Search

Adenylosuccinate synthetase (EC. 6.3.4.4) encoded by purA in Bacillus subtilis, catalyzing the first step of the conversion of IMP to AMP, plays an important role in flux distribution in the purine biosynthetic pathway. In this study, we described the use of site saturation mutagenesis to obtain a desired enzyme activity of adenylosuccinate synthetase and its application in flux regulation. Based on sequence alignment and structural modeling, a library of enzyme variants was created by a semi-rational evolution strategy in position Thr238 and Pro242. Other than purA deletion, the leaky mutation purA(P242N) partially reduced the flux towards AMP derived from IMP and increased the riboflavin synthesis precursor GTP, while also kept the requirement of ATP synthesis for cell growth. PurA(P242N) was introduced into an inosine-producing strain and resulted in an approximately 4.66-fold increase in inosine production, from 0.088±0.009g/L to 0.41±0.051g/L, in minimal medium without hypoxanthine accumulation. These results underline that the directed evolution of adenylosuccinate synthetase could tailor its activities and adjust metabolic flux. This mutation may provide a promising application in purine-based product accumulation, like inosine, guanosine and folate which are directly stemming from purine pathway in B. subtilis.

5-Aminolevulinic acid (ALA) is the precursor for the biosynthesis of tetrapyrroles and has broad agricultural and medical applications. Currently ALA is mainly produced by chemical synthesis and microbial fermentation. Cell free multi-enzyme catalysis is a promising method for producing high value chemicals. Here we reported our work on developing a cell free process for ALA production using thermostable enzymes. Cheap substrates (succinate and glycine) were used for ALA synthesis by two enzymes: 5-aminolevulinic acid synthase (ALAS) from Laceyella sacchari (LS-ALAS) and succinyl-CoA synthase (Suc) from Escherichia coli. ATP was regenerated by polyphosphate kinase (Ppk) using polyphosphate as the substrate. Succinate was added into the reaction system in a fed-batch mode to avoid its inhibition effect on Suc. After reaction for 160min, ALA concentration was increased to 5.4mM. This is the first reported work on developing the cell free process for ALA production. Through further process and enzyme optimization the cell free process could be an effective and economic way for ALA production.

Myostatin (MSTN) gene is described as a negative regulator of the skeletal muscle growth. Controlling MSTN gene expression by genetic manipulation could accelerate the muscle growth and meat production of livestock animals. In the present study, several siRNAs targeting sheep MSTN gene were designed and their interfering efficiency was evaluated in vitro. The present study showed that one of the siRNAs, PSL1, could down-regulate the expression of MSTN significantly. PSL1 was ligated into lentivirus vector, GP-Supersilencing, to construct a siRNA expression lentivirus vector. Fibroblast cells were infected by lentivirus particles and positive cells were isolated by flow cytometry. Nucleus of the positive cell was transferred into enucleated oocytes of sheep. The present study showed that 99.4% of the sorted cells displayed green fluorescence. After enucleation of oocytes with microinjection, about 20% of reconstructed embryos can be developed into morulas, and strong green fluorescence could be observed using a fluorescence microscope. This method can be available to produce transgenic cell line for somatic cell nucleus transfer for transgenic animals.