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Lee, Nahum,Lee, Sang-Hyuk,Baek, Kiheon,Kim, Byung-Gee Springer International 2015 Applied microbiology and biotechnology Vol.99 No.19
<P>Recombinant tyrosinase from Streptomyces avermitilis MA4680, MelC2 (gi:499291317), was heterologously expressed in Escherichia coli BL21 (DE3). The expression level of active MelC2 was increased by the codon-optimized MelC1 caddie protein (KP198295.1). By performing saturation mutagenesis of the Y91 residue of MelC1, it was found that aromatic residues such as Y, F, and W at the 91st position help produce a correctly folded conformation of MelC2. The recombinant MelC2 was utilized as a biocatalyst to convert trans-resveratrol into piceatannol. In order to improve the product yield through suppression of the formation of melanin, a by-product, an increase in the ratio of monooxygenation (k (1)) to dioxygenation (k (2)) of MelC2 is desirable. This was achieved by a combination of protein engineering and regeneration of NADH with glucose dehydrogenase (GDH). Saturation mutagenesis was performed at 15 residues within 8- radius from copper ions of MelC2. A total of 2760 mutants were examined (99.7 % probability for NNK codon) and I41Y, a mutant, was screened. The ratio of k (1) to k (2) of the mutant increased sevenfold on tyrosine and fivefold on resveratrol, when compared to wild-type MelC2. As a result, the overall product yield from 500 mu M resveratrol in 50-mL reaction was 15.4 % (77.4 mu M piceatannol), 1.7 times higher than wild type. When I41Y was incorporated with the NADH regeneration system, the total product yield was 58.0 %, an eightfold increase (290.2 mu M of piceatannol).</P>
Lee, Nahum,Kim, Eun Jung,Kim, Byung-Gee American Chemical Society 2012 ACS CHEMICAL BIOLOGY Vol.7 No.10
<P>Secreted tyrosinase from melanin-forming <I>Streptomyces avermitilis</I> MA4680 was involved in both ortho-hydroxylation and further oxidation of <I>trans</I>-resveratrol, leading to the formation of melanin. This finding was confirmed by constructing deletion mutants of <I>melC</I><SUB><I>2</I></SUB> and <I>melD</I><SUB><I>2</I></SUB> encoding extracellular and intracellular tyrosinase, respectively; the <I>melC2</I> deletion mutant did not produce piceatannol as well as melanin, whereas the <I>melD2</I> deletion mutant oxidized resveratrol and synthesized melanin with the same yields, suggesting that MelC2 is responsible for ortho-hydroxylation of resveratrol. Extracellular tyrosinase (MelC2) efficiently converted <I>trans</I>-resveratrol into piceatannol in the presence of either tyrosinase inhibitors or reducing agents such as catechol, NADH, and ascorbic acid. Reducing agents slow down the dioxygenase reaction of tyrosinase. In the presence of catechol, the regio-specific hydroxylation of <I>trans</I>-resveratrol was successfully performed by whole cell biotransformation, and further oxidation of <I>trans</I>-resveratrol was efficiently blocked. The yield of this ortho-hydroxylation of <I>trans</I>-resveratrol was dependent upon inhibitor concentration. Using 1.8 mg of wild-type <I>Streptomyces avermitilis</I> cells, the conversion yield of 100 μM <I>trans</I>-resveratrol to piceatannol was 78% in 3 h in the presence of 1 mM catechol, indicating 14 μM piceatannol h<SUP>–1</SUP> DCW mg<SUP>–1</SUP> specific productivity, which was a 14-fold increase in conversion yield compared to that without catechol, which is a remarkably higher reaction rate than that of P450 bioconversion. This method could be generally applied to biocatalysis of various dioxygenases.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/acbcct/2012/acbcct.2012.7.issue-10/cb300222b/production/images/medium/cb-2012-00222b_0007.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/cb300222b'>ACS Electronic Supporting Info</A></P>
Kwon, Yong‐,Chan,Oh, In‐,Seok,Lee, Nahum,Lee, Kyung‐,Ho,Yoon, Yeo Joon,Lee, Eun Yeol,Kim, Byung‐,Gee,Kim, Dong‐,Myung Wiley Subscription Services, Inc., A Wiley Company 2013 Biotechnology and bioengineering Vol.110 No.4
<P><B>Abstract</B></P><P>Harnessing the isolated protein synthesis machinery, cell‐free protein synthesis reproduces the cellular process of decoding genetic information in artificially controlled environments. More often than not, however, generation of functional proteins requires more than simple translation of genetic sequences. For instance, many of the industrially important enzymes require non‐protein prosthetic groups for biological activity. Herein, we report the complete cell‐free biogenesis of a heme prosthetic group and its integration with concurrent apoenzyme synthesis for the production of functional P450 monooxygenase. Step reactions required for the syntheses of apoenzyme and the prosthetic group have been designed so that these two separate pathways take place in the same reaction mixture, being insulated from each other. Combined pathways for the synthesis of functional P450 monooxygenase were then further integrated with in situ assay reactions to enable real‐time measurement of enzymatic activity during its synthesis. Biotechnol. Bioeng. 2013; 110: 1193–1200. © 2012 Wiley Periodicals, Inc.</P>
Pandey, Bishnu Prasad,Lee, Nahum,Choi, Kwon-Young,Jung, Eunok,Jeong, Da-hye,Kim, Byung-Gee Elsevier 2011 Enzyme and microbial technology Vol.48 No.4
<P><B>Abstract</B></P><P>Screening of cytochrome P450 monoxygenases responsible for the regiospecific hydroxylation of flavones, isoflavones and chalcones was attempted using a P450 library constructed from <I>Streptomyces avermitilis</I> MA4680, <I>Bacillus</I> and <I>Nocardia farcinica</I> IFM10152 strains. As electron transfer redox partners with the P450s in <I>Escherichia coli</I> system, putidaredoxin reductase (PdR) and putidaredoxin (Pdx) from <I>Pseudomonas putida</I> were used. Among the 50 soluble P450s in the library screened, three cytochrome P450s, i.e. CYP107Y1, CYP125A2 and CYP107P2 from <I>S. avermitilis</I> MA4680 showed good hydroxylation activities towards flavones and isoflavones. However, low product yields prevented us from identifying complete structure of the products. By using <I>S. avermitilis</I> MA4680 as their expression host, further analysis identified that CYP107Y1(SAV2377), CYP125A2(SAV5841) and CYP107P2(SAV4539) showed good regiospecific hydroxylation activities towards genistein (4′,5,7-trihydroxyisoflavone), chrysin (5,7-dihydroxyisoflavone) and apigenin (4′,5,7-dihydroxyisoflavone) to produce 3′,4′,5,7,-tetrahydroxyisoflavone, B-ring hydroxylated 5,7-dihydroxyflavone and 3′,4′,5,7,-tetrahydroxyflavone, respectively. Analyses of the reaction products were performed using HPLC, ESI-MS–MS and GC–MS and 1H NMR.</P>