High-titer production of staurosporine by heterologous expression and process optimization.

Staurosporine is the most well-known member of the indolocarbazole alkaloid family; it can induce apoptosis of many types of cells as a strong protein kinase inhibitor, and is used as an important lead compound for the synthesis of the antitumor drugs. However, the low fermentation level of the native producer remains the bottleneck of staurosporine production. Herein, integration of multi-copy biosynthetic gene cluster (BGC) in well characterized heterologous host and optimization of the fermentation process were performed to enable high-level production of staurosporine. First, the 22.5 kb staurosporine BGC was captured by CRISPR/Cas9-mediated TAR (transformation-associated recombination) from the native producer (145 mg/L), and then introduced into three heterologous hosts Streptomyces avermitilis (ATCC 31267), Streptomyces lividans TK24 and Streptomyces albus J1074 to evaluate the staurosporine production capacity. The highest yield was achieved in S. albus J1074 (750 mg/L), which was used for further production improvement. Next, we integrated two additional staurosporine BGCs into the chromosome of strain S-STA via two different attB sites (vwb and TG1), leading to a double increase in the production of staurosporine. And finally, optimization of fermentation process by controlling the pH and glucose feeding could improve the yield of staurosporine to 4568 mg/L, which was approximately 30-fold higher than that of the native producer. This is the highest yield ever reported, paving the way for the industrial production of staurosporine. KEYPOINTS: • Streptomyces albus J1074 was the most suitable heterologous host to express the biosynthetic gene cluster of staurosporine. • Amplification of the biosynthetic gene cluster had obvious effect on improving the production of staurosporine. • The highest yield of staurosporine was achieved to 4568 mg/L by stepwise increase strategy.

Genome-Directed Discovery of Bicyclic Cinnamoyl-Containing Nonribosomal Peptides with Anticoronaviral Activity from Streptomyces griseus.

Two novel cinnamoyl-containing nonribosomal peptides (CCNPs) grisgenomycin A and B were identified in Streptomyces griseus NBRC 13350 (CGMCC 4.5718) and ATCC 12475, through genome mining using conserved adjacent LuxR family regulators as probes and activators. Notably, grisgenomycins represent a new group of bicyclic decapeptides featuring an unprecedented C-C bond between the tryptophan carbocycle and the cinnamoyl group. A plausible biosynthetic pathway for grisgenomycins was deduced by a bioinformatics analysis. Grisgenomycins exhibited activity against human coronaviruses at the micromolar level.

Discovery of novel septacidin congeners from a high yield heterologous expression strain Streptomyces albus 1597.

Septacidin is an adenine nucleoside antibiotic with antifungal and antitumor activities. During the efforts to construct a better septacidin producer, we obtained a high yield strain S. albus 1597 by putting the biosynthetic gene cluster (BGC) of septacidin under the control of the constitutive strong promoter ermE*. S. albus 1597 could produce new septacidin congeners SEP-538 and SEP-552 with shorter fatty acyl chains. Moreover, SEP-624 with an unprecedented hydroxylated fatty acyl chain was also isolated from this titre improved strain, enriching the diversity of septacidins. SEP-552 showed moderate inhibitory effects against Epidermophyton floccosum 57312 with MIC value 62.5 µM, while SEP-538 and SEP-624 only exhibited weak antifungal activities. The structure-activity relationship investigation revealed that the antifungal activity of septacidins is significantly influenced by the length of and the decoration on their fatty acyl chains.

Streptomyces griseus KJ623766: A Natural Producer of Two Anthracycline Cytotoxic Metabolites ß- and γ-Rhodomycinone.

