Reduction of carboxylic acids by Nocardia aldehyde oxidoreductase requires a phosphopantetheinylated enzyme

Aldehyde oxidoreductase (carboxylic acid reductase (Car)) catalyzes the magnesium-, ATP-, and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes. Heterologous expression of the car gene in Escherichia coli afforded purified recombinant enzyme with a specific activity nearly 50-fold lower than that of purified native Nocardia sp. enzyme. The 5-fold increase in specific activity obtained by incubating purified recombinant Car with CoA and Nocardia cell-free extracts indicated that post-translational phosphopantetheinylation of Car is required for maximum enzyme activity. Nocardia phosphopantetheine transferase (PPTase) expressed in E. coli was isolated and characterized. When incubated with [(3)H]acetyl-CoA and Nocardia PPTase, the labeled acetylphosphopantetheine moiety was incorporated into recombinant Car. Coexpression of Nocardia Car and PPTase in E. coli gave a reductase with nearly 20-fold higher specific activity. Site-directed mutagenesis in which Ser(689) was replaced with Ala resulted in an inactive Car mutant. The results show that Car expressed in Escherichia coli is an apoenzyme that is converted to a holoenzyme by post-translational modification via phosphopantetheinylation. Doubly recombinant resting E. coli cells efficiently reduce vanillic acid to vanillin.     

胆固醇氧化酶基因的克隆及在E.coli中的表达

根据NCBI中报道的BrevibacteriumsterolicumATCC21387胆固醇氧化酶基因序列,采用PCR方法以Brevibacteriumsp.DGCDC-82的基因组为模板,扩增得到了编码胆固醇氧化酶的基因,该基因与来源于BrevibacteriumsterolicumATCC21387的胆固醇氧化酶基因(choB)同源性为98%。将得到的基因定向克隆到pET28a载体中,转化至含有编码argU和proL基因的大肠杆菌BL21-CodonPlus(DE3)-RP中表达。经过IPTG诱导后,经SDS-PAGE检测在约55kD处有一蛋白表达条带,目的蛋白表达量约占总蛋白的16%,经测定酶活为340U/L。     

Biocatalytic synthesis of vanillin

The conversions of vanillic acid and O-benzylvanillic acid to vanillin were examined by using whole cells and enzyme preparations of Nocardia sp. strain NRRL 5646. With growing cultures, vanillic acid was decarboxylated (69% yield) to guaiacol and reduced (11% yield) to vanillyl alcohol. In resting Nocardia cells in buffer, 4-O-benzylvanillic acid was converted to the corresponding alcohol product without decarboxylation. Purified Nocardia carboxylic acid reductase, an ATP and NADPH-dependent enzyme, quantitatively reduced vanillic acid to vanillin. Structures of metabolites were established by (1)H nuclear magnetic resonance and mass spectral analyses.     

Codon preference optimization increases heterologous PEDF expression

Pigment epithelium-derived factor (PEDF) is widely known for its neurotrophic and antiangiogenic functions. Efficacy studies of PEDF in animal models are limited because of poor heterologous protein yields. Here, we redesigned the human PEDF gene to preferentially match codon frequencies of E coli without altering the amino acid sequence. Following de novo synthesis, codon optimized PEDF (coPEDF) and the wtPEDF genes were cloned into pET32a containing a 5' thioredoxin sequence (Trx) and the recombinant Trx-coPEDF or Trx-wtPEDF fusion constructs expressed in native and two tRNA augmented E coli hosts - BL21-CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP, carrying extra copies of tRNAarg,ile,leu and tRNAarg,pro genes, respectively. Trx-PEDF fusion proteins were isolated using Ni-NTA metal affinity chromatography and PEDF purified after cleavage with factor Xα. Protein purity and identity were confirmed by western blot, MALDI-TOF, and UV/CD spectral analyses. Expression of the synthetic gene was ～3.4 fold greater (212.7 mg/g; 62.1 mg/g wet cells) and purified yields ～4 fold greater (41.1 mg/g; 11.3 mg/g wet cell) than wtPEDF in the native host. A small increase in expression of both genes was observed in hosts supplemented with rare tRNA genes compared to the native host but expression of coPEDF was ～3 fold greater than wtPEDF in both native and codon-bias-adjusted E coli strains. ΔGs at -3 to +50 of the Trx site of both fusion genes were -3.9 kcal/mol. Functionally, coPEDF was equally as effective as wtPEDF in reducing oxidative stress, promoting neurite outgrowth, and blocking endothelial tube formation. These findings suggest that while rare tRNA augmentation and mRNA folding energies can significantly contribute to increased protein expression, preferred codon usage, in this case, is advantageous to translational efficiency of biologically active PEDF in E coli. This strategy will undoubtedly fast forward studies to validate therapeutic utility of PEDF in vivo.     

