Animal Safety Test of Bacillus thuringiensis BT Protein

[Objectives]To determine the biological safety of BT protein from Bacillus thuringiensis(Bt)fermentation broth to mammals at high doses.[Methods]Healthy mice were randomly divided into 4 groups with 10 mice in each group.The experimental groups were fed with Bt fermentation supernatant at 0.2,0.6 and 1.0 mL/kg,respectively,once a day for 7 consecutive days.The blank control group was fed normally without intragastric administration.[Results]There was no significant difference in blood routine and blood biochemical analysis between the experimental group and the control group.After intragastric administration,the mice were dissected,and no obvious pathological changes were found;the heart,liver,spleen,lung and kidney were taken to make tissue sections,and no pathological changes were found by microscopic observation.[Conclusions]Mice can tolerate high doses of BT protein from B.thuringiensis fermentation broth.

Degradation of N-Acyl Homoserine Lactone Quorum Sensing Signals by Bacillus thuringiensis AHL Lactonase

Bacterial cells rely on signaling molecules to communicate with others from the same species and induce certain genes in a process known as quorum sensing (QS). A common molecule is N-acyl homoserine lactone (AHL) which is responsible for the expression of virulence and other factors that allow the organisms to compete in a given environment. On the other hand, other bacteria produce certain enzymes such as AHL-lactonase that break down AHL molecules and prevent gene expression of these factors. The aim of this work was to examine the level of degradation of AHL molecules by AHL-lactonase in 62 Bacillus thuringiensis (Bt) strains isolated from Middle Tennessee, Mississippi, and Alabama. N-hexanoyl-homoserine lactone (C6-HSL) and N-3-oxo-hexanoyl homoserine lactone (3-oxo-C6-HSL), which cause Chromobacterium violaceum (CV026) to produce a purple pigment were tested at different concentrations to view the Bt lactonase activity. In addition, PCR was used to test for the presence of the lactonase gene. The results showed that among the 62 Bt strains, there were 58 that possessed the AHL-lactonase (aiiA) gene and 48 strains were able to degrade C6-HSL. At high concentrations of AHL, only 13 strains were able to completely degrade C6-HSL. In addition, degradation of 3-oxo-C6-HSL was weak compared to C6-HSL. The results also revealed that AHL lactonase was thermostable, and it was concluded that the level of degradation varies in Bt strains. Only 13 of the strains studied have potent inhibitory activity against C6-HSL, which may be good to be used in field applications to control agricultural pest.

A Fragment of Cadherin-Like Protein Enhances Bacillus thuringiensis Cry1B and Cry1C Toxicity to Spodoptera exigua(Lepidoptera:Noctuidae)

Cry toxins produced by Bacillus thuringiensis（Bt） are effective biological insecticides against certain insect species.In this study,bioassay results indicated that Cry1B and Cry1C were toxic to Spodoptera exigua.We also identified a cadherin-like gene in S.exigua that could enhance the toxicity of Cry1B and Cry1C.The cadherin-like gene identified from the larvae midgut tissue was cloned by reverse transcription polymerase chain reaction（RT-PCR） and rapid amplification of cDNA ends（RACE）.The full-length cDNA of the gene consisted of 5 220 bp encoding 1 740 amino acid with a predicted molecular mass of 196 kD.BLAST search analysis showed that the predicted amino acid sequence had a high sequence identity to the published sequences of cadherin-like proteins from other Lepidoptera insects.Spatial expression of the cadherin-like gene detected by qRT-PCR analysis revealed that the cadherin-like gene was mainly present in the gut of 4th instar larvae and during different life stages.The results suggested that the commercial development of this synergist has the potential to enhance Cry1B and Cry1C toxicity against Lepidoptera insects.

