Crystallization and phase behavior of 1,3-propanediol esters II. 1,3-propanediol distearate/1,3-propanediol dipalmitate (SS/PP) and 1,3-propanediol distearate/1,3-propanediol dimyristate (SS/MM) binary systems

Polymorphic influences on the phase behavior of two types of binary mixtures of saturated monoacid 1,3-propanediol esters (PADEs), dipalmitate/distearate (PP/SS) and dimyristate/distearate (MM/SS) were examined by X-ray diffraction (XRD), differential scanning calorimetry (DSC), and by solid fat content (SFC), hardness and microscopy measurements. Three stacking modes have been found in the PP/SS binary system. Mixed SS-PP bilayers were detected in all mixtures, SS-SS bilayers in x(PP)=0.0-0.4 mixtures and PP-PP bilayers in x(PP)=0.6-0.1 mixtures. Two different but close beta polymorphs and one beta' polymorph were detected for this system. beta' was only detected in x(PP)=0.5-0.9 mixtures for the mixed bilayers. For the MM/SS binary system, only MM-MM and SS-SS bilayers were detected and both solid phases crystallized in two different beta forms. XRD data evidenced clearly that the MM and SS components were completely immiscible in the solid state. The phase diagrams constructed using DSC data, exhibited a typical eutectic-type phase boundary. The presence of eutectics, the shape of the solidus lines as well as the analysis of the individual enthalpies of melting indicated typical phase separation for both systems. A thermodynamic study based on the Hildebrand equation and using the Bragg-Williams approximation for non-ideality of mixing confirmed the phase separation in the solid phase and suggested that the PP and SS were miscible in the liquid phase and that SS formed an ideal mixing with MM. Avrami analysis of SFC vs. time curves indicated heterogeneous nucleation and spherulitic crystal development from sporadic nuclei, and suggested that the nucleation rate was higher for the mixture at the eutectic composition. The relative hardness was correlated with the enthalpies, the final SFC and the microscopy measurements.     

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.     

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.     

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.     

编码1,3-丙二醇氧化还原酶基因的克隆和表达

采用PCR法克隆了巴氏梭菌（Clostridium pasteurianum）CpN86菌株编码1,3丙二醇氧化还原酶基因（dhaT基因）;完成了dhaT基因测序、表达载体构建和在大肠杆菌中表达;分离和纯化了dhaT基因表达的重组蛋白。实验结果：（1）PCR法克隆的dhaT基因和肺炎克雷伯氏菌Klebsiella pneumoniae菌株dhaT基因的序列同源性为829%;（2）dhaT基因表达蛋白的酶活为108U/mg;（3）dhaT基因表达的蛋白分子量为43kD;（4）Western blot确定了dhaT基因表达的蛋白和 CpN86菌株天然蛋白有相同的抗原反应。     

Cell immobilization for microbial production of 1,3-propanediol

Cell and enzyme immobilization are often used for industrial production of high-value products. In recent years, immobilization techniques have been applied to the production of value-added chemicals such as 1,3-Propanediol (1,3-PDO). Biotechnological fermentation is an attractive alternative to current 1,3-PDO production methods, which are primarily thermochemical processes, as it generates high volumetric yields of 1,3-PDO, is a much less energy intensive process, and generates lower amounts of environmental organic pollutants. Although several approaches including: batch, fed-batch, continuous-feed and two-step continuous-feed were tested in suspended systems, it has been well demonstrated that cell immobilization techniques can significantly enhance 1,3-PDO production and allow robust continuous production in smaller bioreactors. This review covers various immobilization methods and their application for 1,3-PDO production.     

Microbial asymmetric oxidation of 2-butyl-1,3-propanediol

Microbial asymmetric oxidation of 2-butyl-1,3-propanediol was investigated for an efficient synthesis of S- and R-enantiomers of 2-hydroxymethylhexanoic acid (2-HMHA). From an intensive survey of the stocked bacterial strains, Acetobacter pasteurianus IAM 12073 and Pseudomonas putida IFO 3738 were found to show the highest S- and R-2-HMHA-producing activity, respectively. Under optimized conditions, A. pasteurianus (351 mg dry cell weight) and P. putida (642 mg dry cell weight) cells produced 12.0 g l⁻¹ S-2-HMHA with 89% enantiomeric excess (e.e.) at 24 h of incubation and 5.1 g l⁻¹ R-2-HMHA with 94% e.e. at 35 h of incubation from 2-butyl-1,3-propanediol.     

Reactive extraction for downstream separation of 1,3-propanediol

The downstream separation of 1,3-propanediol from dilute aqueous solution was studied. A process combining reversible reaction of 1, 3-propanediol with acetaldehyde to 2-methyl-1,3-dioxane and a simultaneous extraction of the product by organic solvent appears to be technically feasible and attractive. The dioxane yield was 91-92%, the overall conversion of 1,3-propanediol was ca. 98%, and recovery of dioxane into the organic extractant was 75%.     

