OPERON CONSTRUCTION CONTAINING alsS, ilvC, ilvD AND kivd GENES IN Escherichia coli FOR ISOBUTANOL PRODUCTION FROM GLUCOSE

• Main Author: Bagus Kennardi - NIM : 21114014, Gabriel
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/27428
• Institution: Institut Teknologi Bandung

<p align="justify">Isobutanol is a biofuel considered to be a potential gasoline substitute. However, isobutanol production is difficult because there is no native organism that can produce isobutanol. A biosynthetic pathway to produce isobutanol had been designed to utilize pyruvate produced from glucose breakdown by glycolysis in Escherichia coli (E. coli). This biosynthetic pathway consisted of acetolactate-synthase (ALS), ketol-acid reductoisomerase (KARI), dihydroxy-acid dehydratase (DHAD), alpha-ketoisovalerate decarboxylase (KDC) and alcohol dehydrogenase (ADH) enzymes. Since E. coli DH5&#945; do not have ALS, KARI, DHAD and KDC, the genes coding for the protein is needed to be cloned and overexpressed in E. Coli DH5&#945;. Plasmid contains an operon controlled by lac promoter and lac operator consisting of alsS (coded ALS from Bacillus subtilis), ilvC (coded KARI from E. coli MG1655) and ilvD (coded DHAD from E. coli MG1655) genes, obtained from previous research, and operon sequences have been confirmed by DNA sequencing. kivd gene (coding KDC from Lactococcus lactis) was obtained from iGEM 2013 kit. kivd was amplified by PCR and inserted into pJET 1.2 blunt. kivd gene was then added into 3’ end of previous operon using restriction-ligation technique. The plasmid constructed was then transfered into E. coli DH5&#945; using heatshock. The recombinant genes were expressed using IPTG (isopropyl-&#946;-D-1-thiogalactopyranoside) induction. The SDS PAGE results were inconclusive, however isobutanol was detected by Gas Chromatography Mass Spectrometry – Selected Ion Monitoring (GC-MS-SIM) from 48 hours fermentation culture at 30oC (1,17%). An operon regulated by the lac promoter-operator containing four genes for the biosynthesis of isobutanol has been constructed and cloned in E. coli. The isobutanol production was not optimal due to weak expression and repression by glucose, which was used as substrate.<p align="justify">