Choong-Min Kang
California State University, USA
Title: Reduction of feedback inhibition in homoserine kinase (ThrB) enhances the L-threonine biosynthesis
Biography
Biography: Choong-Min Kang
Abstract
L-threonine is a limiting amino acid in livestock diets and its defi ciency results in not only malnutrition of the animals
but also releasing increased volumes of nitrogen compounds to the environment. Addition of L-threonine into livestock
feed is therefore important for healthier animals as well as a cleaner environment. Moreover, L-threonine is utilized in both
pharmaceutical and cosmetic industries. Currently, L-threonine is produced by E. coli, which makes the purifi cation of
L-threonine diffi cult because it produces endotoxins. Th us, we seek to over-produce L-threonine by using Corynebacterium
glutamicum, a GRAS (generally regarded as safe) microorganism. C. glutamicum produces L-threonine from aspartate
through an enzymatic pathway involving aspartate kinase (LysC), aspartate semialdehyde dehydrogenase (Asd), homoserine
dehydrogenase (Hom), homoserine kinase (Th rB) and threonine synthase (Th rC). Among these, LysC, Hom and Th rB are
feedback inhibited by the end-product, L-threonine. Releasing the feedback inhibition in LysC and Hom through mutating their
allosteric site has successfully increased L-threonine biosynthesis. However, it has been unsuccessful to remove the feedback
inhibition in Th rB because L-threonine inhibits the enzyme by competing with L-homoserine (substrate) for the same active
site. To genetically separate the catalytic activity and the feedback inhibition in Th rB of C. glutamicum, we mutated a residue
at the gate of the active site into various amino acids such as hydrophobic residues (Leu and Val) and hydrophilic residue (Ser
and Gly). Enzymatic kinetics with the wild-type and mutant forms of Th rB showed that one specifi c mutation increased Ki
for L-Th reonine about 5-fold while it increased Km for homoserine only 2-fold. More importantly, when we introduced this
mutation into C. glutamicum and E. coli, it increased approximately 20% of L-threonine production. Our approach can be
applied to other metabolic enzymes that are similarly regulated by competitive feedback inhibition.