Identification and Distribution of Bacillus thuringiensis Isolates from Primeval Forests in Yunnan and Hainan Provinces and Northeast Region of China

Ninety-two Bacillus thuringiensis isolates were screened from 683 soil samples collected from tropical and semitropical primeval forests in Yunnan and Hainan provinces of China. Several shapes of crystals, including bipyramidal, square, ovoid, spherical, and amorphous, were observed in the B. thuringiensis isolates. Twenty-six pairs of primers were used to identify 31 holotype cry genes at primary rank of the B. thuringiensis cry gene nomenclature system. The cry gene-types of 92 B. thuringiensis isolates and 33 B. thuringiensis isolates screened from Northeast region of China were identified by PCR-RFLP and SDS-PAGE methods. Fifty-eight isolates harbored cry1 genes, 32 isolates cry2 genes, 12 isolates cry8 genes, 3 isolates cry9 genes, 12 isolates cry11 genes, and 13 isolates cry30 genes. Of the tested isolates, 42 produced no reaction product with 26 pairs of primers and also exhibited no toxicity against 8 insect species tested. The isolate Z2-34 harbored a novel cry30 gene, exhibited insecticidal activity against Aedes albopictus of Dipterans. The accession number of the novel genes in this study is AY916046. Isolation and identification of B. thuringiensis and cry gene are important for investigating the diversity of B. thuringiensis resources and cloning new cry gene.

Toxicity studies for indigenous Bacillus thuringiensis isolates from Malang city,East Java on Aedes aegypti larvae

Objective:To investigate the toxicity of indigenous Bacillus thuringiensis（B.thuringiensis）isolates from Malang City for controlling Aedes aegypti（Ae.aegypti）larvae.Methods:Soil samples were taken from Purwantoro and Sawojajar sub-districts.Bacterial isolation was performed using B.thuringiensis selective media.Phenotypic characteristics of the isolates were obtained with the simple matching method.The growth and prevalence of spores were determined by the Total Plate Count method,and toxicity tests were also performed on the third instar larval stage of Ae.aegypti.The percentage of larval mortality was analysed using probit regression.The LC50was analysed by ANOVA,and the Tukey HSD interval was 95%.Results:Among the 33 selected bacterial isolates,six were obtained（PWR4-31,PWR4-32,SWJ4-2b,SWJ4-4b,SWJ-4k and SWJ5-1）that had a similar phenotype to reference B.thuringiensis.Based on the dendrogram,all of the bacterial isolates were 71%similar.Three isolates that had a higher prevalence of reference B.thuringiensis were PWR4-32,SWJ4-4b and SW5-1,of which the spore prevalence was 52.44%,23.59%,34.46%,respectively.These three indigenous isolates from Malang City successfully killed Ae.aegypti larvae.The PWR4-32 isolates were the most effective at killing the larvae.Conclusions:Six indigenous B.thuringiensis isolates among the 33 bacterial isolates found in the Sawojajar and Purwantoro sub-districts were toxic to the third instar larvae of Ae.aegypti.The PWR4-32 isolates were identical to tbe reference B.thuringiensis and had 88%phenotype similarity.The PWR4-32 isolates had the highest spore prevalence（52.44%）,and the early stationary phase occurred at 36 h.The PWR4-32 isolates were the most effective at killing Ae.aegypti larvae(LC50-72 h=2.3×108 cells/mL).

Homology Modeling of Mosquitocidal Cry30Ca2 of Bacillus thuringiensis and Its Molecular Docking with N-acetylgalactosamine

Abstract Objective To investigate the theoretical model of the three-dimensional structure of mosquitocida Cry3OCa2 and its molecular docking with N-acetylgalactosamine. Methods The theoretical model of Cry30Ca2 was the Cry4Ba. Docking studies were performed N-acetylgalactosamine on the putative receptor. predicted by homology modeling on the structure of to investigate the interaction of Cry3OCa2 with Results Cry3OCa2 toxin is a rather compact molecule composed of three distinct domains and has approximate overall dimensions of 95 by 75 by 60A. Domain I is a helix bundle, Domain Ⅱ consists of three antiparallel β-sheets, Domain Ⅲ is composed of two β-sheets that adopt a 13-sandwich fold. Residue 32111e in loop1, residues 342Gin 343Thr and 345Gin in loop2, residue 393Tyr in loop3 of Cry3OCa2 are responsible for the interactions with GalNAc via 7 hydrogen bonds, 6 of them were related to the oxygen atoms of hydroxyls of the ligand, and one to the nitrogen of the ligand. Conclusion The 3D structure of Cry3OCa2 resembles the previously reported Cry toxin structures but shows still some distinctions. Several residues in the loops of the apex of domain Ⅱ are responsible for the interactions with N-acetylgalactosamine.

