1,3-丙二醇生产的研究进展

作为一种重要的化工材料,1,3-丙二醇凭借其自身的优点,在工业生产中具有很高的应用价值。但其传统的生产方法,操作繁琐、技术难度大、设备投资高,已无法满足对1,3-丙二醇的日益增长的需要;而微生物发酵法又面临着产率低、发酵条件难以控制等弊病。相反,基因工程技术在1,3-丙二醇生产过程中扮演着越来越重要的角色。简要综述了1,3-丙二醇研究及生产工艺的进展。     

1,3-丙二醇发酵液的絮凝预处理研究

研究了天然澄清剂Ⅱ型B组份对1,3-丙二醇发酵液的絮凝预处理效果。首先通过单因素实验和正交实验,确定了影响絮凝的主要因素及其最佳工艺条件：即在pH6．0、用量为0．01g／L、NaCl盐浓为0g／L时,絮凝率可以达到95．97％。然后考察了絮凝预处理对发酵液过滤以及电渗析脱盐的影响,实验结果表明：絮凝预处理能显加快发酵液中固体微粒的沉降,提高过滤速度,其中絮凝样的滤饼湿基、干基重量分别比对照样增加41．13％、51．88％;絮凝预处理还能加快电渗析脱盐速度,与过滤相结合可以代替离心预处理。     

Cloning and Characterization of a Campylobacter jejuni Iron-Uptake Operon

We report that C. jejuni modifies its outer membrane protein (OMP) repertoire when cultivated under iron-limiting conditions such as during incubation with epithelial cells. To identify genes encoding de novo expressed OMPs, a C. jejuni cosmid library was screened with antisera raised against proteins expressed in the presence of epithelial cells. A single clone was identified encoding an 80-kDa antigen. Sequence analysis of subclones identified an operon of three open reading frames (ORFs) encoding proteins that are homologous to the E. coli ferrichrome uptake system encoded by the fhu locus. Under low-iron conditions, C. jejuni expressed the 80-kDa OMP, indicating that its expression is regulated by the presence of iron. Southern blot analysis indicated that six of eleven isolates of C. jejuni harbor a fhuA homolog which, like all other DNA in this region sequenced thus far, is strikingly GC-rich (65%) compared with the C. jejuni genome (35% G+C). Received: 19 June 2000 / Accepted: 30 August 2000     

Molecular Cloning, Co-expression, and Characterization of Glycerol Dehydratase and 1,3-Propanediol Dehydrogenase from Citrobacter freundii

1,3-Propanediol (1,3-PD), an important material for chemical industry, is biologically synthesized by glycerol dehydratase (GDHt) and 1,3-propanediol dehydrogenase (PDOR). In present study, the dhaBCE and dhaT genes encoding glycerol dehydratase and 1,3-propanediol dehydrogenase respectively were cloned from Citrobacter freundii and co-expressed in E. coli. Sequence analysis revealed that the cloned genes were 85 and 77 % identical to corresponding gene of C. freundii DSM 30040 (GenBank No. U09771), respectively. The over-expressed recombinant enzymes were purified by nickel-chelate chromatography combined with gel filtration, and recombinant GDHt and PDOR were characterized by activity assay, kinetic analysis, pH, and temperature optimization. This research may form a basis for the future work on biological synthesis of 1,3-PD.     

产1,3-丙二醇菌株丁酸梭菌的诱变育种

甘油由丁酸梭菌转化成1,3-丙二醇的研究是厌氧条件下进行。为了获得1,3-丙二醇的高产突变株,以丁酸梭菌为出发菌株进行诱变处理。经过硫酸二乙酯（DES）化学诱变得到2株高产正突变株C.but2031和C.but2046,再经过紫外线和亚硝基胍（NTG）复合诱变得到突变株C.but3037。经过初筛、复筛和传代实验,表明其是稳定的突变株。C.but3037的1,3-丙二醇产量由出发菌株的2.2g/L提高到15.7g/L,提高了6.13倍,     

1,3-Propanediol production in a two-step process fermentation from renewable feedstock

In this work, the production of 1,3-propanediol from glucose and molasses was studied in a two-step process using two recombinant microorganisms. The first step of the process is the conversion of glucose or other sugar into glycerol by the metabolic engineered Saccharomyces cerevisiae strain HC42 adapted to high (>200 g l⁻¹) glucose concentrations. The second step, carried out in the same bioreactor, was performed by the engineered strain Clostridium acetobutylicum DG1 (pSPD5) that converts glycerol to 1,3-propanediol. This two-step strategy led to a flexible process, resulting in a 1,3-propanediol production and yield that depended on the initial sugar concentration. Below 56.2 g l⁻¹ of sugar concentration, cultivation on molasses or glucose showed no significant differences. However, at higher molasses concentrations, glycerol initially produced by yeast could not be totally converted into 1,3-propanediol by C. acetobutylicum and a lower 1,3-propanediol overall yield was observed. In our hand, the best results were obtained with an initial glucose concentration of 103 g l⁻¹, leading to a final 1,3-propanediol concentration of 25.5 g l⁻¹, a productivity of 0.16 g l⁻¹ h⁻¹ and 1,3-propanediol yields of 0.56 g g⁻¹ glycerol and 0.24 g g⁻¹ sugar, which is the highest value reported for a two-step process. For an initial sugar concentration (from molasses) of 56.2 g l⁻¹, 27.4 g l⁻¹ of glycerol were produced, leading to 14.6 g l⁻¹ of 1.3-propanediol and similar values of productivity, 0.15 g l⁻¹ h⁻¹, and overall yield, 0.26 g g⁻¹ sugar.     

