Production of poly(3-hydroxybutyrate) by fed-batch culture of filamentation-suppressed recombinant Escherichia coli.

Recombinant Escherichia coli XL1-Blue harboring a high-copy-number plasmid containing the Alcaligenes eutrophus polyhydroxyalkanoate synthesis genes could efficiently synthesize poly(3-hydroxybutyrate) (PHB) in a complex medium containing yeast extract and tryptone but not in a defined medium. One of the reasons for the reduced PHB production in a defined medium was thought to be severe filamentation of cells in this medium. By overexpressing an essential cell division protein, FtsZ, in recombinant E. coli producing PHB, filamentation could be suppressed and PHB could be efficiently produced in a defined medium. A high PHB concentration of 149 g/liter, with high productivity of 3.4 g of PHB/liter/h, could be obtained by the pH-stat fed-batch culture of the filamentation-suppressed recombinant E. coli in a defined medium. It was also found that insufficient oxygen supply at a dissolved oxygen concentration (DOC) of 1 to 3% of air saturation during active PHB synthesis phase did not negatively affect PHB production. By growing cells to the concentration of 110 g/liter and then controlling the DOC in the range of 1 to 3% of air saturation, a PHB concentration of 157 g/liter and PHB productivity of 3.2 g of PHB/liter/h were obtained. For the scale-up studies, fed-batch culture was carried out in a 50-liter stirred tank fermentor, in which the DOC decreased to zero when cell concentration reached 50 g/liter. However, a relatively high PHB concentration of 101 g/liter and PHB productivity of 2.8 g of PHB/liter/h could still be obtained, which demonstrated the possibility of industrial production of PHB in a defined medium by employing the filamentation-suppressed recombinant E. coli.     

Efficient fermentation of Pinus sp. acid hydrolysates by an ethanologenic strain of Escherichia coli.

Process conditions for the acid hydrolysis of pine hemicellulose and cellulose have been described which provide a biocompatible sugar solution. By using an improved strain of recombinant Escherichia coli, strain KO11, hydrolysates supplemented with yeast extract and tryptone nutrients were converted to ethanol with an efficiency of 85% to over 100% on the basis of monomer sugar content (approximately 72 g/liter) and with the production of 35 g of ethanol per liter in 48 h. In the process described, approximately 347 liters of ethanol could be produced per dry metric ton of lignocellulose.     

Efficient fermentation of Pinus sp. acid hydrolysates by an ethanologenic strain of Escherichia coli.

Process conditions for the acid hydrolysis of pine hemicellulose and cellulose have been described which provide a biocompatible sugar solution. By using an improved strain of recombinant Escherichia coli, strain KO11, hydrolysates supplemented with yeast extract and tryptone nutrients were converted to ethanol with an efficiency of 85% to over 100% on the basis of monomer sugar content (approximately 72 g/liter) and with the production of 35 g of ethanol per liter in 48 h. In the process described, approximately 347 liters of ethanol could be produced per dry metric ton of lignocellulose.     

Expression, purification, and characterization of the recombinant calcium-binding equine lysozyme secreted by the filamentous fungus Aspergillus niger: comparisons with the production of hen and human lysozymes.

Equine lysozyme (EqL) has been expressed from a synthetic gene and secreted from a heterologous host, the filamentous fungus Aspergillus niger. By including 100 mM Ca2+ in the growth medium, secreted yields of more than 50 mg/liter could be achieved using polyvinylpyrrolidone (PVP) complete medium. In a soya medium yields of up to 150 mg/liter were achieved. The production of recombinant human lysozyme (HuL) from A. niger with yields of over 40 mg/liter was also achieved using PVP medium. Addition of Ca2+ to the growth medium reduced the yield of both HuL and hen egg white lysozyme (HEWL). Sequence differences between the three lysozymes, EqL, HuL, and HEWL, resulted in different susceptibilities to cleavage by A. niger proteases. An improved procedure for the purification of EqL and HuL from A. niger allowed separation of the proteins from pigments produced by the fungus. Detailed spectroscopic analysis, including 2D 1H NMR, for recombinant EqL and recombinant HuL confirm that both proteins possess their native structure and are purified to homogeneity.     

