| Summary: | 博士 === 國立臺灣大學 === 農業化學系 === 84 === The genes of ask, hom, thrB and hom-thrB amplified from Bre-
vibacterium divaricatum strains by PCR, were cloned in
pSUMN18, and manipulated for the construction of lysine
producers. "Integron"-bearing integrative plasmids were
constructed, and both of the marker-exchange and
integrational mutagenesis were applied for the disruption
of hom, thrB and hom-thrB genes. Several auxotrophic
integrants were obtained from both methods. The integrants
site-specifically inactivated at hom gene were homoserine-
requiring. Those blocked at thrB gene required thre- onine,
and those disrupted at hom and hom-thrB genes were threo- nine
and homoserine auxotrophic. Bothe single and double cross-
over homologous recombination mechanisms were proposed to
expla- in the integration and replacement. The auxotrophic
integrants accumulated1-3% lysine-HCl in the culture broth.
Those disrupted at hom and hom-thrB produced more lysine than
those disrupted at thrB. The integrants derived from double
crossover were extremely stable. Nevertheless, those der- ived
from single crossover were less stable, and the lysine pro-
uction was thus degenerated after repeated transfer.
Amplification of pSUAK-45 containing feedback released ask
gene conferred AEC resistance as well as higher
aspartokinase activity and lysine productivity in its hosts.
When optimized, 12g/L lysine-HCl could be produced in B.
divaricatum ATCC 14020. In fed-batch fermentation of the
integrants, a feeding strate- gy based on the excellent genetic
stability and the quick respon- se of threonine depletion to
the disolved oxygen was conducted. The growth rate and
lysine productivity of the cells were main- tained for a long
time. After 68-hour-cultivation, the lysine-HCl in the culture
broth was 66.6g/L, and the conversion yield to the total sugar
was 31% (g/g).
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