BACKGROUND: This study aimed to produce, purify, structurally elucidate, and explore the biological activities of metabolites produced by Streptomyces (S.) griseus isolate KJ623766, a recovered soil bacterium previously screened in our lab that showed promising cytotoxic activities against various cancer cell lines. METHODS: Production of cytotoxic metabolites from S. griseus isolate KJ623766 was carried out in a 14L laboratory fermenter under specified optimum conditions. Using a 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium-bromide assay, the cytotoxic activity of the ethyl acetate extract against Caco2 and Hela cancer cell lines was determined. Bioassay-guided fractionation of the ethyl acetate extract using different chromatographic techniques was used for cytotoxic metabolite purification. Chemical structures of the purified metabolites were identified using mass, 1D, and 2D NMR spectroscopic analysis. RESULTS: Bioassay-guided fractionation of the ethyl acetate extract led to the purification of two cytotoxic metabolites, R1 and R2, of reproducible amounts of 5 and 1.5 mg/L, respectively. The structures of R1 and R2 metabolites were identified as ß- and γ-rhodomycinone with CD50 of 6.3, 9.45, 64.8 and 9.11, 9.35, 67.3 µg/mL against Caco2, Hela and Vero cell lines, respectively. Values were comparable to those of the positive control doxorubicin. CONCLUSIONS: This is the first report about the production of ß- and γ-rhodomycinone, two important scaffolds for synthesis of anticancer drugs, from S. griseus.

Effective refolding of a cysteine rich glycoside hydrolase family 19 recombinant chitinase from Streptomyces griseus by reverse dilution and affinity chromatography.

Conventional refolding methods are associated with low yields due to misfolding and high aggregation rates or very dilute proteins. In this study, we describe the optimization of the conventional methods of reverse dilution and affinity chromatography for obtaining high yields of a cysteine rich recombinant glycoside hydrolase family 19 chitinase from Streptomyces griseus HUT6037 (SgChiC). SgChiC is a potential biocontrol agent and a reference enzyme in the study and development of chitinases for various applications. The overexpression of SgChiC was previously achieved by periplasmic localization from where it was extracted by osmotic shock and then purified by hydroxyapatite column chromatography. In the present study, the successful refolding and recovery of recombinant SgChiC (r-SgChiC) from inclusion bodies (IB) by reverse dilution and column chromatography methods is respectively described. Approximately 8 mg of r-SgChiC was obtained from each method with specific activities of 28 and 52 U/mg respectively. These yields are comparable to that obtained from a 1 L culture volume of the same protein isolated from the periplasmic space of E. coli BL21 (DE3) as described in previous studies. The higher yields obtained are attributed to the successful suppression of aggregation by a stepwise reduction of denaturant from high, to intermediate, and finally to low concentrations. These methods are straight forward, requiring the use of fewer refolding agents compared with previously described refolding methods. They can be applied to the refolding of other cysteine rich proteins expressed as inclusion bodies to obtain high yields of actively folded proteins. This is the first report on the recovery of actively folded SgChiC from inclusion bodies.

Biotransformation of Erythrodiol for New Food Supplements with Anti-Inflammatory Properties.

Erythrodiol, a typical pentacyclic triterpenic diol in olive oil and its byproduct, olive pomace, frequently appears in food additives for the prevention of cardiovascular diseases because of its antioxidation, anti-inflammatory, and antitumor activities. To develop new derivatives of erythrodiol (1), preparative biotransformations were investigated through Streptomyces griseus ATCC 13273, Penicilium griseofulvum CICC 40293, and Bacillus subtilis ATCC 6633, and ten new (1a-1j) and one known metabolites were isolated. Their structures were elucidated by high resolution electrospray ionization mass spectrometry (HR-ESI-MS) and one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy. Furthermore, relative to 1, most metabolites exhibited lower toxicity and more potent inhibitory activities against nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. In particular, the glycosylated metabolite 1k showed a dramatically increased inhibitory effect with an IC50 value of 2.40 µM, which is even lower than that of quercetin. Thus, biotransformation of erythrodiol is a viable strategy for discovering new triterpenes as food supplements with anti-inflammatory properties.

SrnR from Streptomyces griseus is a nickel-binding transcriptional activator.

