A Streptomyces avermitilis gene encoding a 4-hydroxyphenylpyruvic acid dioxygenase-like protein that directs the production of homogentisic acid and an ochronotic pigment in Escherichia coli.

A 1.5-kb genomic fragment isolated from Streptomyces avermitilis that directs the synthesis of a brown pigment in Escherichia coli was characterized. Since pigment production in recombinant E. coli was enhanced by the addition of tyrosine to the medium, it had been inferred that the cloned DNA might be associated with melanin biosynthesis. Hybridization studies, however, showed that the pigment gene isolated from S. avermitilis was unrelated to the Streptomyces antibioticus melC2 determinant, which is the prototype of melanin genes in Streptomyces spp. Sequence analysis of the 1.5-kb DNA that caused pigment production revealed a single open reading frame encoding a protein of 41.6 kDa (380 amino acids) that resembled several prokaryotic and eukaryotic 4-hydroxyphenylpyruvate dioxygenases (HPDs). When this open reading frame was overexpressed in E. coli, a protein of about 41 kDa was detected. This E. coli clone produced homogentisic acid (HGA), which is the expected product of the oxidation of 4-hydroxyphenylpyruvate catalyzed by an HPD, and also a brown pigment with characteristics similar to the pigment observed in the urine of alkaptonuric patients. Alkaptonuria is a genetic disease in which inability to metabolize HGA leads to increasing concentrations of this acid in urine, followed by oxidation and polymerization of HGA to an ochronotic pigment. Similarly, the production of ochronotic-like pigment in the recombinant E. coli clone overexpressing the S. avermitilis gene encoding HPD is likely to be due to the spontaneous oxidation and polymerization of the HGA accumulated in the medium by this clone.     

Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector.

An integration vector for gene analysis in Streptomyces has been constructed. This vector replicates in Escherichia coli, and integrates into Streptomyces by homologous recombination between a cloned fragment and the genome. To overcome methylation-specific restriction barriers, an E. coli mutant triply defective in DNA methylation was constructed as a source for the integration plasmids. The frequency of integration of pVE616 derivatives into the Streptomyces avermitilis genome was proportional to the size of the cloned DNA. Derivatives of pVE616, containing fragments from pVE650, a plasmid with a 24-kb insert of S. avermitilis DNA, were used in complementation analyses of seven S. avermitilis mutants defective in glycosylation of avermectin (Av). Three complementation groups, located in a 7-kb region, were identified. Derivatives of pVE616, containing fragments from the 18-kb of DNA adjacent to the glycosylation region, were integrated into an Av producer. Av produced from the integrants was substantially reduced, indicating that the 18 kb also encodes gene products which are involved in Av biosynthesis.     

Bleomycin-induced β-lactamase overexpression in Escherichia coli carrying a bleomycin-resistance gene from Streptomyces verticillus and its application to screen bleomycin analogues

Abstract A bleomycin-resistance gene, designated blmA , has been cloned from bleomycin-producing Streptomyces verticillus by Sugiyama et al. (Gene 151 (1994) 11–16). The present study shows that Escherichia coli harboring the blmA -carrying pUC plasmid overproduced β-lactamase, encoded by an ampicillin-resistance gene on the plasmid, when cultured in the presence of bleomycin, which suggests that bleomycin may act as an inducer (or an activator) for the expression of the specific gene in the presence of blmA . We constructed a vector, designated pMAB50, which senses bleomycin and produces a pigment, using blmA and a Streptomyces tyrosinase gene located under the control of β-lactamase promoter: E. coli harboring pMAB50 produced the melanin pigment in the presence of bleomycin-type antibiotics, suggesting that the transformed E. coli can be employed as a reporter organism to screen bleomycin analogues.     

