Proteolytic enzymes from recombinant Streptomyces lividans TK24

Different proteases from the culture fluids of recombinant Streptomyces lividans strains were isolated. Several individual proteases were separated and characterized. A chymotrypsin-chylike activity (CLA) was identified that specifically degrades a fusion protein between the alpha-amylase inhibitor from S. tendae (Tendamistat, HOE467) and proinsulin from the monkey Macaca fascicularis. The effective chemical inhibition of the degrading enzyme is demonstrated.     

Production of aminoglycosides in non-aminoglycoside producing Streptomyces lividans TK24

The pRBM4 cosmid, which harbors a putative cluster of genes spanning a 31.8-kb chromosomal region of the ribostamycin producer Streptomyces ribosidificus ATCC 21294, was heterologously expressed in Streptomyces lividans TK24. ESI-MS/MS, HPLC, and LC-ESI MS analyses showed that the transformation gave rise to ribostamycin production in various culture broths. This is the first report of heterologous aminoglycoside production.     

硫霉素环化酶基因在变铅青链霉菌TK24中的表达及基因定位

将含有硫霉素环化酶基因的重组质粒p6BCl2转化变铅青链霉菌(Streptomyceslividans)TK24,含有p6BCl2的转化子细胞抽提液分别与琉霉素生物合成阻断变株Y,发酵液以及纯化的Y。中间产物经过体外共培养可产生活性物质．化学分析表明与Y,发酵液混合后产生的是硫霉素,与纯化的Y。中间产物混合产生的是一种不稳定的活性物质。说明硫霉素环化酶基因在S.lividans TK24中得到了表达,其产物以Y。中间产物为底物并弥补了Y,中的缺陷。对p6Bcl2中4．5kb外源片段进行了限制酶酶切分析,建立了酶切图谱．利用含硫霉素环化酶基因的S．Lividans TK24转化子体外转化Y,的应用体系,将硫霉素环化酶基因定位在0．9kb Hinc I—Pst I片段上,并证明了硫霉紊环化酶的活性与IPNS同源片段无关。以上实验为进一步研究琉霉素环化酶基因的结构打下了基础。     

Autonomous selection of differentiation mutants of Streptomyces lividans TK24 in continuous culture

Summary Streptomyces lividans TK24 strains transformed with different recombinat derivatives of the vector plasmid pIJ487 were continuously cultivated in a chemostat. The first plasmid derivative contained the determinant for human interferon-alpha-1 (IFN) and the expression-secretion-unit (ESU) for this protein. In the second one a nourseothricin resistance determinant (ntc) was inserted additionally. The chemostat operated mainly at glucose limitation and low dilution rates. The structural and functional stabilities of the plasmids were shown to depend on the selection pressure. The host mutants enriched in the chemostat differed from the parental strain with respect to the growth pattern of aerial and submerged mycelium, the spore formation, and the formation and secretion of pigments and enzymes. Some highly stable host-vector systems could be selected. The plasmids' genotype influenced the growth pattern of the host mutants enriched in the chemostat in dependence on the limitation conditions as well as the stability of plasmid inheritance, plasmid structure and pigment formation in these mutants.     

Identification, isolation and sequencing of the recA gene of Streptomyces lividans TK24

Abstract An internal fragment of the recA gene of Streptomyces cattleya was amplified by the polymerase chain reaction (PCR) employing degenerate oligonucleotide primers. Using this fragment as a hybridization probe, a recA homologous gene could be shown in each tested Streptomyces strain. A 4.4 kb Bam HI fragment which carried the complete recA gene was isolated from Streptomyces lividans TK24. Sequence analysis suggested that the coding region of the recA gene consists of 1122 bp. The highest similarity (∼78%) could be detected to the recA genes of Mycobacterium tuberculosis and Mycobacterium leprae . After fusion with an E. coli promoter the S. lividans recA gene could partially complement an Escherichia coli recA mutant.     

Heterologous expression of SARS-CoV ORF10 and X5 genes in E. coli and Streptomyces lividans TK24

In previous studies a variety of novel accessory genes has been identified that were interspersed among the structural genes of the SARS-CoV (severe acute respiratory syndrome coronavirus) genome. The predicted unknown proteins (PUPs) encoded by the accessory genes, which are considered to be unique to the SARS-CoV genome, might play important roles in the SARS-CoV infection. Two of these genes, called ORF10 and X5, were synthesized and introduced into E. coli and Streptomyces lividans TK24, respectively. SDS-PAGE and Western blot revealed that the ORF10 and X5 genes have been expressed in the two hosts. This is the first report of heterologous expression of ORF10 and X5 genes in E. coli and S. lividans TK24. This work makes it possible to study the structure and potential functions of proteins encoding by these two genes.     