Nickel ions are crucial components for the catalysis of biological reactions in prokaryotic organisms. As an uncontrolled nickel trafficking is toxic for living organisms, nickel-dependent bacteria have developed tightly regulated strategies to maintain the correct intracellular metal ion quota. These mechanisms require transcriptional regulator proteins that respond to nickel concentration, activating or repressing the expression of specific proteins related to Ni(II) metabolism. In Streptomyces griseus, a Gram-positive bacterium used for antibiotic production, SgSrnR and SgSrnQ regulate the nickel-dependent antagonistic expression of two superoxide dismutase (SOD) enzymes, a Ni-SOD and a FeZn-SOD. According to a previously proposed model, SgSrnR and SgSrnQ form a protein complex in which SgSrnR works as repressor, binding directly to the promoter of the gene coding for FeZn-SOD, while SgSrnQ is the Ni(II)-dependent co-repressor. The present work focuses on the determination of the biophysical and functional properties of SgSrnR. The protein was heterologously expressed and purified from Escherichia coli. The structural and metal-binding analysis, carried out by circular dichroism, light scattering, fluorescence and isothermal titration calorimetry, showed that the protein is a well-structured homodimer, able to bind nickel with moderate affinity. DNase I footprinting and ß-galactosidase gene reporter assays revealed that apo-SgSrnR is able to bind its DNA operator and activates a transcriptional response. The structural and functional properties of this protein are discussed relatively to its role as a Ni(II)-dependent sensor.

Cloning and identification of the Frigocyclinone biosynthetic gene cluster from Streptomyces griseus strain NTK 97.

Frigocyclinone is a novel antibiotic with antibacterial and anticancer activities. It is produced by both Antarctica-derived Streptomyces griseus NTK 97 and marine sponge-associated Streptomyces sp. M7_15. Here, we first report the biosynthetic gene cluster of frigocyclinone in the S. griseus NTK 97. The frigocyclinone gene cluster spans a DNA region of 33-kb which consists of 30 open reading frames (ORFs), encoding minimal type II polyketide synthase, aromatase and cyclase, redox tailoring enzymes, sugar biosynthesis-related enzymes, C-glycosyltransferase, a resistance protein, and three regulatory proteins. Based on the bioinformatic analysis, a biosynthetic pathway for frigocyclinone was proposed. Second, to verify the cloned gene cluster, CRISPR-Cpf1 mediated gene disruption was conducted. Mutant with the disruption of beta-ketoacyl synthase encoding gene frig20 fully loses the ability of producing frigocyclinone, while inactivating the glycosyltransferase gene frig1 leads to the production of key intermediate of anti-MRSA anthraquinone tetrangomycin.

Regio- and enantioselective O-demethylation of tetrahydroprotoberberines by cytochrome P450 enzyme system from Streptomyces griseus ATCC 13273.

Tetrahydroprotoberberines (THPBs), a class of naturally occurring isoquinoline alkaloids, contain substituent methoxyl or hydroxyl groups which play a significant role in the pharmacological properties of these molecules. In this study, we report a biocatalytic strategy for selective O-demethylation of THPBs. CYP105D1, a cytochrome P450 from Streptomyces griseus ATCC 13273, exhibited markedly regioselective demethylation of nonhydroxyl-THPBs and monohydroxyl-THPBs on the D-ring. A possible binding mode of THPBs with CYP105D1 was investigated by docking analysis, and the results revealed that the D-rings of THPBs were with the minimum distance to the heme iron. Tetrahydropalmatine was used as a model substrate and enantioselective demethylation was demonstrated. (S)-Tetrahydropalmatine was only demethylated at C-10, while (R)-tetrahydropalmatine was first demethylated at C-10 and then subsequently demethylated at C-9. The kcat/Km value for demethylation of (R)-tetrahydropalmatine by CYP105D1 was 3.7 times greater than that for demethylation of (S)-tetrahydropalmatine. Furthermore, selective demethylation of (S)-tetrahydropalmatine by the CYP105D1-based whole-cell system was demonstrated for the highly efficient production of (S)-corydalmine which has distinct pharmacological applications, such as providing relief from bone cancer pain and reducing morphine tolerance. Moreover, a homologous redox partner was identified to enhance the catalytic efficiency of the CYP105D1-based whole-cell system. This is the first enzymatic characterization of a cytochrome P450 that has regio- and enantioselective demethylation activity of THPBs for application purpose. The cytochrome P450 system could be a promising strategy for selective demethylation in the pharmaceutical industry.

