RESUMO
Several regiospecific enantiomers of hydroxy-(S)-equol (HE) were enzymatically synthesized from daidzein and genistein using consecutive reduction (four daidzein-to-equol-converting reductases) and oxidation (4-hydroxyphenylacetate 3-monooxygenase, HpaBC). Despite the natural occurrence of several HEs, most of them had not been studied owing to the lack of their preparation methods. Herein, the one-pot synthesis pathway of 6-hydroxyequol (6HE) was developed using HpaBC (EcHpaB) from Escherichia coli and (S)-equol-producing E. coli, previously developed by our group. Based on docking analysis of the substrate or products, a potential active site and several key residues for substrate binding were predicted to interpret the (S)-equol hydroxylation regioselectivity of EcHpaB. Through investigating mutations on the key residues, the T292A variant was verified to display specific mono-ortho-hydroxylation activity at C6 without further 3'-hydroxylation. In the consecutive oxidoreductive bioconversion using T292A, 0.95 mM 6HE could be synthesized from 1 mM daidzein, while 5HE and 3'HE were also prepared from genistein and 3'-hydroxydaidzein (3'HD or 3'-ODI), respectively. In the following efficacy tests, 3'HE and 6HE showed about 30â¼200-fold higher EC50 than (S)-equol in both ERα and ERß, and they did not have significant SERM efficacy except 6HE showing 10% lower ß/α ratio response than that of 17ß-estradiol. In DPPH radical scavenging assay, 3'HE showed the highest antioxidative activity among the examined isoflavone derivatives: more than 40% higher than the well-known 3'HD. In conclusion, we demonstrated that HEs could be produced efficiently and regioselectively through the one-pot bioconversion platform and evaluated estrogenic and antioxidative activities of each HE regio-isomer for the first time.
RESUMO
Tyrosinase is generally known as a melanin-forming enzyme, facilitating monooxygenation of phenols, oxidation of catechols into quinones, and finally generating biological melanin. As a homologous form of tyrosinase in plants, plant polyphenol oxidases perform the same oxidation reactions specifically toward plant polyphenols. Recent studies reported synthetic strategies for large scale preparation of hydroxylated plant polyphenols, using bacterial tyrosinases rather than plant polyphenol oxidase or other monooxygenases, by leveraging its robust monophenolase activity and broad substrate specificity. Herein, we report a novel synthesis of functional plant polyphenols, especially quercetin and myricetin from kaempferol, using screened bacterial tyrosinases. The critical bottleneck of the biocatalysis was identified as instability of the catechol and gallol under neutral and basic conditions. To overcome such instability of the products, the tyrosinase reaction proceeded under acidic conditions. Under mild acidic conditions supplemented with reducing agents, a bacterial tyrosinase from Bacillus megaterium (BmTy) displayed efficient consecutive two-step monophenolase activities producing quercetin and myricetin from kaempferol. Furthermore, the broad substrate specificity of BmTy toward diverse polyphenols enabled us to achieve the first biosynthesis of tricetin and 3'-hydroxyeriodictyol from apigenin and naringenin, respectively. These results suggest that microbial tyrosinase is a useful biocatalyst to prepare plant polyphenolic catechols and gallols with high productivity, which were hardly achieved by using other monooxygenases such as cytochrome P450s.
RESUMO
Although bespoke, sequence-specific proteases have the potential to advance biotechnology and medicine, generation of proteases with tailor-made cleavage specificities remains a major challenge. We developed a phage-assisted protease evolution system with simultaneous positive and negative selection and applied it to three botulinum neurotoxin (BoNT) light-chain proteases. We evolved BoNT/X protease into separate variants that preferentially cleave vesicle-associated membrane protein 4 (VAMP4) and Ykt6, evolved BoNT/F protease to selectively cleave the non-native substrate VAMP7, and evolved BoNT/E protease to cleave phosphatase and tensin homolog (PTEN) but not any natural BoNT protease substrate in neurons. The evolved proteases display large changes in specificity (218- to >11,000,000-fold) and can retain their ability to form holotoxins that self-deliver into primary neurons. These findings establish a versatile platform for reprogramming proteases to selectively cleave new targets of therapeutic interest.
