The pro-region of Streptomyces hygroscopicus transglutaminase affects its secretion by Escherichia coli

Streptomyces transglutaminase (TGase) is secreted as a zymogen (pro-TGase) in liquid cultures and is then processed by the removal of its N-terminal region, resulting in active TGase. To date, there is no report describing TGase (or pro-TGase) secretion in Escherichia coli. In this study, the pro-TGase from Streptomyces hygroscopicus was efficiently secreted by E.?coli BL21(DE3) using the TGase signal peptide or the pelB signal peptide. The secreted pro-TGase was efficiently transformed into active TGase by adding dispase to the culture supernatant of the recombinant strains. Mutational analysis showed that deletion of the first six amino acids of the N-terminal of the pro-region reduced the secretion of pro-TGase, and removal of the next 10 amino acids resulted in the formation of insoluble pro-TGase. These results suggest that the pro-region of TGase is essential for its efficient secretion and solubility in E.?coli.     

Characterization of the promoter, signal sequence, and amino terminus of a secreted beta-galactosidase from "Streptomyces lividans"

The gene for a secreted 130-kilodalton beta-galactosidase from "Streptomyces lividans" has been cloned, its promoter, signal sequence, and amino terminal region have been localized, and their nucleotide sequence has been determined. The signal sequence extends over 56 amino acids and shows the characteristic-features of signal sequences, including a hydrophilic amino terminus followed by a hydrophobic core near the signal cleavage site. The secretion of beta-galactosidase depends on the presence of the signal sequence. beta-Galactosidase is the major protein in culture supernatants and extracts of strains expressing the cloned beta-galactosidase gene and represents a valuable tool in the study of protein secretion in Streptomyces spp.     

Regulation and secretion of an extracellular esterase from Streptomyces scabies.

Production of a heat-stable, extracellular esterase by Streptomyces scabies is regulated by zinc ions. The esterase-encoding gene (est) from S. scabies was cloned and expressed in Streptomyces lividans. In S. lividans, expression of the est gene is also regulated by Zn2+, and the esterase is efficiently secreted in this organism. The sequence of the est gene suggests that a 39-amino acid signal peptide is removed during secretion of this protein. Deletion analysis has indicated that the hydrophobic domain of the signal peptide is required for secretion. Gel retardation assays and DNaseI footprinting using an S-30 protein extract from S. scabies have previously identified a specific 23-bp protein-binding site upstream from the est coding sequence. Deletion of this protein-binding sequence significantly decreased expression of the est gene.     

Recombinant production of <Emphasis Type="Italic">Streptococcus equisimilis</Emphasis> streptokinase by <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

Background  

Streptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. It is naturally secreted by several strains of beta-haemolytic streptococci. The low yields obtained in SK production, lack of developed gene transfer methodology and the pathogenesis of its natural host have been the principal reasons to search for a recombinant source for this important therapeutic protein. We report here the expression and secretion of SK by the Gram-positive bacterium Streptomyces lividans. The structural gene encoding SK was fused to the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi) signal sequence or to the Streptomyces lividans xylanase C (xlnC) signal sequence. The native Vsi protein is translocated via the Sec pathway while the native XlnC protein uses the twin-arginine translocation (Tat) pathway.     

人肿瘤坏死因子β(hTNFβ)在变铅青链霉菌中的表达研究

以变铅青链霉菌为宿主研究了人INFβ(hTNFβ)的异源表达。应用链霉菌S.VENEZUELAC cbs762.70分泌产生的枯草杆菌蛋白酶抑制剂vsi基因的启动子、表达调控序列和分泌信号肽序列,分别对hTNFβ进行了直接分泌表达、分泌融合表达和胞内表达。将hTNFβ的cDNA分别直接融合于vsi信号肽序列下游2个氨基酸处、vsi全长基因之后以及vsi起始密码子ATG的下游,获得的表达盒分别克隆至链霉菌高拷贝质粒pIJ486,转化Streptomyces lividans TK24,获得了重组菌株S.lividans(pIJ486-hTNFβ),s.LIVIDANS(PIJ486-vsi-hTNFβ)和S.lividans(pIVPA-hTNFβ)。分别对不同的重组菌株进行摇瓶培养,对其培养的上清液和细胞裂解液进行SDS—PAGE和Westen杂交,结果表明：hTNFβ在重组菌株中均获得了表达,且直接分泌产物和胞内表达产物均具有生物学活性。hTNFβ直接分泌表达产物的分子量约为16kDa,NB培养基中培养48h时表达水平约为0．7mg／L。胞内表达产物分子量与对照重组hTNFβ一致(18．7kDa),但随培养时间的延长远步降解为16kDa,NB培养基中培养48h时的表达水平(25．1mg／L)远高于其直接分泌表达水平。     

