Construction of a Conditionally Asporogenous Bacillus thuringiensis Recombinant Strain Overproducing Cry Protein by Deletion of the leuB Gene.

One of the common shortcomings with Bacillus thuringiensis (Bt) biopesticides in field application is their instability under UV irradiation. In Bt, the leuB gene encodes the 3-isopropylmalate dehydrogenase. In addition to its role in leucine biosynthesis, LeuB would be likely recruited to catalyze the dehydrogenation of malate in the final step of tricarboxylic acid cycle during sporulation. In this study, we constructed a Bt recombinant strain in which the gene leuB was deleted by using the markerless gene deletion system. The ΔleuB mutant strain showed a conditionally asporogenous phenotype while overproducing insecticidal crystal proteins and retaining its insecticidal activity well in both fermentation and LB media. Furthermore, the metabolic regulation mechanisms of LeuB was elucidated by iTRAQ-based quantitative proteomics approach. Evidences from proteomics data suggested that the inhibited supply of pyruvate (carbon source) was an important factor related to the conditionally asporogenous feature of the mutant. Consistently, the mutant regained its ability to sporulate in LB medium by adding 1% glucose or 1% sodium pyruvate. Taken together, our study demonstrated that deletion of the leuB gene resulted in delayed or completely blocked mother cell lysis, allowing the crystals encapsulated within cells, which makes this recombinant strain a good candidate for developing Bt preparations with better UV-stability.

Role of g6pdh and leuB on Lipid Accumulation in Mucor circinelloides.

Mucor circinelloides is a valuable oleaginous filamentous fungus rich in γ-linolenic acid (GLA, 18:3; n-6), which is beneficial for human health. Our previous comparative proteomic analysis between high lipid-producing M. circinelloides WJ11 and low lipid-producing M. circinelloides CBS 277.49 indicated that glucose 6-phosphate dehydrogenase (G6PDH) and ß-isopropylmalate dehydrogenase (IPMDH) were closely involved in lipid accumulation. Transcription analysis suggested that in the strain WJ11, g6pdh1 and g6pdh2, which encode G6PDH, and leuB, which encodes IPMDH, could be the key genes regulating lipid accumulation. To further analyze the effects of these three genes (i.e., g6pdh1, g6pdh2, and leuB) on lipid accumulation, we respectively overexpressed these genes from M. circinelloides WJ11 in defective CBS 277.49 strains in this study. The results showed that overexpression of g6pdh1 and g6pdh2 genes from strain WJ11 increased the fatty acid content of cell dry weight by 23-38 and 41-47%, respectively, compared with the control strain. Furthermore, overexpression of the leuB gene from strain WJ11 increased the fatty acid content of cell dry weight by up to 67-73%. These results suggest that g6pdh1, g6pdh2, and especially leuB genes play important roles in regulating fatty acid synthesis in M. circinelloides.

Targeted inactivation of Salmonella Agona metabolic genes by group II introns and in vivo assessment of pathogenicity and anti-tumour activity in mouse model.

The fight against cancer has been a never-ending battle. Limitations of conventional therapies include lack of selectivity, poor penetration and highly toxic to the host. Using genetically modified bacteria as a tumour therapy agent has gained the interest of scientist from the past few decades. Low virulence and highly tolerability of Salmonella spp. in animals and humans make it as the most studied pathogen with regards to anti-tumour therapy. The present study aims to construct a genetically modified S. Agona auxotroph as an anti-tumour agent. LeuB and ArgD metabolic genes in ΔSopBΔSopD double knockout S. Agona were successfully knocked out using a Targetron gene knockout system. The knockout was confirmed by colony PCR and the strains were characterized in vitro and in vivo. The knockout of metabolic genes causes significant growth defect in M9 minimal media. Quadruple knockout ΔSopBΔSopDΔLeuBΔArgD (BDLA) exhibited lowest virulence among all of the strains in all parameters including bacterial load, immunity profile and histopathology studies. In vivo anti-tumour study on colorectal tumour bearing-BALB/c mice revealed that all strains of S. Agona were able to suppress the growth of the large solid tumour as compared with negative control and ΔLeuBΔArgD (LA) and BDLA auxotroph showed better efficacy. Interestingly, higher level of tumour growth suppression was noticed in large tumour. However, multiple administration of bacteria dosage did not increase the tumour suppression efficacy. In this study, the virulence of BDLA knockout strain was slightly reduced and tumour growth suppression efficacy was successfully enhanced, which provide a valuable starting point for the development of S. Agona as anti-tumour agent.