Dolyemycins A and B, two novel cyclopeptides isolated from Streptomyces griseus subsp. griseus HYS31.

Two novel cyclopeptides with special skeleton, namely, dolyemycins A (1) and B (2) were isolated from Streptomyces griseus subsp. griseus HYS31 by bio-guided isolation. Their structures were elucidated by detailed analysis of spectroscopic data. These two compounds were cyclopeptides containing eleven amino acids including five unusual amino acids (hydroxyglycine, 3-hydroxyleucine, 3-phenylserine, ß-hydroxy-O-methyltyrosine, 2,3-diaminobutyric acid) in both of them and an extra nonprotein amino acids (3-methylaspartic acid) in Dolyemycin B only. Dolyemycins A and B performed antiproliferative activity against human lung cancer A549 cells with IC50 values of 1.0 and 1.2 µM, respectively.

Protection of protein from ruminal degradation by alkali-induced oxidation of chlorogenic acid in sunflower meal.

Lactating ruminants require an adequate supply of absorbable amino acids for the synthesis of milk protein from two sources, that is crude protein (CP) synthesized microbially in the rumen and ruminally undegraded CP (RUP) from feed which can both be digested in the small intestine. Several chemical and physical methods have been identified as being effective in increasing the proportion of RUP of total CP of a feedstuff, yet there is a continuing need for developing and establishing methods which protect feed protein from ruminal degradation with acceptable expenditure of labour and other costs. The objective of this study was to identify and quantify effects of and interactions between chlorogenic acid and protein in solvent-extracted sunflower meal (SFM) as induced by alkali treatment. Response surface methodology was employed to investigate the influence of pH, reaction time and drying temperature on the resulting SFM and, subsequently, its protein value for ruminants estimated from laboratory values. For this purpose, alkali-treated SFM was subjected to a fractionation of feed CP according to the Cornell net carbohydrate and protein system as a basis for estimating RUP at different assumed ruminal passage rates (Kp ). To estimate the intestinal digestibility of the treated SFM and its RUP, a three-step enzymatic in vitro procedure was applied. Alkaline treatment of SFM increased RUP values with factors ranging from approximately 3 (Kp =.08/hr) to 12 (Kp =.02/hr). Furthermore, the intestinal digestibility of the alkali-treated SFM was enhanced by approximately 10% compared to untreated SFM. Increasing pH and reaction time led to both increasing RUP values and intestinal digestibility. In conclusion, a targeted alkaline treatment of naturally occurring compounds in feedstuffs might be a promising approach to provide high-RUP feeds for ruminants which, at the same time, have improved intestinal digestibility values.

High-yield secretory production of stable, active trypsin through engineering of the N-terminal peptide and self-degradation sites in Pichia pastoris.

Streptomyces griseus trypsin (SGT) possesses enzymatic properties similar to mammalian trypsins and has great potential applications in the leather processing, bioethanol, detergent and pharmaceutical industry. Here, a new strategy was reported for improving its stable, active secretory production through engineering of its propeptide and self-degradation sites. By rationally introducing hydrophobic mutations into the N-terminus of SGT Exmt (R145I), replacing the propeptide with FPVDDDDK and engineering the α-factor signal peptide, trypsin production (amidase activity) was improved to 177.85±2.83U·mL-1 in a 3-L fermenter (a 3.75-fold increase). Subsequently, all of the residues involved in autolysis that were identified by mass spectrometry were mutated and the resulting proteins were evaluated. In particular, the variant tbcf (K101A) demonstrated high stability and production (227.65±6.51U·mL-1 and 185.71±5.68mg·L-1, respectively). The recombinant strain constructed here has great potential for large-scale production of active trypsin.

Bioresolution of racemic phenyl glycidyl ether by a putative recombinant epoxide hydrolase from Streptomyces griseus NBRC 13350.

