Cloning, characterization, and high-level expression in Escherichia coli of the Saccharopolyspora erythraea gene encoding an acyl carrier protein potentially involved in fatty acid biosynthesis.

The erythromycin A-producing polyketide synthase from the gram-positive bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus) has evident structural similarity to fatty acid synthases, particularly to the multifunctional fatty acid synthases found in eukaryotic cells. Fatty acid synthesis in S. erythraea has previously been proposed to involve a discrete acyl carrier protein (ACP), as in most prokaryotic fatty acid synthases. We have cloned and sequenced the structural gene for this ACP and find that it does encode a discrete small protein. The gene lies immediately adjacent to an open reading frame whose gene product shows sequence homology to known beta-ketoacyl-ACP synthases. A convenient expression system for the S. erythraea ACP was obtained by placing the gene in the expression vector pT7-7 in Escherichia coli. In this system the ACP was efficiently expressed at levels 10 to 20% of total cell protein. The recombinant ACP was active in promoting the synthesis of branched-chain acyl-ACP species by extracts of S. erythraea. Electrospray mass spectrometry is shown to be an excellent method for monitoring the efficiency of in vivo posttranslational modification of ACPs.     

Novel gene encoding a Ca2+-binding protein and under hexokinase-dependent sugar regulation

A cDNA encoding a predicted 15-kDa protein was earlier isolated from sugar-induced genes in rice embryos (Oryza sativa L.) by cDNA microarray analysis. Here we report that this cDNA encodes a novel Ca2+-binding protein, named OsSUR1 (for Oryza sativa sugar-up-regulated-1). The recombinant OsSUR1 protein expressed in Escherichia coli had 45Ca2+-binding activity. Northern analysis showed that the OsSUR1 gene was expressed mainly in the internodes of mature plants and in embryos at an early stage of germination. Expression of the OsSUR1 gene was induced by sugars that could serve as substrates of hexokinase, but expression was not repressed by Ca2+ signaling inhibitors, calmodulin antagonists and inhibitors of protein kinase or protein phosphatase. These results suggested that Os-SUR1 gene expression was stimulated by a hexokinase-dependent pathway not mediated by Ca2+.     

Studies of Streptomyces reticuli cel-1 (cellulase) gene expression in Streptomyces strains, Escherichia coli, and Bacillus subtilis.

Various streptomyces strains [Streptomyces lividans 66, Streptomyces vinaceus, and Strepotmyces coelicolor A3 (2)] acquired the ability to utilize crystalline cellulose (Avicel) after transformation with a multicopy vector containing the cel-1 gene from Streptomyces reticuli. The expression level in these hosts was two to three times lower than in S. reticuli, indicating the absence of positive regulatory elements. Like S. reticuli, they processed the Avicelase to its catalytic domain and to an enzymatically inactive part. The cel-1 gene with its original upstream region was not expressed within Escherichia coli. When cel-1 had been fused in phase with the lacZ gene, large quantities of the fusion protein were produced in E. coli. However, this protein was enzymatically inactive and proteolytically degraded to a series of truncated forms. As the cellulase (Avicelase) synthesized by S. reticuli is not cleaved by the E. coli proteases, its posttranslational modification is proposed. With Bacillus subtilis as host, the cel-1 gene was expressed neither under its own promoter nor under the control of a strong Bacillus promoter.     

Increasing the efficiency of heterologous promoters in actinomycetes

An Escherichia coli-actinomycete shuttle vector, pCJW93, was constructed which places cloned genes under the control of the thiostrepton-inducible tip promoter from Streptomyces lividans. We also constructed expression vectors bearing the actII-ORF4/PactI activator-promoter system of the actinorhodin biosynthetic pathway of Streptomyces coelicolor. With both types of vector, levels of expression varied widely in different actinomycete strains, indicating different levels of the host factors needed for optimal expression. Deletion of the actII-ORF4 activator gene from one such plasmid in Saccharopolyspora erythraea drastically reduced expression from the cognate actI promoter, showing that host factors are required for optimal production of the activator protein itself. However, a low copy number expression vector pWIZ1 for the polyketide synthase DEBS1-TE, in which the promoter for the activator gene was replaced by the strong heterologous ermE* promoter of S. erythraea directed highly efficient production of polyketide synthase protein in Streptomyces cinnamonensis; and the levels of triketide lactone product found were up to 100-fold greater than were produced by the same plasmid in which actII-ORF4 was expressed from its own promoter. Ensuring appropriate expression of a specific activator protein should enable more convenient and consistent heterologous expression of genes in a broad range of actinomycete hosts.     