Assuntos
Toxinas Botulínicas/metabolismo , Evolução Molecular Direcionada , Engenharia de Proteínas , Animais , Bacteriófago M13/genética , Toxinas Botulínicas/química , Toxinas Botulínicas/genética , Domínio Catalítico , Linhagem Celular , Células Cultivadas , Humanos , Mutação , Neurônios/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Biblioteca de Peptídeos , Domínios Proteicos , Proteínas R-SNARE/metabolismo , Ratos , Seleção Genética , Especificidade por Substrato , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
Tyrosinase is a type 3 copper oxygenase that catalyzes a phenol moiety into ortho-diphenol, and subsequently to ortho-quinone. Diverse tyrosinases have been observed across the kingdom including Animalia, Bacteria, Plantae, and Fungi. Among the tyrosinases, bacterial, and mushroom tyrosinases have been extensively exploited to prepare melanin, ortho-hydroxy-polyphenols, or novel plant secondary metabolites during the past decade. And their use as a biocatalyst to prepare various functional biocompounds have drawn great attention worldwide. Herein, we tailored a bacterial tyrosinase from Bacillus megaterium (BmTy) using circular permutation (CP) engineering technique which is a novel enzyme engineering technique to covalently link original N and C termini and create new termini on the middle of its polypeptide. To construct a smart rationally-designed CP library, we introduced 18 new termini at the edge of each nine loops that link α-helical secondary structure in BmTy. Among the small library, seven functional CP variants were successfully identified and they represented dramatic change in their enzyme characteristics including kinetic properties and substrate specificity. Especially, cp48, 102, and 245 showed dramatically decreased tyrosine hydroxylase activity, behaving like a catechol oxidase. Exploiting the dramatic increased polyphenol oxidation activity of cp48, orobol (3'-hydroxy-genistein) was quantitatively synthesized with 1.48 g/L, which was a 6-fold higher yield of truncated wild-type. We examined their kinetic characters through structural speculation, and suggest a strategy to solubilize the insoluble artificial variants effectively.
Assuntos
Bacillus megaterium/enzimologia , Flavonoides/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Proteínas Mutantes/metabolismo , Polifenóis/metabolismo , Engenharia de Proteínas/métodos , Cinética , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Oxirredução , Conformação ProteicaRESUMO
A potent phytoestrogen, (S)-equol, is a promising isoflavone derivative drawing our great attention owing to its various biological and clinical benefits. Through selective activation of the estrogen receptor ERß or androgen receptor, (S)-equol reduces menopausal symptoms, osteoporosis, skin aging, hair loss, and incidence of prostate or ovarian cancers without adverse effects. Traditional biosynthesis of (S)-equol exploited non-productive natural equol-producing anaerobic bacteria that mainly belong to Coriobacteriaceae isolated from human intestine. Recently, we developed a recombinant Escherichia coli strain which could convert daidzein into (S)-equol effectively under an aerobic condition. However, the yield was limited up to about the 200 mg/L level due to unknown reasons. In this study, we identified that the bottleneck of the limited production was the low solubility of isoflavone (i.e., 2.4 mg/L) in the reaction medium. In order to solve the solubility problem without harmful effect to the whole-cell catalyst, we applied commercial hydrophilic polymers (HPs) and a polar aprotic co-solvent in the reaction medium. Among the examined water-soluble polymers, polyvinylpyrrolidone (PVP)-40k was verified as the most promising supplement which increased daidzein solubility by 40 times and (S)-equol yield up to 1.22 g/L, the highest ever reported and the first g/L level biotransformation. Furthermore, PVP-40k was verified to significantly increase the solubilities of other water-insoluble natural polyphenols in aqueous solution. We suggest that addition of both HP and polar aprotic solvent in the reaction mixture is a powerful alternative to enhance production of polyphenolic chemicals rather than screening appropriate organic solvents for whole-cell catalysis of polyphenols.