Comparison of the Sec and Tat secretion pathways for heterologous protein production by Streptomyces lividans

Streptomyces is an interesting host for the secretory production of recombinant proteins because of its natural ability to secrete high levels of active proteins into the culture broth and the availability of extensive fermentation knowledge. In bacterial expression systems, heterologous protein secretion has, so far, almost exclusively been investigated using signal peptides that direct the secretion to the Sec pathway. In this study, we assessed the possibility of the Streptomyces lividans twin-arginine translocation (Tat) pathway to secrete the human proteins tumor necrosis factor (TNF) alpha and interleukin (IL) 10 by fusing the coding sequences of mature hTNFalpha and hIL10 to the twin-arginine signal peptides of S. lividans xylanase C (XlnC) and Streptomyces antibioticus tyrosinase. Both proteins were secreted and this secretion was blocked in the DeltatatB and DeltatatC single mutants, indicating that the transport of hTNFalpha and hIL10 could be directed through the Tat pathway. Secretion levels of hTNFalpha and hIL10, however, were lower for Tat-dependent than for Sec-dependent transport using the Sec-dependent signal peptide of the Streptomyces venezuelae subtilisin inhibitor. Surprisingly, Sec-dependent transport was enhanced in the tatB deletion strain. This was especially interesting in the case of hIL10, where Sec-dependent transport of hIL10 was at least 15 times higher in the DeltatatB mutant than in the wild-type strain.     

Identification of Streptomyces lividans proteins secreted by the twin-arginine translocation pathway following growth with different carbon sources

Genome-based signal peptide predictions classified Streptomyces coelicolor as the microorganism that secretes the most proteins through the twin-arginine translocation (Tat)-dependent secretion pathway. Availability of a DeltatatC mutant of the closely related strain Streptomyces lividans impaired Tat-dependent protein secretion and enabled identification of many extracellular proteins that are secreted via the Tat pathway. Proteomic techniques were applied to analyze proteins from the supernatants of log-phase cultures. Since the bacterial secretome depends mainly on the carbon sources available during growth, xylose, glucose, chitin, and soil extracts were used. A total of 63 proteins were identified, among which 7 were predicted by the TATscan program, and 20 were not predicted but contained a potential Tat signal motif. Thirteen proteins having no signal sequence could be co-transported by Tat-dependent proteins because the genes that encode these proteins are in close proximity in the genome. Finally, the presence of 23 proteins lacking signal peptides was difficult to explain. More secreted proteins could be identified as Tat substrates in varying carbon sources.     

Production of <Emphasis Type="Italic">Chryseobacterium proteolyticum</Emphasis> protein-glutaminase using the twin-arginine translocation pathway in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The protein glutaminase (PG) secreted by the Gram-negative bacterium Chryseobacterium proteolyticum can deamidate glutaminyl residues in several substrate proteins, including insoluble wheat glutens. This enzyme therefore has potential application in the food industry. We assessed the possibility to produce PG containing a pro-domain in Corynebacterium glutamicum which we have successfully used for production of several kinds of proteins at industrial-scale. When it was targeted to the general protein secretion pathway (Sec) via its own signal sequence, the protein glutaminase was not secreted in this strain. In contrast, we showed that pro-PG could be efficiently produced using the recently discovered twin-arginine translocation (Tat) pathway when the typical Sec-dependent signal peptide was replaced by a Tat-dependent signal sequence from various bacteria. The accumulation of pro-PG in C. glutamicum ATCC13869 reached 183 mg/l, and the pro-PG was converted to an active form as the native one by SAM-P45, a subtilisin-like serine protease derived from Streptomyces albogriseolus. The successful secretion of PG via this approach confirms that the Tat pathway of C. glutamicum is an efficient alternative for the industrial-scale production of proteins that are not efficiently secreted by other systems.     

Twin-Arginine Translocation Pathway in Streptomyces lividans

The recently discovered bacterial twin-arginine translocation (Tat) pathway was investigated in Streptomyces lividans, a gram-positive organism with a high secretion capacity. The presence of one tatC and two hcf106 homologs in the S. lividans genome together with the several precursor proteins with a twin-arginine motif in their signal peptide suggested the presence of the twin-arginine translocation pathway in the S. lividans secretome. To demonstrate its functionality, a tatC deletion mutant was constructed. This mutation impaired the translocation of the Streptomyces antibioticus tyrosinase, a protein that forms a complex with its transactivator protein before export. Also the chimeric construct pre-TorA-23K, known to be exclusively secreted via the Tat pathway in Escherichia coli, could be translocated in wild-type S. lividans but not in the tatC mutant. In contrast, the secretion of the Sec-dependent S. lividans subtilisin inhibitor was not affected. This study therefore demonstrates that also in general in Streptomyces spp. the Tat pathway is functional. Moreover, this Tat pathway can translocate folded proteins, and the E. coli TorA signal peptide can direct Tat-dependent transport in S. lividans.     

