Construction and application of a synthetic promoter library for Corynebacterium glutamicum / 生物工程学报

Promoter is an important genetic tool for fine-tuning of gene expression and has been widely used for metabolic engineering. Corynebacterium glutamicum is an important chassis for industrial biotechnology. However, promoter libraries that are applicable to C. glutamicum have been rarely reported, except for a few developed based on synthetic sequences containing random mutations. In this study, we constructed a promoter library based on the native promoter of odhA gene by mutating the -10 region and the bystanders. Using a red fluorescent protein (RFP) as the reporter, 57 promoter mutants were screened by fluorescence imaging technology in a high-throughput manner. These mutants spanned a strength range between 2.4-fold and 19.6-fold improvements of the wild-type promoter. The strongest mutant exhibited a 2.3-fold higher strength than the widely used strong inducible promoter Ptrc. Sequencing of all 57 mutants revealed that 55 mutants share a 1-4 bases shift (4 bases shift for 68% mutants) of the conserved -10 motif "TANNNT" to the 3' end of the promoter, compared to the wild-type promoter. Conserved T or G bases at different positions were observed for strong, moderate, and weak promoter mutants. Finally, five promoter mutants with different strength were employed to fine-tune the expression of γ-glutamyl kinase (ProB) for L-proline biosynthesis. Increased promoter strength led to enhanced L-proline production and the highest L-proline titer of 6.4 g/L was obtained when a promoter mutant with a 9.8-fold higher strength compared to the wild-type promoter was used for ProB expression. The use of stronger promoter variants did not further improve L-proline production. In conclusion, a promoter library was constructed based on a native C. glutamicum promoter PodhA. The new promoter library should be useful for systems metabolic engineering of C. glutamicum. The strategy of mutating native promoter may also guide the construction of promoter libraries for other microorganisms.

Gas chromatography-mass spectrometry (GC-MS) and its application in metabonomics / 生物工程学报

Metabonomics involves the unbiased quantitative and qualitative analysis of the complete set of metabolites present in cells, body fluids and tissues (the metabolome) based on modern analytic technique with high throughput, high sensitivity, and high resolution. Gas chromatography-mass spectrometry (GC-MS) is used to gain qualitative results of detected metabolites for biological samples as it provides superior distinguishability, detection sensitivity and integrated standard mass spectrometry library. In this article, the historic developments of GC-MS and its application in metabonomics in the past several years were reviewed. Firstly, the classification and the derivative methods of GC-MS were introduced. Subsequently, sample pretreatment process, qualitative and quantitative analysis and data analysis during detecting metabolites by GC-MS were introduced, then its application in microorganism, plant and disease diagnosis was systematically summarized. Finally, the problems in metabonomics study based on GC-MS and the research prospect in the future were discussed.