Cloning and expression in Escherichia coli, Bacillus subtilis, and Streptococcus sanguis of a gene for staphylokinase--a bacterial plasminogen activator

Summary The gene coding for the bacterial plasminogen activator staphylokinase was cloned from the Staphylococcus aureus phage 42D, a serogroup F phage used for lysotyping, onto the standard Escherichia coli plasmid vector pACYC184. The coding and flanking sequences of the sak42D gene were largely identical to those of a sak gene cloned from the serologically different S. aureus phage SøC (Sako and Tsuchida 1983). Subcloning of a 2.5 kb phage 42D DNA fragment onto plasmid pGB3631 allowed the sak42D gene to be introduced into the gram-positive hosts Bacillus subtilis and Streptococcus sanguis. The sak42D gene was expressed and secreted most efficiently by B. subtilis cells (25 g/ml of culture supernatant) reduced in exoprotease production. In this host expression and secretion of Sak was initiated at the early growth phase and continued through the logarithmic phase. Formation of Sak was, however, also observed with the other cloning hosts. The Sak elaborated by the heterologous hosts was serologically identical with authentic Sak derived from S. aureus.     

Expression and Secretion of an Acid-Stable <Emphasis Type="Italic">α</Emphasis>-Amylase Gene in Bacillus Subtilis by <Emphasis Type="Italic">SacB</Emphasis> Promoter and Signal Peptide

Alpha amylase gene from Bacillus licheniformis was mutated by site-directed mutagenesis to improve its acid stability. The mutant gene was expression in Bacillus subtilis under the control of the promoter of sacB gene which was followed by either the α-amylase leader peptide of Bacillus licheniformis or the signal peptide sequence of sacB gene of Bacillus subtilis. Both peptides efficiently directed the secretion of α-amylase from the recombinant B. subtilis cells. The extracellular α-amylase activities in two recombinants were 1001 and 2012 U ml⁻¹, respectively. The purity of the recombinant product was confirmed by SDS-PAGE.     

A T7 promoter-specific, inducible protein expression system forBacillus subtilis

The adaptation and application of theEscherichia coli T7 RNA polymerase system for regulated and promoter-specific gene expression inBacillus subtilis is reported. The expression cassette used inBacillus subtilis was tightly regulated and T7 RNA polymerase (T7 RNAP) appeared 30 min after induction. The efficiency of T7 promoter-specific gene expression inB. subtilis was studied using one secretory and two cytosolic proteins of heterologous origin. The accumulation ofE. coli -galactosidase, as well as a 1,4--glucosidase fromThermoanaerobacter brockii inB. subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity. The-amylase ofThermoactinomyces vulgaris, a secretory protein, was found to accumulate in the culture supernatant up to levels of about 70 mg/l 10–20 h after T7 RNAP induction, but was also deposited in cellular fractions. The addition of rifampicin inhibited-amylase secretion, but unexpectedly, after a short period, also prevented its further (intra)cellular accumulation     

Propeptide of Bacillus subtilis amylase enhances extracellular production of human interferon-α in Bacillus subtilis

The Gram-positive bacterium, Bacillus subtilis and related species are widely used industrially as hosts for producing enzymes. These species possess a high potential to produce secreted proteins into the culture medium. Nevertheless, the secretion of heterologous proteins by these species is frequently inefficient. In this study, the human interferon-α2b (hIFN-α2b) was used as a heterologous model protein, to investigate the effect of B. subtilis AmyE propeptide in enhancing the secretion of heterologous proteins in B. subtilis. We found that the secretion production and activity of hIFN-α2b with AmyE propeptide increased by more than threefold, compared to that without AmyE propeptide. The maximum amount of secreted hIFN-α2b with propeptide was 14.8 ± 0.6 μg ml⁻¹. In addition, the pro-hIFN-α2b bioactivity reached 5.4 ± 0.5 × 10⁷ U mg⁻¹, which is roughly the same level as that of the non-propeptide hIFN-α2b. These results indicated that AmyE propeptide enhanced the secretion of the hIFN-α2b protein from B. subtilis. This study provides a useful method to enhance the extracellular production of heterologous proteins in B. subtilis.     

