Title:
METHOD FOR SCREENING AND QUANTIFYING ISOPRENE BIOSYNTHESIS ENZYME ACTIVITY
Document Type and Number:
WIPO Patent Application WO/2014/129808
Kind Code:
A1
Abstract:
The present invention is a novel technology of performing high-throughput screening of various enzymatic activities with high sensitivity using artificial genetic circuits. More specifically, the invention is a novel screening and quantifying methods of isoprene biosynthesis enzymes activity using an artificial genetic circuit capable of sensing isoprene. The artificial genetic circuit (IspGESS) is comprising a gene encoding a isoprene-sensing transcriptional regulator which recognizes isoprene, at least one reporter gene such fluorescence protein-encoding genes, a isoprene-sensing transcriptional regulator binding region and promoters for genes encoding isoprene-sensing transcriptional regulators and reporter proteins. IspGESS detects isoprene liberated from many enzymatic reactions and allows to measure the activity of reporter genes, such as fluorescent reporter genes and antibiotic resistance genes. This system is shown that it is a widely applicable tool for high throughput and quantitative screening of isoprene biosynthesis enzymes and MEP/MVA pathway enzymes. Thus, the invention can be advantageously used in the protein engineering technology for enzyme modification. Particularly, it can quantitatively investigate enzymatic activity, and thus can be applied to molecular evolution technology.
Inventors:
LEE SEUNG-GOO (KR)
LEE DAE HEE (KR)
KIM SEONG KEUN (KR)
KIM SEO HYEON (KR)
GAM JONGSIK (KR)
RHA EUGENE (KR)
KIM HASEONG (KR)
CHOI SU-LIM (KR)
CHOI EUISUNG (KR)
BINDU SUBHADRA (KR)
LEE DAE HEE (KR)
KIM SEONG KEUN (KR)
KIM SEO HYEON (KR)
GAM JONGSIK (KR)
RHA EUGENE (KR)
KIM HASEONG (KR)
CHOI SU-LIM (KR)
CHOI EUISUNG (KR)
BINDU SUBHADRA (KR)
Application Number:
PCT/KR2014/001360
Publication Date:
August 28, 2014
Filing Date:
February 20, 2014
Export Citation:
Assignee:
KOREA RES INST OF BIOSCIENCE (KR)
International Classes:
C12Q1/25; C12Q1/68
Foreign References:
| US20120238470A1 | 2012-09-20 |
Other References:
YANG ET AL.: "Enhancing production of bio-isoprene using hybrid MVA pathway and isoprene synthase in E. coli", PLOS ONE, vol. 7, no. 4, 27 April 2012 (2012-04-27), pages 1 - 7, XP055046587, DOI: doi:10.1371/journal.pone.0033509
XUE ET AL.: "Enhancing isoprene production by genetic modification of the 1-deoxy-d-xylulose-5-phosphate pathway in Bacillus subtilis", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 77, no. 7, 4 February 2011 (2011-02-04), pages 2399 - 2405, XP055046378, DOI: doi:10.1128/AEM.02341-10
ZHAO ET AL.: "Biosynthesis of isoprene in Escherichia coli via methylerythritol phosphate (MEP) pathway", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 90, no. 6, 6 April 2011 (2011-04-06), pages 1915 - 1922
SHARKEY ET AL.: "Evolution of the isoprene biosynthetic pathway in kudzu", PLANT PHYSIOLOGY, vol. 137, no. 2, February 2005 (2005-02-01), pages 700 - 712, XP002552522, DOI: doi:10.1104/pp.104.054445
DATABASE genbank [O] 14 August 2008 (2008-08-14), Database accession no. CAL69918.1
XUE ET AL.: "Enhancing isoprene production by genetic modification of the 1-deoxy-d-xylulose-5-phosphate pathway in Bacillus subtilis", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 77, no. 7, 4 February 2011 (2011-02-04), pages 2399 - 2405, XP055046378, DOI: doi:10.1128/AEM.02341-10
ZHAO ET AL.: "Biosynthesis of isoprene in Escherichia coli via methylerythritol phosphate (MEP) pathway", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 90, no. 6, 6 April 2011 (2011-04-06), pages 1915 - 1922
SHARKEY ET AL.: "Evolution of the isoprene biosynthetic pathway in kudzu", PLANT PHYSIOLOGY, vol. 137, no. 2, February 2005 (2005-02-01), pages 700 - 712, XP002552522, DOI: doi:10.1104/pp.104.054445
DATABASE genbank [O] 14 August 2008 (2008-08-14), Database accession no. CAL69918.1
Attorney, Agent or Firm:
LEE, Cheo Young et al. (Gangnam-gu, Seoul 135-748, KR)
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