Highly efficient biotransformation of eugenol to ferulic acid and further conversion to vanillin in recombinant strains of Escherichia coli

The vaoA gene from Penicillium simplicissimum CBS 170.90, encoding vanillyl alcohol oxidase, which also catalyzes the conversion of eugenol to coniferyl alcohol, was expressed in Escherichia coli XL1-Blue under the control of the lac promoter, together with the genes calA and calB, encoding coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase of Pseudomonas sp. strain HR199, respectively. Resting cells of the corresponding recombinant strain E. coli XL1-Blue(pSKvaomPcalAmcalB) converted eugenol to ferulic acid with a molar yield of 91% within 15 h on a 50-ml scale, reaching a ferulic acid concentration of 8.6 g liter(-1). This biotransformation was scaled up to a 30-liter fermentation volume. The maximum production rate for ferulic acid at that scale was 14.4 mmol per h per liter of culture. The maximum concentration of ferulic acid obtained was 14.7 g liter(-1) after a total fermentation time of 30 h, which corresponded to a molar yield of 93.3% with respect to the added amount of eugenol. In a two-step biotransformation, E. coli XL1-Blue(pSKvaomPcalAmcalB) was used to produce ferulic acid from eugenol and, subsequently, E. coli(pSKechE/Hfcs) was used to convert ferulic acid to vanillin (J. Overhage, H. Priefert, and A. Steinbüchel, Appl. Environ. Microbiol. 65:4837-4847, 1999). This process led to 0.3 g of vanillin liter(-1), besides 0.1 g of vanillyl alcohol and 4.6 g of ferulic acid liter(-1). The genes ehyAB, encoding eugenol hydroxylase of Pseudomonas sp. strain HR199, and azu, encoding the potential physiological electron acceptor of this enzyme, were shown to be unsuitable for establishing eugenol bioconversion in E. coli XL1-Blue.     

Dehydrogenation of ribitol with Gluconobacter oxydans: production and stability of L-ribulose

l-Ribulose is an important chiral lead molecule used for the synthesis of, among others, l-ribose, a high-value rare sugar used in the preparation of antiviral drugs. These drugs--nucleoside-analogues--gain importance in the treatment of severe viral diseases, like those caused by the HIV or hepatitis virus. In this study, factors that may have an impact on l-ribulose production with Gluconobacter oxydans and on the stability of l-ribulose were investigated. A bioconversion-type process, using washed resting cells, was chosen to produce l-ribulose from ribitol. In this process, the cell production and bioconversion phase were separated. The former was first optimized and a maximum cell mass of 1.5 g CDWL(-1) could be produced. For the bioconversion phase, the aeration level of the system proved to be one of the most critical factors; a maximal production rate of 15.7 g L(-1)h(-1) or 5.9 g(g CDW)(-1)h(-1) of l-ribulose could be reached. Furthermore, resting cells were found capable of completely converting ribitol solutions of up to 300 g L(-1) within 30 h, although the kinetics indicated a rather low affinity of the dehydrogenase enzymes for the substrate.     

Production of D-p-hydroxyphenylglycine by N-carbamoyl-D-amino acid amidohydrolase-overproducing Escherichia coli strains.

The N-carbamoyl-D-amino acid amidohydrolase (D-carbamoylase) gene from Agrobacterium radiobacter NRRL B11291 has been successfully cloned and expressed in Escherichia coli. Subcloning of the D-carbamoylase gene into different types of vectors and backgrounds of E. coli strains showed that the optimal expression level of D-carbamoylase was achieved in a ColE1-derived plasmid with a 150-fold increase in specific enzyme activity compared to that in a pSC101-derived plasmid. In addition, the recombinant plasmids were very stable in the E. coli strain ATCC11303 but not in JCL1258 tested here. Employing the recombinant E. coli strain DH5alpha/pAH61 for D-p-hydroxyphenylglycine production showed that the cell was capable of transforming N-carbamoyl-D-hydroxylphenylglycine to D-p-hydroxyphenylglycine with a molar conversion yield of 100% and a production rate of 1.9 g/(L h). In comparison with A. radiobacter NRRL B11291, this productivity approximates a 55-fold increase in D-hydroxyphenylglycine production. This result suggests the potential application of recombinant E. coli strains for the transformation reaction.     

Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli

Lactose and all of the major sugars (glucose, xylose, arabinose, galactose, and mannose) present in cellulose and hemicellulose were converted to ethanol by recombinant Escherichia coli containing plasmid-borne genes encoding the enzymes for the ethanol pathway from Zymomonas mobilis. Environmental tolerances, plasmid stability, expression of Z. mobilis pyruvate decarboxylase, substrate range, and ethanol production (from glucose, lactose, and xylose) were compared among eight American Type Culture Collection strains. E. coli ATCC 9637(pLO1297), ATCC 11303(pLO1297), and ATCC 15224(pLO1297) were selected for further development on the basis of environmental hardiness and ethanol production. Volumetric ethanol productivities per hour in batch culture were 1.4 g/liter for glucose (12%), 1.3 g/liter for lactose (12%), and 0.64 g/liter for xylose (8%). Ethanol productivities per hour ranged from 2.1 g/g of cell dry weight with 12% glucose to 1.3 g/g of cell dry weight with 8% xylose. The ethanol yield per gram of xylose was higher for recombinant E. coli than commonly reported for Saccharomyces cerevisiae with glucose. Glucose (12%), lactose (12%), and xylose (8%) were converted to (by volume) 7.2% ethanol, 6.5% ethanol, and 5.2% ethanol, respectively.     

Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli.

Lactose and all of the major sugars (glucose, xylose, arabinose, galactose, and mannose) present in cellulose and hemicellulose were converted to ethanol by recombinant Escherichia coli containing plasmid-borne genes encoding the enzymes for the ethanol pathway from Zymomonas mobilis. Environmental tolerances, plasmid stability, expression of Z. mobilis pyruvate decarboxylase, substrate range, and ethanol production (from glucose, lactose, and xylose) were compared among eight American Type Culture Collection strains. E. coli ATCC 9637(pLO1297), ATCC 11303(pLO1297), and ATCC 15224(pLO1297) were selected for further development on the basis of environmental hardiness and ethanol production. Volumetric ethanol productivities per hour in batch culture were 1.4 g/liter for glucose (12%), 1.3 g/liter for lactose (12%), and 0.64 g/liter for xylose (8%). Ethanol productivities per hour ranged from 2.1 g/g of cell dry weight with 12% glucose to 1.3 g/g of cell dry weight with 8% xylose. The ethanol yield per gram of xylose was higher for recombinant E. coli than commonly reported for Saccharomyces cerevisiae with glucose. Glucose (12%), lactose (12%), and xylose (8%) were converted to (by volume) 7.2% ethanol, 6.5% ethanol, and 5.2% ethanol, respectively.     

High-level production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch culture of recombinant Escherichia coli.

Fermentation strategies for production of high concentrations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] with different 3-hydroxyvalerate (3HV) fractions by recombinant Escherichia coli harboring the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes were developed. Fed-batch cultures of recombinant E. coli with the pH-stat feeding strategy facilitated production of high concentrations and high contents of P(3HB-co-3HV) in a chemically defined medium. When a feeding solution was added in order to increase the glucose and propionic acid concentrations to 20 g/liter and 20 mM, respectively, after each feeding, a cell dry weight of 120.3 g/liter and a relatively low P(3HB-co-3HV) content, 42.5 wt%, were obtained. Accumulation of a high residual concentration of propionic acid in the medium was the reason for the low P(3HB-co-3HV) content. An acetic acid induction strategy was used to stimulate the uptake and utilization of propionic acid. When a fed-batch culture and this strategy were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 141.9 g/liter, 88.1 g/liter, 62.1 wt%, and 15.3 mol%, respectively. When an improved nutrient feeding strategy, acetic acid induction, and oleic acid supplementation were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 203.1 g/liter, 158.8 g/liter, 78.2 wt%, and 10.6 mol%, respectively; this resulted in a high level of productivity, 2.88 g of P(3HB-co-3HV)/liter-h.     