A gene cluster for biosynthesis of the sesquiterpenoid antibiotic pentalenolactone in Streptomyces avermitilis

Streptomyces avermitilis, an industrial organism responsible for the production of the anthelminthic avermectins, harbors a 13.4 kb gene cluster containing 13 unidirectionally transcribed open reading frames corresponding to the apparent biosynthetic operon for the sesquiterpene antibiotic pentalenolactone. The advanced intermediate pentalenolactone F, along with the shunt metabolite pentalenic acid, could be isolated from cultures of S. avermitilis, thereby establishing that the pentalenolactone biosynthetic pathway is functional in S. avermitilis. Deletion of the entire 13.4 kb cluster from S. avermitilis abolished formation of pentalenolactone metabolites, while transfer of the intact cluster to the pentalenolactone nonproducer Streptomyces lividans 1326 resulted in production of pentalenic acid. Direct evidence for the biochemical function of the individual biosynthetic genes came from expression of the ptlA gene (SAV2998) in Escherichia coli. Assay of the resultant protein established that PtlA is a pentalenene synthase, catalyzing the cyclization of farnesyl diphosphate to pentalenene, the parent hydrocarbon of the pentalenolactone family of metabolites. The most upstream gene in the cluster, gap1 (SAV2990), was shown to correspond to the pentalenolactone resistance gene, based on expression in E. coli and demonstration that the resulting glyceraldehyde-3-phosphate dehydrogenase, the normal target of pentalenolactone, was insensitive to the antibiotic. Furthermore, a second GAPDH isozyme (gap2, SAV6296) has been expressed in E. coli and shown to be inactivated by pentalenolactone.     

Cloning and analysis of a DNA fragment stimulating avermectin production in various Streptomyces avermitilis strains

To isolate a gene for stimulating avermectin production, a genomic library of Streptomyces avermitilis ATCC 31267 was constructed in Streptomyces lividans TK21 as the host strain. An 8.0-kb DNA fragment that significantly stimulated actinorhodin and undecylprodigiosin production was isolated. When wild-type S. avermitilis was transformed with the cloned fragment, avermectin production increased approximately 3.5-fold. The introduction of this fragment into high-producer (ATCC 31780) and semi-industrial (L-9) strains also resulted in an increase of avermectin production by more than 2.0- and 1.4-fold, respectively. Subclones were studied to locate the minimal region involved in stimulation of pigmented-antibiotic and avermectin production. An analysis of the nucleotide sequence of the entire DNA fragment identified eight complete and one incomplete open reading frame. All but one of the deduced proteins exhibited strong homology (68 to 84% identity) to the hypothetical proteins of Streptomyces coelicolor A3(2). The orfX gene product showed no significant similarity to any other protein in the databases, and an analysis of its sequence suggested that it was a putative membrane protein. Although the nature of the stimulatory effect is still unclear, the disruption of orfX revealed that this gene was intrinsically involved in the stimulation of avermectin production in S. avermitilis.     

透明颤菌血红蛋白在肉桂地链霉菌中的表达对基因细胞生长及抗生素合成的影响

肉桂地链霉菌（S.cinnamonensis)是莫能菌素（Monensin)的产生菌,大肠杆菌－链霉菌穿梭表达载体pHZ1252中的透明颤菌血红蛋白基因（vhb)位于硫链丝菌素诱导启动子ＰtipA之下,它在肉桂地链霉菌中的结构不稳定,,发生了重组缺失,缺失的片段包括大肠杆菌质粒部分vhb基因。但来自阿维链霉菌(S.avermitilis)中缺失了大肠杆菌质粒部分却保留了完整的vhb基因及tipA启动子的pHZ1252,可在肉桂地链霉菌中稳定复制,不再发生缺失,经硫链丝菌素诱导表达出了有生物活性的ＶＨb蛋白,摇瓶发酵实验证明,ＶＨb蛋白在氧限条件下可明显促进肉桂地链霉菌的菌体生长和抗生素合成。     

Molecular cloning and expression of the glucose/xylose isomerase gene from Streptomyces sp. NCIM 2730 in Escherichia coli

Abstract A partial genomic library of Streptomyces sp. NCIM 2730 was constructed in Escherichia coli using pUC8 vector and screened for the presence of the d-glucose/xylose isomerase (GXI) gene using an 18-mer mixed oligonucleotide probe complementary to a highly conserved six-amino acid sequence of GXI from actinomycetes. Eight clones which hybridized with the radiolabelled oligoprobe showed the ability to complement xylose isomerase-defective E. coli mutants. The restriction map of the insert from one (pMSG27) of the eight GXI-positive clones showing detectable GXI activity was constructed. GXI-deficient strains of E. coli were able to utilize xylose as the sole carbon source for their growth upon transformation with pMSG27. E. coli JM105 (pMSG27) and E. coli JC1553 (pMSG27) were inducible by IPTG suggesting that the expression of the cloned gene was under the control of the lacZ promoter. Western blot analysis revealed that the cloned gene is expressed as a fusion protein of M _r 110. This is the first report of expression of a catalytically active GXI from Streptomyces in Escherichia coli .     

