Stoichiometric analysis and experimental investigation of glycerol–glucose co-fermentation in Klebsiella pneumoniae under microaerobic conditions

The ratio between two substrates is an important parameter in microbial co-fermentation, such as 1,3-propanediol production from glycerol by Klebsiella pneumoniae using glucose as the cosubstrate. In this study, the glycerol–glucose cometabolism by K. pneumoniae is stoichiometrically analyzed according to energy (ATP), reducing equivalent (NADH₂) and product balances. The theoretical analysis reveals that the yield of 1,3-propanediol to glycerol under microaerobic conditions depends not only on the ratio of glucose to glycerol initially added, but also on the molar fraction of reducing equivalent oxidized completely by molecular oxygen in tricarboxylic acid (TCA) cycle (δ) and the molar fraction of TCA cycle in acetyl-CoA metabolism (γ). The maximum ratio of 0.32 mol glucose per mol glycerol is needed to convert glycerol completely to 1,3-propanediol under anaerobic conditions if glycerol neither enters oxidation pathways nor forms biomass. The ratio can be reduced under microaerobic conditions. The experimental results of batch cultures demonstrate that the biomass concentration and yield of 1,3-propanediol on glycerol could be enhanced by using glucose as a co-substrate. The theoretical analysis reveals the relationship between yield of 1,3-propanediol to glycerol, ratio of glucose to glycerol and respiratory quotient (RQ). These results are helpful for the experimental design and control.     

Inactivation of <Emphasis Type="Italic">dhaD</Emphasis> and <Emphasis Type="Italic">dhaK</Emphasis> abolishes by-product accumulation during 1,3-propanediol production in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

1,3-Propanediol (1,3-PD) can be used for the industrial synthesis of a variety of compounds, including polyesters, polyethers, and polyurethanes. 1,3-PD is generated from petrochemical and microbial sources. 1,3-Propanediol is a typical product of glycerol fermentation, while acetate, lactate, 2,3-butanediol, and ethanol also accumulate during the process. Substrate and product inhibition limit the final concentration of 1,3-propanediol in the fermentation broth. It is impossible to increase the yield of 1,3-propanediol by using the traditional whole-cell fermentation process. In this study, dhaD and dhaK, the genes for glycerol dehydrogenase and dihydroxyacetone kinase, respectively, were inactivated by homologous recombination in Klebsiella pneumoniae. The dhaD/dhaK double mutant (designated TC100), selected from 5,000 single or double cross homologous recombination mutants, was confirmed as a double cross by using polymerase chain reaction. Analysis of the cell-free supernatant with high-performance liquid chromatography revealed elimination of lactate and 2,3-butanediol, as well as ethanol accumulation in TC100, compared with the wild-type strain. Furthermore, 1,3-propanediol productivity was increased in the TC100 strain expressing glycerol dehydratase and 1,3-PDO dehydrogenase regulated by the arabinose P_BAD promoter. The genetic engineering and medium formulation approaches used here should aid in the separation of 1,3-propanediol from lactate, 2,3-butanediol, and ethanol and lead to increased production of 1,3-propanediol in Klebsiella pneumoniae.     

Determination of kinetic parameters of 1,3-propanediol fermentation by Clostridium diolis using statistically optimized medium

1,3-propanediol (1,3-PD) is a chemical compound of immense importance primarily used as a raw material for fiber and textile industry. It can be produced by the fermentation of glycerol available abundantly as a by-product from the biodiesel plant. The present study was aimed at determination of key kinetic parameters of 1,3-PD fermentation by Clostridium diolis. Initial experiments on microbial growth inhibition were followed by optimization of nutrient medium recipe by statistical means. Batch kinetic data from studies in bioreactor using optimum concentration of variables obtained from statistical medium design was used for estimation of kinetic parameters of 1,3-PD production. Direct use of raw glycerol from biodiesel plant without any pre-treatment for 1,3-PD production using this strain investigated for the first time in this work gave results comparable to commercial glycerol. The parameter values obtained in this study would be used to develop a mathematical model for 1,3-PD to be used as a guide for designing various reactor operating strategies for further improving 1,3-PD production. An outline of protocol for model development has been discussed in the present work.     

