Youming Zhang

Improving the insecticidal activity by expression of a recombinant cry1Ac gene with chitinase-encoding gene in acrystalliferous Bacillus thuringiensis.

In order to improve the insecticidal activity, the chitinase gene from tobacco (Nicotiana tabacum) endochitinase and the cry1Ac gene from Bacillus thuringiensis were cloned into the vector pHT315 and designated as pHUAccB5 plasmid. The constructed transcriptional fusion was attempted under the control of the native cry1Ac promoter. Plasmid pHUAccB5 was introduced into B. thuringiensis acrystalliferous by electroporation. Analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot, the transformant XBU-HUAccB5 produced 130–kDa Cry1Ac protein and 30-kDa chitinase protein. During the chitinase active analysis, the transformant, XBU-HUAccB5 chitinase active, reached 7.5 U/mL at 72 h, and was 5 times higher than the HTX-42 and 6 times higher than the parent strains. When the insecticidal activity of the transformant was evaluated against Helicoverpa armigera Hubner, the XBU-HUAccB5 toxicity was 11.30 times higher than the transformant HTX-42 expressed single cry1Ac at 48 h and was 18.76 times higher at 72 h.

Recent advances in the biochemistry of spinosyns.

Spinosyn and its analogs, produced by Saccharopolyspora spinosa, are the active ingredients in a family of insect control agents. They are macrolides with a 21-carbon, 12-membered tetracyclic lactones that are attached to two deoxysugars, tri-O-methylrhamnose and forosamine. Labeling studies, analysis of the biosynthetically blocked mutants, and the genetic identification of the spinosyn gene cluster have provided detailed information concerning the mechanism of spinosyn biosynthesis and have enabled combinatorial biosynthesis of a large group of new spinosyns. The following developments have recently impacted the field of spinosyn biology: (1) A second-generation spinosyn called spinetoram (XDE-175) was launched in late 2007; it is a semisynthesized spinosyn derivative produced through the modification of 3′-O-methyl group of rhamnose and the double bond between C5 and C6 of spinosyn J and L. This molecule was shown to have improved insecticidal activity, enhanced duration of control, and an expanded pest spectrum. (2) A new class of spinosyns, the butenyl-spinosyns, was discovered from Saccharopolyspora pogona. The butenyl-spinosyns are similar to spinosyns, but differ in the length of the side chain at C-21. In addition to structural similarities with the spinosyns, the butenyl-spinosyns exhibit a high level of similarity in insecticidal activity to spinetoram. (3) Spinosyn analogs, 21-cyclobutyl-spinosyn A and 21-cyclobutyl-spinosyn D were generated by metabolic engineering of the spinosyn biosynthetic gene cluster. They showed better insecticidal activities against cotton aphid and tobacco budworm than that of spinosyn A and D. Future progress toward the development of more potent spinosad analogs, as well as enhancements in production yields will likely result from these recent advances in the genetics and biochemistry of spinosyns.

Escherichia coli Nissle 1917 Targets and Restrains Mouse B16 Melanoma and 4T1 Breast Tumors through Expression of Azurin Protein

ABSTRACT Many studies have demonstrated that intravenously administered bacteria can target and proliferate in solid tumors and then quickly be released from other organs. Here, we employed the tumor-targeting property of Escherichia coli Nissle 1917 to inhibit mouse B16 melanoma and 4T1 breast tumors through the expression of azurin protein. For this purpose, recombinant azurin-expressing E. coli Nissle 1917 was developed. The levels of in vitro and in vivo azurin secretion in the engineered bacterium were determined by immunochemistry. Our results demonstrated that B16 melanoma and orthotopic 4T1 breast tumor growth were remarkably restrained and pulmonary metastasis was prevented in immunocompetent mice. It is worth noting that this therapeutic effect partially resulted from the antitumor activity of neutrophils and lymphocytes due to inflammatory responses caused by bacterial infections. No toxicity was observed in the animal during the experiments. This study indicates that E. coli Nissle 1917 could be a potential carrier to deliver antitumor drugs effectively for cancer therapy.

Increase in Insecticidal Toxicity by Fusion of the cry1Ac Gene from Bacillus thuringiensis with the Neurotoxin Gene hwtx-I

A fusion gene was constructed by combining the cry1Ac gene of Bacillus thuringiensis strain 4.0718 with a neurotoxin gene, hwtx-1, which was synthesized chemically. In this process, an enterokinase recognition site sequence was inserted in frame between two genes, and the fusion gene, including the promoter and the terminator of the cry1Ac gene, was cloned into the shuttle vector pHT304 to obtain a new expression vector, pXL43. A 138-kDa fusion protein was mass-expressed in the recombinant strain XL002, which was generated by transforming pXL43 into B. thuringiensis acrystalliferous strain XBU001. Quantitative analysis indicated that the expressed protein accounted for 61.38% of total cellular proteins. Under atomic force microscopy, there were some bipyramidal crystals with a size of 1.0xa0×xa02.0xa0μm. Bioassay showed that the fusion crystals from recombinant strain XL002 had a higher toxicity than the original Cry1Ac crystal protein against third-instar larvae of Plutella xylostella, with an LC50 (after 48xa0h) value of 5.12xa0μg/mL. The study will enhance the toxicity of B. thuringiensis Cry toxins and set the groundwork for constructing fusion genes of the B. thuringiensiscry gene and other foreign toxin genes and recombinant strains with high toxicity.