Stress-activated expression of a Streptomyces pristinaespiralis multidrug resistance gene (ptr) in various Streptomyces spp. and Escherichia coli

A promoter which controls expression of the pristinamycin multidrug resistance gene ( ptr ) in Streptomyces pristinaspiralis could be induced by physiological stresses in both Streptomyces spp. and Escherichia coli . In S. pristinaspiralis , the ptr promoter ( Pptr ) was induced by pristinamycin I (PI) or pristinamycin II (PII). Streptomyces lividans was adopted as a convenient heterologous host for studies of Pptr regulation since it has no known pristinamycin biosynthetic genes. Two key regulatory features were documented in these studies: many (19 of 70) antibiotics and chemicals with no common targets or structural features induced the Pptr ; induction with PI was most efficient during a transition phase when antibiotic biosynthetic genes are switched on. In Streptomyces coelicolor, Pptr activity was similarly inducible by PI and not dependent on sigma factors HrdA, HrdC, or HrdD. In E. coli, Pptr cloned in the bifunctional promoter probe vector plJ2839 was functional and activated upon entry into stationary phase in the absence of exogenous inducer. Finally, gel-retardation studies demonstrated a Pptr -binding protein in S. lividans (where its activity was PI-inducible), S. coelicolor and S. pristinaespiralis . The fact that this activity was not detected in E. coli suggested the existence of another regulatory system perhaps also present in Streptomyces .     

Calcium/calmodulin-dependent protein kinase types II and IV differentially regulate CREB-dependent gene expression.

Phosphorylation of CREB (cyclic AMP [cAMP]- response element [CRE]-binding protein) by cAMP-dependent protein kinase (PKA) leads to the activation of many promoters containing CREs. In neurons and other cell types, CREB phosphorylation and activation of CRE-containing promoters can occur in response to elevated intracellular Ca2+. In cultured cells that normally lack this Ca2+ responsiveness, we confer Ca(2+)-mediated activation of a CRE-containing promoter by introducing an expression vector for Ca2+/calmodulin-dependent protein kinase type IV (CaMKIV). Activation could also be mediated directly by a constitutively active form of CaMKIV which is Ca2+ independent. The CaMKIV-mediated gene induction requires the activity of CREB/ATF family members but is independent of PKA activity. In contrast, transient expression of either a constitutively active or wild-type Ca2+/calmodulin-dependent protein kinase type II (CaMKII) fails to mediate the transactivation of the same CRE-containing reporter gene. Examination of the subcellular distribution of transiently expressed CaMKIV and CaMKII reveals that only CaMKIV enters the nucleus. Our results demonstrate that CaMKIV, which is expressed in neuronal, reproductive, and lymphoid tissues, may act as a mediator of Ca(2+)-dependent gene induction.     

透明颤菌血红蛋白基因在阿维链霉菌中的表达

将含有自身启动子的透明颤菌血红蛋白基因（ ｖｈｂ） 克隆至大肠杆菌—链霉菌穿梭质粒载体ｐＩＪ６５３ 中构建成表达载体ｐ ＷＹ１０１ 和ｐ ＷＹ１０２ ,用它们转化阿维菌素（ａｖｅｒｍｅｃｔｉｎｓ） 产生菌———阿维链霉菌（ Ｓｔｒｅｐｔｏｍｙｃｅｓ ａｖｅｒｍｉｔｉｌｉｓ） ,经Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ 分析并未检测到ｖｈｂ 基因表达,但用穿梭载体ｐＨＺ１２５２（ 其中的ｖｈｂ 基因位于受硫链丝菌素诱导的链霉菌强启动子ＰｔｉｐＡ之下） 转化阿维链霉菌并经硫链丝菌素诱导,则在该菌中表达出了有活性的ＶＨｂ 蛋白。ｐＨＺ１２５２ 在阿维链霉菌中发生了重组缺失,但缺失的ｐＨＺ１２５２ 上仍含有完整的ｖｈｂ 基因及诱导型强启动子,且可在阿维链霉菌中稳定遗传,却不能再转化大肠杆菌。     