Development of a two-step process for production of 3-hydroxypropionic acid from glycerol using <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> and <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis>

In this work, a two-step process was developed for the production of 3-hydroxypropionic acid from glycerol. In the first step, glycerol was converted to 1,3-propanediol by Klebsiella pneumonia. In the second step, the 1,3-propanediol was converted into 3-hydroxypropionic acid by Gluconobacter oxydans. In a 7.0 L bioreactor, the whole process took 54 h, consumed 480 g glycerol and produced 242 g 3-hydroxypropionic acid. The conversion rate of glycerol to 3-hydroxypropionic acid was 50.4 % (g g^?1). The final concentration of 3-hydroxypropionic acid arrived 60.5 g L^?1. The process was effective for 3-HP production from glycerol and it might provide a new approach to the biosynthesis of 3-HP from a cheap starting material. Moreover, in this paper, it was first reported that the by-product of 3-hydroxypropionic acid production from 1,3-propandeiol was acrylic acid.     

Construction of stress-induced metabolic pathway from glucose to 1,3-propanediol in <Emphasis Type="Italic">Escherichia coli</Emphasis>

1,3-Propanediol is an important chemical widely used in polymer production, but its availability is being restricted owing to its expensive synthesis. The aim of this study was to engineer an Escherichia coli strain that can produce 1,3-propanediol directly from glucose. We successfully constructed a stress-induced metabolic pathway from glucose to 1,3-propanediol in recombinant E. coli by the expression of gpd1 and gpp2 genes from Saccharomyces cerevisiae and dha operon from Klebsiella pneumoniae, respectively. Batch cultivation of the recombinant E. coli showed that 12.1 g/L 1,3-propanediol was accumulated in the culture without using any inducer.     

Enhanced 1,3-propanediol production in recombinant <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> carrying the gene <Emphasis Type="Italic">yqh</Emphasis>D encoding 1,3-propanediol oxidoreductase isoenzyme

The yqhD gene from Escherichia coli encoding 1,3-propanediol oxidoreductase isoenzyme (PDORI) and the tetracycline resistant gene (tetR) from plasmid pHY300PLK were amplified by PCR. They were inserted into vector pUC18, yielding the recombinant expression vector pUC18-yqhD-tetR. The recombinant vector was then cloned into Klebsiella pneumoniae ME-308. The overexpression of PDORI in K. pneumoniae surprisingly led to higher 1,3-propanediol production. The final 1,3-propanediol concentration of recombinant K. pneumoniae reached 67.6 g/l, which was 125.33% of that of the original strain. The maximum activity of recombinant PDORI converting 3-HPA to 1,3-PD reached 110 IU/mg after induction by IPTG at 31°C during the fermentation, while it was only 11 IU/mg under the same conditions for the wild type strain. The K _m values of the purified PDORI for 1,3-propanediol and NADP were 12.1 mM and 0.15 mM, respectively. Compared with the original strains, the concentration of the toxic intermediate 3-hydroxypropionaldehyde during the fermentation was also reduced by 22.4%. Both the increased production of 1,3-propanediol and the reduction of toxic intermediate confirmed the significant role of 1,3-propanediol oxidoreductase isoenzyme from E. coli in converting 3-hydroxypropionaldehyde to 1,3-propanediol for 1,3-PD production.     

