758 Ramesh malothu. et al. / International Journal of Biological & Pharmaceutical Research. 2012; 3(6): 758-761. e- ISSN 0976 - 3651 Print ISSN 2229 - 7480 International Journal of Biological & Pharmaceutical Research Journal homepage: www.ijbpr.com OVER EXPRESSION OF ASPARTOKINASE GENE IN CORYNEBACTERIUM GLUTAMICUM FOR HIGHER PRODUCTION LEVELS OF L-LYSINE Ramesh Malothu 1 , Meenu Singh 3 , Adarsh G 1 , M.Abdul Rajak 2 , S.V. Satyanarayana 4 and D. Muralidhara Rao* 2 1 Genetic Engineering Laboratory, Department of Biotechnology, JNTU Kakinada-UCEV, Andhra Pradesh, India. 2 Bio Process laboratory, Department of Biotechnology, S. K. D University, Anantapur, Andhra Pradesh, India. 3 Research Scholar, Department of Pharmacy, NIMS University, Shoba Nagar, Jaipur. 4 Department of Chemical Engineering, JNTUA-CEA, Anantapur, Andhra Pradesh, India. ABSTRACT The Identification of novel target genes to improve the lysine production is a big task for researchers. The present research work deals with the over expression of Aspartokinase gene in PBR322 plasmid present under a constitutive promoter. Aspartokinase upon overexpression in recombinant Corynebacterium glutamicum ATCC 13032 cells exhibited greater L-lysine production from 2.9 mg/mL by traditional method to 3.5mg/mL after 96 h of fermentation period. The study revealed that the optimum parameters were: temperature of 30°C, 7.5 pH, agitation speed of 200 rpm, 1 vvm air flow rate and glucose concentration of 90g/l to achieve maximum yield of L-lysine. Key Words: Corynebacterium glutamicum, over expression, L-lysine, Recombinant Plasmid. INTRODUCTION L – Lysine is the third most frequently produced amino acid in a large industrial scale. (Meenusingh et al., 2011). Today L-Lysine production involves fermentation in very large fermentation tanks, often exceeding hundreds of cubic meters, which makes it challenging to obtain homogeneity in the fermenter and to maintain sufficient mass-transfer rates. Normally traditional stirred-tank reactors are used since they allow for a high specific power intake, which makes it possible to obtain high oxygen transfer rates (Kelle et al., 2005). To optimize lysine yield and productivity the process is normally run as a fed-batch or repeated fed-batch process. Attempts have been made to develop a continuous process (Hirao et al., 1989). Corresponding Author D. Muralidhara Rao Email: muralidhararao@yahoo.com However, due to numerous practical aspects such as sterility and strain stability in industrial scale, continuous strain production is generally not applied (Kelle et al., 2005).The common practice of developing amino acid overproducing strains by mutagenesis and selection is a very well established technique (Rowlands, 1984). Mutagenic procedures were optimized in terms of the mutagen used and the dose applied. Selection procedures were designed to allow maximum expression and detection of the desirable mutant types. So far the improvement of amino acid-producing Corynebacterium glutamicum strains has mainly been carried out by an iterative procedure of mutagenesis and selection. Overexpression or deletion of genes in microorganisms via recombinant DNA techniques is the most powerful method for the construction of strains with the desired genotype. Defined improvements were added to strains generated by random mutagenesis. These improvements usually involved the introduction of feedback resistant biosynthetic genes or the IJBPR