A Cry1Ac toxin variant generated by directed evolution has enhanced toxicity against Lepidopteran insects.

Cry1Ac insecticidal crystal proteins produced by Bacillus thuringiensis (Bt) have become an important natural biological agent for the control of lepidopteran insects. In this study, a cry1Ac toxin gene from Bacillus thuringiensis 4.0718 was modified by using error-prone PCR, staggered extension process (StEP) shuffling combined with Red/ET homologous recombination to investigate the insecticidal activity of delta-endotoxin Cry1Ac. A Cry1Ac toxin variant (designated as T524N) screened by insect bioassay showed increased insecticidal activity against Spodoptera exigua larvae while its original insecticidal activity against Helicoverpa armigera larvae was still retained. The mutant toxin T524N had one amino acid substitution at position 524 relative to the original Cry1Ac toxin, and it can accumulate within the acrystalliferous strain Cry-B and form more but a little smaller bipyramidal crystals than the original Cry1Ac toxin. Analysis of theoretical molecular models of mutant and original Cry1Ac proteins indicated that the mutation T524N located in the loop linking β16–β17 of domain III in Cry1Ac toxin happens in the fourth conserved block which is an arginine-rich region to form a highly hydrophobic surface involving interaction with receptor molecules. This study showed for the first time that single mutation T524N played an essential role in the insecticidal activity. This finding provides the biological evidence of the structural function of domain III in insecticidal activity of the Cry1Ac toxin, which probably leads to a deep understanding between the interaction of toxic proteins and receptor macromolecules.

Assessment of protoxin composition of Bacillus thuringiensis strains by use of polyacrylamide gel block and mass spectrometry

Assessment of protoxin composition in Bacillus thuringiensis parasporal crystals is principally hampered by the fact that protoxins in a single strain usually possess high sequence homology. Therefore, new strategies towards the identification of protoxins have been developed. Here, we established a powerful method through embedding solubilized protoxins in a polyacrylamide gel block coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of in-gel-generated peptides for protoxin identification. Our model study revealed that four protoxins (Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa) and six protoxins (Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, Cyt1Aa, and Cyt2Ba) could be rapidly identified from B. thuringiensis subsp. kurstaki HD1 and subsp. israelensis 4Q2-72, respectively. The experimental results indicated that our method is a straightforward tool for analyzing protoxin expression profile in B. thuringiensis strains. Given its technical simplicity and sensitivity, our method might facilitate the present screening program for B. thuringiensis strains with new insecticidal properties.

Improved Insecticidal Toxicity by Fusing Cry1Ac of Bacillus thuringiensis with Av3 of Anemonia viridis

Av3, a neurotoxin of Anemonia viridis, is toxic to crustaceans and cockroaches but inactive in mammals. In the present study, Av3 was expressed in Escherichia coli Origami B (DE3) and purified by reversed-phase liquid chromatography. The purified Av3 was injected into the hemocoel of Helicoverpa armigera, rendering the worm paralyzed. Then, Av3 was expressed alone or fusion expressed with the Cry1Ac in acrystalliferous strain Cry−B of Bacillus thuringiensis. The shape of Cry1Ac was changed by fusion with Av3. The expressed fusion protein, Cry1AcAv3, formed irregular rhombus- or crescent-shaped crystalline inclusions, which is quite different from the shape of original Cry1Ac crystals. The toxicity of Cry1Ac was improved by fused expression. Compared with original Cry1Ac expressed in Cry−B, the oral toxicity of Cry1AcAv3 to H. armigera was elevated about 2.6-fold. No toxicity was detected when Av3 wasxa0expressed in Cry−B alone. The present study confirmed that marine toxins could be used in bio-control and implied that fused expression with other insecticidal proteins could be an efficient way for their application.