Isolation and Characterization of Clostridium butyricum DSM 5431 Mutants with Increased Resistance to 1,3-Propanediol and Altered Production of Acids

Clostridium butyricum mutants were isolated from the parent strain DSM 5431 after mutagenesis with N-methyl-N(prm1)-nitro-N-nitrosoguanidine and two selection procedures: osmotic pressure and the proton suicide method. Isolated mutants were more resistant to glycerol and to 1,3-propanediol (1,3-PD) than was the wild type, and they produced more biomass. In batch culture on 62 g of glycerol per liter, the wild type produced more acetic acid than butyrate, with an acetate/butyrate ratio of 5.0, whereas the mutants produced almost the same quantities of both acids or more butyrate than acetate with acetate/butyrate ratios from 0.6 to 1.1. The total acid formation was higher in the wild-type strain. Results of analysis of key metabolic enzymatic activities were in accordance with the pattern of fermentation product formation: either the butyrate kinase activity increased or the acetate kinase activity decreased in cell extracts of the mutants. A decreased level of the hydrogenase and NADH-ferredoxin activities concomitant with an increase in ferredoxin-NAD(sup+) reductase activities supports the conclusion that the maximum percentage of NADH available and used for the formation of 1,3-PD was higher for the mutants (97 to 100%) than for the wild type (70%). In fed-batch culture, at the end of the fermentation (72 h for the wild-type strain and 80 to 85 h for the mutants), 44% more glycerol was consumed and 50% more 1,3-PD was produced by the mutants than by the wild-type strain.     

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.     

1,3-Propanediol and its copolymers: research, development and industrialization

1,3-Propanediol (PDO), is now taking the transition from a traditional "specialty chemical" to a "commodity chemical". The market for PDO is growing rapidly as the technology develops. With the advancing PDO production technology, polytrimethylene terephthalate (PTT) as a new type of polyester has been applied in carpet and textile fibers, monofilaments, films, and nonwoven fabrics, and in the engineering thermoplastics area, because PTT has unique properties compared to other polymers such as polyethylene terephthalate (PET) and polybutylene terephthalate (PBT). Responding to the environmental and sustainability factors, one- or two-step fermentation technology for PDO production has attracted people's attention. A novel flexible process for PDO production by using aerobic fermentation from glycerol or glucose has been developed and demonstrated with a facility capacity of 4000 t/year in a pilot plant. By using engineered Escherichia coli, 135 g/L PDO was obtained with glucose as feedstock. Since the bio-process of PDO production consumes 40% less energy and reduces greenhouse gas emissions by 20% versus petroleum-based propanediol, the bio-based PTT is more environmentally friendly and sustainable compared with the fossil fuel-based polymers, which made PTT more attractive with good prospects for the future.     

混合菌发酵1,3-丙二醇的初步研究

将表达酿酒酵母3-磷酸甘油脱氢酶基因(GPD1)和3-磷酸甘油酯酶基因(HOR2)的质粒PSE-gpd1-hor2转化到甘油激酶基因(glpK)和甘油脱氢酶基因(gldA)双缺失的大肠杆菌JM109C中,构建产甘油的工程菌JM109C/PSE-gpd1-hor2.接种JM109C/pSE-gpd1-hor2和Klebsiella在含1%葡萄糖的摇瓶发酵培养基中37℃发酵56 h,1,3-丙二醇的最高产量为1.28 g/L,葡萄糖摩尔转化率为37.5%;在30 L发酵罐中发酵68 h,1,3-丙二醇的最高产量为24.09 g/L,葡萄糖摩尔转化率为38.0%;5 g/L的乙酸、乳酸,10 g/L的乙醇分别使1,3-丙二醇的产量降低了91.41%、54.68%和51.56%.     

1,3-Propanediol production by Klebsiella pneumoniae under different aeration strategies

1,3-Propanediol production by Klebsiella pneumoniae was studied in batch cultures under N2 flow and four airflow systems. Different byproducts were formed under different aeration conditions. An anaerobic/aerobic combined fed-batch culture was developed giving 70 g 1,3-propanediol l(-1) and 16 g 2,3-butanediol l(-1) with total diol yield of 0.6 mol(-1) glycerol.     