Optimization of culture conditions for 1,3-propanediol production from crude glycerol by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> using response surface methodology

To produce 1,3-propanediol (1,3-PD) from crude glycerol, cultivation conditions were optimized by response surface methodology (RSM) based on a 2⁵ factorial central composite design (CCD). RSM was adopted to derive a statistical model for the individual and interactive effects of crude glycerol, (NH₄)₂SO₄, pH, cultivation time and temperature on the production of 1,3-PD. Optimal conditions for maximum 1,3-PD production were as follows: crude glycerol, 35 g/L; (NH₄)₂SO₄, 8 g/L; pH, 7.37; cultivation time, 10.8 h; temperature, 36.88°C. Under these optimal conditions, the design expert presented the maximal numerical solution with a predicted 1,3-PD production level of up to 13.74 g/L. The experimental production of 1,3-PD yielded 13.8 g/L, which was in close agreement with the model prediction.     

Examining the feasibility of bulk commodity production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli is currently used by many research institutions and companies around the world as a platform organism for the development of bio-based production processes for bulk biochemicals. A given bulk biochemical bioprocess must be economically competitive with current production routes. Ideally the viability of each bioprocess should be evaluated prior to commencing research, both by metabolic network analysis (to determine the maximum theoretical yield of a given biocatalyst) and by techno-economic analysis (TEA; to determine the conditions required to make the bioprocess cost-competitive). However, these steps are rarely performed. Here we examine theoretical yields and review available TEA for bulk biochemical production in E. coli. In addition, we examine fermentation feedstocks and review recent strain engineering approaches to achieve industrially-relevant production, using examples for which TEA has been performed: ethanol, poly-3-hydroxybutyrate, and 1,3-propanediol.     

基于甘油的1,3-丙二醇生物合成的代谢局限及其改造策略

粗甘油是生物柴油生产中的主要副产物,一些微生物可将甘油转化为重要化工原料1,3-丙二醇(1,3-PD),而利用这些微生物野生菌株生物合成1,3-PD会存在一些局限性,如底物抑制、产物抑制等。文中从1,3-丙二醇的甘油生物转化途径与这些局限性出发,总结了生物合成中存在的问题,并针对这些问题提出了一些基于基因敲除或基因过表达等基因工程技术的改造方法,综述了利用基因工程菌生物转化甘油生成1,3-丙二醇的最新研究进展。     

Microbial production of 1,3-propanediol: Recent developments and emerging opportunities

1,3-Propanediol, a valuable bifunctional molecule, can be produced from renewable resources using microorganisms. It has several promising properties for many synthetic reactions, particularly for polymer and cosmetic industries. By virtue of being a natural product, relevant biochemical pathways can be harnessed into fermentation processes to produce 1,3-propanediol. Various strategies for the microbial production of 1,3-propanediol are reviewed and compared in this article with their promises and constraints. Furthermore, genetic and metabolic engineering could significantly improve product yields and overcome the limitations of fermentation technology. Present review gives an overview on 1,3-propanediol production by wild and recombinant strains. It also attempts to encompass the various issues concerned in utilization of crude glycerol for 1,3-propanediol production, with particular emphasis laid on biodiesel industries. This review also summarizes the present state of strategies studied for the downstream processing and purification of biologically produced 1,3-propanediol. The future prospect of 1,3-propanediol and its potential as a major bulk chemical are discussed under the light of the current research.     

Construction of a novel recombinant Escherichia coli strain capable of producing 1,3–propanediol and optimization of fermentation parameters by statistical design

Summary The structural gene yqhD from a wild-type Escherichia coli encoding 1,3-propanediol oxidoreductase isoenzyme and the structural gene dhaB from Citrobacter freundii encoding glycerol dehydratase were amplified by using the PCR method. The temperature control expression vector pHsh harboring the yqhD and dhaB genes was transformed into E. coli JM109 to yield the recombinant strain E. coli JM109 (pHsh-dhaB-yqhD). The response surface method (RSM) was then applied to further optimize the fermentation condition of the recombinant strain. A mathematical model was then developed to show the effect of each medium composition and their interactions on the production of 1,3-propanediol by recombinant strain E. coli JM109. The model estimated that a maximal yield of 1,3-propanediol (43.86 g/l) could be obtained when the concentrations of glycerol, yeast extract and vitamin B₁₂ were set at 61.8 g/l, 6.2 g/l and 49 mg/l, respectively; and the fermentation time was 30 h. These predicted values were also verified by validation experiments. Compared with the values obtained by other runs in the experimental design, the optimized medium resulted in a significant increase in the yield of 1,3-propanediol. The yield and productivity under the optimal parameters and process can reach 43.1 g/l and 1.54 g/l/h. Maximum 1,3-propanediol yield of 41.1 g/l was achieved in a 5-l fermenter using the optimized medium. This makes the engineered strain have potential application in the conversion of glycerol to 1,3-propanediol on an industrial scale.     