Complete genome sequence of Bacillus thuringiensis Bt185, a potential soil insect biocontrol agent

Bacillus thuringiensis Bt185 and its insecticidal spectrum-expanded engineering strains are considered as potential biocontrol agents to soil insect Holotrichia parallela,Holotrichia oblita or Anomala corpulenta.Here we reported the complete genome of strain Bt185,it harbors eight plasmids,and plasmid p BT1850294 carries three cry8 genes.

Differentiation Between Bacillus thuringiensis and Bacillus cereus by 16S rDNA-PCR and ERIC-PCR

16S rDNA and ERIC （Enterobacteia Repetitive Intergenic Consensus Sequences） based on PCR method were tested for the effectiveness of the differentiation of B. thuringiensis and B. cereus. 16S rDNA-PCR primers were designed based on the sequence difference in variable regions of B. cereus 16S rDNA and B. thuringiensis 16S rDNA, 16S rDNA-PCR showed no obvious difference between B. cereus and B. thuringiensis. The only difference was that one 1600-bp amplificon could be obtained from all the three B. Cereus strains, and none amplificon from any B. thuringiensis strains. ERIC was optimized based on previous reports. The genonlic DNA was used for the template of ER1C-PCR, and the following DNA fingerprints were analyzed by the agarose gel electrophoresis. The results showed that DNA fingerprint of three B. thuringiensis strains had a unique amplicon less than 100-bp, while DNA fingerprint of three B. cereus＂ strains had none. Moreover, DNA fingerprint of B. cereus showed a 700-bp amplicon, but didn＇t have any DNA fingerprints ofB. thuringiensis genome. Therefore, ERIC-PCR technique should be able to be used for the differentiation of B. thuringiensis and B. cereus.

Penetration of a Single Domain of Bacillus thuringiensis Cry1Ie-Domain I to a Lipid Membrane In vitro

Domain I of the activated Crystal protein from Bacillus thuringiensis has a seven a-helix bundle structure, which is responsible for membrane channel formation in its insecticidal mechanism. Crylle is toxic to Asian corn borer, Ostrinia furnacalis （Guen6e）, and plays important roles in insect biological control. The domain I from Crylle has been expressed and purified in its normal conformation, as embedded in the full length homologous toxin structure. The membrane insertion ability of this single domain was compared with the full length homologous toxin using a monolayer insertion experiment. The results indicated that the Crylle-domain I had the ability to insert into the lipid monolayer, and this ability is greater than that of the IE648 toxin. However, the state of insertion is not stable and remains for only a short period of time. The Crylle-domain I plays no role in receptor binding as it had a nonspecific binding with the brush border membrane vesicles of the Asian corn borer.

Biodegradation of Kirkuk light crude oil by <i>Bacillus thuringiensis</i>, Northern of Iraq

This study was conducted to identify the viability of Bacillus thuringiensis bacterial on a bio-degradation process for Kirkuk light crude oil. The viable count of Bacillus thuringiensis showed great capability on the biodegradation of crude oil. These bacteria exhibit the ability to dismantle crude oil through clear emulsion layer of crude oil. And they have a good efficiency to dismantle hydrocarbon compounds by 80%, and total biomass reaches to 5 g/l, while the amount of emulsion reaches to 2.3 g/l. For more evidences on the biodegradation action of Bacillus thuringiensis which have been supported by using the technology of gas-Chromatography which confirms the occurring of biodegradation process. The visual examination of gas-Chromatography shows the disappearance of a number of chemicals, as well as decrease in peak area for some material.

Evaluation of Bacillus thuringiensis to Control Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) under Laboratory Conditions

The tomato leaf miner, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) is among the most destructive pests that attack tomato in many countries. In this study, the efficiency of three suspensions (106;105;104 cell/ml) of Bacillus thuringiensis var. Kurstaki was tested on T. absoluta larvae 1st, 2nd, 3rd and 4th instar was assessed to study the effect of these suspensions on larval mortality. Results showed that the concentration 106 cell/ml resulted in the highest mortality of all instars larvae with an average mortality of 20%, 22.66%, 18.66% and 23.33% was recorded for the first, second, third and fourth instar, respectively. The greatest percentage of mortality occurred in the third day after the larvae fed with leaves treated with suspension B. thuringiensis.