E. coli Nissle 1917-Derived Minicells for Targeted Delivery of Chemotherapeutic Drug to Hypoxic Regions for Cancer Therapy

Purpose: Systemic administration of free chemotherapeutic drugs leads to severe toxic effects, and physiological characteristics of solid tumors restrain the drugs from reaching the hypoxic regions. E. coli Nissle 1917 (EcN) has been known to penetrate the barrier and proliferate in the interface between the viable and necrotic regions of tumors. This study aimed to fabricate a nanoscale minicell via genetic engineering of EcN for targeted delivery of chemotherapeutic drugs to the hypoxic regions of tumors for cancer therapy. Methods: A large number of minicells were produced by knocking out the minCD gene and enhancing the minE expression in EcN. Then, a pH (low) insertion peptide (pHLIP) was displayed on the membrane surface through protein display technology to endow the cells with the ability to target the acidic microenvironments of tumors. The acidic-microenvironment targeting ability and therapeutic effect of the engineered minicells with chemotherapeutic drugs was thoroughly evaluated by using breast cancer cells and an orthotopic model of breast tumor. Results: The EcN-derived minicells displaying pHLIP could be directly extracted from the fermentation broth and used for delivering chemotherapeutic drugs without any further modification. Targeting of doxorubicin (DOX)-loaded minicells to cancer cells via pHLIP resulted in rapid internalization and drug release in acidic media. Importantly, the pHLIP-mosaic minicells successfully invaded the necrotic and hypoxic regions of orthotopic breast cancers where free chemotherapeutic drugs could never get to because of vascular insufficiency and high interstitial fluid pressure. This invasion resulted in significant regression of an orthotopic breast tumor in a mouse model, while no seriously pathogenic effects were observed during the animal experiments. Conclusions: This study provides a novel strategy for the fabrication of tumor-targeting carriers via genetic engineering based on biomaterials with the ability to penetrate hypoxic regions of tumors, high biocompatibility and low toxicity.

Heterologous expression and antitumor activity analysis of syringolin from Pseudomonas syringae pv. syringae B728a

BackgroundSyringolin, synthesized by a mixed non-ribosomal peptide synthetase/polyketide synthetase in Pseudomonas syringae pv. syringae (Pss) B728a, is a novel eukaryotic proteasome inhibitor. Meanwhile, directly modifying large fragments in the PKS/NRPS gene cluster through traditional DNA engineering techniques is very difficult. In this study, we directly cloned the syl gene cluster from Pss B301D-R via Red/ET recombineering to effectively express syringolin in heterologous hosts.ResultsA 22xa0kb genomic fragment containing the sylA–sylE gene cluster was cloned into the pASK vector, and the obtained recombinant plasmid was transferred into Streptomyces coelicolor and Streptomyces lividans for the heterologous expression of syringolin. Transcriptional levels of recombinant syl gene in S. coelicolor M145 and S. lividans TK24 were evaluated via RT-PCR and the production of syringolin compounds was detected via LC–MS analysis. The extracts of the engineered bacteria showed cytotoxic activity to B16, 4T1, Meth-A, and HeLa tumor cells. It is noteworthy that the syringolin displayed anticancer activity against C57BL/6 mice with B16 murine melanoma tumor cells. Together, our results herein demonstrate the potential of syrinolin as effective antitumor agent that can treat various cancers without apparent adverse effects.ConclusionsThis present study is the first to report the heterologous expression of the entire syl gene cluster in Streptomyces strains and the successful expression of syringolin in both S. coelicolor M145 and S. lividans TK24. Syringolin derivatives demonstrated high cytotoxicity in vitro and in vivo. Hence, this paper provided an important foundation for the discovery and production of new antitumor compounds.

Escherichia coli Nissle 1917 engineered to express Tum-5 can restrain murine melanoma growth

Tumor growth and metastasis depend on angiogenesis. Thus, inhibiting tumor angiogenesis has become promising cancer therapeutic strategy in recent years. Tumstatin is a more powerful angiogenesis inhibitor than endostatin. Anti-angiogenic active fragment encoding amino acids 45–132 (Tum-5) of tumstatin was subcloned into four different inducible expression vectors and successfully solubly expressed in Escherichia coli BL21 (DE3) in this study. Subsequently, an anaerobic inducible expression vector was constructed under Vitreoscilla hemoglobin gene promoter Pvhb in E. coli Nissle 1917 (EcN). The secretory expression of Tum-5 in the engineered bacterium was determined in vitro and in vivo by Western blot or immunochemistry. The anti-tumor effect detection demonstrated that EcN could specifically colonize the tumor, and B16 melanoma tumor growth was remarkably restrained by EcN (Tum-5) in mice bearing B16 melanoma tumor. Abundant infiltrating inflammatory cells were observed in tumor areas of the EcN-treated group through hematoxylin and eosin staining, with a relatively reduced expression of endothelial marker platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) by immunofluorescence in tumor sections of EcN (Tum-5)-treated mice. No significant morphological differences were observed in the liver, kidney and spleen between EcN-treated mice and the control group, indicating that EcN was cleared by the immune system and did not cause systemic toxicity in mice. These findings demonstrated that the gene delivery of Tum-5 to solid tumors could be an effective strategy for cancer therapy.