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.     

Microbial production of 1,3-propanediol

1,3-Propanediol (1,3-PD) production by fermentation of glycerol was described in 1881 but little attention was paid to this microbial route for over a century. Glycerol conversion to 1,3-PD can be carried out by Clostridia as well as Enterobacteriaceae. The main intermediate of the oxidative pathway is pyruvate, the further utilization of which produces CO₂, H₂, acetate, butyrate, ethanol, butanol and 2,3-butanediol. In addition, lactate and succinate are generated. The yield of 1,3-PD per glycerol is determined by the availability of NADH₂, which is mainly affected by the product distribution (of the oxidative pathway) and depends first of all on the microorganism used but also on the process conditions (type of fermentation, substrate excess, various inhibitions). In the past decade, research to produce 1,3-PD microbially was considerably expanded as the diol can be used for various polycondensates. In particular, polyesters with useful properties can be manufactured. A prerequisite for making a “green” polyester is a more cost-effective production of 1,3-PD, which, in practical terms, can only be achieved by using an alternative substrate, such as glucose instead of glycerol. Therefore, great efforts are now being made to combine the pathway from glucose to glycerol successfully with the bacterial route from glycerol to 1,3-PD. Thus, 1,3-PD may become the first bulk chemical produced by a genetically engineered microorganism. Received: 12 January 1999 / Received revision: 9 March 1999 / Accepted: 14 March 1999     

Klebsiella pneumoniae 1,3-propanediol:NAD+ oxidoreductase.

Fermentative utilization of glycerol, a more reduced carbohydrate than aldoses and ketoses, requires the disposal of the two extra hydrogen atoms. This is accomplished by sacrificing an equal quantity of glycerol via an auxiliary pathway initiated by glycerol dehydratase. The product, 3-hydroxypropionaldehyde, is then reduced by 1,3-propanediol NAD+:oxidoreductase (1,3-propanediol dehydrogenase; EC 1.1.1.202), resulting in the regeneration of NAD+ from NADH. The pathway for the assimilation of glycerol is initiated by an NAD-linked dehydrogenase. In Klebsiella pneumoniae the two pathways are encoded by the dha regulon which is inducible only anaerobically. In this study 1,3-propanediol:NAD+ oxidoreductase was purified from cells grown anaerobically on glycerol. The enzyme was immunochemically distinct from the NAD-linked glycerol dehydrogenase and was an octamer or hexamer of a polypeptide of 45,000 +/- 3,000 daltons. When tested as a dehydrogenase, only 1,3-propanediol served as a substrate; no activity was detected with ethanol, 1-propanol, 1,2-propanediol, glycerol, or 1,4-butanediol. The enzyme was inhibited by chelators of divalent cations. An enzyme preparation inhibited by alpha,alpha'-dipyridyl was reactivated by the addition of Fe2+ or Mn2+ after removal of the chelator by gel filtration. As for glycerol dehydrogenase, 1,3-propanediol oxidoreductase is apparently inactivated by oxidation during aerobic metabolism, under which condition the enzyme becomes superfluous.     

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。     

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.     

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.     

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。     

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.     

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.     

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%.     

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.     

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。     

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.     

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)     

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.     

Metabolic engineering for the microbial production of 1,3-propanediol

Improvements in the biological production of 1,3-propanediol, a key component of an emerging polymer business, have been realized. Utilizing genes from natural strains that produce 1,3-propanediol from glycerol, metabolic engineering has enabled the development of a recombinant strain that utilizes the lower cost feedstock D-glucose. This accomplishment bodes well for future metabolic engineering efforts and, ultimately, for increased societal benefit obtained through the production of chemicals from renewable resources.     

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.