Assuntos
Equol/biossíntese , Microbiologia Industrial/métodos , Solventes/química , Escherichia coli/genética , Isoflavonas/química , Água/químicaRESUMO
Equols are isoflavandiols formed by reduction of soy isoflavones such as daidzein and genistein by gut microorganisms. These phytoestrogens are of interest for their various biological effects. We report biosynthesis from genistein to (-)-5-hydroxy-equol in recombinant E. coli expressing three reductases (daidzein reductase DZNR, dihidrodaidzein reductase DHDR, tetrahydrodaidzein reductase THDR) and a racemase (dihydrodaidzein racemase, DDRC) originating from the gut bacterium, Slackia isoflavoniconvertens. The biosynthesized 5-hydroxy-equol proved as an optically negative enantiomer, nonetheless it displayed an inverse circular dichroism spectrum to (S)-equol. Compartmentalized expression of DZNR and DDRC in one E. coli strain and DHDR and THDR in another increased the yield to 230 mg/L and the productivity to 38 mg/L/h. If the last reductase was missing, the intermediate spontaneously dehydrated to 5-hydroxy-dehydroequol in up to 99 mg/L yield. This novel isoflavene, previously not known to be synthesized in nature, was also detected in this biotransformation system. Although (S)-equol favors binding to human estrogen receptor (hER) ß over hERα, (-)-5-hydroxy-equol showed the opposite preference. This study provides elucidation of the biosynthetic route of (-)-5-hydroxy-equol and measurement of its potent antagonistic character as a phytoestrogen for the first time.
Assuntos
Actinobacteria/enzimologia , Vias Biossintéticas , Equol/metabolismo , Escherichia coli/metabolismo , Genisteína/metabolismo , Isoflavonas/metabolismo , Fitoestrógenos/metabolismo , Actinobacteria/genética , Actinobacteria/metabolismo , Biotransformação , Equol/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Isoflavonas/genética , Oxirredutases/genética , Oxirredutases/metabolismo , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
(S)-Equol, a gut bacterial isoflavone derivative, has drawn great attention because of its potent use for relieving female postmenopausal symptoms and preventing prostate cancer. Previous studies have reported on the dietary isoflavone metabolism of several human gut bacteria and the involved enzymes for conversion of daidzein to (S)-equol. However, the anaerobic growth conditions required by the gut bacteria and the low productivity and yield of (S)-equol limit its efficient production using only natural gut bacteria. In this study, the low (S)-equol biosynthesis of gut microorganisms was overcome by cloning the four enzymes involved in the biosynthesis from Slackia isoflavoniconvertens into Escherichia coli BL21(DE3). The reaction conditions were optimized for (S)-equol production from the recombinant strain, and this recombinant system enabled the efficient conversion of 200 µM and 1 mM daidzein to (S)-equol under aerobic conditions, achieving yields of 95% and 85%, respectively. Since the biosynthesis of trans-tetrahydrodaidzein was found to be a rate-determining step for (S)-equol production, dihydrodaidzein reductase (DHDR) was subjected to rational site-directed mutagenesis. The introduction of the DHDR P212A mutation increased the (S)-equol productivity from 59.0 mg/liter/h to 69.8 mg/liter/h in the whole-cell reaction. The P212A mutation caused an increase in the (S)-dihydrodaidzein enantioselectivity by decreasing the overall activity of DHDR, resulting in undetectable activity for (R)-dihydrodaidzein, such that a combination of the DHDR P212A mutant with dihydrodaidzein racemase enabled the production of (3S,4R)-tetrahydrodaidzein with an enantioselectivity of >99%.