Biocatalytic synthesis of flavones and hydroxyl-small molecules by recombinant Escherichia coli cells expressing the cyanobacterial CYP110E1 gene

Background: Cyanobacteria possess several cytochrome P450s, but very little is known about their catalytic functions. CYP110 genes unique to cyanaobacteria are widely distributed in heterocyst-forming cyanobacteria including nitrogen-fixing genera Nostoc and Anabaena. We screened the biocatalytic functions of all P450s from three cyanobacterial strains of genus Nostoc or Anabaena using a series of small molecules that contain flavonoids, sesquiterpenes, low-molecular-weight drugs, and other aromatic compounds. Results: Escherichia coli cells carrying each P450 gene that was inserted into the pRED vector, containing the RhFRed reductase domain sequence from Rhodococcus sp. NCIMB 9784 P450RhF (CYP116B2), were co-cultured with substrates and products were identified when bioconversion reactions proceeded. Consequently, CYP110E1 of Nostoc sp. strain PCC 7120, located in close proximity to the first branch point in the phylogenetic tree of the CYP110 family, was found to be promiscuous for the substrate range mediating the biotransformation of various small molecules. Naringenin and (hydroxyl) flavanones were respectively converted to apigenin and (hydroxyl) flavones, by functioning as a flavone synthase. Such an activity is reported for the first time in prokaryotic P450s. Additionally, CYP110E1 biotransformed the notable sesquiterpene zerumbone, anti-inflammatory drugs ibuprofen and flurbiprofen (methylester forms), and some aryl compounds such as 1-methoxy and 1-ethoxy naphthalene to produce hydroxylated compounds that are difficult to synthesize chemically, including novel compounds. Conclusion: We elucidated that the CYP110E1 gene, C-terminally fused to the P450RhF RhFRed reductase domain sequence, is functionally expressed in E. coli to synthesize a robust monooxygenase, which shows promiscuous substrate specificity (affinity) for various small molecules, allowing the biosynthesis of not only flavones (from flavanones) but also a variety of hydroxyl-small molecules that may span pharmaceutical and nutraceutical industries

ON THE REACTION OF BROMINE UPON HISTIDINE AND TYROSINE

It was investigated by MILLAR that tyrosine was readily converted into dibromtyrosine by the action of bromine, and that by means of this reaction tyrosine could be determined in mixtures of amides and aminoacids. PLIMMER and EAVES (1913) applied this reaction with some modification to the determination of tyrosine in proteins. Lately PLIMMER and PHILLIPS (1924) studied this reaction more closely, and applied it to the determination of tyrosine and histidine. A sample acidified with HCl was treated with some excess of potassium bromide and sodium bromate. And after bromination some potassium iodide was quickly added and the liberated iodine was titrated with standard sodium thiosulphate solution. They described that by bromination of the cleavage products of proteins, histidine can be estimated in the phosphptungstic acid precipitate and tyrosine in the filtrate, and that both histidine and tyrosine absorbe two atomes of bromine, respectively. It is obvious that PLIMMER and PHILLIPS\u27 method cannot be used in the presence of cystine since OKUDA (1916), THRUN and TROWBRIDGE (1918) have already shown that one molecule of cystine reacts with IO atoms of bromine in acid solution. And PLIMMER and PHILLIPS also found that cystine reacts with bromine. But even in absence of cystine, I failed to justify the findings of PLIMMER and his coworkers (EAVES and PHILLIPS) about tyrosine and histidine. According to my experiments tyrosine and histidine react with more than two atoms of bromine, dependig on the temperature and time of bromination. Thus we may conclude that their methods for the determination of tyrosine and histidine are not satisfactry.近頃 PLIMMER 及び PHILLIPS (6) は臭素化によりてヒスチヂン及チロシンを蛋白質中に定量する方法を發表したり。但し此方法はシスチンの存在に於ては使用され難き事は既に奥田 (4), THRUN 及び TROWBRIDGE (8) がシスチンの1分子は10原子の臭素と作用することを示したる文献に徴するも明なりと雖余輩はシスチンの不在に於て此方法が使用され得べきや否やを確めんとして此研究を行ひたり

ヒスチヂン及びチロシンに対する臭素の反應に就て

It was investigated by MILLAR that tyrosine was readily converted into dibromtyrosine by the action of bromine, and that by means of this reaction tyrosine could be determined in mixtures of amides and aminoacids. PLIMMER and EAVES (1913) applied this reaction with some modification to the determination of tyrosine in proteins. Lately PLIMMER and PHILLIPS (1924) studied this reaction more closely, and applied it to the determination of tyrosine and histidine. A sample acidified with HCl was treated with some excess of potassium bromide and sodium bromate. And after bromination some potassium iodide was quickly added and the liberated iodine was titrated with standard sodium thiosulphate solution. They described that by bromination of the cleavage products of proteins, histidine can be estimated in the phosphptungstic acid precipitate and tyrosine in the filtrate, and that both histidine and tyrosine absorbe two atomes of bromine, respectively. It is obvious that PLIMMER and PHILLIPS' method cannot be used in the presence of cystine since OKUDA (1916), THRUN and TROWBRIDGE (1918) have already shown that one molecule of cystine reacts with IO atoms of bromine in acid solution. And PLIMMER and PHILLIPS also found that cystine reacts with bromine. But even in absence of cystine, I failed to justify the findings of PLIMMER and his coworkers (EAVES and PHILLIPS) about tyrosine and histidine. According to my experiments tyrosine and histidine react with more than two atoms of bromine, dependig on the temperature and time of bromination. Thus we may conclude that their methods for the determination of tyrosine and histidine are not satisfactry.近頃 PLIMMER 及び PHILLIPS (6) は臭素化によりてヒスチヂン及チロシンを蛋白質中に定量する方法を發表したり。但し此方法はシスチンの存在に於ては使用され難き事は既に奥田 (4), THRUN 及び TROWBRIDGE (8) がシスチンの1分子は10原子の臭素と作用することを示したる文献に徴するも明なりと雖余輩はシスチンの不在に於て此方法が使用され得べきや否やを確めんとして此研究を行ひたり