Secretory Expression of Nattokinase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> YF38 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Nattokinase producing bacterium, B. subtilis YF38, was isolated from douchi, using the fibrin plate method. The gene encoding this enzyme was cloned by polymerase chain reaction (PCR). Cytoplasmic expression of this enzyme in E. coli resulted in inactive inclusion bodies. But with the help of two different signal peptides, the native signal peptide of nattokinase and the signal peptide of PelB, active nattokinase was successfully expressed in E. coli with periplasmic secretion, and the nattokinase in culture medium displayed high fibrinolytic activity. The fibrinolytic activity of the expressed enzyme in the culture was determined to reach 260 urokinase units per micro-liter when the recombinant strain was induced by 0.7 mmol l⁻¹ isopropyl-β-D- thiogalactopyranoside (IPTG) at 20°C for 20 h, resulting 49.3 mg active enzyme per liter culture. The characteristic of this recombinant nattokinase is comparable to the native nattokinase from B. subtilis YF38. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases.     

Cloning and expression inEscherichia coli of the sucrase gene fromBacillus subtilis

Summary A recombinant cosmid carrying the sucrase gene (sacA) was obtained from a colony bank ofE. coli harboring recombinant cosmids representative of theB. subtilis genome. It was shown that thesacA gene is located in a 2 kbEcoRI fragment and that the cloned sequence is homologous to the corresponding chromosomal DNA fragment. A fragment of 2 kb containing the gene was subcloned in both orientations in the bifunctional vector pHV33 and expression was further looked for inB. subtilis andE. coli. Complementation of asacA mutation was observed in Rec⁺ and Rec^- strains ofB. subtilis. Expression of sucrase was also demonstrated inE.coli, which is normally devoid of this activity, by SDS-polyacrylamide gel electrophoresis, specific immunoprecipitation and assay of the enzyme in crude extracts. The specific activity of the enzyme depended on the orientation of the inserted fragment. The saccharolytic activity was found to be cryptic inE. coli since the presence of the recombinant plasmids did not allow the transport of [U¹⁴C] sucrose and the growth of the cells.It was shown also that the recombinant cosmid contained part of the neighboring locus (sacP) which corresponds to a component of the PEP-dependent phosphotransferase system of sucrose transport ofB. subtilis.     

Effect of aPMR1 disruption on the processing of heterologous glycoproteins secreted in the yeastSaccharomyces cerevisiae

TheSaccharomyces cerevisiae PMR1 gene encodes a Ca²⁺-ATPase localized in the Golgi. We have investigated the effects ofPMR1 disruption inS. cerevisiae on the glycosylation and secretion of three heterologous glycoproteins, human α₁-antitrypsin (α₁-AT), human antithrombin III (ATHIII), andAspergillus niger glucose oxidase (GOD). Thepmr1 null mutant strain secreted larger amounts of ATHIII and GOD proteins per a unit cell mass than the wild type strain. Despite a lower growth rate of thepmr1 mutant, two-fold higher level of human ATHIII was detected in the culture supernatant from thepmr1 mutant compared to that of the wild-type strain. Thepmr1 mutant strain secreted α₁-AT and the GOD proteins mostly as core-glycosylated forms, in contrast to the hyperglycosylated proteins secreted in the wild-type strain. Furthermore, the core-glycosylated forms secreted in thepmr1 mutant migrated slightly faster on SDS-PAGE than those secreted in themnn9 deletion mutant and the wild type strains. Analysis of the recombinant GOD with anti-α1,3-mannose antibody revealed that GOD secreted in thepmr1 mutant did not have terminal α1,3-linked mannoses unlike those secreted in themnn9 mutant and the wild type strains. The present results indicate that thepmr1 mutant, with the super-secretion phenotype, is useful as a host system to produce recombinant glycoproteins lacking high-mannose outer chains.     

Efficient constitutive expression of thermostable 4-α-glucanotransferase in Bacillus subtilis using dual promoters

4-α-Glucanotransferases possess strong transglycosylation activity which has been used in various carbohydrate chemistry fields. Due to safety issues of the recombinant enzymes we chose Bacillus subtilis as an expression host to produce a thermostable 4-α-glucanotransferase from Thermus scotoductus (TSαGT). The HpaII promoter in the Gram-positive bacterial vector pUB110 was used first to express TSαGT gene in B. subtilis. However, the activity of TSαGT in B. subtilis was only 4% of that in our previous Escherichia coli system. Two expression systems constructed by sequential alignment of another constitutive promoter for either α-amylase from B. subtilis NA64 or maltogenic amylase from Bacillus licheniformis downstream of the HpaII promoter elevated the TSαGT productivity by 11- and 12-fold, respectively, compared to the single HpaII promoter system. In conclusion, the dual promoter systems in this study were much better than the single promoter system to express the TSαGT gene in B. subtilis.     

