Search

Biotransformation of L-lysine (L-Lys) to L-pipecolic acid (L-PA) using lat-expressing Escherichia coli has been reported (Fujii et al., Biosci. Biotechnol. Biochem., 66, 622-627 (2002)). The rate-limiting step of this biotransformation seemes to be the transport of L-Lys into cells. To improve the L-PA production rate, we attempted to increase the rate of L-Lys uptake. E. coli BL21 carrying a plasmid with lat and lysP (pRH125) caused a 5-fold increase in the rate of L-PA production above the level of cells carrying a plasmid with lat (pRH124). Moreover, E. coli BL21 carrying a plasmid with lat, lysP, and yeiE (pRH127) caused a 6.4-fold increase in the rate of L-PA production above the level of cells carrying pRH124. Our results from RT-PCR experiments and the sequence similarity of YeiE to LysR transcriptional regulators suggest the possibility that yeiE expression induces lysP expression. The amplification of lysP, or rather both lysP and yeiE, increases the rate of L-PA production using lat-expressing E. coli.

The enzyme involved in the reduction of delta1-piperideine-6-carboxylate (P6C) to L-pipecolic acid (L-PA) has never been identified. We found that Escherichia coli JM109 transformed with the lat gene encoding L-lysine 6-aminotransferase (LAT) converted L-lysine (L-Lys) to L-PA. This suggested that there is a gene encoding "P6C reductase" that catalyzes the reduction of P6C to L-PA in the genome of E. coli. The complementation experiment of proC32 in E. coli RK4904 for L-PA production clearly shows that the expression of both lat and proC is essential for the biotransformation of L-Lys to L-PA. Further, We showed that both LAT and pyrroline-5-carboxylate (P5C) reductase, the product of proC, were needed to convert L-Lys to L-PA in vitro. These results demonstrate that P5C reductase catalyzes the reduction of P6C to L-PA. Biotransformation of L-Lys to L-PA using lat-expressing E. coli BL21 was done and L-PA was accumulated in the medium to reach at an amount of 3.9 g/l after 159 h of cultivation. It is noteworthy that the ee-value of the produced pipecolic acid was 100%.

L-Lysine 6-aminotransferase (LAT) is an enzyme involved in L-lysine catabolism in a wide range of living organisms. LAT from Flavobacterium lutescens IFO3084 was purified, and its structural gene (lat) was cloned, sequenced and expressed in Escherichia coli. Native PAGE analysis of purified LAT gave a single band corresponding to a molecular weight of about 110,000. lat encoded a protein of 493 amino acids with a deduced molecular weight of 53,200, which is very close to that of purified LAT determined on SDS-PAGE. Expression of lat in E. coli revealed that lat encodes a single subunit protein leading to LAT activity. These data suggested that LAT from F. lutescens IFO3084, like most other aminotransferases, is derived from a single ORF and is active as a homodimer.