A second generation snp-derived Escherichia coli-Streptomyces shuttle expression vector that is generally transferable by conjugation

An Escherichia coli-Streptomyces shuttle vector (pJN100) was constructed, by inserting an origin of transfer (oriT), derived from the E. coli broad host range plasmid RK2, into pANT1202, a high-copy-number vector for gene expression in Streptomyces. The resulting conjugably transferable vector contains the pANT1202-derived SnpR (LysR-like protein) activated snpA promoter that drives strong heterologous expression of proteins. We initially demonstrated that plasmid pJN100 was transferred with high frequency (10(-5-7) exconjugants per recipient) into several Streptomyces strains that were refractory to transformation by other means. Plasmid pJN100 was also shown to be stable in E. coli and Streptomyces. We confirmed functional protein expression by using a pJN100 derivative to complement a mutant of Streptomyces griseus with a disrupted chromosomal copy of the gene nonM, a gene encoding an essential reductase in the nonactin biosynthesis gene cluster. High levels of protein expression were confirmed using Western blotting to assess the production of the serine esterase NonR, an enzyme responsible for nonactin resistance in the nonactin producer S. griseus.     

Equilibrium dialysis measurements of the Ca2+-binding properties of recombinant radish vacuolar Ca2+-binding protein expressed in Escherichia coli

Vacuoles of radish (Raphanus sativus) contained a Ca2+-binding protein (RVCaB) of 43 kDa. We investigated the Ca2+-binding properties of the protein. RVCaB was expressed in Escherichia coli and was purified from an extract by ion-exchange chromatography, nitrocellulose membrane filtration, and gel-filtration column chromatography. Ca2+-binding properties of the recombinant protein were examined by equilibrium dialysis with 45Ca2+ and small dialysis buttons. The protein was estimated to bind 19Ca2+ ions per molecule with a Kd for Ca2+ of 3.4 mM. Ca2+ was bound to the protein even in the presence of high concentrations of Mg2+ or K+. The results suggested that the protein bound Ca2+ with high ion selectivity, high capacity, and low affinity.     

Streptomyces hygroscopicus谷氨酰胺转胺酶酶原基因的鉴定及在大肠杆菌中的表达

[目的]鉴定来源于吸水链霉菌的谷氨酰胺转胺酶基因;研究其在大肠杆菌系统的克隆与表达;分析该酶与其同源酶的活性中心氨基酸序列.[方法]从本实验室筛选的吸水链霉菌(Streptomyces hygroscopicus;CCTCC M203062)发酵液中,分离纯化得到谷氨酰胺转胺酶酶原(pro-MTGase),测得N-端前十个氨基酸序列并与其它链霉菌来源的相应基因序列比较设计引物,扩增得到pro-MTGase 基因,将该基因插入到表达载体pET-20b( )信号肽pelB下游,构建分泌型表达载体pET/pro-MTG,并转化不同的大肠杆菌宿主BL21(DE3)和Rosetta(DE3)pLysS.[结果]获得了pro-MTGase的完整基因序列,多重碱基序列比对表明其与S.platensis和S.caniferus的pro-MTGase基因同源性高达92%.利用Rosetta(DE3)pLysS通过降温至24℃诱导策略,获得部分胞外表达的酶原.SDS-PAGE显示,胞外表达重组蛋白的分子量约为44kDa,与吸水链霉菌表达的天然酶原相符.诱导4 h后发酵液中的重组酶原经胰蛋白酶活化为成熟酶后测得最高酶活为0.24U/mL.[结论]该研究是对吸水链霉菌的谷氨酰胺转胺酶基因的首次报道,也是国内首次利用大肠杆菌实现pro-MTGase的胞外可溶性表达.     