Development of a method to produce hemoglobin in a bioreactor culture of Escherichia coli BL21(DE3) transformed with a plasmid containing Plesiomonas shigelloides heme transport genes and modified human hemoglobin genes

We describe a method for production of recombinant human hemoglobin by Escherichia coli grown in a bioreactor. E. coli BL21(DE3) transformed with a plasmid containing hemoglobin genes and Plesiomonas shigelloides heme transport genes reached a cell dry weight of 83.64 g/liter and produced 11.92 g/liter of hemoglobin in clarified lysates.     

Metabolic engineering of a 1,2-propanediol pathway in Escherichia coli

1,2-Propanediol (1,2-PD) is a major commodity chemical that is currently derived from propylene, a nonrenewable resource. A goal of our research is to develop fermentation routes to 1,2-PD from renewable resources. Here we report the production of enantiomerically pure R-1,2-PD from glucose in Escherichia coli expressing NADH-linked glycerol dehydrogenase genes (E. coli gldA or Klebsiella pneumoniae dhaD). We also show that E. coli overexpressing the E. coli methylglyoxal synthase gene (mgs) produced 1,2-PD. The expression of either glycerol dehydrogenase or methylglyoxal synthase resulted in the anaerobic production of approximately 0.25 g of 1,2-PD per liter. R-1,2-PD production was further improved to 0.7 g of 1,2-PD per liter when methylglyoxal synthase and glycerol dehydrogenase (gldA) were coexpressed. In vitro studies indicated that the route to R-1,2-PD involved the reduction of methylglyoxal to R-lactaldehyde by the recombinant glycerol dehydrogenase and the reduction of R-lactaldehyde to R-1, 2-PD by a native E. coli activity. We expect that R-1,2-PD production can be significantly improved through further metabolic and bioprocess engineering.     

改造大肠杆菌合成海藻糖途径以高效合成海藻糖

海藻糖是相容性溶质的一种,因其具有多种生物学功能,在食品、化妆品、药品以及器官移植等方面均有很广泛应用。然而近几年生产海藻糖主要集中在使用酶催化的方法,虽然这种方法的转化效率高,但是却存在着副产物的问题,难以得到高纯度的海藻糖产品,严重制约了海藻糖的应用。本文通过基因工程技术在大肠杆菌Escherichia coli中构建了海藻糖高效合成新途径,通过全细胞催化合成海藻糖。利用PCR技术在哈氏噬纤维菌Cytophaga hutchinsonii中克隆获得海藻糖双功能合成酶基因(tpsp),采用E.coli pTac-HisA高效表达载体,实现海藻糖双功能合成酶基因(tpsp)高效表达,利用高效表达菌株进行全细胞催化,将葡萄糖高效转化为海藻糖。结果表明C.hutchinsonii海藻糖合成酶基因(tpsp)在E.coli中成功实现表达,该酶能够在胞内将葡萄糖高效转化为海藻糖,并将其转运到胞外,实现海藻糖的高效率合成,海藻糖的产量提高到1.2 g/L,相对转化率为21%。当将此高产菌株在发酵罐中进行转化时,海藻糖的产量达到13.3 g/L,葡萄糖的相对转化率达到48.6%。采用C.hutchinsonii海藻糖合成酶基因高效表达并且应用于海藻糖全细胞合成催化在国内外尚属首次报道,海藻糖的转化率及产率都已达到文献报道最高水平,本研究为开拓海藻糖生产新技术奠定了基础。     

Effects of environmental conditions on xylose fermentation by recombinant Escherichia coli

In batch fermentations, optimal conversion of xylose to ethanol by recombinant Escherichia coli was obtained under the following conditions: 30 to 37 degrees C, pH 6.4 to 6.8, 0.1 to 0.2 M potassium phosphate buffer, and xylose concentrations of 8% or less. A yield of 39.2 g of ethanol per liter (4.9% ethanol by volume) was observed with 80 g of xylose per liter, equivalent to 96% of the maximum theoretical yield. Maximal volumetric productivity was 0.7 g of ethanol per liter per h in batch fermentations and 30 g of ethanol per liter per h in concentrated cell suspensions (analogous to cell recycling).     

Effects of environmental conditions on xylose fermentation by recombinant Escherichia coli.