Investigations into the ability of the peptide,HAL18, to interact with bacterial membranes

Halocidin was isolated from hemocytes, Halocynthia aurantium as a heterodimeric peptide consisting of two α-helical subunits, Hal15 and Hal18. Hal18 was shown to have antibacterial properties against Bacillus subtilis (MLC = 15 μM) and Escherichia coli (MLC = 100 μM). The peptide was shown to produce stable monolayers, which were characteristic of α-helical peptides predicted to orientate parallel to the surface of the interface. Constant area assays showed that Hal18 was surface active (4 μM) inducing surface pressure changes >30 mN m⁻¹ characteristic of membrane interactive peptides. The peptide induced stable surface pressure changes in monolayers that were mimetic of B. subtilis membranes (circa 7 mN m⁻¹) and E. coli membrane-mimics (circa 4 mN m⁻¹). Hal18 inserted readily into zwitterionic DOPE and anionic DOPG monolayers inducing surface pressure changes circa 8 mN m⁻¹ in both cases, providing evidence that interaction is not headgroup specific. Thermodynamic analysis of compression isotherms showed that the presence of Hal18 destabilised B. subtilis membranes (ΔG _Mix > 0), which is in contrast to its stabilising effect on E. coli lipid extract implying the differential antimicrobial efficacy may be driven by lipid packing.     

High level expression of a recombinant phospholipase C from <Emphasis Type="Italic">Bacillus cereus</Emphasis> in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Twenty-two Bacillus cereus strains were screened for phospholipase C (PLC, EC 3.1.4.3) activity using p-nitrophenyl phosphorylcholine as a substrate. Two strains (B. cereus SBUG 318 and SBUG 516) showed high activity at elevated temperatures (>70°C) at acidic pH (pH 3.5–6) and were selected for cloning and functional expression using Bacillus subtilis. The genes were amplified from B. cereus DNA using primers based on a known PLC sequence and cloned into the expression vector pMSE3 followed by transformation into B. subtilis WB800. On the amino acid level, one protein (PLC318) was identical to a PLC described from B. cereus, whereas PLC516 contained an amino acid substitution (E173D). PLC production using the recombinant strains was performed by an acetoin-controlled expression system. For PLC516, 13.7 U g⁻¹ wet cell weight was determined in the culture supernatant after 30 h cultivation time. Three purification steps resulted in pure PLC516 with a specific activity of 13,190 U mg⁻¹ protein.     

Comparison of the cell surface barrier and enzymatic modification system inBrevibacterium flavum andB. lactofermentum

To investigate impediments to plasmid transformation inBrevibacterium flavum BF4 andB. lactofermentum BL1, cell surface barriers were determined by measuring growth inhibition whilst enzymatic barriers were determined by comparing DNA methylation properties.B. lactofermentum was more sensitive to growth inhibition by glycine thanB. flavum. Release of cellular proteins during sonication was more rapid forB. lactofermentum than forB. flavum. Plasmid DNA (pCSL17) isolated fromB. flavum transformed recipient McrBC⁺ strains ofEscherichia coli with lower efficiency than McrBC⁻.McrBC digestion of this DNA confirmed thatB. flavum contain methylated cytidines in the target sequence ofMcrBC sequences butB. lactofermentum contained a different methylation pattern. DNA derived from theB. lactofermentum transformed recipient EcoKR⁺ strains ofE. coli with lower efficiency than EcoKR⁻, indicating the presence of methylated adenosines in the target sequence of EcoK sequences. The present data describe the differences in the physical and enzymatic barriers between two species of corynebacteria and also provide some insight into the successful foreign gene expression in corynebacteria.     

