Department of Molecular Science and Technology

Contents.

L-Histidine  Amino acids are commercially important as they are widely used in food, pharmaceutical, cosmetics and animal feed industries with annual market growth rate of 5-7% worldwide.  L-Histidine is one of the 20 standard proteinogenic amino acids present in proteins of all living organisms.  L-Histidine is an essential amino acid present in the human body and other mammals, and its inclusion through diet is necessary for normal metabolic functions.  L-Histidine plays a key role in production of red and white blood cells. It has been reported that rheumatoid arthritis sufferers have abnormally low blood levels of L-histidine  L-Histidine has a neurotransmitter or a neuromodulator function in the mammalian central nervous system and it is present in many proteins controlling the transmission of metal elements.

The biosynthetic pathway of L-histidine in E.coli, regulations involved, and the strategies for constructing the L-histidine producing base strains

Construction of engineered E.coli strains for the production of L-Histidine.  Feedback inhibition of ATP phosphoribosyltransferase were removed by SDM.  Histidine promoter replacement.  Enhanced production of L-Histidine by coamplification of the hisG and hisDCBHAFI.  Transcriptome analysis of the L-Histidine production strain.  Cell growth inhibition test.(L-Histidine tolerance test)  Engineering of L-Histidine transport system.  Feedback inhibition of ATP phosphoribosyltransferase were removed by SDM.  Construction of cloning vector that is constitutive tac, trc promoter  Flask cultivation / Batch fermentation of W3110 that harboring mutant hisG and quantification of L-histidine in medium.  Cell growth inhibition test using L-Histidine.

L-histidine concentration Fermentation : 433mg/L Flask cultivation : 411mg/L

L-Histidine concentration 2.65mM

 Promoter and mutant hisG replacement.

 Promoter use that is not affected to regulation by glucose. Constitutive tac promoterConstitutive trc promoter -Cloning of hisG mutants. -Analysis of promoter strength and compare with original vector by GFP. -Problem : RBS + Spacer

 Cell growth inhibition test using histidine alalogue.  Study of transcriptome analysis of the amino acid production strain. L-Histidine analogue

L-Histidine Synthetic RNA devices to expedite the evolution of metabolite- producing microbes, Jina-Yang et al., 2013

Synthetic RNA devices to expedite the evolution of metabolite- producing microbes, Jina-Yang et al., 2013

 Crocin (C 44 H 64 O 24 ) is a natural carotenoid chemical compound that is found in the flowers crocus and gardenia. It is the diester formed from the disaccharide gentiobiose and the dicarboxylic acid crocetin. It has a deep red color and forms crystals with a melting point of 186 °C. When dissolved in water, it forms an orange solution.  Crocin is the chemical ingredient primarily responsible for the color of saffron.  Crocin has been shown to be a potent antioxidant. It has also been shown to have an anticarcinogenic action. In rodents, crocin has been shown to have antidepressant properties and one study reports aphrodisiac properties.

A C35 Carotenoid Biosynthetic Pathway. D. Umeno et al, 2003

Evolution of a Pathway to Novel Long-Chain Carotenoid. D. Umeno et al, 2004

CrtN, CrtP Original pathway for production of crocetin Alternative pathway for production of crocetin Engineering of enzymes for production of Crocetin in E.coli

Creation of Novel C 20 Carotenoid Biosynthetic Pathway.  C 35, C 45, C 50 등의 unnatural 한 carotenoid 를 CrtM 의 mutation 을 이용하여 E.coli 내에서 확보한 논문을 바탕으로 C 20 의 carotenoid 를 만드는 새로운 pathway 를 만들고 CrtN / CrtI 를 이용하여 이중결합을 생성하고, CrtP 로 양 끝을 aldehyde 기를 붙임으로서, E.coli 내에서 Crocetin dialdehyde 를 확보함이 그 목적.  pUCM_CrtM* Library (Library : 9240, Mutant : 109 )

- Cloning of trGPP synthase from pJBEI-6409.