In batch fermentations, optimal conversion of xylose to ethanol by recombinant Escherichia coli was obtained under the following conditions: 30 to 37 degrees C, pH 6.4 to 6.8, 0.1 to 0.2 M potassium phosphate buffer, and xylose concentrations of 8% or less. A yield of 39.2 g of ethanol per liter (4.9% ethanol by volume) was observed with 80 g of xylose per liter, equivalent to 96% of the maximum theoretical yield. Maximal volumetric productivity was 0.7 g of ethanol per liter per h in batch fermentations and 30 g of ethanol per liter per h in concentrated cell suspensions (analogous to cell recycling).     

Overproduction of recombinant ribulose 1,5-bisphosphate carboxylase/oxygenase from Synechococcus sp. strain PCC6301 in glucose-controlled high-cell-density fermentations by Escherichia coli K-12.

A predictive and feedback glucose feed controller, previously developed for nutrient-sufficient growth of Escherichia coli to high cell densities, was used to produce large quantities of a heterologous, cyanobacterial recombinant hexadecameric (L8S8) protein, ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) in E. coli. Culture and plasmid stability conditions were optimized to yield the production of approximately 1 g of soluble, active recombinant RubisCO per liter. Recombinant RubisCO also was produced in lactose-induced high-cell-density fermentation of E. coli K-12.     

三酶级联催化L-苏氨酸生产L-2-氨基丁酸

L-2-氨基丁酸(L-ABA)是一种重要的化工原材料和手性医药中间体,为了实现L-ABA的高效生产,本研究在大肠杆菌EscherichiacoliBL21(DE3)中分别表达大肠杆菌来源的苏氨酸脱氨酶(Threonine deaminase,TD)、苏云金芽孢杆菌来源的亮氨酸脱氢酶(Leucine dehydrogenase,LDH)和博伊丁假丝酵母来源的甲酸脱氢酶(Formatedehydrogenase,FDH),构建体外级联酶催化反应实现L-苏氨酸向L-ABA的转化,体系中TD、LDH和FDH添加最适比例为1∶1∶0.2。为了简化生产工艺,将3种酶在一株菌E. coli 3FT+L中共表达并实现上述配比,在30 L发酵罐中用E. coli 3FT+L全细胞转化12 h,L-ABA的产量为68.5 g/L,底物L-苏氨酸的摩尔转化率达到99.0%。该工艺路线绿色高效,为未来大规模生产L-ABA提供借鉴。     

过量表达苹果酸脱氢酶对大肠杆菌NZN111产丁二酸的影响

大肠杆菌NZN111是敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸-甲酸裂解酶的编码基因 (pflB) 的工程菌,厌氧条件下由于辅酶NAD(H) 的不平衡导致其丧失了代谢葡萄糖的能力。构建了苹果酸脱氢酶的重组菌大肠杆菌NZN111/pTrc99a-mdh,在厌氧摇瓶发酵过程中通过0.3 mmol/L的IPTG诱导后重组菌的苹果酸脱氢酶 (Malate dehydrogenase,MDH) 酶活较出发菌株提高了14.8倍,NADH/NAD+的比例从0.64下降到0.26,同时NAD+和NADH浓度分别     

Expression and purification of the synthetic preS1 gene of Hepatitis B Virus with preferred Escherichia coli codon preference

To produce high levels of hepatitis B virus (HBV) preS1 protein at low cost, a DNA fragment encoding the preS1 region, residues 1-119, of HBV adr subtype was synthesized by overlapping-PCR according to Escherichia coli (E. coli) B preferred codon usage. The synthetic preS1 gene (spreS1) was cloned into the bacterial expression vector pET-30a and transferred into the expression strain E. coli BL21(DE3). Recombinant preS1 protein with an N-terminal His6 tag was expressed at high levels in soluble form, yielding about 44% of the total cellular protein. This technique overcomes problems that existed in previously reported expression systems of preS1 or its epitope, i.e., low-level expression or expression in inclusion bodies. Using this His-tagged preS1 expression system, recombinant protein was purified by single-step affinity chromatography on a Ni-NTA column resulting in a yield was about 28 mg recombinant protein per liter culture. Furthermore, Western blotting and indirect ELISA analysis demonstrate that the reactivity of preS1-specific antibody is comparable between the recombinant and commercialized preS1 protein. Thus, our improved expression system could be used for practical, low-cost large-scale production of recombinant preS1 without refolding steps.