In order to produce enantiomerically pure epoxides for the synthesis of value-added chemicals, a novel putative epoxide hydrolase (EH) sgeh was cloned and overexpressed in pET28a/Escherichia coli BL21(DE3). The 1047 bp sgeh gene was mined from Streptomyces griseus NBRC 13350 genome sequence. The recombinant hexahistidyl-tagged SGEH was purified (16.6-fold) by immobilized metal-affinity chromatography, with 90% yield as a homodimer of 100 kDa. The recombinant E. coli whole cells overexpressing SGEH could kinetically resolve racemic phenyl glycidyl ether (PGE) into (R)-PGE with 98% ee, 40% yield, and enantiomeric ratio (E) of 20. This was achieved under the optimized reaction conditions i.e. cell/substrate ratio of 20:1 (w/w) at pH 7.5 and 20 °C in 10% (v/v) dimethylformamide (DMF) in a 10 h reaction. 99% enantiopure (R)-PGE was obtained when the reaction time was prolonged to 12 h with a yield of 34%. In conclusion, an economically viable and environment friendly green process for the production of enantiopure (R)-PGE was developed by using wet cells of E. coli expressing recombinant SGEH.

Efficient production of nonactin by Streptomyces griseus subsp. griseus.

Here we report the production of the cyclic macrotetrolide nonactin from the fermentation culture of Streptomyces griseus subsp. griseus. Nonactin is a member of a family of naturally occurring cyclic ionophores known as the macrotetrolide antibiotics. Our fermentation procedure of Streptomyces griseus was performed at 30 °C and 200 rev·min(-1) for 5 days on a rotary shaker. Diaion HP-20 and Amberlite XAD-16 were added to the fermentation medium. Isolated yield of nonactin was up to 80 mg·L(-1) using our methodology. Nonactin is commonly known as an ammonium ionophore and also exhibits antibacterial, antiviral, and antitumor activities. It is also widely used for the preparation of ion-selective electrodes and sensors. Chemical synthesis of nonactin has been achieved by some groups; however, overall yields are very low, making efficient biosynthesis an attractive means of production.

TmcN is involved in ATP regulation of tautomycetin biosynthesis in Streptomyces griseochromogenes.

The regulatory mechanism of tautomycetin (TMC) biosynthesis remains largely unknown, although it has been of great interest to the pharmaceutical industry. Our previous study showed that intracellular adenosine triphosphate (inATP) level is negatively correlated with secondary metabolite biosynthesis in various Streptomyces spp. In this study, by exogenous treatment of ATP, we also found a negative correlation between TMC biosynthesis and inATP level in Streptomyces griseochromogenes (S. griseochromogenes). However, the underlying mechanism remains unclear. TmcN, a pathway-specific transcriptional regulator of TMC biosynthetic genes, was previously revealed as a large ATP-binding LuxR (LAL) family protein. The predicted amino acid sequence of TmcN shows highly conserved Walker A and B binding motifs, which suggest an ATPase function of TmcN. We therefore hypothesized that the ATPase domain of TmcN may play a role in sensing endogenous pool of ATP, and is thus involved in the ATP regulation of TMC biosynthesis. To test the hypothesis, we first explored the key residue that affects the ATPase activity of TmcN by amino acid sequence alignment and structural simulation. After that, we disrupted tmcN gene in S. griseochromogenes, and the tmcN or site-direct-mutated tmcN were re-introduced to get the complementary and ATPase domain disrupted strains. The transcription level of tmcN, TMC yield, and inATP, as well as the effect of ATP on TMC production of different mutants were evaluated. Deletion of tmcN or site-direct mutation of ATPase domain of TmcN in S. griseochromogenes significantly reduced the TMC production, and it was not affected by exogenous ATP treatment. In addition, a relatively high level of inATP was detected in tmcN deletion and site-direct mutation strains. Our results here suggested that TmcN, especially its ATPase domain, is involved in consuming of endogenous ATP pool and thus plays pivotal role in connecting the primary and secondary metabolite in S. griseochromogenes.