Biological Activity of Bacillus Thuringiensis (Berliner) Strains on Larvae and Adults of Ceratitis Capitata (Wiedemann) (Diptera: Tephritidae)

The objective of this study was to evaluate the efficiency of Moroccan Bt strains against neonate larvae, third instar larvae and emerged adults of Ceratitis capitata. This Mediterranean fruit fly causes serious damages to Argan forest and other agricultural plants. There is no successful control program of this pest fly in the endemic Argan forest in Morocco. A single-dose test was performed on neonate larvae (25 μL/g) and adult (333.33 μL/g), when three doses of Bt toxins (50 μL/g, 100 μL/g and 150 μL/g) were tested against third instar of C. capitata. Among the twenty-six Bt strains examined, local Bt13.4 and Bt A7 strains showed highest toxicity levels against larvae and adults, when compared to the reference strain, Bacillus thuringiensis subsp. israelensis HD567 “code 4Q1”, and commercial product “Skeetal”. One hundred percent mortality was observed against neonate larvae after 7 days of application by Bt 13.4 toxin. Third instar larvae were very susceptible to Bt A7 and Bt M-Ag 21.6 strains with 68% mortality (Lethal Concentration: LC50 = 1.115) at a dose of 150 μL/g. The Bt A7 strain was also highly toxic to adults with 81.66% of mortality after 7 days of application. This study demonstrated that some of our collection Bt strains can contribute to integrated C. capitata management system with strong biological control components.

An Overview on the Crystal Toxins from <i>Bacillus thuringiensis</i>

Strains of Bacillus thuringiensis (Bt) are known to produce crystalline proteins (δ-endotoxins) concomitantly with sporulation during their stationary phase of growth, which are demonstrated as lethal to lepidopeterous, coleopeterous and dipterous insects in addition to mites, nematodes, protozoa and flukes. Upon ingestion, the δ-nascent endotoxin is an inactive protoxin complex of (Cry alone or Cry and Cyt toxins together) high molecular mass, which is cleaved upon ingestion into the active component proteins at the high alkaline environments in the digestive tract of these agricultural pests. Conventionally, Bt-crystals are being produced employing submerged or liquid fermentation techniques in commercial media, but recently many workers have used solid-state fermentation strategy for the enhanced production of Bt-toxin at low cost. Apart from δ-endotoxin, some isolates of Bt produce another class of insecticidal small molecules called β-exotoxin (thuringiensin), which may be harmful to humans. Moreover, resistance to Bt developed in various target pest is yet another concern for Bt-industry. Following a brief introduction, this review addresses various toxins produced by various strains of Bt, Bt production media and media formulations with emphasis to solid-state fermentation, general structure of Cry toxin, its mode of action, target pests, bioassay, resistance to Bt toxins and resistance management. Briefly, this review would provide the readers an overview on the general aspects of Bt toxin, its general structure and mechanism of action.

Assessment of Two <i>Trichogramma</i>Species with <i>Bacillus thuringiensis</i>var. krustaki for the Control of the Tomato Leafminer <i>Tuta absoluta</i>Meyrick (Lepidoptera: Gelechiidae) in Iran

In this study, we investigated the efficiency of T. brassicae and T. embryophagum in combination with bacterial suspension of B. thuringiensis against the tomato leafminer T. absoluta eggs in cage inside greenhouse (semi field) experiments. Four treatments were used (T. brassicae or T. embryophagum + T. absoluta) (T1), (B. thuringiensis + T. absoluta) (T2), (T. brassicae or T. embryophagum + B. thuringiensis + T. absoluta) (T3) and control (T4). The lowest number of T. absoluta mines (6.1, 0.5 mine per plant) were recorded in T3 for T. brassicae and T. embryophagum were significantly lower than those of all other treatments which were followed by T1 and T2, while the highest number of mines per plant (50.70) were recorded in control (T4). In addition, the parasitism rate, adults’ emergence, the number of females and adult longevity of two parasitoids were investigated. According to the obtained results, the highest parasitism rate was obtained for T. embryophagum when treated with Bt reared in the T. absoluta eggs (31.18%). However, no significant differences were detected between T. brassicae and T. embryophagum in mortality and adult emergence rates were found when they were treated with/ without Bt reared in the T. absoluta eggs in cage inside greenhouse. Also, the longevity of T. embryophagum was significantly better than T. brassicae p = 0.000. This is the first study to investigate T. embryophagum in cage inside greenhouse for parasitizing the eggs of T. absoluta and results of present study suggested that T. embryophagum with Bt could be more efficient for biocontrol of T. absoluta.