Cloning of a xylanase gene from Aspergillus usamii and its expression in Escherichia coli

The complete gene xyn// that encodes endo-1,4-beta-xylanase secreted by Aspergillus usamii E001 was cloned and sequenced. The coding region of the gene is separated by only one intron. It encodes 184 amino acid residues of a protein with a calculated molecular weight of 19.8kDa plus a signal peptide of 27 amino acids. The amino acid sequence of the xyn// gene has higher similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The mature peptide encoding cDNA was subcloned into pET-28a(+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL, and xylanase activity was measured. The expressed fusion protein was analyzed by SDS-PAGE and a new specific band with molecular weight of about 20kDa was found when induced by IPTG. Enzyme activity assay verified the recombinant protein as a xylanase. A maximum activity of 49.6Umg(-1) was obtained from cellular extract of E. coli BL21-CodonPlus (DE3)-RIL harboring pET-28a-xyn//. The xylanase had optimal activity at pH 4.6 and 50 degrees C. This is the first report on the cloning of a xylanase gene from A. usamii.     

Vanillin production using <Emphasis Type="Italic">Escherichia coli</Emphasis> cells over-expressing isoeugenol monooxygenase of <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

The isoeugenol monooxygenase gene of Pseudomonas putida IE27 was inserted into an expression vector, pET21a, under the control of the T7 promoter. The recombinant plasmid was introduced into Escherichia coli BL21(DE3) cells, containing no vanillin-degrading activity. The transformed E. coli BL21(DE3) cells produced 28.3 g vanillin/l from 230 mM isoeugenol, with a molar conversion yield of 81% at 20°C after 6 h. In the reaction system, no accumulation of undesired by-products, such as vanillic acid or acetaldehyde, was observed.     

海栖热袍菌极耐高温木聚糖酶基因xynB64在大肠杆菌中的融合表达

来源于极端嗜热菌海栖热袍菌(Thermotoga maritima MSB8)的木聚糖酶B具有极高的热稳定性,在饲料、造纸、能源和食品医药行业具有巨大应用潜力。携带酶基因xynB64的pET28a(+)重组载体在宿主大肠杆菌BL21(DE3)中诱导表达,重组酶活力较低。更换宿主为携带稀有tRNA基因的大肠杆菌:BL21-CodonPlus(DE3)-RIPL和Rosetta(DE3)后,酶活力分别提高了197%和277%,但是后者中的表达会形成部分包涵体。宿主菌为大肠杆菌Rosetta(DE3),更换载体为4种融合表达载体pET32a(+)、pET42a(+)、pET43.1a(+)和pMAL-c2X进行表达,重组酶分别融合了Trx、GST、Nus和MBP标签。其中Rosetta(DE3)/pMAL-c2X-xynB64表达酶活力最高,相当于Rosetta(DE3)/pET28a-xynB64表达酶的88%,而且目的酶表达量占全细胞蛋白的40%,几乎不形成包涵体。     

Coenzyme precursor-assisted expression of a cholesterol oxidase from <Emphasis Type="Italic">Brevibacterium</Emphasis> sp. in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene (choB _b ), encoding cholesterol oxidase from Brevibacterium sp. CCTCC M201008, was cloned and sequenced by PCR (GenBank accession number: DQ345780). The gene consists of 1653 base pairs and encodes a protein of 551 amino acids. ChoB _b exhibited a homology of 98% with cholesterol oxidase gene from Brevibacterium sterolicum ATCC 21387. The cholesterol oxidase gene, cloned in the vector pET-28a, was over-expressed in Escherichia coli BL21–CodonPlus (DE3)-RP grown at 23°C in Luria-Bertani medium containing 50 μM riboflavin, the precursor of the FAD coenzyme of the enzyme. A maximum activity of 3.7 U/mg was obtained from cell free extract of E. coli BL21-CodonPlus (DE3)-RP harboring the pET-28a-choB_b.     