Increase in xylanase production by Streptomyces lividans through simultaneous use of the Sec- and Tat-dependent protein export systems

Xylanase B1 (XlnB1) from Streptomyces lividans is a protein consisting of two discrete structural and functional units, an N-terminal catalytic domain and a C-terminal substrate binding domain. In the culture medium, two forms of xylanase B are present, namely, XlnB1 and XlnB2, the latter of which corresponds to the catalytic domain of XlnB1 deprived of the substrate binding domain. Both forms of the xylanase have the same activity on xylan. The enzyme is secreted through the Sec-dependent pathway with a better yield of XlnB1 than XlnB2. Interestingly, XlnB2 exhibits 80% identity with XlnC which is secreted exclusively through the Tat-dependent pathway. To demonstrate whether XlnB1 and XlnB2 could also be secreted through the Tat-dependent pathway, the Tat-targeting xlnC signal sequence was fused to the structural genes of xlnB1 and xlnB2. Both XlnB1 and XlnB2 were secreted through the Tat-dependent pathway, but XlnB2 was better produced than XlnB1. As XlnB1 and XlnB2 could be better secreted through the Sec- and Tat-dependent systems, respectively, a copy of the structural gene of xlnB1 fused to a Sec signal sequence and a copy of the structural gene of xlnB2 fused to a Tat signal sequence were inserted into the same plasmid under the control of the xlnA promoter. The transformant produced xylanase activity which corresponded approximately to the sum of activities of the individual strain producing xylanase by either the Sec- or Tat-dependent secretion system. This indicated that both secretion systems are functional and independent of each other in the recombinant strain. This is the first report on the efficient secretion of a protein using two different secretion systems at the same time. Assuming that the protein to be secreted could be properly folded prior to and after translocation via the Tat- and Sec-dependent pathways, respectively, the simultaneous use of the Sec- and Tat-dependent pathways provides an efficient means to increase the production of a given protein.     

Heterologous expression and secretion of a Streptomyces scabies esterase in Streptomyces lividans and Escherichia coli.

The esterase gene from Streptomyces scabies FL1 was cloned and expressed in Streptomyces lividans on plasmids pIJ486 and pIJ702. In S. lividans, the esterase gene was expressed during later stages of growth and was regulated by zinc, as is seen with S. scabies. The 36-kDa secreted form of the esterase was purified from S. lividans. N-terminal amino acid sequencing indicated that the processing site utilized in S. lividans for the removal of the signal sequence was the same as that recognized for processing in S. scabies. Western blots (immunoblots) revealed the presence of a 40-kDa precursor form of the esterase in cytoplasmic extracts. A 23-amino-acid deletion was introduced into the putative signal sequence for the esterase. When this deleted form of the esterase was expressed in S. lividans, a cytoplasmic 38-kDa precursor protein was produced but no secreted esterase was detected, suggesting the importance of the deleted sequence for efficient processing and secretion. The esterase gene was also cloned into the pUC119 plasmid in Escherichia coli. By using the lac promoter sequence, the esterase gene was expressed, and the majority of the esterase was localized to the periplasmic space.     

Characteristics of the surface-located carbohydrate-binding protein CbpC from Streptomyces coelicolor A3(2)

Streptomyces coelicolor A32 produces a 35.6-kDa carbohydrate-binding protein (named CbpC) in the presence of cellobiose, cellulose or chitin as sole carbon source. The protein was found secreted (a typical signal sequence was present at the N-terminus) and linked to the peptidoglycan layer of the mycelia. At its C-terminal end a putative cell-wall sorting signal was identified, consisting of (1) Streptomyces specific recognition site for a transpeptidase (LAETG instead of generic LPXTP consensus), (2) a hydrophobic region and (3) a tail of positively charged residues. The deletion of this sorting signal abolished the cell-wall attachment because the resulting CbpC-form was found extracellular. After purification this protein was shown to interact strongly with crystalline cellulose; different crystalline chitin-forms were recognised moderately and chitosan not. As demonstrated by analysing further truncated CbpC-forms a glycine-aspartate/serine rich region, which separates the carbohydrate-binding module from the sorting signal, plays an important role in protein stability.     