Characterization of a unique methyl-specific restriction system in Streptomyces avermitilis.

Streptomyces avermitilis contains a unique restriction system that restricts plasmid DNA containing N6-methyladenine or 5-methylcytosine. Shuttle vectors isolated from Escherichia coli RR1 or plasmids isolated from modification-proficient Streptomyces spp. cannot be directly introduced into S. avermitilis. This restriction barrier can be overcome by first transferring plasmids into Streptomyces lividans or a modification-deficient E. coli strain and then into S. avermitilis. The transformation frequency was reduced greater than 1,000-fold when plasmid DNA was modified by dam or TaqI methylases to contain N6-methyladenine or by AluI, HhaI, HphI methylases to contain 5-methylcytosine. Methyl-specific restriction appears to be common in Streptomyces spp., since either N6-methyladenine-specific or 5-methylcytosine-specific restriction was observed in seven of nine strains tested.     

Improving fatty acid production in Escherichia coli through the overexpression of malonyl coA-acyl carrier protein transacylase

The microbial biosynthesis of free fatty acid, which can be used as precursors for the production of fuels or chemicals from renewable carbon sources, has attracted significant attention in recent years. Free fatty acids can be produced by introducing an acyl-carrier protein (ACP) thioesterase (TE) gene into Escherichia coli. The first committed step of fatty acid biosynthesis is the conversion of acetyl-CoA to malonyl-CoA by an adenosine triphosphate (ATP)-dependent acetyl-CoA carboxylase followed by the conversion of malonyl-CoA to malonyl-ACP through the enzyme malonyl CoA-acyl carrier protein transacylase (MCT; FabD). The E. coli fabD gene encoding MCT has been cloned and studied. However, the effect of FabD overexpression in a fatty acid overproducing strain has not been examined. In this study, we examined the effect of FabD overexpression in a fatty acid overproducing strain carrying an acyl-ACP TE. Specifically, the effect of overexpressing a fabD gene from four different organisms on fatty acid production was compared. The strains carrying a fabD gene from E. coli, Streptomyces avermitilis MA-4680, or Streptomyces coelicolor A3(2) improved the free fatty acid production; these three strains produced more free fatty acids, about 11% more, than the control strain. The strain carrying a fabD gene from Clostridium acetobutylicum ATCC 824, however, produced similar quantities of free fatty acids as the control strain. In addition, the three FabD overexpressed strains also have higher fatty acid/glucose yields. The results suggested that FabD overexpression can be used to improve free fatty acid production by increasing the malonyl-ACP availability.     

Genome mining in Streptomyces. Elucidation of the role of Baeyer-Villiger monooxygenases and non-heme iron-dependent dehydrogenase/oxygenases in the final steps of the biosynthesis of pentalenolactone and neopentalenolactone

The pentalenolactone biosynthetic gene clusters have been cloned and sequenced from two known producers of the sesquiterpenoid antibiotic pentalenolactone, Streptomyces exfoliatus UC5319 and Streptomyces arenae TU?469. The recombinant enzymes PenE and PntE, from S. exfoliatus and S. arenae, respectively, catalyze the flavin-dependent Baeyer-Villiger oxidation of 1-deoxy-11-oxopentalenic acid (7) to pentalenolactone D (8). Recombinant PenD, PntD, and PtlD, the latter from Streptomyces avermitilis, each catalyze the Fe(2+)-α-ketoglutarate-dependent oxidation of pentalenolactone D (8) to pentalenolactone E (15) and pentalenolactone F (16). Incubation of PenD, PntD, or PtlD with the isomeric neopentalenolactone D (9) gave PL308 (12) and a compound tentatively identified as neopentalenolactone E (14). These results are corroborated by analysis of the ΔpenD and ΔpntD mutants of S. exfoliatus and S. arenae, respectively, both of which accumulate pentalenolactone D but are blocked in production of pentalenolactone as well as the precursors pentalenolactones E and F. Finally, complementation of the previously described S. avermitilis ΔptlE ΔptlD deletion mutant with either penE or pntE gave pentalenolactone D (8), while complemention of the ΔptlE ΔptlD double mutant with pntE plus pntD or penE plus pntD gave pentalenolactone F (16).     

