链霉菌酪氨酸酶基因的克隆与表达

本文综述了近年来对链霉菌酪氨酸酶基因的研究。由酪氨酸酶合成黑色素是链霉菌属各个种的共同特性,并受到酪氨酸酶基因的控制。链霉菌几个种的酪氨酸酶基因已克隆到,其产黑色素的特性使之作为重要的标记基因得以广泛应用,同时,该基因在链霉菌的不同种及不同属的微生物中得到了高水平的表达。链霉菌酪氨酸酶基因表达调控的分子机制也得到较深入的研究。     

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.     

The minimal replicon of a Streptomycete plasmid produces an ultrahigh level of plasmid DNA

A functional map of Streptomyces coelicolor plasmid SCP2* was deduced from derivatives constructed by in vitro deletions. Functions were analyzed on bifunctional shuttle plasmids that contained pBR322 for selection and replication in Escherichia coli and fragments of SCP2* for replication in Streptomyces griseofuscus C581 and strains of Streptomyces lividans. The aph gene for neomycin resistance from Streptomyces fradiae and the tsr gene for thiostrepton resistance from Streptomyces azureus were incorporated as selectable antibiotic resistance markers in streptomycetes. An 11.8-kb sequence bounded by EcoRI and KpnI restriction sites contains the information for self-transfer and normal replication of the plasmid. A 5.9-kb EcoRI-SalI fragment contains all of the information for normal replication. Partial digestion generated a 2.2-kb Sau3A fragment that is sufficient for replication but it produces ten times higher plasmid copy number than the basic replicon. pHJL400 and PHJL401 are useful shuttle vectors containing the moderate-copy-number streptomycete plasmid combined with the E. coli plasmid pUC19. A 1.4-kb BclI-Sau3A fragment with an additional internal BclI site contains the minimal replicon but it produces 1000 times higher plasmid copy number than the basic replicon. pHJL302 is a useful shuttle vector containing the ultrahigh-copy-number streptomycete plasmid combined with the E. coli plasmid pUC19.     

Structural stability of heterologous genes cloned in Streptomyces plasmid pIJ702

A recombinant plasmid pWCL1 containing Streptomyces plasmid pIJ702, E. coli plasmid pUC12, and hepatitis B viral surface antigen (HBsAg) gene was stably maintained in E. coli, but exhibited structural instability in S. lividans 1326. The deletions were found ranging from 2.75 to 5.65 kilobases (kb) and most of them occurred within the melanin (mel) gene of pIJ702, resulting in the loss of part of the mel gene sequence plus the insert. The removal of the pUC12 sequence from pWCL1 eliminated the instability. However, pUC12 alone inserted in either orientation on pIJ702 also caused the deletion in S. lividans 1326. The results indicated that the structural instability of hybrid plasmid of pIJ702 depended on the interaction between the mel sequence and the inserted sequence.     

尤马马杜拉放线菌染色体复制区的克隆及功能分析

尤马马杜拉放线菌(Actinomadura yumaensis)NRRL12515产生马杜拉霉素,用于防治禽类球虫病。试验以放线菌dnaA与dnaN基因保守区设计的简并引物进行PCR扩增,获得了包含尤马马杜拉放线菌的染色体复制区oriC的片段,并进行了序列分析和复制功能的研究。尤马马杜拉放线菌染色体的oriC全长为919碱基对,含有14个DnaA盒子和2个AT富含区,DnaA盒子的保守序列是(T/C)(T/C)GTCC(A/C)CA,与已发表的3个属的放线菌染色体oriC的序列特征不同。携带该oriC片段的大肠杆菌质粒可以在天蓝色链霉菌中复制并以低拷贝方式遗传,表明这是一段有复制功能的序列。比较来自放线菌4个属的oriC,发现以oriC序列和以16S rRNA基因序列构建的进化树十分相似,表明oriC序列也可以体现放线菌物种之间的关系。     

Gene disruption and gene replacement in Streptomyces via single stranded DNA transformation of integration vectors.

For the isolation of single stranded plasmid DNA, various E. coli and E. coli-Streptomyces shuttle plasmids were equipped with the phage f1 replication origin. The transformation of some representative Streptomyces species with plasmid vectors occurred irrespective of whether single or double stranded DNA was used. In contrast, the transformation of Streptomyces was 10 to 100 times more efficient when an integration vector was in the single stranded form as opposed to the double stranded form. Streptomyces viridochromogenes was transformed by single stranded DNA integration vectors in order to replace the pat by the tsr gene and generate mutants unable to synthesize phosphinothricin-tripeptide (PTT).     

Shuttle vectors for cloning recombinant DNA in Escherichia coli and Streptomyces griseofuscus C581.

The replicon of the Streptomyces plasmid SCP2 was located on a 5.9-kilobase EcoRI-SalI restriction fragment. The SCP2 replicon was combined with Escherichia coli plasmid pBR322 and genes specifying neomycin resistance and thiostrepton resistance in streptomycetes to construct shuttle vectors that are useful for cloning in E. coli and streptomycetes.     