Heterologous expression of the alpha-amylase inhibitor gene cloned from an amplified genomic sequence of Streptomyces tendae.

The coding region for a secreted proteinaceous inhibitor of the human alpha-amylase (tendamistat; HOE 467) was identified by using a synthetic oligonucleotide probe. The gene is part of a 37-kilobase amplified genomic sequence found in an overproducing mutant of Streptomyces tendae. After subcloning, sequence analysis revealed an open reading frame of 312 base pairs preceded by a putative ribosome-binding site. The reading frame is 30 codons longer than necessary for the mature protein. This sequence coded for an amino-terminal extension of tendamistat and shows typical features of a signal peptide. After being cloned into Streptomyces vector plasmids and transformed to the heterologous host, Streptomyces lividans TK24, the gene was expressed, and the alpha-amylase inhibitor was correctly processed and secreted into the culture medium. The amount of secreted protein was dependent on the gene dosage and on the promoter arrangement.     

Effect of protease mutations on the production of xylanases in Streptomyces lividans

Three protease mutants--7 (tap-), 12 (tap-, ssp-), and 17 (multiple mutations)--of Streptomyces lividans were tested for their influence on protein secretion. Streptomyces lividans grown in xylan secretes 3 xylanases (A, B, and C). Xylanases A (XlnA) and B (XlnB) are secreted by the Sec pathway, whereas xylanase C (XlnC) is secreted by the Tat pathway. The production of XlnA and XlnC was affected in the mutants, suggesting that the mutations interfered with both Sec- and Tat-secretion systems. However, the processing rate for the Sec and Tat precursor was similar to the wild-type strain, indicating that the mutations had no direct effect on secretion. Streptomyces lividans naturally produced 2 forms of XlnB: XlnB1, which contains the catalytic and the xylan-binding domains, and XlnB2, which contains the catalytic domain only. There was no change from the wild-type strain in the ratio of XlnB1/XlnB2 produced by the mutants, indicating that these proteases are not involved in this process. Although XlnA1, partially truncated in its xylan-binding domain, was rapidly degraded to its catalytic domain (XlnA2) in the wild-type strain, the rate of conversion was reduced in the 3 mutants, indicating that the proteases participated to some extent in this proteolytic process.     

Protein secretion in gram-negative bacteria. The extracellular metalloprotease B from Erwinia chrysanthemi contains a C-terminal secretion signal analogous to that of Escherichia coli alpha-hemolysin

The secretion signal of extracellular metalloprotease B that is secreted without a signal peptide by the Gram-negative phytopathogenic bacterium Erwinia chrysanthemi is shown by deletion and gene fusion analyses to be located within the last 40 C-terminal amino acids. Secretion of a peptide containing only this region of the protease requires the same three secretion factors (PrtD, PrtE, and PrtF) that were previously shown to be required for the secretion of the full-length protease. This secretion signal can also be recognized, albeit inefficiently, by the analogous secretion machinery of alpha-hemolysin, another protein with a C-terminal secretion signal that is secreted by some strains of the Gram-negative bacterium Escherichia coli. The secretion signal was fused to an internal 200-amino acid fragment from the sequence of the cytoplasmic protein amylomaltase to promote its specific secretion by the protease secretion pathway. Almost exactly the same sequence as that identified as the protease B secretion signal was also found at the C terminus of metalloprotease C that is also secreted by E. chrysanthemi.     

Protein secretion biotechnology using Streptomyces lividans: large-scale production of functional trimeric tumor necrosis factor alpha.

We evaluated the feasibility of large-scale production of biopharmaceuticals expressed as heterologous polypeptides from the Gram-positive bacterium Streptomyces lividans. As a model protein we used murine tumor necrosis factor alpha (mTNFalpha). mTNFalpha fused C-terminally to the secretory signal peptide of the subtilisin-inhibitor protein from Streptomyces venezuelae. Under appropriate fermentation conditions, significant amounts of mature mTNFalpha (80-120 mg/L) can be recovered from spent growth media. Efficient downstream processing allowing rapid purification of mTNFalpha from culture supernatants was developed. Importantly, the protein is recovered from the spent growth medium in its native trimeric state as judged by biophysical analysis. Further, mTNFalpha secreted by S. lividans is significantly more active in an in vitro apoptosis tissue culture assay than a corresponding polypeptide produced in Escherichia coli. This pilot study provides the first validation of S. lividans protein secretion as an alternative bioprocess for large-scale production of oligomeric proteins of potential therapeutic value.