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。     

阿维链霉菌Streptomyces avermitilis碱性脂肪酶LpsA2的表达和酶学性质分析

【目的】本研究将推测的阿维链霉菌(Streptomyces avermitilis)脂肪酶基因lpsA2在大肠杆菌(Escherichia coli)中进行异源表达及系统的酶学性质分析。【方法】提取阿维链霉菌基因组,设计特异性引物,PCR扩增脂肪酶基因lpsA2,使其在大肠杆菌中异源表达,利用6个组氨酸标签纯化脂肪酶LpsA2,并进行酶学性质分析;对LpsA2进行序列比对和进化分析。【结果】氨基酸序列比对显示LpsA2具有脂肪酶典型的由Ser、His和Asp构成的活性部位,即Ser130-Asp221-His25,其中Ser位于保守的五肽结构(Gly128-His129-Ser130-Gln131-Gly132)中;分子系统学分析显示,LpsA2是脂肪酶第一家族亚家族成员(Subfamily I.7);实验测得纯化的重组脂肪酶LpsA2的最适反应pH为8.0,最适反应温度为50℃;最适底物为对硝基苯酚豆蔻酸酯;在10℃-50℃范围内该酶的激活自由能为6.3 kcal/mol;1 mmol/L Co2+、Hg2+、Zn2+可使酶活性提高至250%以上;15%的二甲基甲酰胺和二甲基亚砜使酶活分别提高至110.7%和138%;0.1%和1%的Span-20可使酶活性分别提高至352.7%和189.7%。【结论】本研究对推测的来源于S.avermitilis的脂肪酶基因lpsA2进行了异源表达和酶学功能鉴定,不仅为脂肪酶的研究积累了更多数据,也为具有优良性能的脂肪酶生物工程菌的筛选奠定了基础,更为其在食品加工、药物合成等工业生产中的应用提供了依据。     

Cloning of regulator genes controlling fimbrial production by enterotoxigenic Escherichia coli

Sequences regulating production of fimbriae were cloned from two enterotoxigenic Escherichia coli strains. One cloned region, from E. coli 0.25.H42, controlled expression of coli surface-associated (CS) antigen 4, whereas the function of the other, from E. coli 0167.H5, was unclear. Both regulators were related to the cfaD gene that controls expression of colonization factor antigen I (CFA/I) although low stringency conditions were required to show significant hybridization between cfaD and the regulatory fragment from E. coli 0167. The cloned regulatory genes promoted expression of CFA/I, CS1, CS2 and CS4 antigens but the levels of production in the presence of the 0167 regulator were lower than those promoted by the CS4 regulator or cfaD.     

Cloning and characterization of a Streptomyces single module type non-ribosomal peptide synthetase catalyzing a blue pigment synthesis

In the present study, we cloned a gene, designated bpsA, which encodes a single module type non-ribosomal peptide synthetase (NRPS) from a D-cycloserine (DCS)-producing Streptomyces lavendulae ATCC11924. A putative oxidation domain is significantly integrated into the adenylation domain of the NRPS, and the condensation domain is absent from the module. When S. lividans was transformed with a plasmid carrying bpsA, the transformed cells produced a blue pigment, suggesting that bpsA is responsible for the blue pigment synthesis. However, to produce the blue pigment in Escherichia coli, the existence of the 4'-phosphopantetheinyl transferase (PPTase) gene from Streptomyces was necessary, in addition to bpsA. The chemical structure of the pigment was determined as 5,5'-diamino-4,4'-dihydroxy-3,3'-diazadiphenoquinone-(2,2'), called indigoidine. The bpsA gene product, designated BPSA, was overproduced in an E. coli host-vector system and purified to homogeneity, demonstrating that the recombinant enzyme prefers L-Gln as a substrate. The in vitro experiment using L-Gln also showed that the blue pigment was formed by the purified BPSA only when the enzyme was phosphopantetheinylated by adding a Streptomyces PPTase purified from E. coli cells. Each site-directed mutagenesis experiment of Lys(598), Tyr(601), Ser(603), and Tyr(608), which are seen in the oxidation domain of BPSA, suggests that these residues are essential for the binding of FMN to the protein and the synthesis of the blue pigment.     