Cloning, expression in Escherichia coli and nucleotide sequence of a tetracycline-resistance gene from Streptomyces rimosus

Determinants of tetracycline resistance have been cloned from two different tetracycline-producing industrial strains of Streptomyces into Streptomyces lividans using the plasmid vector pUT206. Three plasmids, pUT250 and pUT260 with a 9.5 and a 7.5 kb insert respectively of Streptomyces rimosus DNA, and pUT270 with a 14.0 kb insert of Streptomyces aureofaciens DNA, conferring resistance to tetracycline, have been isolated. By in vitro sub-cloning, a similar fragment of 2.45 kb containing the tetracycline resistance gene (tet347) was further localized on these plasmids. The S. rimosus gene has been cloned into Escherichia coli and expressed under the control of lambda pL or Lpp promoters. Differential protein extraction of E. coli cells revealed the presence of an additional membrane-embedded protein in tetracycline-resistant cells. On the basis of available restriction endonuclease maps, the tet347 gene is probably identical to the tetB gene from S. rimosus recently identified by T. Ohnuki and co-workers as responsible for the reduced accumulation of tetracycline. The nucleotide sequence of a 2052 bp DNA fragment containing the TcR structural gene from S. rimosus has been determined. The amino acid sequence of the tet347 protein (Mr35818) deduced from the nucleotide sequence shows a limited but significant homology to other characterized tetracycline transport acting determinants from pathogenic bacteria.     

Characterization of the human calmodulin-like protein expressed in Escherichia coli.

The protein-coding region of an intronless human calmodulin-like gene [Koller, M., & Strehler, E. E. (1988) FEBS Lett. 239, 121-128] has been inserted into a pKK233-2 expression vector, and the 148-residue, M(r) = 16,800 human protein was purified to apparent homogeneity by phenyl-Sepharose affinity chromatography from cultures of Escherichia coli JM105 transformed with the recombinant vector. Several milligrams of the purified protein were obtained from 1 L of bacterial culture. A number of properties of human CLP were compared to those of bacterially expressed human calmodulin (CaM) and of bovine brain CaM. CLP showed a characteristic Ca(2+)-dependent electrophoretic mobility shift on SDS-polyacrylamide gels, although the magnitude of this shift was smaller than that observed with CaM. CLP was able to activate the 3',5'-cyclic nucleotide phosphodiesterase to the same Vmax as normal CaM, albeit with a 7-fold higher Kact. In contrast, the erythrocyte plasma membrane Ca(2+)-ATPase could only be stimulated to 62% of its maximal CaM-dependent activity by CLP. CLP was found to contain four Ca(2+)-binding sites with a mean affinity constant of 10(5) M-1, a value about 10-fold lower than that for CaM under comparable conditions. The highly tissue-specifically-expressed CLP represents a novel human Ca(2+)-binding protein showing characteristics of a CaM isoform.     

Mutation and cloning of eryG, the structural gene for erythromycin O-methyltransferase from Saccharopolyspora erythraea, and expression of eryG in Escherichia coli.

A mutant strain derived by chemical mutagenesis of Saccharopolyspora erythraea (formerly known as Streptomyces erythreus) was isolated that accumulated erythromycin C and, to a lesser extent, its precursor, erythromycin D, with little or no production of erythromycin A or erythromycin B (the 3"-O-methylation products of erythromycin C and D, respectively). This mutant lacked detectable erythromycin O-methyltransferase activity with erythromycin C, erythromycin D, or the analogs 2-norerythromycin C and 2-norerythromycin D as substrates. A 4.5-kilobase DNA fragment from S. erythraea originating approximately 5 kilobases from the erythromycin resistance gene ermE was identified that regenerated the parental phenotype and restored erythromycin O-methyltransferase activity when transformed into the erythromycin O-methyltransferase-negative mutant. Erythromycin O-methyltransferase activity was detected when the 4.5-kilobase fragment was fused to the lacZ promoter and introduced into Escherichia coli. The activity was dependent on the orientation of the DNA relative to lacZ. We have designated this genotype eryG in agreement with Weber et al. (J.M. Weber, B. Schoner, and R. Losick, Gene 75:235-241, 1989). It thus appears that a single enzyme catalyzes all of the 3"-O-methylation reactions of the erythromycin biosynthetic pathway in S. erythraea and that eryG codes for the structural gene of this enzyme.     

