Monday, July 30, 2007
P42
Production of L-lysine from methanol in Methylophilus methylotrophus: Isolation of new lysE allele encoding a possible exporter for L-lysine/L-arginine in methylotrophs
Hisashi Yasueda, Institute of Life Sciences, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, 210-8681, Japan and Yoshiya Gunji, Fermentation and Biotechnology Labs., Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, 210-8681, Japan.
As an alternative raw material for amino acid fermentation, we are planning to use methanol. We have developed an L-lysine producer strain from an obligate methylotroph, M. methylotrophus, in which a putative L-lysine exporter LysE24, originating from LysE of Corynebacterium glutamicum, was employed for efficient excretion of L-lysine. lysE24 was a spontaneously mutated lysE gene, and the lysE24 allele induced L-lysine production even in the wild-type strain of M. methylotrophus. In addition, M. methylotrophus containing lysE24 gene displayed a tolerance to AEC (S-(2-aminoethyl)-L-cysteine, an L-lysine analog) in the growth. Since lysE24 had a frame-shift mutation in the middle of open reading frame (ORF) in the original lysE locus, we speculated that LysE24 is working in the combined form of the divided polypeptides.
To further analyze the structure and function relationship of the putative L-lysine exporter, we attempted to acquire other lysE alleles encoding functional exporters in methylotroph. We mutagenized a wild type lysE expression plasmid with hydroxylamine and introduced the plasmid into M. methylotrophus. Among the AEC-tolerant transformants, we could isolate new lysE mutant genes which induced M. methylotroph to produce L-lysine and L-arginine in the culture medium. The change of Gly to Ser at 56th of the amino acid residue in the LysE protein was commonly detected in these mutant genes. In addition, these lysE mutant genes could also induce L-lysine and L-arginine production in another methylotroph, Methylobacillus glycogenes. The results from these studies will be discussed.
To further analyze the structure and function relationship of the putative L-lysine exporter, we attempted to acquire other lysE alleles encoding functional exporters in methylotroph. We mutagenized a wild type lysE expression plasmid with hydroxylamine and introduced the plasmid into M. methylotrophus. Among the AEC-tolerant transformants, we could isolate new lysE mutant genes which induced M. methylotroph to produce L-lysine and L-arginine in the culture medium. The change of Gly to Ser at 56th of the amino acid residue in the LysE protein was commonly detected in these mutant genes. In addition, these lysE mutant genes could also induce L-lysine and L-arginine production in another methylotroph, Methylobacillus glycogenes. The results from these studies will be discussed.
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