Extending the diversity of cytochrome P450 enzymes by DNA family shuffling

Rosic, Nedeljka; Huang, Weiliang; Johnston, Wayne A.; DeVoss, James J.; Gillam, Elizabeth M. J.

doi:10.1016/j.gene.2007.01.031

Cited by 29 publications

(18 citation statements)

References 39 publications

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“…7). This is consistent with the lack of library parental contamination inherent in the restriction enzyme-mediated shuffling protocol due to introduction of a size-selective filtration step to recover digested fragments as well as efficient fragmentation using restriction enzymes Rosic et al, 2007). In comparison, at least 13.8% of the previous DNaseI-mediated shuffled 1A library appeared to show parental hybridization patterns, a value that may have been elevated due to linkage between probed segments (Abécassis et al, 2000).…”

Section: Discussionsupporting

confidence: 73%

A Shuffled CYP1A Library Shows Both Structural Integrity and Functional Diversity

Johnston

Huang²,

Voss³

et al. 2007

Drug Metabolism and Disposition

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ABSTRACT:The cytochrome P450 enzymes (P450s) that mediate mammalian xenobiotic metabolism are highly versatile monooxygenases, which show wide and overlapping substrate ranges but generally poor catalytic rates. Re-engineering of these P450s may enable the development of useful biocatalysts for industrial applications. In the current study, restriction enzyme-mediated DNA family shuffling was used to create a library from human CYP1A1 and CYP1A2. Among sequenced clones (four randomly selected and eight functional clones), 5.9 ؎ 2.3 crossovers and 1.5 ؎ 1.5 spontaneous mutations (mean ؎ S.D.) were detected per mutant. A high level of structural integrity as well as diverse functionality were found, with 53% of clones expressed at significant levels (>50 nM P450 hemoprotein) and 23% of clones showing activity on one or more of the following compounds: luciferin 6-chloroethyl ether (luciferin-CEE), luciferin 6-methyl ether (luciferin-ME), 6-deoxyluciferin (luciferin-H), the ethylene glycol ester of luciferin 6-methyl ether, 7-ethoxyresorufin, and p-nitrophenol (PNP). Different activity profiles were seen with higher specific activity on individual compounds (e.g., clone 22; 9 times the CYP1A1 specific activity toward luciferin-CEE), novel activities (e.g., clone 35; activity toward luciferin-H and PNP), and broadening of substrate range observed in particular clones (e.g., clone 9; activity toward both selective substrates luciferin-ME and luciferin-CEE as well as toward luciferin-H and PNP). In summary, forms were found with distinct and novel activity profiles, despite the relatively small number of mutants examined. In addition, the whole-cell metabolic assays described here provide simple, high-throughput methods useful for screening larger libraries.Enzymes are exquisitely specific biocatalysts, able to modify even complex organic chemicals with a high degree of stereo-and regioselectivity by virtue of their complex, stereoselective, three-dimensional structures. Directed or artificial evolution is being used to engineer enzymes with novel catalytic and physicochemical properties that can be used as biocatalysts for industrial applications. The development of enzyme libraries relies upon the ability to navigate a practically infinite sequence space of possible enzymes while identifying novel and desired characteristics in library clones. DNA family shuffling is a powerful technique that allows sparse sampling of a large region of the sequence space, whereas techniques based on random mutagenesis either explore more limited sequence space close to the parental starting point or are prohibitively massive in scope (Crameri et al., 1998).Cytochrome P450 (P450) enzymes are ideal starting points for directed evolution. The need for organisms to clear a diversity of xenobiotics has given several mammalian P450s the capability to metabolize a wide range of foreign chemicals via monooxygenation, with considerable regiospecificity on even large and complex molecules (Gillam, 2005). Despite this diversity of activity, th...