Use of modified BL21(DE3) Escherichia coli cells for high-level expression of recombinant peanut allergens affected by poor codon usage

We previously cloned a panel of peanut allergens by phage display technology. Examination of the codons used in these sequences indicated that most of the cDNAs contain an excess of the least used codons in Escherichia coli, namely AGG/AGA, that correspond to a minor tRNA, the product of the dnaY gene. To achieve high-level expression of the peanut allergens, the cDNAs were subcloned into an expression vector of the pET series (Novagen) in order to produce (His)(10)-tagged fusion proteins in conventional E. coli BL21(DE3) cells. The peanut allergens Ara h 1, Ara h 2, and Ara h 6 with an AGG/AGA codon content of 8-10% were only marginally expressed, whereas the peanut profilin Ara h 5, with an AGG/AGA codon content of only 0.8%, was efficiently expressed in these cells. Hence, by using modified BL21(DE3) E. coli cells, namely BL21-CodonPlus(DE3)-RIL cells (Stratagene) with extra copies of E. coli argU, ileY, and leuW tRNA genes, it was possible to attain high-level expression of the proteins affected by rare codon usage. IPTG-induced expression of several recombinant peanut allergens, such as Ara h 1, Ara h 2, and Ara h 6, was greatly increased in these special cells compared to the expression yield achieved by conventional E. coli hosts. The purification of the soluble and the insoluble fraction of Ara h 2 was performed by metal-affinity chromatography and yielded a total of about 30 mg (His)(10)-tagged recombinant protein per liter of culture of transformed BL21(DE3)CodonPlus-RIL cells. This is over 100 times more than achieved by production of Ara h 2 in conventional BL21(DE3) cells.     

Nocardia sp. carboxylic acid reductase: cloning, expression, and characterization of a new aldehyde oxidoreductase family

We have cloned, sequenced, and expressed the gene for a unique ATP- and NADPH-dependent carboxylic acid reductase (CAR) from a Nocardia species that reduces carboxylic acids to their corresponding aldehydes. Recombinant CAR containing an N-terminal histidine affinity tag had K(m) values for benzoate, ATP, and NADPH that were similar to those for natural CAR, and recombinant CAR reduced benzoic, vanillic, and ferulic acids to their corresponding aldehydes. car is the first example of a new gene family encoding oxidoreductases with remote acyl adenylation and reductase sites.     

诺卡氏菌酰胺酶基因的分子克隆及在大肠杆菌中表达的初步研究

酰胺酶是一种重要的工业酶。利用生物信息学手段,在和已知酰胺酶基因序列分析比对的基础上,首次从Ncordiasp.YS-2002中成功地克隆得到酰胺酶基因ami,并对其基因序列及氨基酸序列的性质进行了分析。结果表明,所得酰胺酶基因ami片段大小共为1446bp,由启动子区、阅读框和回文结构终止区三部分构成。序列分析和进化树分析表明,Ncordiasp.YS-2002酰胺酶是一种比较特殊的酰胺酶,不含大多数酰胺酶共同具有的保守区序列。进一步将酰胺酶基因连接到pET-28a( )上,转入大肠杆菌BL21(DE3)中筛选获得重组菌株PEAB。酶活测定结果表明重组菌具有酰胺酶酶活,但较低,其原因可能是因为大量表达的产物主要以包涵体的形式存在。     

极端嗜热菌海栖热袍菌α-葡萄糖醛酸酶的高效表达和重组酶的纯化

α-葡萄糖醛酸酶作为木聚糖降解的限速酶之一,在木聚糖类半纤维素的生物转化中起着重要的作用。海栖热袍菌Thermotoga maritima是一个嗜极端高温的厌氧细菌,其产生的极耐热性酶类具有非常可观的工业应用前景。但热袍菌属Thermotoga的基因在大肠杆菌中的表达一般较困难。研究了T. maritima中的极耐热性α葡萄糖醛酸酶基因在大肠杆菌不同菌株中的表达水平及纯化技术。结果表明,稀有密码子AGA、AGG和AUA限制了该基因在大肠杆菌中的表达,在大肠杆菌BL21-CodonPlus(DE3)RIL可得到高效表达,重组蛋白表达量达20%,比酶活比野生菌株提高5倍;重组蛋白经热处理和金属Ni²⁺的亲和层析提纯后,达到了电泳纯,提纯倍数为5.1倍,收率为55.1%。对重组菌诱导表达条件的研究表明,营养丰富的TB培养基有助于重组菌的生长, 重组菌生长至OD₆₀₀为0.7～0.8时添加IPTG诱导5h后重组蛋白的表达量最高。     

