Research
High-throughput screening techniques for high-value small molecules can find intensive applications in the studies of biosynthesis of these molecules. The production of small molecules from the biosynthetic pathways can be impacted by the activities of key enzymes and the balance of the expression levels of pathway enzymes. Elegant high-throughput screening techniques for small molecules can greatly aid the optimization processes of the biosynthetic pathways for improving biosynthetic efficiency.
1-Deoxynojirimycin (1-DNJ) is one of the iminosugars. Known as α-glucosidase inhibitors, 1-DNJ and its derivatives have shown potential therapeutic effects on diabetes, HIV infection as well as Gaucher’s disease. 1-DNJ is mainly found in plants. However, low amounts are found in some microorganisms. The majority of small molecules are not ready to be easily detected with colorimetric or fluorescent screening techniques, and 1-DNJ is one of them. Therefore, a simple, sensitive and cost-effective technique that allows high-throughput screening of endogenous production 1-DNJ is essential in the study of 1-DNJ biosynthesis.
Researchers in Prof. Shuang-Yan Tang’s group have developed a solid-phase high-throughput screening technique based on the inhibitory effects of 1-DNJ on the hydrolysis activity of the β-glycosidase LacS from Sulfolobussolfataricus toward X-GAL.
The expression of the TYB gene cluster from Bacillus spp. in E.coli led to 1-DNJ production. The TYB gene cluster contained the gabT1, yktc1 and gutB1 genes. The lacS gene was integrated into the E. coli BW25113 chromosome, resulting in strain BWLacS, which gave blue-color colonies growing on LB agar plate supplemented with X-GAL. When BWLacS cells expressing the TYB gene cluster were plated onto the agar plates described above, the cells producing higher titers of 1-DNJ showed lighter blue (or whiter) color compared with lower producers.
This high-throughput screening method was used to optimize the TYB gene cluster for higher production of 1-DNJ. The random mutagenesis library of the key enzyme GutB1, a putative oxidoreductase, was constructed and screened. A mutant strain with 54% increase in 1-DNJ production was obtained. In order to optimize the expression levels of the pathway enzymes, a mutagenesis library of the intergenic regions of the TYB gene cluster was generated and screened. A mutant strain with 68% increase of 1-DNJ production was selected.
These results have demonstrated the effectiveness of the high-throughput screening technique for 1-DNJ. This technique also has potential applications in the screening of inhibitors of glycosidases.
The work was supported by grants from Ministry of Science and Technology of China and the Natural Science Foundation of China. The results were published in Scientific Reports.
http://www.nature.com/srep/2015/150224/srep08563/full/srep08563.html
High-throughput screening technique for 1-deoxynojirimycin (Image from Prof. TANG's lab)
Contact:
Dr. Shuang-Yan Tang
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering，Institute Of Microbiology，Chinese Academy of Sciences，100101，Beijing, China
E-mail: tangsy@im.ac.cn