Plasmid cloning vectors that integrate site-specifically in Streptomyces spp

Cloning vectors based on the Streptomyces ambofaciens plasmid pSAM2 and the streptomycete phage phi C31 were developed for use in Streptomyces spp. These vectors replicate in Escherichia coli but integrate by site-specific recombination in Streptomyces spp. Both pSAM2-based and phi C31-based vectors transformed a number of different Streptomyces spp; however, the phi C31-based vectors consistently transformed at higher frequencies than pSAM2-based vectors. Southern analysis indicated that the phi C31-based vectors integrated at a unique site in the S. ambofaciens chromosome, while the pSAM2-based vectors gave complex patterns which could indicate structural instability or use of multiple loci. Both types of vectors utilize the apramycin (Am)-resistance gene which can be selected in E. coli and Streptomyces spp. with either Am or the commercially available antibiotic Geneticin (G418).     

Development of integrative vectors for Actinomycetes--producers of antibiotics]

The integrative vectors pSU 475 and pSU 476 with variable numbers of copies per genome were developed for antibiotic producing actinomycetes. For this, the amplifying sequence AUD-Sr 1 of Streptomyces rimosus and the BamHIB fragment of the eSA 1 genetic element from Streptomyces antibioticus were used. The eSA 1 fragment was an element required for integration of a vector to the actinomycete chromosomes since it was homologous with the chromosomal DNAs of S. lividans, S. erythraeus and S. antibioticus. At the first stage the AUD-Sr 1 sequence within the actinomycete plastid pSU 23 was cloned by the vector pUC 19 to E coli. In that experiment the 12.4-kb plasmid pSU 449 was isolated. At the second stage the BamHIB-fragment of the eSA 1 element was incorporated into the resultant hybrid plasmid pSU 449. The 16.5-kb hybrid plasmids pSU 475 and pSU 476 were isolated. In these plasmids the BamHIB fragment of eSA 1 was present in two orientations. The developed vectors were useful in cloning DNA to S. lividans and S. erythraeus.     

A cloned ompR-like gene of Streptomyces lividans 66 suppresses defective melC1, a putative copper-transfer gene

Expression of tyrosinase in Streptomyces requires functional MelC1 protein, which is postulated to transfer copper to apotyrosinase. We have previously isolated a mutant of Streptomyces lividans, HT32, that phenotypically suppressed mutations in cloned melC1 (H.-C. Tseng and C. W. Chen, in preparation). Plasmid pLUS132, containing an ATG to ATA transition at the initiation codon of melC1, was used for cloning the suppressor gene from HT32. A 1687 bp suppressor DNA was isolated that contained two characteristic Streptomyces coding sequences: a 217-amino-acid open reading frame (cutR) and a truncated open reading frame (cutS) downstream. Subcloning analysis attributed the phenotypic suppression activity to the putative cutR gene from HT32. The putative CutR exhibited similarity to the response regulator OmpR of the osmoregulatory signal-transduction system in Escherichia coli. The truncated CutS resembled, to a lesser degree, the N-terminus of EnvZ, the histidine protein kinase counterpart of OmpR. DNA hybridizing to the cloned cutR-cutS sequence was detected in 16 other Streptomyces species. We postulate that the putative cutR-cutS operon regulates copper metabolism in Streptomyces.     

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.     

Construction and shuttling of novel bifunctional vectors for Streptomyces spp. and Escherichia coli.

Shuttle vectors for gene transfer between Streptomyces spp. and Escherichia coli have been constructed by fusion of an artificial multicopy E. coli replicon and DNA fragments of pIJ702. Stable transfer to Streptomyces lividans was obtained. Marked differences in transformation efficiency were observed when plasmid DNA isolated from E. coli GM119 was used instead of that from strain HB101.     

Cloning and expression of a Streptomyces fradiae neomycin resistance gene in Escherichia coli

A Streptomyces fradiae DNA sequence, which codes for a neomycin phosphotransferase, has been subcloned from the Streptomyces recombinant plasmid pIJ2 [a chimera between the Streptomyces plasmid SLP1.2 and chromosomal DNA containing a neomycin (Nm) resistance gene] into the BamHI restriction enzyme site of pHV14. Three different recombinant plasmids (pWHR1, pWHR2, pWHR3) have been isolated which transform Escherichia coli to Nm resistance. Southern transfer hybridization experiments show that the recombinant plasmids contain the cloned Streptomyces Nm resistance gene, and lysates of E. coli containing the recombinant plasmids were shown to have Nm phosphotransferase activity, demonstrating that a gene from Streptomyces can be expressed in E. coli.     

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.     

Construction of pRES18 and pRES19, Streptomyces-Escherichia coli shuttle vectors carrying multiple cloning sites

Abstract We developed two Streptomyces-Escherichia coli shuttle vectors. The plasmid pRES102, consisting of the essential region of pRES1 and the thiostrepton resistance gene ( tsr ) fragment of pIJ702, was combined with the E. coli plasmid vector pUC18 or pUC19. The resulting shuttle vectors, designated pRES18 and pRES19, respectively, have relatively compact size (6.25 kb), low copy number, multiple cloning sites reciprocally arranged in opposite directions, and selection markers for both Streptomyces ( tsr ) and E. coli (β-lactamase ( bla ) and β-galactosidase ( lacZ )). These shuttle vectors are capable of carrying DNA fragments as long as 10 kb, of being maintained in S. griseus, S. lavendulae and S. lividans , and are compatible with pIJ702.