Enhancement of pipecolic acid production by the expression of multiple lysine cyclodeaminase in the Escherichia coli whole-cell system
Yeong Hoon HanTae Rim ChoiYe Lim ParkJun Yong ParkHun suk SongHyun Joong KimSun‐Mi LeeSol Lee ParkHye Soo LeeShashi Kant BhatiaRanjit GuravYung‐Hun Yang
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Abstract:
Pipecolic acid, a non-proteinogenic amino acid, is a metabolite in lysine metabolism and a key chiral precursor in local anesthesia and macrolide antibiotics. To replace the environmentally unfriendly chemical production or preparation procedure of pipecolic acid, many biological synthetic routes have been studied for a long time. Among them, synthesis by lysine cyclodeaminase (LCD), encoded by pipA, has several advantages, including stability of enzyme activity and NAD+ self-regeneration. Thus, we selected this enzyme for pipecolic acid biosynthesis in a whole-cell bioconversion. To construct a robust pipecolic acid production system, we investigated important conditions including expression vector, strain, culture conditions, and other reaction parameters. The most important factors were introduction of multiple pipA genes into the whole-cell system and control of agitation. As a result, we produced 724 mM pipecolic acid (72.4% conversion), and the productivity was 0.78 g/L/h from 1 M L-lysine after 5 days. This is the highest production reported to date.Keywords:
Pipecolic acid
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Abstract— Employing both the intraventricular and intraperitoneal injection techniques, 14 C‐ l ‐lysine at non‐overloading concentrations was found to be metabolized to l ‐ 14 C‐pipecolic acid at significantly high levels in the rat. Labeled pipecolic acid in the brain and liver was only found at rather low levels 24 h after intraperitoneal administration of 14 C‐ l ‐lysine regardless of non‐labeled lysine metabolite overload. A marked enhancement of pipecolic acid labeling was only found in the brain when 14 C‐ l ‐lysine was intraventricularly administered to animals under various lysine metabolite overloads. While overloading doses of non‐labeled saccharopine or α‐aminoadipate did not significantly alter the labeling patterns of pipecolic acid in the brain, liver or urine when 14 C‐ l ‐lysine was intraperitoneally administered, pipecolate overloading markedly reduced labeled pipecolic acid levels in the brain, liver and urine. These results indicate: pipecolic acid formation is subject to product inhibition, and saccharopine is not in the pathway of pipecolic acid synthesis from l ‐lysine. The labeling pattern of lysine metabolites was not significantly affected by the overloading injection of pipecolic acid when 14 C‐ l ‐lysine was intraventricularly administered suggesting a blood‐brain barrier for pipecolate. Besides 14 C‐pipecolic acid, labeled α‐aminoadipic acid was also found at significant levels mostly in the brain. Labeled saccharopine was not detected in any tissues or urine samples analyzed. The 14 C‐ l ‐lysine metabolic pattern of the newborn rats did not seem to be any different from the adult rats, i.e. labeled pipecolic acid was also detected in substantial quantities in the brain, liver and urine 5 h after injection. 14 C‐ d ‐Lysine was mainly metabolized to l ‐ 14 C‐pipecolic acid through either route of administration. These experimental evidences indicate that the pipecolic acid‐forming pathway is a significant route for lysine metabolism in the rat, and that the rat brain probably utilizes this pathway mainly for lysine metabolism. The present study also discusses the potential neurological significance of the pipecolic acid pathway in relation to the major lysine metabolic pathway (the saccharopine pathway).
Pipecolic acid
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STUDIES ON WHEAT PLANTS USING CARBON-14 COMPOUNDS: XIX. OBSERVATIONS ON THE METABOLISM OF LYSINE-C14
When generally labelled lysine-C 14 or α-aminoadipic acid-6-C 14 was administered to wheat seedlings 48% and 57%, respectively, of the carbon-14 was recovered in water-soluble materials. An additional 39% of the lysine carbon-14 was found in the insoluble residue whereas with α-aminoadipic acid-6-C 14 only 11% of the carbon-14 was in the residue. When lysine-C 14 was administered, lysine, pipecolic acid, and α-aminoadipic acid had high specific activities while glutamic acid and some related substances contained significant amounts of carbon-14. By contrast, when α-aminoadipic acid-6-C 14 was used as tracer the lysine and pipecolic acid isolated were weakly labelled, although α-aminoadipic acid of very high specific activity was recovered from the tissues. Appreciable carbon-14 was also found in the glutamic acid, 63% of this being in position-5.The data are taken as evidence that α-aminoadipic acid and pipecolic acid are on the pathway of lysine metabolism, with acetate being a product of further degradation. The results provide no evidence that α-aminoadipic acid can serve as a precursor to lysine.
Pipecolic acid
Glutamic acid
Residue (chemistry)
Carbon-14
Carbon fibers
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STUDIES ON WHEAT PLANTS USING CARBON-14 COMPOUNDS: XIX. OBSERVATIONS ON THE METABOLISM OF LYSINE-C14
When generally labelled lysine-C 14 or α-aminoadipic acid-6-C 14 was administered to wheat seedlings 48% and 57%, respectively, of the carbon-14 was recovered in water-soluble materials. An additional 39% of the lysine carbon-14 was found in the insoluble residue whereas with α-aminoadipic acid-6-C 14 only 11% of the carbon-14 was in the residue. When lysine-C 14 was administered, lysine, pipecolic acid, and α-aminoadipic acid had high specific activities while glutamic acid and some related substances contained significant amounts of carbon-14. By contrast, when α-aminoadipic acid-6-C 14 was used as tracer the lysine and pipecolic acid isolated were weakly labelled, although α-aminoadipic acid of very high specific activity was recovered from the tissues. Appreciable carbon-14 was also found in the glutamic acid, 63% of this being in position-5.The data are taken as evidence that α-aminoadipic acid and pipecolic acid are on the pathway of lysine metabolism, with acetate being a product of further degradation. The results provide no evidence that α-aminoadipic acid can serve as a precursor to lysine.
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