Sulfate reduction and anaerobic glycerol degradation by a mixed microbial culture

Anaerobic glycerol degradation by a mixed microbial culture from a fermenter fed with industrial alcohol distillation waste water, was investigated in the absence or presence of sulfate, at 37°C and at a constant pH of 7.2. In the absence of sulfate, glycerol utilization was found to be characterized by the transient formation of 1,3-propanediol prior to propionate and acetate accumulation. In the presence of sulfate, 1,3-propanediol production was minor, and the carbon balance reflected a considerable accumulation of intermediate(s). A study of the role of sulfate reduction and methanogenesis on anaerobic 1,3-propanediol degradation showed that consumption of this substrate by the mixed microbial culture required a terminal electron acceptor. The number of fermentative and sulfate-reducing bacteria with glycerol or 1,3-propanediol as carbon and energy source revealed that sulfate-reducing bacteria outcompete fermentative bacteria for these substrates. The possible ecological role of sulfate-reducing bacteria in the metabolism of these reduced substrates is discussed.     

Isolation and characterization of microorganisms able to produce 1,3-propanediol under aerobic conditions

Microbial fermentation under strictly anaerobic conditions has been conventionally used for the production of 1,3-propanediol, a key raw material required for the synthesis of polytrimethylene terephthalate (PTT) and other polyester fibers. In the current study, we have identified eight strains of microorganism which are able to produce 1,3-propanediol under aerobic condition. Those strains were isolated from garden soil, which were enriched by culturing in LB medium with glycerol added under aerobic condition. The identities of those strains were established based on their 16S rRNA sequences and physiological characteristics. Results indicated 6 strains are Citrobacter freundii and 2 strains are Klebsiella pneumoniae subsp Penumoniae. One of Klebsiella pneumoniae subsp Penumoniae strains, designated as TUAC01, demonstrated comparable levels of 1,3-propanediol oxidoreductase, glycerol dehydratase and glycerol dehydrogenase activity to the anaerobic microorganisms described in the literature. Accordingly, in larger scales (5 l) fed-batch culture the TUAC01 strain showed a remarkable 1,3-propanediol producing potency under aerobic conditions. 60.1 g/l of 1,3-propanediol was yield after 42 h incubation in an agitating bioreactor; and in air-lift bioreactor 66.3 g/l of 1,3-propanediol was yield after 58.5 h incubation. The aerobic ferment process, reduced the product cost and made the biological method of 1,3-propanediol production more attractive.     

Production of 1,3-propanediol from Glycerol by Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Using Incompatible Plasmids System

1,3-Propanediol (1,3-PD) has numerous applications in polymers, cosmetics, foods, lubricants, and medicines as a bifunctional organic compound. The genes for the production of 1,3-PD in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, and gdrAB, which encodes glycerol dehydratase reactivating factor, are naturally under the control of different promoters and are transcribed in different directions. These genes were coexpressed in E. coli using two incompatible plasmids (pET28a and pET22b) in the presence of selective pressure. The recombinant E. coli coexpressed the glycerol dehydratase, 1,3-propanediol oxidoreductase and reactivating factor for the glycerol dehydratase at high levels. In a fed-batch fermentation of glycerol and glucose, the recombinant E. coli containing these two incompatible plasmids consumed 14.3 g/l glycerol and produced 8.6 g/l 1,3-propanediol. In the substitution case of yqhD (encoding alcohol dehydrogenase from E. coli) for dhaT, the final 1,3-propanediol concentration of the recombinant E. coli could reach 13.2 g/l.     