Modification of length, hydrophobic properties and electric charge of Bacillus subtilis alpha-amylase signal peptide and their different effects on the production of secretory proteins in B. subtilis and Escherichia coli cells

Summary Bacillus subtilis -amylase signal peptide, which consists of 33 amino acids, is functional in Escherichia coli cells.Lysine, glutamic acid, leucine, leucyl-leucine, or leucyl-leucyl-leucine was inserted between positions 28 and 29 of the -amylase signal peptide using site directed mutagenesis. DNAs encoding the wild-type and modified signal peptides were then fused in-frame to DNAs encoding the mature regions of the -lactamase of pBR322 and a thermostable -amylase. The secretion of -lactamase in E. coli cells was more inhibited by the modified signal peptides than that in B. subtilis cells, although the degree of inhibition varied and the inhibitory effect of each signal peptide was found to be similar in the two strains. In contrast, the difference in the inhibitory effect of each modified signal peptide was no longer detected in the case of the production of thermostable -amylase, except for the insertion of glutamic acid. Nearly 50% of thermostable -amylase in the precursor form was accumulated in the intracellular fraction of E. coli cells containing the DNAs for the modified signal peptides. The insertion of glutamic acid inhibited the secretion of the two enzymes in both B. subtilis and E. coli cells.     

Optimization of staphylokinase production inBacillus subtilis using inducible and constitutive promoters

Staphylokinase (SAK) was produced inB. subtillis using two different promoter systems,i.e. the P43 andsacB promoters. To maximize SAK expression inB. subtilis, fermentation control strategies for each promoter were examined. SAK, under P43, a vegetative promoter transcribed mainly by σ^B containing RNA polymerase, was overexpressed at low dissolved oxygen (D.O.) levels, suggesting that thesigB operon is somewhat affected by the energy charge of the cells. The expression of SAK at the 10% D.O. level was three times higher than that at the 50% D.O. level. In the case ofsacB, a sucrose-inducible promoter, sucrose feeding was used to control the induction period and induction strength. Since sucrose is hydrolyzed by two sucrose hydrolyzing enzymes in the cell and culture broth, the control strategy was based on replenishing the loss of sucrose in the culture. With continuous feeding of sucrose, WB700 (pSAKBQ), which contains the SAK gene undersacB promoter, yieldedca. 35% more SAK than the batch culture. These results present efficient promoter-dependent control strategies inB. subtilis host system for foreign protein expression.     

Characterization of an L-arabinose isomerase from Bacillus subtilis

An isolated gene from Bacillus subtilis str. 168 encoding a putative isomerase was proposed as an L-arabinose isomerase (L-AI), cloned into Escherichia coli, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,491 bp, capable of encoding a polypeptide of 496 amino acid residues. The gene was overexpressed in E. coli and the protein was purified using nickel-nitrilotriacetic acid chromatography. The purified enzyme showed the highest catalytic efficiency ever reported, with a k _cat of 14,504 min⁻¹ and a k _cat/K _m of 121 min⁻¹ mM⁻¹ for L-arabinose. A homology model of B. subtilis L-AI was constructed based on the X-ray crystal structure of E. coli L-AI. Molecular dynamics simulation studies of the enzyme with the natural substrate, L-arabinose, and an analogue, D-galactose, shed light on the unique substrate specificity displayed by B. subtilis L-AI only towards L-arabinose. Although L-AIs have been characterized from several other sources, B. subtilis L-AI is distinguished from other L-AIs by its high substrate specificity and catalytic efficiency for L-arabinose.     

Optimal Production of Poly-γ-glutamic Acid by Metabolically Engineered Escherichia coli

Metabolically-engineered Escherichia coli strains were developed by cloning poly-γ-glutamic acid (γ-PGA) biosynthesis genes, consisting of pgsB, pgsC and pgsA, from Bacillus subtilis The metabolic and regulatory pathways of γ-PGA biosynthesis in E. coli were analyzed by DNA microarray. The inducible trc promoter and a constitutive promoter (P_HCE) derived from the d-amino acid aminotransferase (D-AAT) gene of Geobacillus toebii were employed. The constitutive HCE promoter was more efficient than inducible trc promoter for the expression of γ-PGA biosynthesis genes. DNA microarray analysis showed that the expression levels of several NtrC family genes, glnA, glnK, glnG, yhdX, yhdY, yhdZ, amtB, nac, argT and cbl were up-regulated and sucA, B, C, D genes were down-regulated. When (NH₄)₂SO₄ was added at 40 g/l into the feeding solution, the final γ-PGA concentration reached 3.7 g/l in the fed-batch culture of recombinant E. coli/pCOpgs.     