Debottlenecking the 1,3-propanediol pathway by metabolic engineering

The history of 1,3-propanediol (1,3-PD) conversion from being a specialty chemical to being a bulk chemical illustrates that the concerted effort of different metabolic engineering approaches brings the most successful results. In order to metabolically tailor the 1,3-PD production pathway multiple strategies have been pursued. Knocking-out genes responsible for by-products formation, intergeneric transfer and overexpression of the genes directly involved in the pathway, manipulation with internal redox balance, introduction of a synthetic flux control point, and modification of the substrate mechanism of transport are some of the strategies applied. The metabolic engineering of the microbial 1,3-PD production exploits both native producers and microorganisms with acquired ability to produce the diol via genetic manipulations. Combination of the appropriate genes from homologous and heterologous hosts is expected to bring a desired objective of production of 1,3-PD cheaply, efficiently and independently from non-renewable resources. The state-of-the-art of the 1,3-PD pathway metabolic engineering is reviewed in this paper.     

微生物发酵法生产1,3-丙二醇的研究新进展

1,3-丙二醇(1,3-PD)是一种重要的化工原料,广泛应用于医药、化工、食品及化妆品等行业,同时1,3-PD是合成聚对苯二甲酸丙二酯(PTT)的重要单体,市场需求量逐年增多。基于生态友好型、生产安全和可持续发展的要求,利用微生物转化可再生资源来生产1,3-PD受到了人们的广泛重视。综述了微生物发酵法生产1,3-PD的菌株、代谢途径、发酵和下游分离工艺及其新进展,并对工业生产中利用生物技术生产1,3-PD的未来前景和挑战进行了探讨。     

Serinol (2-Amino-1,3-propanediol) and 3-Amino-1,2-propanediol in Soybean Nodules

The compound X, which had previously been found to be accumulatedin the soybean nodules formed by infection with wild-type H₂-uptakenegative Bradyrhizobium japonicum strains, was identified asserinol (2-amino-1,3-propanediol) by means of elementary analysis,infrared spectrometry, ¹H-nuclear magnetic resonance, ¹³C-nuclearmagnetic resonance, high-performance liquid chromatography andgas chromatography/mass spectrometry. During the process ofpurification of compound X, it was also elucidated that 3-amino-1,2-propanediolwas present in the soybean nodules as a minor component. (Received January 6, 1986; Accepted June 16, 1986)     

Biological characterization of Sorona polymer from corn-derived 1,3-propanediol

The Sorona family of polymers, based on corn-derived 1,3-propanediol, have recently been developed as novel bio-based materials for use in plastics, films, and fibers. In the present study, Sorona polymers were investigated for their effects on cell survival and inflammatory cell activation using in vitro mouse cell cultures. Cytotoxicity of Sorona polymers was evaluated by placing material samples in direct contact with 3T3 fibroblast cells. Both Sorona plastic and Sorona films were non-cytotoxic to fibroblasts. The inflammatory potential of Sorona samples was evaluated by exposing J774 macrophage cells to material samples, and measuring TNF-α release from macrophages. Sorona plastic and Sorona films did not elicit inflammatory TNF-α release from macrophages. These results indicate that Sorona polymers are non-cytotoxic and non-inflammatory. While the 1,3-propanediol component of Sorona 3GT is manufactured in a bacterial fermentation process, the absence of an inflammatory response to Sorona film and Sorona plastic is highly encouraging. The results are significant for the design of materials that utilize bio-based polymers.     

Use of oxidoreduction potential as an indicator to regulate 1,3-propanediol fermentation by Klebsiella pneumoniae

Anaerobic fermentation was relatively difficult to optimize due to lack of monitoring parameters. In this paper, a new method was reported using extracellular oxidoreduction potential (ORP) to monitor 1,3-propanediol (1,3-PD) biosynthesis process by Klebsiella pneumoniae. In batch fermentation, cell growth, 1,3-propanediol production and by-products distribution were studied at four different ORP levels: 10, −140, −190 and −240 mV. From the results, the ORP level of −190 mV was preferable, which resulted in fast cell growth and high 1,3-propanediol concentration. The NAD⁺/NADH ratio was determined at different ORP levels, and a critical NAD⁺/NADH ratio of 4 was defined to divide fermentation environments into two categories: relatively oxidative environment (NAD⁺/NADH>4) and relatively reductive environment (NAD⁺/NADH<4). The former was correlative with high 1,3-propanediol productivity and high specific growth rate. The mechanism of ORP regulation was discussed. It is suggested that ORP regulation of fermentation might be due to its influence on the ratio of NAD⁺/NADH, which determined metabolic flux. Furthermore, a batch fermentation of modulating ORP following a profile in different levels corresponding to different fermentation stage was tested. The 1,3-PD concentration was 22.3% higher than that of constant ORP fermentation at −190 mV. Therefore, ORP is a valuable parameter to monitor and control anaerobic fermentation production.     

