A comparison of the process issues in expressing the same recombinant enzyme periplasmically in Escherichia coli and extracellularly in Streptomyces lividans.

The choice of a host for the production of a biological molecule will have a significant effect on isolation and purification procedures employed. This paper makes a comparison between the production of a single enzyme, a recombinant alpha-amylase, in Escherichia coli and Streptomyces lividans, on a small scale. It defines the differences in the cultivation and in the isolation stages and also describes the impact of the expression system on later downstream processing steps. At the cultivation stage, the specific productivity of the E. coli in units per gram per hour is four times that of the S. lividans while the total biomass yields are of the same order. The initial volume for downstream processing of S. lividans is six-fold larger and the total protein released into the extracellular medium is three times greater than E. coli, however, the recoverable yield from the E. coli is a fifth of that obtained from the S. lividans and requires three additional stages prior to chromatography. Even with these stages the final specific activity is 64% of the S. lividans. The results indicate the need to consider the whole process when making such comparisons.     

High-level overproduction of <Emphasis Type="Italic">Thermus</Emphasis> enzymes in <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

Biotechnology needs to explore the capacity of different organisms to overproduce proteins of interest at low cost. In this paper, we show that Streptomyces lividans is a suitable host for the expression of Thermus thermophilus genes and report the overproduction of the corresponding proteins. This capacity was corroborated after cloning the genes corresponding to an alkaline phosphatase (a periplasmic enzyme in T. thermophilus) and that corresponding to a beta-glycosidase (an intracellular enzyme) in Escherichia coli and in S. lividans. Comparison of the production in both hosts revealed that the expression of active protein achieved in S. lividans was much higher than in E. coli, especially in the case of the periplasmic enzyme. In fact, the native signal peptide of the T. thermophilus phosphatase was functional in S. lividans, being processed at the same peptide bond in both organisms, allowing the overproduction and secretion of this protein to the S. lividans culture supernatant. As in E. coli, the thermostability of the expressed proteins allowed a huge purification factor upon thermal denaturation and precipitation of the host proteins. We conclude that S. lividans is a very efficient and industry-friendly host for the expression of thermophilic proteins from Thermus spp.     

Expression of a Streptomyces plasmid promoter in Escherichia coli

A 166-bp DNA fragment from the Streptomyces multicopy plasmid pIJ101 with in vivo promoter activity both in Streptomyces lividans and in Escherichia coli was isolated. The start point of the RNA transcribed from this fragment, determined by high resolution S1 nuclease mapping, was the same in S. lividans and in E. coli. This suggests that the E. coli RNA polymerase recognizes the same sequence determinants and chooses the point of initiation of RNA synthesis in the same way as the corresponding S. lividans enzyme. The putative promoter sequence shows good homology to the E. coli promoter consensus sequence in the '-35' region but poor homology in the '-10' region.     

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.     

Restriction of two-replicon shuttle Escherichia coli-Streptomyces plasmids in Streptomyces lividans strain 66

The shuttle Escherichia coli - Streptomyces plasmids were used to transform S. lividans 66. Plasmid DNAs isolated from this strain transform it 10-1000-fold more efficiently than DNAs from E. coli. Rare transformant cured from most restricted plasmid is more efficient recipient of plasmid DNA from E. coli and has the property of R +/- M+ mutant. Restriction in S. lividans 66 correlates with the appearance in DNA from E. coli of the sites susceptible to Scg2I restriction endonuclease. The latter was isolated earlier from recombinant strain Rcg2, a hybrid between S. griseus Kr. 15 and S. coelicolor A3(2). Scg2I possesses the recognition sequence CCTAGG, like EcoRII, MvaI and Eco dcm methylase. The DNA resistant to Scg2I cleavage retained this ability after in vitro modification by EcoRII methylase. So, the resistance of DNA to Scg2I cleavage is not connected with methylation at 4th and 5th position of second cytosine in the recognition sequence. Neither restriction of plasmid DNA in S. lividans 66 is dependent on dcm modification in E. coli, though its dependence on dam modification is not excluded. It is assumed that the restriction in S. lividans 66 is specified by endonuclease analogous to Scg2I.     

