Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris

Morawski, Birgit; Lin, Zhanglin; Cirino, Pat; Joo, Hyun; Bandara, Geethani; Arnold, Frances H.

doi:10.1093/protein/13.5.377

Cited by 122 publications

(112 citation statements)

References 45 publications

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“…Some incompatibilities between the expressed gene and heterologous host, such as codon usage or the recognition of signal sequences, can be overcome by changing the gene sequence. Thus, achieving functional expression is a good target for directed evolution (13,42,43).Laccases, like other ligninolytic enzymes, are notoriously difficult to express in nonfungal systems. The laccase from Myceliophthora thermophila (MtL) used in this work was previously expressed only in Aspergillus oryzae (6).…”

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Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

Bülter

Alcalde

Sieber

et al. 2003

Appl Environ Microbiol

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Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k cat , the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.Directed evolution by random mutagenesis and recombination followed by screening or selection is a valuable tool for the engineering of enzymes (2,3,16,61). Functional gene expression in a suitable host is a prerequisite for directed evolution. Considering transformation efficiency, stability of plasmid DNA, and growth rate, Escherichia coli and Saccharomyces cerevisiae are best suited for these experiments. Heterologous expression in these hosts, however, is often limited by differences in the expression systems from the native organism (50). Different codon usage, missing chaperones, and posttranslational modifications such as disulfide bridges or glycosylation can all cause low expression levels and misfolded proteins that are degraded or driven into inclusion bodies (20). Finding the bottlenecks of a specific expression system requires consideration of many possibilities whose impact is hard to predict. Some incompatibilities between the expressed gene and heterologous host, such as codon usage or the recognition of signal sequences, can be overcome by changing the gene sequence. Thus, achieving functional expression is a good target for directed evolution (13,42,43).Laccases, like other ligninolytic enzymes, are notoriously difficult to express in nonfungal systems. The laccase from Myceliophthora thermophila (MtL) used in this work was previously expressed only in Aspergillus oryzae (6). Expression in S. cerevisiae has been reported for other laccase genes (11,33,34,60). Kojima et al. demonstrated expression of...

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Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

Bülter

Alcalde

Sieber

et al. 2003

Appl Environ Microbiol

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259

170

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“…The only reasonable candidate for a bond with the 5-methyl was Leu 37 . This residue has not been assigned any function in the HRP active site, but directed evolution experiments have shown that its mutation into an isoleucine improves enzymatic activity (28). Modeling of a glutamate or an aspartate (an isoleucine steric analog) at this site gave the nearest approach distances of 1.5 and 2.1 Å, respectively (Fig.…”

Section: Structure Superposition and Choice Of Mutations-mpomentioning

confidence: 99%

Horseradish Peroxidase Mutants That Autocatalytically Modify Their Prosthetic Heme Group

Colas

Montellano

2004

Journal of Biological Chemistry

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The mammalian peroxidases, including myeloperoxidase and lactoperoxidase, bind their prosthetic heme covalently through ester bonds to two of the heme methyl groups. These bonds are autocatalytically formed. No other peroxidase is known to form such bonds. To determine whether features other than an appropriately placed carboxylic acid residue are important for covalent heme binding, we have introduced aspartate and/or glutamic acid residues into horseradish peroxidase, a plant enzyme that exhibits essentially no sequence identity with the mammalian peroxidases. Based on superposition of the horseradish peroxidase and myeloperoxidase structures, the mutated residues were Leu 37 , Phe 41 , Gly 69 , and Ser 73 . The F41E mutant was isolated with no covalently bound heme, but the heme was completely covalently bound upon incubation with H 2 O 2 . As predicted, the modified heme released from the protein was 3-hydroxymethylheme. The S73E mutant did not covalently bind its heme but oxidized it to the 8-hydroxymethyl derivative. The hydroxyl group in this modified heme derived from the medium. The other mutations gave unstable proteins. The rate of compound I formation for the F41E mutant was 100 times faster after covalent bond formation, but the reduction of compound I to compound II was similar with and without the covalent bond. The results clearly establish that an appropriately situated carboxylic acid group is sufficient for covalent heme attachment, strengthen the proposed mechanism, and suggest that covalent heme attachment in the mammalian peroxidases relates to peroxidase biology or stability rather than to intrinsic catalytic properties.Mammalian peroxidases share a unique feature, the formation of two or three covalent bonds to their prosthetic heme 1 group, not found in the peroxidases of other organisms. The two common bonds in all mammalian peroxidases are ester links between a conserved glutamate and aspartate and the heme 1-methyl and 5-methyl substituents, respectively. In MPO a third bond is forged between a methionine and the ␤ carbon of the 2-vinyl substituent (1). Mutagenesis studies with LPO have demonstrated that at least one of the two ester bonds is required for catalytic activity (2). Interestingly, a single covalent link between the heme and protein is also found in most members of the CYP4A, -4B, and -4F classes of cytochrome P450 enzymes (3-7). In contrast to LPO, however, mutagenesis studies show that the heme-protein covalent bond is not essential for the catalytic activity of at least some of these P450 enzymes (8, 6). A long known example of heme covalent binding is provided by cytochrome c, in which a cysteine residue is covalently bound to each of the two porphyrin vinyl groups. Although many hypotheses have been formulated concerning the functional advantages of the links in cytochrome c, including bending of the heme, increasing the heme affinity (9), and increasing the stability of the methionyl-Fe(II) coordination (10), the question has yet to be definitively answered.Despite m...

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“…To maintain the glycosylation pattern of the surface-bound HRP, we used the same yeast host for heterologous expression of the soluble enzyme. Because HRP is expressed poorly in this yeast (14,22), we used a strain of , discovered in each round of evolution. Red, yellow, blue, and green bar colors represent substrates 1, 2, 3, and 4, respectively; hatched and solid bars designate surface-bound and soluble HRP, respectively.…”

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confidence: 99%

“…The L and D letters designate the direction of enantiopreference; the Roman numerals after the letters define the round of directed evolution; the r and/or s letters after the Roman numerals indicate whether these variants were isolated from the random or saturated mutagenesis libraries, respectively. Mutations: LIr (Arg178Gln), LIs (Phe68Leu, Gly69Ala, Asn72Glu, Ser73Leu, Ala74Tyr) (14), LIrs (LIr ϩ LIs), LIIr (LIrs ϩ Gln147Arg), LIII (LIIr ϩ Asn158Asp), DIs (Phe68Glu, Gly69Pro, Asn72Lys) (14), DIIs (DIs ϩ Asn137Arg, Ala140His, Phe142Lys, Phe143Met), and DIII (DIIs ϩ Ser167Ile).…”

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Highlylanddenantioselective variants of horseradish peroxidase discovered by an ultrahigh-throughput selection method

Antipov¹,

Cho

Wittrup

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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A highly efficient selection method for enhanced enzyme enantioselectivity based on yeast surface display and fluorescenceactivated cell sorting (FACS) is developed and validated. Its application to horseradish peroxidase has resulted in enzyme variants up to 2 orders of magnitude selective toward either substrate enantiomer at will. These marked improvements in enantioselectivity are demonstrated for the surface-bound and soluble enzymes and rationalized by computational docking studies. directed evolution ͉ enzyme design ͉ molecular modeling ͉ redox enzymes ͉ stereoselectivity

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Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris

Cited by 122 publications

References 45 publications

Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

Horseradish Peroxidase Mutants That Autocatalytically Modify Their Prosthetic Heme Group

Highlylanddenantioselective variants of horseradish peroxidase discovered by an ultrahigh-throughput selection method

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