Stability of plasmid vector plJ303 in Streptomyces lividans TK24 during laboratory-scale fermentations

Plasmid plJ303 stability in Streptomyces lividans cultures has been studied by measuring plasmid copy number under various growth conditions. An increase in mean plasmid copy number was normally seen during early rapid growth in both shaken culture and stirred vessel fermentations at 28 degrees C. Maximum copy numbers were consistently attained in early stationary phase followed by a decline (of variable amount) upon further incubation. The imposition of environmental stress (high growth temperature, i.e., 37 degrees C, and low dissolved oxygen tension, i.e., <5% air saturation) led to a plasmid copy number of zero and a 50% reduction, respectively. Interestingly, the relative proportions of plasmid topoisomers changed with time since progressively more supercoiled forms were observed throughout the stationary phase. Plasmid dimers were also observed in some cultures, and no evidence of structural plasmid instability was found. In general, this host-vector system seemed remarkably stable under normal growth conditions. However, copious organic acid production by the host was observed and was thought to be undesirable for good heterologous gene expression of a secreted protein. (c) 1993 John Wiley & Sons, Inc.     

Heterologous mevalonate production in Streptomyces lividans TK23

Mevalonate is a ubiquitous biosynthetic intermediate of terpenoids and is used as a moisturizer in cosmetics and a chemical for biochemical research. In this study, we have achieved a heterologous production of this useful compound by expression in Streptomyces lividans TK23 of 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA reductase genes, which were cloned from Streptomyces sp. strain CL190.     

amlC,Another amylolytic gene maps close to the amlB locus in Streptomyces lividans TK24

The region located upstream of the α-amylase gene (amlB) of Streptomyces lividans TK24 (Yin et al., 1997) contains a 2978-bp-long ORF divergent from amlB, and designated amlC. amlC Encodes a 993 amino acid (aa) protein with a calculated molecular weight of 107.054 kDa. On the basis of sequence similarity as well as enzymatic activity, AmlC is likely to belong to the 1,4-α-d-glucan glucanohydrolase family. amlC is transcribed as a unique 3 kb leaderless monocistronic mRNA. Primer extension experiments allowed the identification of promoter sequences that do not resemble the typical eubacterial promoter sequences. amlC was successfully disrupted and was mapped at approx. 700 kb from a chromosomal end of S. lividans TK24, 100 kb on the right of the amplifiable unit AUD1 (Volff et al., 1996). Nevertheless, amlC disruption seemed to be accompanied by extensive rearrangements of the 2500-kb DraI–II fragment of the chromosome.     

人肿瘤坏死因子基因在变铅青链霉菌中的克隆与表达

以质粒ｐＩＪ４８６为载体,将来源于质粒ｐＨＴ１的肿瘤坏死坏因子（ＴＮＦ－ａ）ｃＤＮＡ克隆至变铅青链霉菌,以新霉素（３０μｇ／ｍｌ）为选择标记,获得了数百转化子,实验表明Ｎｏ．７转化子ｓ．ｌｉｖｉｄａｎｓ ＴＫ５４－ＨＴ所含重组质粒ｐＩＪＴ７已克隆有ＴＮＦｃＤＮＡ。Ｌ９２９细胞毒实验结果表明该转化子ＴＮＦ表达量可达１０＾８活性单位／升以上,中和实验确证其表达产物为人ＴＮＦ－ａ,ＳＤＳ－ＰＡＧＥ表明克     

Increased heterologous production of the antitumoral polyketide mithramycin A by engineered Streptomyces lividans TK24 strains

Mithramycin A is an antitumor compound used for treatment of several types of cancer including chronic and acute myeloid leukemia, testicular carcinoma, hypercalcemia and Paget’s disease. Selective modifications of this molecule by combinatorial biosynthesis and biocatalysis opened the possibility to produce mithramycin analogues with improved properties that are currently under preclinical development. The mithramycin A biosynthetic gene cluster from Streptomyces argillaceus ATCC12956 was cloned by transformation assisted recombination in Saccharomyces cerevisiae and heterologous expression in Streptomyces lividans TK24 was evaluated. Mithramycin A was efficiently produced by S. lividans TK24 under standard fermentation conditions. To improve the yield of heterologously produced mithramycin A, a collection of derivative strains of S. lividans TK24 were constructed by sequential deletion of known potentially interfering secondary metabolite gene clusters using a protocol based on the positive selection of double crossover events with blue pigment indigoidine-producing gene. Mithramycin A production was evaluated in these S. lividans strains and substantially improved mithramycin A production was observed depending on the deleted gene clusters. A collection of S. lividans strains suitable for heterologous expression of actinomycetes secondary metabolites were generated and efficient production of mithramycin A with yields close to 3 g/L, under the tested fermentation conditions was achieved using these optimized collection of strains.

     

Cloning and expression of an alpha-amylase gene from Streptomyces thermoviolaceus CUB74 in Escherichia coli JM107 and S. lividans TK24

A gene coding for a thermostable extracellular alpha-amylase, carried by a 5.7 kb BamHI chromosomal DNA fragment isolated from Streptomyces thermoviolaceus strain CUB74, was cloned into Escherichia coli JM107 using, as a cloning vector, the high-copy-number plasmid pUC8. E. coli containing a recombinant plasmid pQR300 expressed the amylase gene and exported the enzyme into the periplasmic space and the culture medium. The amylase protein expressed by E. coli had the same molecular mass (50 kDa) as that expressed by the Streptomyces parent strain, which suggests that the enzyme is processed similarly by both strains. The amylase gene was also cloned into Streptomyces lividans TK24 using pIJ702 as vector. The enzyme was stable at 70 degrees C when CaCl2 was present.     