Marine-Derived Secondary Metabolite, Griseusrazin A, Suppresses Inflammation through Heme Oxygenase-1 Induction in Activated RAW264.7 Macrophages.

A new secondary metabolite, named griseusrazin A (1), was isolated from the marine-derived bacterium Streptomyces griseus subsp. griseus. The structure of the compound was determined by analysis of spectroscopic data including MS, COSY, HSQC, HMBC, and (15)N-HMBC data. Griseusrazin A (1) inhibited the production of inflammatory mediators, such as prostaglandin E2 and nitric oxide, which was mediated through the suppression of the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The production of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α, in the LPS-stimulated cells was also effectively blocked by griseusrazin A (1). Furthermore, this anti-inflammatory activity of 1 was linked to its inhibitory effects against the nuclear translocation of NF-κB p50 and p65, as wells as NF-κB binding activity. In the further study to elucidate the anti-inflammatory mechanism, 1 was shown to induce heme oxygenase-1 (HO-1) expression through the enhancement of nuclear translocation of nuclear factor E2-related factor 2. Furthermore, the anti-inflammatory activity of 1 in the LPS-stimulated cells was partially reversed by an HO inhibitor, tin protoporphyrin. These results indicate that the anti-inflammatory effect of 1 is associated with Nrf2-mediated HO-1 expression.

Grisemycin, a Bridged Angucyclinone with a Methylsulfinyl Moiety from a Marine-Derived Streptomyces sp.

Grisemycin (1), the first sulfur angucyclinone with an unusual ether-bridged system, was isolated from a marine-derived Streptomyces griseus strain M268. Its novel, here cage-like, structure was determined by spectroscopic analysis and single-crystal X-ray diffraction. Compound 1 exhibited modestly selective activity against the HL-60 cell line with an IC50 value of 31.54 µM. Futhermore, the absolute stereochemistry of kiamycin (2), an 1,12-epoxybenz[a]anthracene, previously obtained from the same strain, was established by X-ray diffraction analysis.

Improved Production of Active Streptomyces griseus Trypsin with a Novel Auto-Catalyzed Strategy.

N-terminal sequences play crucial roles in regulating expression, translation, activation and enzymatic properties of proteins. To reduce cell toxicity of intracellular trypsin and increase secretory expression, we developed a novel auto-catalyzed strategy to produce recombinant trypsin by engineering the N-terminus of mature Streptomyces griseus trypsin (SGT). The engineered N-terminal peptide of SGT was composed of the thioredoxin, glycine-serine linker, His6-tag and the partial bovine trypsinogen pro-peptide (DDDDK). Furthermore, we constructed a variant TLEI with insertion of the artificial peptide at N-terminus and site-directed mutagenesis of the autolysis residue R145. In fed-batch fermentation, the production of extracellular trypsin activity was significantly improved to 47.4 ± 1.2 U·ml(-1) (amidase activity, 8532 ± 142.2 U·ml(-1) BAEE activity) with a productivity of 0.49 U·ml(-1)·h(-1), which was 329% greater than that of parent strain Pichia pastoris GS115-SGT. This work has significant potential to be scaled-up for microbial production of SGT. In addition, the N-terminal peptide engineering strategy can be extended to improve heterologous expression of other toxic enzymes.

Iminimycin A, the new iminium metabolite produced by Streptomyces griseus OS-3601.

A new natural product, designated iminimycin A, was isolated from the cultured broth of a streptomycin-producing microbial strain, Streptomyces griseus OS-3601, via a physicochemical screening method using HP-20, silica gel and ODS column chromatographies and subsequent preparative HPLC. Iminimycin A is an indolizidine alkaloid, containing of an unusual iminium group and a cyclopropane ring with a triene side chain. The absolute configuration of iminimycin A was elucidated by NMR studies and electronic circular dichroism analysis. Iminimycin A shows anti-bacterial activity against Bacillus subtilis, Kocuria rhizophila and Xanthomonas campestris pv. orizae, and cytotoxic activity against HeLa S3 and Jurkat cells with IC50 values of 43 and 36 µM, respectively.