Studies on purification of 1,3-propanediol by molecular distillation

The separation of 1,3-propanediol using molecular distillation has been studied. The effects of operating temperature and feed flow rate through a sequential distillation strategy were investigated. The optimal experimental temperature was at 70°C for separating 1,3-propanediol and the by-product 2,3-butanediol. Meanwhile, the volume flow rate was 10 mL/min. As a result, the recovery of 1,3-propanediol and 2,3-butanediol were 87.6 and 87.5%, respectively. Furthermore, the integrated separation characteristic of 1,3-propanediol was evaluated through macrolevel and micro-level models. The separation factors of 1,3-propanediol versus 2,3-butanediol and glycerol were 0.11 and 1.07, respectively, affirming that the separation of 1,3-propanediol by molecular distillation was feasible.     

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。     

Glycerol conversion to 1,3-propanediol by newly isolated clostridia

Summary From pasteurized mud and soil samples glycerol-fermenting clostridia that produced 1,3-propanediol, butyrate and acetate were obtained. The isolates were taxonomically characterized and identified as Clostridium butyricum. The most active strain, SH1 = DSM 5431, was able to convert up to 110 g/l of glycerol to 56 g/l of 1,3-propanediol in 29 h. A few Clostridium strains from culture-collections (3 out of 16 of the C. butyricum group) and some isolates of Kutzner from cheese samples were also able to ferment glycerol, but the final concentration and the productivity of 1,3-propanediol was lower than in strain SH1. Strain SH1 grew well in a pH range between 6.0 and 7.5, with a weak optimum at 6.5, and was stimulated by sparging with N₂. Best overall productivity was obtained in fed-batch culture with a starting concentration of 5% glycerol. In all fermentations the yield of 1,3-propanediol in relation to glycerol was higher than expected from NADH production by acid formation. On the other hand the H₂ production was lower than expected, if per mole of acetyl coenzyme A one mole of H₂ is released. The observations point to a substantial transfer of reducing potential from ferredoxin to NAD, which finally results in increased 1,3-propanediol production.     

Formaldehyde metabolism by Escherichia coli. Carbon and solvent deuterium incorporation into glycerol, 1,2-propanediol, and 1,3-propanediol

Escherichia coli were grown on 14.3% uniformly 13C-labeled glucose as the sole carbon source and challenged anaerobically with 90% 13C-labeled formaldehyde. The major multiply labeled metabolites were identified by 13C NMR spectroscopy to be glycerol and 1,2-propanediol, and a minor metabolite was shown to be 1,3-propanediol. In each case, formaldehyde is incorporated only into the C1 position. A novel form of 13C NMR isotope dilution analysis of the major products reveals that all the 1,2-diol C1 is formaldehyde derived but that about 40% of the glycerol C1 is derived from bacterial sources. Glycerokinase converted the metabolite [1-13C]glycerol to equal amounts of [3-13C]glycerol 3-phosphate and [1-13C]glycerol 3-phosphate, demonstrating that the metabolite is racemic. When [13C]formaldehyde incubation was carried out in H2O/D2O mixtures, deuterium incorporation was detected by beta- and gamma-isotope shifts. The 1,3-diol is deuterium labeled only at C2 and only once, while the 1,2-diol and glycerol are each labeled independently at both C2 and C3; C3 is multiply labeled. Deuterium incorporation levels are different for each metabolite, indicating that the biosynthetic pathways probably diverge early.     

An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside.

Acyclic nucleoside analogues with carboxamido- or nitro-substituted heterocyclic bases have been evaluated for their possible use as universal bases in oligodeoxynucleotides. The acyclic moiety endows the constructs with enough flexibility to allow good base stacking. The 5-nitroindazole analogue afforded the most stable duplexes among the acyclic derivatives with the least spread in Tm versus the four natural bases. In spite of the acyclic moiety, stabilities are comparable with those of duplexes incorporating the recently described 5-nitroindole nucleoside analogue, but considerably exceed those for the 3-nitropyrrole analogue.     

Fermentation of glycerol to 1,3-propanediol: use of cosubstrates

Three fermentable substances, glucose, 1,2-ethanediol and 1,2-propanediol were checked as cosubstrates for the fermentation of glycerol by Clostridium butyricum and Citrobacter freundii with the aim of achieving a complete conversion of glycerol to 1,3-propanediol. Glucose was fermented by C. butyricum mainly to acetate, CO₂ and reducing equivalents in the presence of glycerol and contributed markedly to the 1,3-propanediol yield. However, because of relatively slow growth on glucose, complete conversion was not achieved. If the two glycols were used as cosubstrates for glycerol fermentation, the 1,3-propanediol yield did not increase but dimished considerably, as they were converted to more reduced products, i.e. alcohols instead of acids. From 1,2-propanediol 2-propanol was formed in addition to 1-propanol. The ratio of the propanols was dependent on the culture conditions.     

补料分批发酵过程中两阶段发酵生产1,3-丙二醇

在补料分批发酵过程中提高比生长速率不仅减少乙醇、甲酸的生成,而且提高1,3-丙二醇的得率和比生产速率.发酵后期甘油的浓度在15～26 g/L时有利于提高1,3-丙二醇的生产.采取在发酵前期控制菌体较高比生长速率和发酵后期控制适宜甘油浓度相结合的策略,有效地提高了1,3-丙二醇的生产,降低副产物乳酸和乙醇的生成.