Larvicidal efficacy of Catharanthus roseus Linn.(Family:Apocynaceae)leaf extract and bacterial insecticide Bacillus thuringiensis against Anopheles stephensi Liston.

Objective:To explore the larvicidal activity of Catharanthus roseus(C.roseus) leaf extract and Raeillus thuringiensis(B.thuringiensis) against the malarial vector Anopheles stephensi(An. stephensi),when being used alone or together.Methods:The larvicidal activity was assayed at various concentrations under the laboratory and field conditions.The LC_(50) and LC_(90) values of the C.roseus leaf extract were determined by probit analysis.Results:The plant extract showed larvicidal effects after 24 h of exposure;however,the highest larval mortality was found in the petroleum ether extract of C.roseus against the first to fourth instars larvae with LC_(50)=3.34,4.48, 5.90 and 8.17 g/L,respectively;B.thuringiensis against the first to fourth instars larvae with LC_(50)=1.72.1.93.2.17 and 2.42 g/L.respectively:and the combined treatment with LC_(50)=2.18.2.41. 2.76 and 3.22 g/L,respectively.No mortality was observed in the control.Conclusions:The petroleum ether extract of C.roseus extract and B.thuringiensis have potential to be used as ideal eco-friendly agents for the control of An.stephensi in vector control programs.The combined treatment with this plant crude extract and bacterial toxin has better larvicidal efficacy against An.stephensi.

Does cotton bollworm show cross-resistance to the Bacillus thuringiensis toxins Cry1Ac and Cry2Ab? A mini review

Since 1996, transgenic Bacillus thuringiensis(Bt) cotton has been commercially grown in numerous countries in an effort to stem the losses caused by key lepidopteran pests. However, the development of pest resistance to Bt toxins has jeopardized the continued utilization of Bt cotton. As a strategy designed to circumvent the development of resistance, Bt cotton varieties expressing two or more toxins targeting the same pest have been introduced. Nevertheless, from the perspective of long-term planting of Bt cotton, the potential risk of cross-resistance to these Bt toxins is a threat that cannot be ignored. In this paper, we review current research(including that based on the analysis of protein binding sites and resistance genes) on the resistance of cotton bollworm(Helicoverpa armigera) to the Bt toxins Cry1 Ac and Cry2 Ab and the interrelationship between these toxins. On the basis of existing evidence, we assume that the actions of Cry1 Ac and Cry2 Ab against cotton bollworm are not completely independent, and then propose the "resistance-associated gene mutation potential hypothesis". Although the mechanisms underlying the resistance of pests to Bt toxins are yet to be comprehensively elucidated, this hypothesis could undoubtedly have important implications for adopting "pyramid" strategy in the future. Further research is recommended to devise strategies to retard the development of H. armigera resistance to Bt cotton, either using different Bt toxins or their various combinations.

Identification of similar transcriptional regulatory mechanisms in multiple cry genes in Bacillus thuringiensis HD12

Bacillus thuringiensis subspecies morrisoni strain HD12, whose genome harbors multiple insecticidal protein-encoding genes, includes eight cry genes, as indicated by genome sequencing. This strain produces crystals that are toxic to lepidopteran species. These crystal inclusions were purified by sucrose gradients and sodium dodecyl sulphate-polyacrylamide gel electrophoresis （SDS-PAGE）, followed by liquid chromatography-mass spectrometry, and found to contain five proteins （Cry1Da, Cry1Ae, Cry1Bb, Cry1Fb, and CrylJa）. The transcriptional activities of the cry1Da, cry1Ae, cry1Bb, cry1Fb, and cry11Ja promoters indicated that transcription of crylDa is controlled by SigE; however, the other four cry genes were found to be controlled by both SigE and SigK. The activities of the crylJa and crylFb promoters were the strongest among the five genes studied. These promoters were therefore used to direct the expression of the Cry1Ac- and Cry2Ab-encoding genes concurrently in a single strain. Our findings indicate that promoters with the same transcriptional profile may be used to direct the expression of different cry genes in one strain. Our results are expected to be valuable for the construction of strains with efficient expression of multiple cry genes in order to overcome current limitations associated with the application of B. thuringiensis-based insecticides.

