Production of Lysine by Lactobacilli or Aspergillus Ficuum

Abstract

In the animal feed industries, there is a global need for adding certain nutritional ingredients to augment deficits usually associated with plant-based materials. As a result, the industrial practices require direct addition of ingredients such as amino acids and vitamins. One of the key ingredients in this context is lysine. Alternately, the same goal can be achieved indirectly through in situ co-culturing of microorgan-isms. The focus of this thesis was genetic improvement of bacterial and /or fungal mutants, which could over-produce lysine. The accumulation of free lysine during microbial growth serves this end based on de-regulation of the lysine biosynthetic pathway. Microorganisms used in this thesis were nine species of lactobacilli and Aspergillus ficuum. Having in mind the highly complex nutritional requirements of lacto-bacilli, the assessment of possible lysine auxotrophy was performed. No lysine auxotrophs were found and the choice of Lactobacillus plantarum as the working species among nine others was based on its higher growth rate in minimal medium. Selection of mutants that overproduced lysine was carried out in the minimal medium supplemented with the following lysine analogs: S-aminoethyl-L-cysteine (AEC), DL-aspartic acid-ƒÒ-hydroxamate (DL-ASP), ƒÒ -fluoropyruvic-acid (FPA), L-lysine hydroxamate (LHX) and diaminopimelic acid (DAP). In L. plantarum, LHX was shown to be the most potent inhibitor; although, the bacterium demonstrated high resistance to all the analogs tested. The inhibition by LHX was obtained only after significant alteration of the minimal medium M3. Furthermore, the mutant # 34, resistant to 2 mM of LHX, secreted only 4.52 £gM of lysine in M3. To address the question of low lysine yield obtained by L. plantarum, thorough study of the regulation of aspartokinase (AK) was performed. It was found that AK exists as four isozymes, threonine sensitive, methionine sensitive and two lysine sensitive isozymes. Activity differed with respect to the growth stage of L. plantarum. Beside lysine, threonine and methionine have influenced the repression of AK isozymes, which suggested that effective lysine over-production could be obtained only if AK is simultaneously resistant to threonine and methionine analogs. In the case of A. ficuum, mutant #5-10 secreted 29.25 £gM of lysine in the minimal medium, which was approximately 30 % higher than that of the wild type. DL-ASP was found as the most potent inhibitor only after the conidia were soaked for 8 h in 0.03 % Tween 80. Ammonium phosphate as a nitrogen source enhanced lysine secretion in A. ficuum compared to five other nitrogen sources tested.