Studies On Pathway Of Methylxanthines Degradation In P.Putida CT25 And Screening Of Recombinants With High-caffeine-tolerant Capability

Caffeine biodegradation is a hopeful new tech for producing low-caffeine tea products. Base on the previous achievement of high-caffeine tolerant Pseudomonas putida CT25, caffeine degradation pathway has been illustrated through comparing the metabolization efficiency of various methylxanthines; Many high-caffeine tolerant recombinants with insertion of CT25 DNA fragments have been screened and the caffeine degradation gene clusters have been obtained, The major results are as follows:(1) P putida CT25 can completely degrade the caffeine in medium with average rate of 0.043 g/L/h when 2.5 g/L caffeine is used as sole carbon and nitrogen source, and its growth log phase is found at 28-58h.(2) Many methylxanthines can be metabolized by the P. putida CT25 when these compounds has been used as sole C- & N-sources in medium, however, their degradation rates are quite different and the order of the rate is xanthine> 7-methylxanthine≈theobromine> caffeine> theophylline> 1-methylxanthine. Futhermore, the degradation rate of xanthine was 2 times of theobromine (7-methylxanthine),3 times of caffeine,8 times of theophylline, indicating that N-1 demethylation might be the rate-limiting step of methylxanthines utilization by P. putida CT25.(3) The degradation products change with various methylxanthines metabolized by P. putida CT25. Caffeine might mainly be degraded through demethylation ways, namely, caffeineâ†' theobromine (with a small amount of theophylline)â†'7-methylxanthineâ†'xanthineâ†' uric acid. The metabolization of theobromine and 7-methylxanthine is similar as caffeine. Degradation of 1-methylxanthine might primarily be through the oxidation pathway, namely,1-methylxanthineâ†' 1-methyl uric acid; while theophylline might be degraded through three ways, including oxidation and N-demethylation ways, as well as combination of N-demethylation and oxidation pathway.(4)The enzyme activity has been mainly detected in the crude protein (44.34%) extracted from bacterial cells instead of from the incubation medium(1.5%), indicating the caffeine degradation occurs in cell matrixes instead of in cultivation medium., Degradation efficiencies of theobromine and 7-methylxanthine are around 65% by extracted enzymes, higher than that of caffeine and theophylline (around 45%). Metabolic process of caffeine and theophylline by the extracted enzymes can be described as(5) Five proteins have been found significantly increase expression in caffeine-incubated P. putida CT25 compared with in peptone-incubated bacterial, and their molecular weight are around 28kDa,32kDa,35kDa,40kDa and 65kDa, respectively.(6) Thirty two positive recombinants inserted with genomic fragment of P. putida CT25 have been achieved when 5250 clones are screened in the medium with 2.5g/L caffeine, sixteen inserted fragments have been sequenced. The inserted fragment includes the gene cluster encoding the proteins related to chemotactic response, signal transduction, transcriptional regulation, protein secretion, methyl transfer, redox and ion transportation. Recombinant 11-14-ban-all and 15-18-ban-d13 (with inserted fragment encoding the enzymes for caffeine N-demthylation) have been found tolerant to lOg/L caffeine; while 15-18-ban-c14 and 87-90-ban-p22 (with inserted fragment encoding the enzymes for redox process) tolerant 7.5g/L caffeine.