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Section: Discussionsupporting

confidence: 73%

A Shuffled CYP1A Library Shows Both Structural Integrity and Functional Diversity

Johnston

Huang²,

Voss³

et al. 2007

Drug Metabolism and Disposition

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“…In addition to indigo, different kinds of indigoid pigments, including indirubin can also be produced by E. coli expressing indole oxygenases (O'Connor et al 1997;O'Connor and Hartmans 1998;Nakamura et al 2001;Meyer et al 2002;Choi et al 2003;Furuya et al 2004;Lim et al 2005;McClay et al 2005;Rui et al 2005). Moreover, human cytochrome P450 and bacterial cytochrome P450 proteins have been engineered to produce a variety of indigoids in E. coli (Gillam et al 1999;Li et al 2000;Nakamura et al 2001;Celik et al 2005;Wu et al 2005;Rosic et al 2007;Rosic 2009). …”

Section: Introductionmentioning

confidence: 99%

Identification and characterization of an indigo-producing oxygenase involved in indole 3-acetic acid utilization by Acinetobacter baumannii

Lin

Chen

Huang

et al. 2012

Antonie van Leeuwenhoek

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Acinetobacter baumannii harbours a gene cluster similar to the iac locus of Pseudomonas putida 1290, which can catabolize the plant hormone indole 3-acetic acid (IAA) as an energy source. However, there has been no evidence showing that IAA can be utilized by A. baumannii. This study showed that A. baumannii can grow in M9 minimal medium containing IAA as the sole carbon source. A mutagenesis study indicated that iacA, encoded in the iac locus of A. baumannii, is involved in the catabolism of IAA. As shown by western blotting analysis, the IacA protein was detected in A. baumannii grown in M9 minimal medium with IAA but not with pyruvate, suggesting that the expression of iacA is regulated by the presence of IAA. In vitro studies have shown that IacA can oxidize indole, an IAA-like molecule, converting it to indoxyl, which spontaneously dimerises to form indigo. In this study, we show that the crude extracts from either wild-type A. baumannii or Escherichia coli overexpressing IacA can oxidize IAA. These results imply that the iac gene cluster of A. baumannii is involved in IAA degradation and that the iacA gene is upregulated when cells encounter IAA in their native environments.

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“…This evolutionary method involves the controlled fragmentation of source DNA either by DNAase I (Stemmer, 1994a;Zhao and Arnold, 1997) or common restriction enzymes prior to a primer-less PCR gene assembly (Fig. 3) (Kikuchi et al, 1999;Kaper et al, 2002;Rosic et al, 2007). A number of additional, more intricate techniques, although applied on a less frequent basis, have also been devised for the creation of libraries, such as ITCHY (incremental truncation for the creation of hybrid enzymes) (Ostermeier and Lutz, 2003), RACHITT (random chimeragenesis on transient templates) (Coco et al, 2001) and random circular permutation (Qian et al, 2007); see the indicated references for details.…”

Section: Directed Evolution Techniquesmentioning

confidence: 99%

Starch and α-glucan acting enzymes, modulating their properties by directed evolution

Kelly

Dijkhuizen

Leemhuis

2009

Journal of Biotechnology

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a b s t r a c tStarch is the major food reserve in plants and forms a large part of the daily calorie intake in the human diet. Industrially, starch has become a major raw material in the production of various products including bio-ethanol, coating and anti-staling agents. The complexity and diversity of these starch based industries and the demand for high quality end products through extensive starch processing, can only be met through the use of a broad range of starch and ␣-glucan modifying enzymes. The economic importance of these enzymes is such that the starch industry has grown to be the largest market for enzymes after the detergent industry. However, as the starch based industries expand and develop the demand for more efficient enzymes leading to lower production cost and higher quality products increases. This in turn stimulates interest in modifying the properties of existing starch and ␣-glucan acting enzymes through a variety of molecular evolution strategies. Within this review we examine and discuss the directed evolution strategies applied in the modulation of specific properties of starch and ␣-glucan acting enzymes and highlight the recent developments in the field of directed evolution techniques which are likely to be implemented in the future engineering of these enzymes.

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Extending the diversity of cytochrome P450 enzymes by DNA family shuffling

Cited by 29 publications

References 39 publications

A Shuffled CYP1A Library Shows Both Structural Integrity and Functional Diversity

A Shuffled CYP1A Library Shows Both Structural Integrity and Functional Diversity

Identification and characterization of an indigo-producing oxygenase involved in indole 3-acetic acid utilization by Acinetobacter baumannii

Starch and α-glucan acting enzymes, modulating their properties by directed evolution

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