Biosynthesis of indole-3-acetic acid (IAA) via the indole-3-pyruvic acid pathway involves three kinds of enzymes; indole-3-pyruvic acid decarboxylase encoded by ipdC, aminotransferase encoded by aspC, and indole-3-acetaldehyde dehydrogenase encoded by iad1. The ipdC, aspC, and iad1 from Enterobacter...
Biosynthesis of indole-3-acetic acid (IAA) via the indole-3-pyruvic acid pathway involves three kinds of enzymes; indole-3-pyruvic acid decarboxylase encoded by ipdC, aminotransferase encoded by aspC, and indole-3-acetaldehyde dehydrogenase encoded by iad1. The ipdC, aspC, and iad1 from Enterobacter cloacae ATCC 13047, Escherichia coli, and Ustilago maydis, respectively, were cloned and expressed under control of several promoters in E. coli and Corynebacterium glutamicum. According to SDS-PAGE and enzyme activity, IpdC and Iad1 showed good expressions under the control of Ptac, whereas, AspC was efficiently expressed by Psod in E. coli. The activities of IpdC, AspC, and Iad1 from the crude extracts of recombinant E. coli Top 10 were 215.6, 5.7, and 272.1 nmol/min/mg-protein, respectively. The recombinant E. coli DH5α expressing IpdC, AspC, and Iad1 produced about 1.1 g/l of IAA and 0.13 g/l of tryptophol (TOL) after 48 hr of cultivation in LB medium with 2 g/l tryptophan. To improve IAA production, a tnaA gene mediating indole formation from tryptophan was deleted. As a result, E. coli IAA68 with expression of the three genes produced 1.8 g/l of IAA, which is a 1.6-fold increase compared to wild-type DH5α harboring the same plasmids. Moreover, the complete conversion of tryptophan to IAA was achieved by E. coli IAA68. Finally, E. coli IAA68 produced 3.0 g/l of IAA after 24 hr cultivation in LB medium supplemented with 4 g/l of tryptophan. While, C. glutamicum expressing IpdC linked with PilvC produced the highest concentration of tryptophol, indicating IpdC under the control of ilvC promoter is functioning very well in C. glutamicum. According to analyses of SDS-PAGE and enzyme activities, AspC and Iad1 were efficiently expressed by sod promoter and their activities in C. glutamicum were 5.8 and 168.5 nmol/min/mg-protein, respectively. C. glutamicum with pCIAI68 expressing IpdC, AspC, and Iad1 produced 0.68 g/l of IAA from 2 g/l of tryptophan at flask fermentation. Furthermore, recombinant cells with pCIAI68 accumulated 7.3 g/l of IAA in fermentation medium with 10 g/l of tryptophan at 5-L fermentor, which is the highest concentration of IAA when compared with previous reports.
Biosynthesis of indole-3-acetic acid (IAA) via the indole-3-pyruvic acid pathway involves three kinds of enzymes; indole-3-pyruvic acid decarboxylase encoded by ipdC, aminotransferase encoded by aspC, and indole-3-acetaldehyde dehydrogenase encoded by iad1. The ipdC, aspC, and iad1 from Enterobacter cloacae ATCC 13047, Escherichia coli, and Ustilago maydis, respectively, were cloned and expressed under control of several promoters in E. coli and Corynebacterium glutamicum. According to SDS-PAGE and enzyme activity, IpdC and Iad1 showed good expressions under the control of Ptac, whereas, AspC was efficiently expressed by Psod in E. coli. The activities of IpdC, AspC, and Iad1 from the crude extracts of recombinant E. coli Top 10 were 215.6, 5.7, and 272.1 nmol/min/mg-protein, respectively. The recombinant E. coli DH5α expressing IpdC, AspC, and Iad1 produced about 1.1 g/l of IAA and 0.13 g/l of tryptophol (TOL) after 48 hr of cultivation in LB medium with 2 g/l tryptophan. To improve IAA production, a tnaA gene mediating indole formation from tryptophan was deleted. As a result, E. coli IAA68 with expression of the three genes produced 1.8 g/l of IAA, which is a 1.6-fold increase compared to wild-type DH5α harboring the same plasmids. Moreover, the complete conversion of tryptophan to IAA was achieved by E. coli IAA68. Finally, E. coli IAA68 produced 3.0 g/l of IAA after 24 hr cultivation in LB medium supplemented with 4 g/l of tryptophan. While, C. glutamicum expressing IpdC linked with PilvC produced the highest concentration of tryptophol, indicating IpdC under the control of ilvC promoter is functioning very well in C. glutamicum. According to analyses of SDS-PAGE and enzyme activities, AspC and Iad1 were efficiently expressed by sod promoter and their activities in C. glutamicum were 5.8 and 168.5 nmol/min/mg-protein, respectively. C. glutamicum with pCIAI68 expressing IpdC, AspC, and Iad1 produced 0.68 g/l of IAA from 2 g/l of tryptophan at flask fermentation. Furthermore, recombinant cells with pCIAI68 accumulated 7.3 g/l of IAA in fermentation medium with 10 g/l of tryptophan at 5-L fermentor, which is the highest concentration of IAA when compared with previous reports.
주제어
#indole-3-acetic acid aminotransferase indole-3-purivic acid decarboxylase indole-3-acetaldehyde dehydrogenase functional expression Escherichia coli Corynebacterium glutamicum
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