Dielectric barrier discharge plasma as a novel approach for improving 1,3-propanediol production in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Dielectric barrier discharge plasma was used to generate a stable strain of Klebsiella pneumoniae (designated to as Kp-M2) with improved 1,3-propanediol production. The specific activities of glycerol dehydrogenase, glycerol dehydatase and 1,3-propanediol oxidoreductase in the crude cell extract increased from 0.11, 9.2 and 0.15 U mg⁻¹, respectively, for wild type to 0.67, 14.4 and 1.6 U mg⁻¹ for Kp-M2. The glycerol flux of Kp-M2 was redistributed with the flux to the reductive pathway being increased by 20% in batch fermentation. The final 1,3-propanediol concentrations achieved by Kp-M2 in batch and fed-batch fermentations were 19.9 and 76.7 g l⁻¹, respectively, which were higher than those of wild type (16.2 and 49.2 g l⁻¹). The results suggested that dielectric barrier discharge plasma could be used as an effective approach to improve 1,3-propanediol production in K. pneumoniae.     

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     

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。     

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.     

Pathway and kinetic analysis of 1,3-propanediol production from glycerol fermentation by Clostridium butyricum

Stoichiometric analysis is applied to continuous glycerol fermentation by Clostridium butyricum to calculate theoretical maximum yields and to predict preferred pathways under different conditions. The upper limits of product concentration and productivity as a function of dilution rate in continuous culture is also predicted from product inhibition kinetic. The theoretical maximum propanediol yield (0.72 mol/mol glycerol) which is calculated for a culture without hydrogen and butyric acid formation agrees well with the experimental maximum value (around 0.71 mol/mol). Comparisons of experimental results (product concentration and productivity) with theoretical calculations and those of the glycerol fermentation by Klebsiella pneumoniae reveal that the production of 1,3-propanediol by C. butyricum is far below the optimum performance available with the present strain. One of the reasons is the relatively high formation of butyric acid under the culture conditions so far applied. The distribution of reducing equivalents to propanediol and hydrogen is also suboptimal. The utilization of the reducing power from pyruvate oxidation for propanediol production is about 60–70% of the theoretical maximum under the present experimental conditions.     

Production of 1,3-propanediol, 2,3-butanediol and ethanol by a newly isolated Klebsiella oxytoca strain growing on biodiesel-derived glycerol based media

The production of 1,3-propanediol, 2,3-butanediol and ethanol was studied, during cultivations of strain Klebsiella oxytoca FMCC-197 on biodiesel-derived glycerol based media. Different kinds of glycerol feedstocks and experimental conditions had an important impact upon the distribution of metabolic products; production of 1,3-propanediol was positively influenced by stable pH conditions and by the absence of N₂ gas infusions throughout the fermentation. Thus, during batch bioreactor fermentations conducted at increasing glycerol concentrations, 1,3-propanediol at 41.3 g/L and yield ～47% (w/w) was achieved at initial glycerol concentration ～120 g/L. At even higher initial glycerol media (150 and 170 g/L), growth was not ceased, but 1,3-propanediol production declined. During fed-batch fermentation under optimal experimental conditions, 126 g/L of glycerol were converted into 50.1 g/L of 1,3-propanediol. In this experiment, also 25.2 g/L of ethanol (conversion yield ～20%, w/w) were formed. A batch-bioreactor culture was performed under non-sterilized conditions and the 1,3-propanediol production was almost equivalent to the sterilized process. Concerning 2,3-butanediol formation, the most detrimental parameter was the absence of N₂ sparging and as a result, no 2,3-butanediol was produced. The presence of glucose as co-substrate seriously enhanced 2,3-butanediol production; when commercial glucose was employed as sole substrate, 32.1 g/L of 2,3-butanediol were formed.