Cloning and expression of a nitroaryl reductase gene from Streptomyces aminophilus strain MCMB411 in E. coli JM109 and Streptomyces lividans TK64

A partial genomic library was prepared in E. coli JM109 using pBR322 as vector and 2.4 kb Sau 3A I chromosomal fragment, encoding a nitroaryl reductase (nbr A) gene, from Streptomyces aminophilus strain MCMB 411. From the library, 2.4 kb fragment was recloned in E. coli JM109 and S. lividans TK64 using pUC18 and pIJ702 as vectors respectively. The recombinant plasmids pSD103 and pSD105 expressed the reductase gene and exported the enzyme in periplasmic space of E. coli and in cytoplasm of S. lividans TK64. The proteins expressed by E. coli and S. lividans had the same molecular mass (70 kD) as that expressed by parent strain, which suggested that the enzyme was processed similarly by all strains. Activities of the enzymes cloned in E. coli JM109 and S. lividans TK64 containing recombinant plasmids pSD103 and pSD105 respectively were optimum at 30 degrees C and pH 9 and requirement of cofactors was same as that of the parent strain.     

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.     

Molecular cloning and expression in Streptomyces lividans of a proteinous alpha-amylase inhibitor (HaimII) gene from Streptomyces griseosporeus

The gene encoding a proteinous alpha-amylase inhibitor (HaimII) of Streptomyces griseosporeus YM-25 has been cloned in Escherichia coli K12 using a deoxyinosine-containing synthetic oligonucleotide as the probe. A 1.6 kilobases BamHI fragment was confirmed to hybridize with the probe and subcloned in an E. coli-S. lividans shuttle vector. The plasmid clone was transferred into S. lividans by transformation. An appreciable amount of alpha-amylase inhibitor activity was found in the culture medium of S. lividans harboring the plasmid. As the specificity was indistinguishable from that of HaimII produced by the original S. griseosporeus strain, we concluded that the HaimII protein was synthesized in S. lividans and excreted into the medium.     

Hyperexpression and analysis of choB encoding cholesterol oxidase of Brevibacterium sterolicum in Escherichia coli and Streptomyces lividans.

We examined the expression of choB, encoding cholesterol oxidase of Brevibacterium sterolicum ATCC 21387, in Escherichia coli JM105 and Streptomyces lividans TK23 using various deletion DNA fragments within the 5'-flanking region. The enzyme activity could be detected intracellularly in E. coli only when the 5'-flanking region was reduced to less than 256-bp and choB was transcribed by the lac promoter. A large amount of the enzyme were produced as inactive inclusion bodies when ChoB protein was fused with the NH2-terminal portion of LacZ protein. In contrast, choB with more than 256-bp of the 5'-flanking region was efficiently expressed in S. lividans TK23, and about 85 times as much of the active enzyme (170 U/ml) was secreted into the culture filtrate as with B. sterolicum in flask culture. These results suggest that the promoter of choB exist within 256-bp of the 5'-flanking region and can be efficiently recognized by the RNA polymerase of S. lividans. The characteristics of the enzyme purified from the culture filtrate of the S. lividans transformant and that of B. sterolicum were identical although the NH2-terminal amino acid sequence of the enzyme from the S. lividans transformant was 6 amino acids shorter than that from B. sterolicum.     

Saccharomyces cerevisiae pyruvate kinase Pyk1 is PKA phosphorylation substrate in vitro

Factor C is an unusual extracellular protein capable of inducing cytodifferentiation in certain Streptomyces strains. The protein is produced by Streptomyces griseus 45H at such a low amount that the study of its mode of action was hindered by the shortage of purified protein. We report here the expression of C-terminally hexa-His-tagged factor C in Streptomyces lividans and Escherichia coli. Expression in S. lividans is low while in E. coli it is relatively high, yielding about 5--10 mg of biologically fully active protein per liter culture.     

Functional large-scale production of a novel Jonesia sp. xyloglucanase by heterologous secretion from Streptomyces lividans

The gene encoding a novel xyloglucanase (Xeg) belonging to family 74 glycoside hydrolases was isolated from a Jonesia sp. strain through functional screening in Escherichia coli. The encoded xyloglucanase is a protein of 972 aminoacyl residues with a 23 residue aminoterminal signal peptide. Over-expression of Xeg in B. subtilis or E. coli failed. In contrast, Xeg was successfully over-expressed and secreted in Streptomyces lividans TK24. To this end Xeg was fused C-terminally to the secretory signal peptide of the subtilisin inhibitor protein (vsi) from Streptomyces venezuelae. The native Xeg signal peptide derived from Jonesia sp. is only poorly functional in S. lividans. Under optimal growth conditions, significant amounts of mature Xeg (100-150 mg/l) are secreted in the spent growth media. A protocol to rapidly purify Xeg to homogeneity from culture supernatants was developed. Biophysical and biochemical analyses indicate that the enzyme is intact, stable and fully functional. Xeg is the longest heterologous polypeptide shown to be secreted from S. lividans. This study further validates use of S. lividans for production of active heterologous proteins and demonstrates that heterologous polypeptides of up to 100 kDa are also tractable by this system.     

