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Study of regulation of branched-chain amino acid biosynthesis in C.glutamicum and construction of valine producing strain
Holátko, Jiří ; Pátek, Miroslav (advisor) ; Petříček, Miroslav (referee) ; Svobodová, Jaroslava (referee)
L-valine is an essential amino acid with a great medical and industoial importance and the need for efficient bacterial producersof this amino acid is increasing. Corynebacteriumglutamicum is a suitable organism for construction of such productionstrainsandthusbiosynthesisofvaline and expressionofgenes involved in its pathway are intensively studed. In this PhD. thesis, analysis of regulationand modulationof expressionwas carriedout with: (l) genes,which code for common enzymesof all branched-chainamino acids; (2) genes,whose products are involved in the competing biosynthesis pathways of L-isoleucine and L-leucine; and (3) the genethatdeterminesthe key enzyme in the catabolism of pyruvate. Results were used for construction of C. glutamicum production shains with deregulatedbiosynthesisof valine, restrictedbiosynthesisof isoleucine and leucine and optimum distributionof intermediatesof valine. The production strains were manipulated via modification of promoters in the chromosome. Promoters of the ilvA, leuA, ilvD, ilvE, and aceE genes were modified by site-directedmutagenesisof their -10 regions. All these alterationsincreased production of valine by the modified strains. Introduction of weak P-ilvA and P-leuA promotersinto the C. glutamicumchromosomeresultedin an intrinsic lowJevel biosynthesisof...
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