变铅青链霉菌TK24 secE启动子表达特性的研究

通过PCR扩增得到变铅青链霉菌（Streptomyces lividans)TK24 secE基因上游496bp的片段,其序列与S.coelicolor secE启动子序列同源性为99.8%。将该序列克隆到以儿茶酚加氧酶基因（xylE)为报告基因的链霉菌启动子探测质粒pIJ4083上,并转化S.lividans TK24原生质体,获得了重组菌株S.lividans [pIJ4083-secE]。S.lividans[pIJ4083-secE]菌株发酵结果表明,secE启动子为强启动子,活性与vsi基因启动子相当。secE启动子的表达在对数生长期达到高峰,平台期下降;28℃发酵培养时secE启动子活性远高于37℃发酵培养;比较了不同发酵培养基Phage,NB和CM中secE启动子的活性;实验结果还表明培养基中葡萄糖含量对secE启动子的表达有抑制作用。     

变铅青链霉菌异柠檬酸脱氢酶的异源表达及序列分析

通过同源性引物成功扩增和克隆了变铅青链霉菌(Streptomyces lividans)TK54的异柠檬酸脱氢酶(isocitrate dehydrogenase, IDH) (简称SlIDH)基因icd (GenBank登录号为EU661252).icd的起始密码子为GTG,GC含量为69.55 %,显示了链霉菌基因的高GC含量特征,实现了SlIDH在E.coli中的异源高效表达.0.5 mmol/L的IPTG为最佳诱导条件.SlIDH的分子量约为80 kD.在Mn~(2+)或Mg~(2+)条件下,SlIDH以NADP~+为辅酶时的活性分别为7.94 U/mg及4.00 U/mg,以NAD~+为辅酶时的活性分别为0.58 U/mg及0.27 U/mg,SlIDH更偏爱以NADP~+为辅酶.与不同种属单体IDH的氨基酸序列比对显示,SlIDH与单体IDH的序列一致性均在60 %以上.因此本工作首次以实验性证据初步鉴定了SlIDH为NADP-依赖型单体IDH.本工作为进一步探索单体IDH的结构与功能以及单体IDH与同源二聚体IDH的进化关系奠定了基础.     

Molecular cloning and characterization of an rRNA operon in Streptomyces lividans TK21.

The number of rRNA genes in Streptomyces lividans was examined by Southern hybridization. Randomly labeled 23 and 16S rRNAs were hybridized with BamHI, BglII, PstI, SalI, or XhoI digests of S. lividans TK21 DNA. BamHi, BglII, SalI and XhoI digests yielded six radioactive bands each for the 23 and 16S rRNAs, whereas PstI digests gave one band for the 23S rRNA and one high-intensity band and six low-density bands for the 16S rRNA. The 7.4-kilobase-pair BamHI fragment containing one of the rRNA gene clusters was cloned into plasmid pBR322. The hybrid plasmid, pSLTK1, was characterized by physical mapping, Southern hybridization, and electron microscopic analysis of the R loops formed between pSLTK1 and the 23 and 16S rRNAs. There were at least six rRNA genes in S. lividans TK21. The 16 and 23S rRNA genes were estimated to be about 1.40 and 3.17 kilobase pairs, respectively. The genes for the rRNAs were aligned in the sequence 16S-23S-5S. tRNA genes were not found in the spacer region or in the context of the rRNA genes. The G + C content of the spacer region was calculated to be approximately 58%, in contrast to 73% for the chromosome as a whole.     

Enzymatic characterization of a monomeric isocitrate dehydrogenase from Streptomyces lividans TK54

Isocitrate dehydrogenase (IDH) is one of the key enzymes in the citric acid cycle, which involves in providing energy and biosynthetic precursors for metabolism. Here, we report for the first time the enzymatic characterization of a monomeric NADP⁺-dependent IDH from Streptomyces lividans TK54 (SlIDH). The icd gene (GenBank database accession number EU661252) encoding IDH was cloned and overexpressed in Escherichia coli. The molecular mass of SlIDH was about 80 kDa, typical of a monomeric NADP-IDH, and showed high amino acid sequence identity with known monomeric IDHs. The optimal activity of the 6His-tagged SlIDH was found at pH values 8.5 (Mn²⁺) and 9.0 (Mg²⁺), and the optimal temperature was around 46 °C. Heat-inactivation studies showed that about 50% SlIDH activity was preserved at 38 °C after 20 min of incubation. The recombinant SlIDH displayed a 62,000-fold (k_cat/K_m) preference for NADP⁺ over NAD⁺ with Mn²⁺, and a 85,000-fold greater specificity for NADP⁺ than NAD⁺ with Mg²⁺. Therefore, SlIDH is a divalent cation-dependent monomeric IDH with remarkably high coenzyme preference for NADP⁺.