Evaluation of Cuban Bacillus thuringiensis(Berliner,1911)(Bacillales:Bacillacea)isolates with larvicidal activity against Aedes aegypti(Linnaeus,1762)(Diptera:Culicidae)

Objective:To evaluate 11 Cuban native Bacillus(B.)thuringiensis isolates in order to select one with the best larvicidal activity against Aedes(Ae.)aegypti and low cytotoxicity.Methods:The cry and cyt genes of the isolates(A21,A51,L95,L910,M29,R84,R85,R87,R89,U81 and X48)were amplified by PCR.The influence of organic matter and NaCl on the larvicidal activity was tested by bioassays.Cytotoxicity was assayed on peritoneal macrophages of BALB/c mice.Results:The cyt1(Aa,Ab,Ba),cyt2,cry4aA,cry4Ba,cry11(Aa,Ba,Bb)and cry10 genes were identified in all native Cuban isolates.The larvicidal activity(LC_(90))of seven isolates was affected by the presence of organic matter in the water,while A21,A51,L910,R84,U81 and X48 had better LC_(50),LC_(90),LC_(95) than the 266/29-Ⅶ-98 control strain.The LC_(50) of two isolates was affected by the presence of NaCl and A21,A51,R85 isolate had better larvicidal activity than the 266/29-Ⅶ-98 control strain.In terms of toxicity against macrophages,the extracts of nine isolates were less cytotoxic than the control strains.Conclusions:Native isolate A21 had the main virulence factors against Ae.aegypti larvae,displayed a good larvicidal activity in presence of different factors related with Ae.aegypti breeding sites,and had low citotoxicity against macrophages.These results can contribute to the improvement of existing biological control strategies and the development of new biolarvicides.

Polyhedrosis Virus in Bacillus thuringiensis

This study was conducted to build a recombinant strain with highly insecticidal activity and a wide host range by using the crylAc and p74 gene. Firstly, the p74 gene was amplified from the genosome ofAutographa californica multicapsid nucleopolyhedrovirus. The crylAc gene and the terminator gene of crylAc, named crylAct, were amplified from the plasmid of Bt 4.0718 strain. Three T vectors, named pTp74, pT1Ac, and pT1Act which held the aimed gene p74, cry1Ac, and crylAct, respectively, and two middle vectors, named pTp74Act and pTIAcp74 which held the aimed fusion gene p74-crylAct and cry lAc-p74, respectively, were built by using pMDI 8-T. Then pTiAcp74 and the shuttle plasmid were digested and linked and an expressing-vector pH1Acp74 was built. Finally, pH1Acp74 was transformed into the acrystalliferous strain XBU001 and the aimed recombinant strain XBU-H1Acp74 was obtained. The expression of Bt transformant XBU-H1Acp74 was analyzed by SDS-PAGE which showed XBU-H1Acp74 could produce 130 kDa CrylAc protein and 50 kDa P74 protein. The insecticidal activity of transformant against Spodoptera exigua was evaluated compared with the contrast strains HTX-42 （only crylAc gene was transformed into XBU001） after autolysis. The LCs0 of HTX-42 was higher than that of the XBU-H IAcp74＇s, which implied that P74 could increase the efficacy and range of Bt Cry toxins in insect control. The fusion gene of crylAc and p74 were constructed successfully which will be served as the foundation lbr constructing the fusion genes of Bt cry gene and other foreign genes.

Influence of Formate on Bioactivity Material-thuringiensin Synthesized by Bacillus thuringiensis YBT-032

The biological method to synthesize thuringiensin and the influence of formate on thuringiensin biosynthesis were investigated. Addition of 1.00 g/L formate to growth medium of bacillus thuringiensis YBT-032 resulted in significant enhancements in productions of citrate, α -ketoglutarate, intracellular adenine and thuringiensin. These results demonstrate that added formate attends metabolism of cell, facilitates carbon metabolic flux in tricarboxylic acid cycle and hexose monophosphate pathway. As a carbon source, formate facilitates cell growth, increases glucose consumption and enhances the ability of cell to synthesis adenine analogues, and subsequently thuringiensin. Thuringiensin production rate significantly enhanced from 6.44 to 8.46 mg·g^-1 · h and transformation ratio from glucose to thuringiensin increased by 43.30%.