L-脯氨酸-4-羟化酶的异源表达和合成反式-4-羟基-L-脯氨酸的条件优化

L-脯氨酸-4-羟化酶(L-Proline-4-hydroxylase,P4H)是依赖α-酮戊二酸(α-KG)和Fe2+的双加氧酶成员之一,在反式-4-羟基-L-脯氨酸(trans-4-hydroxy-L-proline,t-4Hyp)等重要手性化合物的生物合成中发挥关键作用。本研究构建了来源于Bradyrhizobium japonicum USDA 6的P4H重组大肠杆菌Escherichia coli BL21(DE3)/p ET-28b-p4h BJ,SDS-PAGE和酶活检测结果表明,该菌株具有表达可溶性P4H和催化合成t-4Hyp的能力。通过优化,确定了该重组菌全细胞催化合成t-4Hyp较优的反应体系和条件:10 m L p H 6.5 80 mmol/LMES缓冲液、9 mmol/L L-Pro,6 mmol/L L-抗坏血酸,6 mmol/Lα-KG,0.8 mmol/L Fe SO4·7H2O,反应温度为35℃;在20 g/L湿细胞的催化反应中,t-4Hyp的合成量达到34.86 mg/L,比优化前(17.53 mg/L)提高了98.86%。该工作为进一步利用P4H生物催化法合成t-4Hyp奠定了一定的技术基础。     

Use of Modified BL21(DE3) Escherichia coli Cells for High-Level Expression of Recombinant Peanut Allergens Affected by Poor Codon Usage

We previously cloned a panel of peanut allergens by phage display technology. Examination of the codons used in these sequences indicated that most of the cDNAs contain an excess of the least used codons in Escherichia coli, namely AGG/AGA, that correspond to a minor tRNA, the product of the dnaY gene. To achieve high-level expression of the peanut allergens, the cDNAs were subcloned into an expression vector of the pET series (Novagen) in order to produce (His)₁₀-tagged fusion proteins in conventional E. coli BL21(DE3) cells. The peanut allergens Ara h 1, Ara h 2, and Ara h 6 with an AGG/AGA codon content of 8–10% were only marginally expressed, whereas the peanut profilin Ara h 5, with an AGG/AGA codon content of only 0.8%, was efficiently expressed in these cells. Hence, by using modified BL21(DE3) E. coli cells, namely BL21-CodonPlus(DE3)-RIL cells (Stratagene) with extra copies of E. coli argU, ileY, and leuW tRNA genes, it was possible to attain high-level expression of the proteins affected by rare codon usage. IPTG-induced expression of several recombinant peanut allergens, such as Ara h 1, Ara h 2, and Ara h 6, was greatly increased in these special cells compared to the expression yield achieved by conventional E. coli hosts. The purification of the soluble and the insoluble fraction of Ara h 2 was performed by metal-affinity chromatography and yielded a total of about 30 mg (His)₁₀-tagged recombinant protein per liter of culture of transformed BL21(DE3)CodonPlus-RIL cells. This is over 100 times more than achieved by production of Ara h 2 in conventional BL21(DE3) cells.     

Efficacy evaluation of live Escherichia coli expression Brucella P39 protein combined with CpG oligodeoxynucleotides vaccine against Brucella melitensis 16M, in BALB/c mice

Brucella is gram-negative bacteria responsible for brucellosis in a wide variety of animals and humans. BALB/c mice were immunized with live Escherichia coli expression the p39 gene of Brucella melitensis, a gene coding for the periplasmic binding protein. Mice were injected with either E. coli BL21 (DE3) pEt15b or E. coli BL21 (DE3) pEt15b-p39 alone or adjuvanted with either CpG oligodeoxynucleotides (CpG ODN) or non-CpG ODN. E. coli BL21 (DE3) pEt15b-p39 with CpG ODN or with non-CpG ODN mice groups showed a significant IFN-γ production and T-cell proliferation as a reaction to P39 antigen. In addition, antibody responses (IgG, IgG1 and IgG2a), were only found in these two mice groups. A higher level of protection against B. melitensis 16M were observed in mice immunized with E. coli BL21 (DE3) pEt15b-p39 and CpG ODN comparing with those immunized with E. coli BL21 (DE3) pEt15b-p39 alone or with non-CpG ODN. No protection against B. melitensis 16M was observed in mice immunized with E. coli BL21 (DE3) pEt15b alone or with the adjuvant. Rev.1 protection at 4 and 8 weeks post-challenge was more effective than that observed with E. coli BL21 (DE3) pEt15b-p39 and CpG ODN.     

