively, which were 17.8%, 25.0% and 17.7% higher than that from glycerol alone.