基于2-C-甲基-D-赤藓糖醇-4磷酸途径的产紫槐二烯大肠杆菌构建及其补糖策略优化

2-C-甲基-D-赤藻糖醇-4-磷酸(2-methyl-D-erythritol-4-phosphate, MEP) 途径是大肠杆菌Escherichiacoli 唯一的萜类前体合成途径,研究表明它比甲羟戊酸(Mevalonate, MVA)途径具有更高的理论产率。但目前有关MEP 途径的调控所知非常有限,故单独强化MEP 途径对萜类异源合成产量的提高效果并不理想。研究中通过引入外源MEP 途径基因强化E. coli 萜类合成的遗传改造策略和发酵过程补糖控制优化,尝试更有效地释放MEP 途径的潜力,建立青蒿素前体——紫槐二烯的高密度发酵过程。研究结果表明共表达阿维链霉菌Streptomyces avermitilis dxs2 基因和枯草芽胞杆菌Bacillus subtilis idi 基因可使紫槐二烯的摇瓶发酵产量比野生菌株提高12.2 倍。随后针对该菌株建立了高密度发酵过程,发现稳定期的中前期(24?72 h) 是产物合成的关键期,通过稳定期补糖速率的调整,明显改善了产物合成速度,使紫槐二烯的产量从2.5 g/L 提高到了4.85 g/L,但不影响产物积累的周期。考虑到72 h 后菌体老化可能会影响产物合成,进一步采取了调整对数期的补糖速率控制菌体生长的策略,使紫槐二烯的产量达到6.1 g/L。研究结果为基于MEP 途径的萜类异源合成工程菌构建及其发酵工艺的建立奠定了基础。     

阿维链霉菌孢子色素生物合成对阿维菌素产量的影响

以野生型阿维链霉茵NRRL8165为出发菌株,用PCR方法克隆孢子色素基因簇直系同源基因(whiEa)侧翼片段,并构建基因置换载体pHL643.将pilL643跨属接合转移进入阿维链霉菌NRRL8165,通过置换载体和染色体之间的同源双交换,对染色体上的whiEa基因簇进行置换,得到3株阿泊拉霉素抗性、硫链丝菌素敏感的重组菌株,均表现为孢子色素合成缺陷.通过Southern杂交分析,证明whiEa基因簇被置换.通过摇瓶发酵和HPLC检测,发现whiEa基因簇置换菌株所产阿维菌素产量明显提高,表明孢子色素与阿维菌素生物合成之间可能有竞争底物的现象.     

Characterization of the Streptomyces clavuligerus argC gene encoding N-acetylglutamyl-phosphate reductase: expression in Streptomyces lividans and effect on clavulanic acid production.

The argC gene of Streptomyces clavuligerus encoding N-acetylglutamyl-phosphate reductase (AGPR) has been cloned by complementation of argC mutants Streptomyces lividans 1674 and Escherichia coli XC33. The gene is contained in an open reading frame of 1,023 nucleotides which encodes a protein of 340 amino acids with a deduced molecular mass of 35,224 Da. The argC gene is linked to argE, as shown by complementation of argE mutants of E. coli. Expression of argC from cloned DNA fragments carrying the gene leads to high levels of AGPR in wild-type S. lividans and in the argC mutant S. lividans 1674. Formation of AGPR is repressed by addition of arginine to the culture medium. The protein encoded by the argC gene is very similar to the AGPRs of Streptomyces coelicolor, Bacillus subtilis, and E. coli and, to a lesser degree, to the homologous enzymes of Saccharomyces cerevisiae and Anabaena spp. A conserved PGCYPT domain present in all the AGPR sequences suggests that this may be the active center of the protein. Transformation of S. clavuligerus 328, an argC auxotroph deficient in clavulanic acid biosynthesis, with plasmid pULML30, carrying the cloned argC gene, restored both prototrophy and antibiotic production.     

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。