大肠杆菌-链霉菌高效接合载体的构建及其应用

以链霉菌质粒SCP2^*的衍生质粒pHJL400为基础,构建了能够在大肠杆菌到链霉菌之间进行高效接合转移的质粒DGH112。pGH112含有在大肠杆菌和链霉菌中复制起始位点,以及分别在大肠杆菌和链霉菌中进行筛选的抗性标记。用pGH112转化Escherichia coli ET12567(pUZ8002)后,与天蓝链霉菌(Streptomyces coelicolor A3(2))、除虫链霉菌(Streptomyces avermitilis)、变铅青链霉菌(Streptomyces lividans TK54)、毒三素链霉菌(Streptomyces toxytricini NRRL15443)、委内瑞拉链霉菌(Streptomyces．vertezuelae ISP5230)和红色糖多孢菌(Saccharopolypora erythraea)进行接合,发现本构建的pGH112与pKC1139相比,接合转移效率较高,稳定性好,而且宿主范围较广。把组成型启动子ermE^*与绿色荧光蛋白基因(gfp)克隆到本构建的pGH112,通过接合转移到链霉菌中,gfp获得表达,证明其可以用作基因接合转移的有效工具载体,这为研究链霉菌的基因功能创造了有利条件。     

The novel murine Ca2+-binding protein,Scarf, is differentially expressed during epidermal differentiation

Calcium (Ca2+) signaling-dependent systems, such as the epidermal differentiation process, must effectively respond to variations in Ca2+ concentration. Members of the Ca2+-binding proteins play a central function in the transduction of Ca2+ signals, exerting their roles through a Ca2+-dependent interaction with their target proteins, spatially and temporally. By performing a suppression subtractive hybridization screen we identified a novel mouse gene, Scarf (skin calmodulin-related factor), which has homology to calmodulin (CaM)-like Ca2+-binding protein genes and is exclusively expressed in differentiating keratinocytes in the epidermis. The Scarf open reading frame encodes a 148-amino acid protein that contains four conserved EF-hand motifs (predicted to be Ca2+-binding domains) and has homology to mouse CaM, human CaM-like protein, hClp, and human CaM-like skin protein, hClsp. The functionality of Scarf EF-hand domains was assayed with a radioactive Ca2+-binding method. By Southern blot and computational genome sequence analysis, a highly related gene, Scarf2, was found 15 kb downstream of Scarf on mouse chromosome 13. The functional Scarf Ca2+-binding domains suggest a role in the regulation of epidermal differentiation through the control of Ca2+-mediated signaling.     

Ca(2+)-binding properties of the platelet glycoprotein IIb ligand-interacting domain.

Glycoprotein (GP) IIb is the alpha subunit of platelet integrin GPIIb-IIIa. Analysis of the primary structure of this subunit has indicated the presence of four stretches of amino acid residues that are highly conserved among various integrin alpha subunits and that have been suggested to be putative calcium-binding sites. To verify the Ca(2+)-binding capacity of these conserved domains and their implication in integrin adhesive functions, a fragment corresponding to the amino acid sequence of GPIIb from positions 171 to 464 was expressed. The nucleotide sequence coding for this GPIIb domain was generated by polymerase chain reaction, cloned into the pTG1924 expression vector, and expressed in Escherichia coli strain TGE901. The recombinant protein was purified by gel exclusion chromatography and used in equilibrium dialysis experiments. The results demonstrate that the four binding sites can be occupied by Ca2+. Two classes of binding sites can be detected, including two sites with a Kd of 30 microns and two sites of lower affinity with a Kd of 120 microns. Interaction of Ca2+ with these two classes of sites was inhibited by a large excess of Mg2+ or Mn2+, suggesting that these cations are competitive for the same sites on GPIIb. Thus, the four Ca(2+)-binding sites of GPIIb are not similar and exhibit different affinities for divalent ions. To verify the functional implication of these Ca(2+)-binding sites, the effect of Ca2+ on the binding of fibrinogen to the recombinant protein was analyzed using a solid-phase assay. The results indicate that optimal fibrinogen binding occurs when the four calcium-binding sites are occupied and establish the functional importance of this Ca(2+)-binding domain in the ligand-binding activity of GPIIb.     

