Investigation Of Allosteric Regulation Of Dihydrodipicolinate Synthase For Rational Design Of Bacterial Strains For Lysine Bioproduction

L-lysine is one of the most important essential amino acids in human and animalnutrition. L-lysine is widely used as an animal feed additive with a huge marketvolume of more than1.5million tons per year. The most widely used method forL-lysine production in industry is bioproduction by fermentation. However, there is amajor bottleneck in the bioproduction of lysine, namely aspartate kinase Ⅲ(AKⅢ)and dihydrodipicolinate synthase (DHDPS) are feedback inhibited by L-lysine in vivo.DHDPS is the first reaction specific for the biosynthesis of L-lysine. Ingram-negative bacteria such as Escherichia coli, DHDPS is allosterically inhibited byL-lysine and thus becomes one of the most important limiting factors for L-lysineproduction in this microorganism. However, in most gram-positive bacteria, DHDPSis not inhibited by L-lysine at all. The underlying mechanism for this difference is notclear. A deeper understanding of it is desired for metabolical engineering of L-lysinebioproduction. The major work and achievemenchts of this thsis toward this goal areas follows:1. Sequence and structure comparisons were used to investigate the key determinantsof feedback regulation in two industrially important DHDPSs, the L-lysine-sensitiveDHDPS from E. coli (eDHDPs) and L-lysine-insensitive DHDPS fromCorynebacterium Glutamicum (cgDHDPs) Feedback inhibition of E. coli DHDPSwas successfully alleviated after substitution of the residues around the inhibitor’sbinding sites with those of C. glutamicum DHDPS. In agreement with the prediction,the mutations H56K and E84T totally destroyed the inhibition of E. coli DHDPS byL-lysine. Mutations at point49or51partly disrupted the inhibition, but caused someactivity loss of the enzyme.2. To investigate whether the loss of L-lysine inhibition in cgDHDPS are only due tothe mutations of residues within the L-lysine binding sites, different cgDHDPSmutants were constructed. Mutagenesis of the lysine binding sites of C. glutamicumDHDPS according to E. coli DHDPS didn’t recover the expected feedback inhibitionbut an activation of DHDPS by L-lysine, probably due to differences in the allosteic signal transduction in the DHDPS of these two organisms.3. E. coli DHDPS mutants were over-expressed in E. coli for improved L-lysineoverproduction. Over-expression of L-lysine-insensitive E. coli DHDPS mutants in E.coli MG1655or BW25113resulted in an improvement of L-lysine production yieldby67%in shake flask cultures and47%in fermentation in bioreactor.