产1,3-丙二醇菌株的诱变和筛选

为提高克雷伯氏肺炎杆菌产1,3-丙二醇的能力,以离子束、紫外线和氯化锂为复合诱变法,建立了产酸圈和产物耐受相结合的平板筛选方法,获得可耐受高浓度1,3-丙二醇并且副产物中乙醇含量较少的优良突变菌株2株。与出发菌株相比,两株高产突变菌株Klebsiella pneumoniae LM 03和Klebsiella pneumoniae LM05的1,3-丙二醇产量分别提高了33% 和30% ,达到66.74 g/L和65.12 g/L;乙醇产量分别降低了38% 和24% ,降低为6.59 g/L和8.05 g/L。同时测定了诱变前后还原途径中甘油脱水酶（GDHt）和1,3-丙二醇氧化还原酶（PDOR）的酶活变化,研究表明诱变对GDHt有明显的促进作用,而对PDOR的影响不明显。该诱变和筛选方法目标明确、易操作、效率高,在1,3-PD工业规模的生物法生产中将具有良好的应用价值,而且对于其他具有工业应用价值的菌株筛选工作也具有一定的借鉴意义。     

1,3-Propanediol production and tolerance of a halophilic fermentative bacterium, Halanaerobium saccharolyticum subsp. saccharolyticum

1,3-Propanediol (1,3-PD) is widely used in polymer industry in production of polyethers, polyesters and polyurethanes. In this article, a study on 1,3-PD production and tolerance of Halanaerobium saccharolyticum subsp. saccharolyticum is presented. 1,3-PD production was optimized for temperature, vitamin B(12) and acetate concentration. The highest 1,3-PD concentrations and yields (0.6 mol/mol glycerol) were obtained at vitamin B?? concentration 64 μg/l and an inverse correlation between 1,3-PD and hydrogen production was observed with varying vitamin B?? concentrations. In the studied temperature range and initial acetate concentrations up to 10 g/l, no significant variations were observed in 1,3-PD production. High initial acetate (29-58 g/l) was observed to cause slight decrease in 1,3-PD concentrations produced but no effects on 1,3-PD yields (mol/mol glycerol). Initial 1,3-PD concentrations inhibited the growth of H. saccharolyticum subsp. saccharolyticum. When initial 1,3-PD concentration was raised from 1g/l to 57 g/l, a decrease of 12% to 75%, respectively, in the highest optical density was observed.     

Characterization of the glnK-amtB Operon of Azotobacter vinelandii

To determine whether in Azotobacter vinelandii the P_II protein influences the regulation of nif gene expression in response to fluxes in the ammonium supply, the gene encoding P_II was isolated and characterized. Its deduced translation product was highly similar to P_II proteins from other organisms, with the greatest degree of relatedness being exhibited to the Escherichia coli glnK gene product. A gene designated amtB was found downstream of and was cotranscribed with glnK as in E. coli. The AmtB protein is similar to functionally characterized ammonium transport proteins from a few other eukaryotes and one other prokaryote. glnK and amtB comprise an operon. Attempts to isolate a stable glnK mutant strain were unsuccessful, suggesting that glnK, like glnA, is an essential gene in A. vinelandii. amtB mutants were isolated, and although growth on limiting amounts of ammonium was similar in the mutant and wild-type strains, the mutants were unable to transport [¹⁴C]methylammonium.     

Genome Sequence of Klebsiella pneumoniae LZ, a Potential Platform Strain for 1,3-Propanediol Production

Klebsiella pneumoniae LZ is a bacterium isolated from soil which can produce 1,3-propanediol from glycerol. Here we present a 5,431,750-bp assembly of its genome sequence. We annotated 9 coding sequences (CDSs) responsible for glycerol fermentation to 1,3-propanediol, 19 CDSs encoding glycerol utilization, and 134 CDSs related to its virulence and defense.     

1,3-Propanediol:NAD+ oxidoreductases of Lactobacillus brevis and Lactobacillus buchneri.

In the cofermentation of glycerol with a sugar by Lactobacillus brevis and Lactobacillus buchneri, a 1,3-propanediol:NAD+ oxidoreductase provides an additional method of NADH disposal. The enzyme has been purified from both L. brevis B22 and L. buchneri B190 and found to have properties very similar to those reported for the enzyme from Klebsiella pneumoniae. The enzymes required Mn2+ and are probably octamers with a molecular mass of 350 kDa. Although not absolutely specific for 1,3-propanediol when tested as dehydrogenases, the enzymes have less than 10% activity with glycerol, ethanol, and 1,2-propanediol. These properties contrast sharply with those of a protein isolated from another Lactobacillus species (L. reuteri) that ferments glycerol with glucose and previously designated a 1,3-propanediol dehydrogenase.     

Production of 1,3-Propanediol from Glycerol by Clostridium acetobutylicum and Other Clostridium Species

Glycerol was fermented with the production of 1,3-propanediol as the major fermentation product by four strains of Clostridium acetobutylicum, six of C. butylicum, two of C. beijerinckii, one of C. kainantoi, and three of C. butyricum. 1,3-Propanediol was identified by its retention times in gas chromatography and high-pressure liquid chromatography and by its mass spectrum. During growth of C. butylicum B593 in a chemostat culture at pH 6.5, 61% of the glycerol fermented was converted to 1,3-propanediol. When the pH was decreased to 4.9, growth and 1,3-propanediol production were substantially reduced.