The Influence Of Trehalose Synthesis On The Relative Environmental Tolerance And Glutamate Production In Corynebacterium Glutamicum ATCC 13032

Environmental tolerance of microbe has closely relationship to the microbial fermention of amino,and the trehalose is an important factor to the environmental tolerance of microbial cells.In this research, An OtsAB pathway deficient mutant ATCC 13032ΔotsAB and an otsAB genes over-expressing strainATCC13032(pECotsAB) were constructed from Corynebacterium glutamicum ATCC 13032 .The intracellular trehalose contents of these two mutants and wild type strain cultured in different conditions were measured, and the environmental tolerance of these strains to osmolality ,temperature and glucose was compared.we also compared the glutamate synthesis capacity of these three strain cultured in mediums for glutamate producing with different glucose concentrations.The major research content and results are summarized as follows:(1)First, an OtsAB pathway deficient mutant ATCC 13032ΔotsAB was constructed from Corynebacterium glutamicum ATCC 13032 by SOEing-PCR and homologous recombination; and an otsAB genes over-expressing strain was constructed by using Corynebacterium glutamicum-E.Coli shuttle vector pEC-XK99E at the same time. By real time quantitative PCR,gene knockout and overexpression results were verified.(2)At different culture temperature, NaCl concentrations and glucose concentrations,the intracellular trehalose contents and the growth rate of ATCC 13032 and the mutant strains were compared.The results show that,otsAB gene knockout and overexpression of C.glutamicum significantly affect its intracellular trehalose synthesis capability,and affect its tolerance to high temperature,high salt and high glucose enviroment.High intracellular trehalose synthesis capablility can increase the tolerance of C.glutamicum to the environment.(3)The ATCC 13032 and its mutant strains were cultured in mediums for glutamate producing with different glucose concentrations by shaking flask, and the concentrations of glutamate were detection by HPLC, The results showed that,at the fermentation medium with different glucose concentrations, the change of intracellular trehalose content has significant impact on the growth rate and glutamate production of Corynebacterium glutamicum, especially in the higher glucose concentration conditions, ATCC 13032 ( pECotsAB) showed significant growth and glutamate production advantages.