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Link to original content: https://pubmed.ncbi.nlm.nih.gov/25708517
Design and application of a novel high-throughput screening technique for 1-deoxynojirimycin - PubMed Skip to main page content
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. 2015 Feb 24:5:8563.
doi: 10.1038/srep08563.

Design and application of a novel high-throughput screening technique for 1-deoxynojirimycin

Peixia Jiang  1 , Shanshan Mu  2 , Heng Li  2 , Youhai Li  1 , Congmin Feng  1 , Jian-Ming Jin  3 , Shuang-Yan Tang  1
Affiliations

Design and application of a novel high-throughput screening technique for 1-deoxynojirimycin

Peixia Jiang et al. Sci Rep. .

Abstract

High-throughput screening techniques for small molecules can find intensive applications in the studies of biosynthesis of these molecules. A sensitive, rapid and cost-effective technique that allows high-throughput screening of endogenous production of the natural iminosugar 1-deoxynojirimycin (1-DNJ), an α-glucosidase inhibitor relevant to the pharmaceutical industry, was developed in this study, based on the inhibitory effects of 1-DNJ on the activity of the β-glycosidase LacS from Sulfolobus solfataricus. This technique has been demonstrated effective in engineering both the key enzyme and the expression levels of enzymes in the 1-DNJ biosynthetic pathway from Bacillus atrophaeus cloned in E. coli. Higher biosynthetic efficiency was achieved using directed evolution strategies.

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Figures

Figure 1
Figure 1. The proposed biosynthetic pathway of 1-DNJ, adapted from Horenstein.
Figure 2
Figure 2. Inhibition of LacS activity by 1-DNJ.
(a) In vitro inhibition of oNPG hydrolysis. (b) In vivo inhibition of X-GAL hydrolysis. Strain BWLacS expressing LacS was grown on LB agar supplemented with 1 mM L-arabinose, 40 μg·mL−1 X-GAL and 0 (left) or 0.5 mM (right) 1-DNJ.
Figure 3
Figure 3. The 1-DNJ production of strain BWLacS harboring various plasmids cultured for 14 h.
Figure 4
Figure 4. The specific activity of wild type () and mutant () GutB1 on various substrates.
Figure 5
Figure 5. The sfGFP fluorescence measured from strain BWLacS expressing sfGFP-fused Yktc1 or GutB1 in both wild-type (pDNJ6, ) and mutant (pM13, ) TYB gene clusters.
See this image and copyright information in PMC

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