Cloning and expression of an actinokinase gene from a thermophilic Streptomyces in Escherechia coli

A thermophilic Streptomyces megasporus strain SD5, could secrete a new fibrinolytic (actinokinase) at 55 degrees C. The gene (ackS) encoding actinokinase was isolated from the chromosomal DNA of S. megasporus SD5 and cloned in different hosts and vectors. The expression was obtained in E. coli JM109 using Cla I linearized pBR322 as vector (pSR 500). The recombinant E. coli containing pSR 500 expressed active actinokinase but the expression was low and the recombinant was unstable in liquid culture. Deletion analysis revealed that removal of Bam H I-Sal I fragment from down stream and Cla I-EcoRI from upsream enhanced the stability and expression of ackS in both solid and liquid media. For over expresion, the ackS gene was cloned in E. coli C 600 using Bam HI linearized pT7-7. This seemed to be the most suitable host vector system. The recombinant and native form of actinokinase exhibited similar characteristics. Actinokinase was the first thrombolytic enzyme from a thermophile to be cloned and over expressed in a mesophilic heterologous expression system.     

Analysis of genes involved in 6-deoxyhexose biosynthesis and transfer in Saccharopolyspora erythraea

Glycosylation represents an attractive target for protein engineering of novel antibiotics, because specific attachment of one or more deoxysugars is required for the bioactivity of many antibiotic and antitumour polyketides. However, proper assessment of the potential of these enzymes for such combinatorial biosynthesis requires both more precise information on the enzymology of the pathways and also improved Escherichia coli-actinomycete shuttle vectors. New replicative vectors have been constructed and used to express independently the dnmU gene of Streptomyces peucetius and the eryBVII gene of Saccharopolyspora erythraea in an eryBVII deletion mutant of Sac. erythraea. Production of erythromycin A was obtained in both cases, showing that both proteins serve analogous functions in the biosynthetic pathways to dTDP-L-daunosamine and dTDP-L-mycarose, respectively. Over-expression of both proteins was also obtained in S. lividans, paving the way for protein purification and in vitro monitoring of enzyme activity. In a further set of experiments, the putative desosaminyltransferase of Sac. erythraea, EryCIII, was expressed in the picromycin producer Streptomyces sp. 20032, which also synthesises dTDP-D-desosamine. The substrate 3-alpha-mycarosylerythronolide B used for hybrid biosynthesis was found to be glycosylated to produce erythromycin D only when recombinant EryCIII was present, directly confirming the enzymatic role of EryCIII. This convenient plasmid expression system can be readily adapted to study the directed evolution of recombinant glycosyltransferases.     

一个热诱导穿梭表达载体的构建及其在链霉菌中的应用

为探索大肠杆菌λ噬菌体表达调控元件在链霉菌中的应用,构建了一个链霉菌大肠杆菌穿梭表达载体pHZ1080,并将来自链霉菌FR-008的聚酮合酶(PKS)基因置于其中的λ噬菌体启动子P_R下游,得到表达PKS的穿梭质粒pHZ1067。与在大肠杆菌中一样,该质粒在变铅青链霉菌中也受热诱导表达100kD的PKS蛋白;表达的PKS蛋白可由SDSPAGE和Western-blot实验检测到。PKS在链霉菌中的热诱导表达表明,构建的载体也能用于链霉菌诱导表达外源基因。  