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。     

Extracellular production of Streptomyces lividans acetyl xylan esterase A in Escherichia coli for rapid detection of activity

Acetyl xylan esterase A (AxeA) from Streptomyces lividans belongs to a large family of industrially relevant polysaccharide esterases. AxeA and its truncated form containing only the catalytically competent domain, AxeA(tr), catalyze both the deacetylation of xylan and the N-deacetylation of chitosan. This broad substrate specificity lends additional interest to their characterization and production. Here, we report three systems for extracellular production of AxeA(tr): secretion from the native host S. lividans with the native signal peptide, extracellular production in Escherichia coli with the native signal peptide, and in E. coli with the OmpA signal peptide. Over five to seven days of a shake flask culture, the native host S. lividans with the native signal peptide secreted AxeA(tr) into the extracellular medium in high yield (388 mg/L) with specific activity of 19 U/mg corresponding to a total of 7000 U/L. Over one day of shake flask culture, E. coli with the native secretion signal peptide produced 84-fold less in the extracellular medium (4.6 mg/L), but the specific activity was higher (100 U/mg) corresponding to a total of 460 U/L. A similar E. coli culture using the OmpA signal peptide, produced 10mg/L with a specific activity of 68 U/mg, corresponding to a total of 680 U/L. In 96-well microtiter plates, extracellular production with E. coli gave approximately 30 and approximately 86 microg/mL in S. lividans. Expression in S. lividans with the native signal peptide is best for high level production, while expression in E. coli using the OmpA secretion signal peptide is best for high-throughput expression and screening of variants in microtiter plate format.     

Purification and characterization of the Streptomyces lividans initiator protein DnaA.

The Streptomyces lividans DnaA protein (73 kDa) consists, like the Escherichia coli DnaA protein (52 kDa), of four domains. The larger size of the S. lividans protein is due to an additional stretch of 120 predominantly acidic amino acids within domain II. The S. lividans protein was overproduced as a His-tagged fusion protein. The purified protein (isoelectric point, 5.7) has a weak ATPase activity. By DNase I footprinting studies, each of the 17 DnaA boxes (consensus sequence, TTGTCCACA) in the S. lividans oriC region was found to be protected by the DnaA fusion protein. Purified mutant proteins carrying a deletion of the C-terminally located helix-loop-helix (HLH) motif or with amino acid substitutions in helix A (L577G) or helix B (R595A) no longer interact with DnaA boxes. A substitution of basic amino acids in the loop of the HLH motif (R587A or R589A) entailed the formation of S. lividans mutant DnaA proteins with little or no capacity for binding to DnaA boxes. Thus, like in E. coli, the C-terminally located domain IV is absolutely necessary for the specific binding of DnaA. A mutant protein lacking a stretch of acidic amino acids corresponding to domain II is not affected in its DNA binding capacity. Whether the acidic domain II interacts with accessory proteins remains to be elucidated.     

Heterologous expression of Rhodococcus opacus L-amino acid oxidase in Streptomyces lividans

L-amino acid oxidase (L-AAO) from Rhodococcus opacus is a highly enantioselective enzyme with a broad substrate specificity that catalyses the oxidation of L-amino acids to keto acids. The lao-gene (AY053450) from R. opacus was cloned into different Escherichia coli and Streptomyces lividans expression vectors. Expression in E. coli resulted in the accumulation of insoluble protein, but S. lividans was a suitable host for the heterologous production of L-AAO. When using the thiostrepton-inducible vector pIlaao, a specific activity of 0.18 Umg(-1) was obtained in the crude extract of S. lividans 1326. For the vector pUlaao, which contains the constitutive ermEp(*) promoter, a specific activity of 0.05 Umg(-1) was reached. Both the wild type and the recombinant L-AAO were purified to homogeneity. The expression systems described here now allow the structural and biochemical analysis of the L-AAO using genetic engineering methods.     

Site-specific degradation of Streptomyces lividans DNA during electrophoresis in buffers contaminated with ferrous iron.

Streptomyces lividans DNA contains a modification which makes it susceptible to double-strand cleavage during electrophoresis in buffers contaminated with ferrous iron (which may be present in some batches of EDTA). The cleavage of the DNA is site-specific and the average fragment size resulting from limit digestion of total S. lividans DNA is about 6kb. DNA from Streptomyces coelicolor A3(2) and several other Streptomyces strains, and from E. coli, is not cleaved under the same conditions. A S. lividans mutant has been isolated which lacks the DNA modification. We suspect that many reports of "poor" preparations of S. lividans plasmids may be due to the above effect.