An efficient transformation method for <Emphasis Type="Italic">Bacillus subtilis</Emphasis> DB104

Bacillus subtilis strains are used for extracellular expression of enzymes (i.e., proteases, lipases, and cellulases) which are often engineered by directed evolution for industrial applications. B. subtilis DB104 represents an attractive directed evolution host since it has a low proteolytic activity and efficient secretion. B. subtilis DB104 is hampered like many other Bacillus strains by insufficient transformation efficiencies (≤10³ transformants/μg DNA). After investigating five physical and chemical transformation protocols, a novel natural competent transformation protocol was established for B. subtilis DB104 by optimizing growth conditions and histidine concentration during competence development, implementing an additional incubation step in the competence development phase and a recovery step during the transformation procedure. In addition, the influence of the amount and size of the transformed plasmid DNA on transformation efficiency was investigated. The natural competence protocol is “easy” in handling and allows for the first time to generate large libraries (1.5 × 10⁵ transformants/μg plasmid DNA) in B. subtilis DB104 without requiring microgram amounts of DNA.     

Desulfurization of dibenzothiophene by Bacillus subtilis recombinants carrying dszABC and dszD genes

The desulfurization (dsz) genes from Rhodococcus erythropolis DS-3 were successfully integrated into the chromosomes of Bacillus subtilis ATCC 21332 and UV1 using an integration vector pDGSDN, yielding two recombinant strains, B. subtilis M29 and M28 in which the integrated dsz genes were expressed efficiently under the promoter, Pspac. The dibenzothiophene (DBT) desulfurization efficiency of M29 was 16.2 mg DBT l⁻¹ h⁻¹ at 36 h, significantly higher than that of R. erythropolis DS−3 and B. subtilis M28 and also showed no product inhibition. The interfacial tension of the supernatant fermented by M29 varied from 48 mN m⁻¹ to 4.2 mN m⁻¹, lower than that of the recombinant strain, M28, reveals that the biosurfactant secreted from M29 may have an important function in the DBT desulfurization process.     

Comparison of microbial hosts and expression systems for mammalian CYP1A1 catalysis

Mammalian cytochrome P450 enzymes are of special interest as biocatalysts for fine chemical and drug metabolite synthesis. In this study, the potential of different recombinant microorganisms expressing rat and human cyp1a1 genes is evaluated for such applications. The maximum specific activity for 7-ethoxyresorufin O-deethylation and gene expression levels were used as parameters to judge biocatalyst performance. Under comparable conditions, E. coli is shown to be superior over the use of S. cerevisiae and P. putida as hosts for biocatalysis. Of all tested E. coli strains, E. coli DH5α and E. coli JM101 harboring rat CYP1A1 showed the highest activities (0.43 and 0.42 U g_CDW⁻¹, respectively). Detection of active CYP1A1 in cell-free E. coli extracts was found to be difficult and only for E. coli DH5α, expression levels could be determined (41 nmol g_CDW⁻¹). The presented results show that efficient expression of mammalian cyp1a1 genes in recombinant microorganisms is troublesome and host-dependent and that enhancing expression levels is crucial in order to obtain more efficient biocatalysts. Specific activities currently obtained are not sufficient yet for fine chemical production, but are sufficient for preparative-scale drug metabolite synthesis.     

Alkaloid profile and antimicrobial activity of <Emphasis Type="Italic">Lupinus angustifolius</Emphasis> L. alkaloid extract

Purpose of the present study was to evaluate alkaloid profile of the aerial parts of Lupinus angustifolius growing in Turkey by capillary gas chromatography-mass spectrometry (GC-MS). Fifteen alkaloids were identified by capillary GC-MS. 13α-Hydroxylupanine (50.78%) and lupanine (23.55%) were determined as the main alkaloids in the aerial parts of L. angustifolius. Ammodendrine, isoangustifoline, tetrahydrorhombifoline, angustifoline, α-isolupanine, 5,6-dehydrolupanine, 11,12-dehydrolupanine, 13α-acetoxylupanine, 13α-isovaleroyloxylupanine, 13α-valeroyloxylupanine, 13α-tigloyloxylupanine, 13α-cis-cinnamoyloxylupanine and 13α-cis-cinnamoyloxy-17-oxolupanine were identified as the minor alkaloids of the plant. Furthermore, antibacterial and antifungal activities of L. angustifolius alkaloid extract were tested against standard strains of the following bacteria; Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus as well as the fungi; Candida albicans and C. krusei. The alkaloid extract showed significant activity on B. subtilis, S. aureus and P. aeruginosa while it was weakly active on E. coli. On the other hand, the extract possessed moderate activity against C. albicans and C. krusei.