猪瘟兔化弱毒株E2基因的原核表达及间接ELISA的初步建立

猪瘟病毒E2蛋白C端含有一段30多个疏水性氨基酸组成的跨膜区域(Transmembraneregion,TMR),用RTPCR和巢式PCR分别扩增了含不同长度TMR的猪瘟兔化弱毒E2基因,并克隆入pGEX4T1的MCS中,构建了原核表达载体pGEXTE2339(无TMR)、pGEXTE2355(1TMR)、pGEXTE2375(3TMR)。因E2基因含有大量大肠杆菌稀有密码子,选择了BL21CodonPlus(DE3)RP作为表达受体菌,结果表明pGEXTE2339、pGEXTE2355以包涵体的形式正确表达了目的蛋白,而pGEXTE2375没有明显表达。并利用pGEXTE2339、pGEXTE2355表达的融合蛋白初步建立了间接ELISA方法。     

Large increase in Leuconostoc citreum KM20 dextransucrase activity achieved by changing the strain/inducer combination in an E. coli expression system

A recombinant putative dextransucrase (DexT) was produced from Leuconostoc citreum KM20 as a 160 kDa protein, but its productivity was very low (264 U/l). For optimization, we examined enzyme activity in 7 Escherichia coli strains with inducer molecules such as lactose or IPTG. E. coli BL21-CodonPlus(DE3)-RIL exhibited the highest enzyme activity with lactose. Finally, DexT activity was remarkably increased by 12-fold under the optimized culture conditions of a cell density to start induction (OD???) of 0.95, a lactose concentration of 7.5 mM, and an induction temperature of 17 degrees C. These results may effectively apply to the heterologous expression of other large DexT genes.     

Stereospecific synthesis of (R)-2-hydroxy carboxylic acids using recombinant E. coli BL21 overexpressing YiaE from Escherichia coli K12 and glucose dehydrogenase from Bacillus subtilis

The yiaE gene from Escherichia coli K12 was functionally expressed in E. coli BL21 using an IPTG inducible pET expression system (2.1 U/mg), and YiaE was purified to a specific activity of 18 U/mg. The purified enzyme catalyzes reduction of various aromatic and aliphatic 2-oxo carboxylic acids to the corresponding (R)-2-hydoxy carboxylic acids using NADPH. For practical applications, the problem of NADPH recycle was effectively solved by using recombinant E. coli overexpressing YiaE and glucose dehydrogenase from Bacillus subtilis in the same cell. The recombinant E. coli was used to prepare (R)-phenyllactic acid and (R)-2-hydroxy-4-phenylbutanoic acid from the corresponding 2-oxo carboxylic acids (98% ee) while the alpha-carbonyl group of 2,4-dioxo-4-phenylbutyric acid was reduced regio- and stereospecifically to give (R)-2-hydroxy-4-oxo-4-phenylbutyric acid (97% ee) in quantitative yields. The cells could be recycled for 3 days at room temperature in 100 mM phosphate buffer (pH 7.0) without loss of activity, which reduced to 70% after 1 week.     

生物转化法制备L-天冬酰胺

探索生物转化法制备L-天冬酰胺的技术与工艺。通过分子生物学方法,克隆来源于大肠杆菌(Escherichia coli, E.coli)JM109的天冬酰胺合成酶A基因asnA,并于E. coli BL21(DE3)中表达,利用构建的E.coli基因工程菌E.coli BL21(DE3)/pET28a(+)-asnA全细胞高密度催化L-天冬氨酸生产L-天冬酰胺,以PITC柱前衍生-高效液相检测底物和产物。表达的蛋白质分子质量约为37kDa,与预期大小相符,比酶活力为1786.6U/g。L-天冬氨酸转化率为95.8%,L-天冬酰胺产量可达126.5g/L,生产速率为15.81g/(L·h)。结果表明,已成功构建高效表达天冬酰胺合成酶A基因工程菌株,并用于催化L-天冬氨酸转化生产L-天冬酰胺,解决了L-天冬酰胺生物转化生产工艺中ATP成本过高的难题,为L-天冬酰胺制备提供新的绿色途径。