Highly<scp>l</scp>and<scp>d</scp>enantioselective variants of horseradish peroxidase discovered by an ultrahigh-throughput selection method

Antipov, Eugene; Cho, Art E.; Wittrup, K. Dane; Klibanov, Alexander M.

doi:10.1073/pnas.0809851105

Cited by 47 publications

(49 citation statements)

References 32 publications

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“…Larger increases in k cat may require modified selection or screening strategies that explicitly couple survival with multiple turnover kinetics, perhaps by integrating our system with in vitro compartmentalization. Despite the widespread use of yeast display in the evolution of binding interactions (18), to the best of our knowledge, sortase A is only the third enzyme to be evolved using yeast display, in addition to horseradish peroxidase (30,31) and an esterase catalytic antibody (32). Our results highlight the attractive features of yeast display that offer significant advantages for enzyme evolution, including quality control mechanisms within the secretory pathway that ensure display of properly folded proteins and compatibility with FACS (18).…”

Section: Discussionmentioning

confidence: 87%

A general strategy for the evolution of bond-forming enzymes using yeast display

Chen

Dorr

Liu

2011

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

511

584

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The ability to routinely generate efficient protein catalysts of bondforming reactions chosen by researchers, rather than nature, is a long-standing goal of the molecular life sciences. Here, we describe a directed evolution strategy for enzymes that catalyze, in principle, any bond-forming reaction. The system integrates yeast display, enzyme-mediated bioconjugation, and fluorescence-activated cell sorting to isolate cells expressing proteins that catalyze the coupling of two substrates chosen by the researcher. We validated the system using model screens for Staphylococcus aureus sortase A-catalyzed transpeptidation activity, resulting in enrichment factors of 6,000-fold after a single round of screening. We applied the system to evolve sortase A for improved catalytic activity. After eight rounds of screening, we isolated variants of sortase A with up to a 140-fold increase in LPETG-coupling activity compared with the starting wild-type enzyme. An evolved sortase variant enabled much more efficient labeling of LPETG-tagged human CD154 expressed on the surface of HeLa cells compared with wild-type sortase. Because the method developed here does not rely on any particular screenable or selectable property of the substrates or product, it represents a powerful alternative to existing enzyme evolution methods.

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Section: Discussionmentioning

confidence: 87%

A general strategy for the evolution of bond-forming enzymes using yeast display

Chen

Dorr

Liu

2011

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

511

584

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“…For the first generation of evolution, we use two previously created libraries (10,30). In the first, the HRP gene is randomly mutated using epPCR at a rate of one to three mutations per gene.…”

Section: Methodsmentioning

confidence: 99%

“…By contrast, it is much more challenging to select for other functionalities, such as catalysis (8). Cells also can be used to link genotype and phenotype; both growth-based selections and screening cells by FACS can potentially access libraries larger than 10 8 , but the range of reactions is very limited, either to survival or a cell-bound product, respectively (3,9,10). Genotype and phenotype can also be linked by compartmentalizing individual elements of the library in the aqueous drops of a water-in-oil emulsion (9).…”

Section: Editor-in-chiefmentioning

confidence: 99%

Ultrahigh-throughput screening in drop-based microfluidics for directed evolution

Agresti

Antipov

Abate

et al. 2010

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

Self Cite

1,012

960

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The explosive growth in our knowledge of genomes, proteomes, and metabolomes is driving ever-increasing fundamental understanding of the biochemistry of life, enabling qualitatively new studies of complex biological systems and their evolution. This knowledge also drives modern biotechnologies, such as molecular engineering and synthetic biology, which have enormous potential to address urgent problems, including developing potent new drugs and providing environmentally friendly energy. Many of these studies, however, are ultimately limited by their need for even-higher-throughput measurements of biochemical reactions. We present a general ultrahigh-throughput screening platform using drop-based microfluidics that overcomes these limitations and revolutionizes both the scale and speed of screening. We use aqueous drops dispersed in oil as picoliter-volume reaction vessels and screen them at rates of thousands per second. To demonstrate its power, we apply the system to directed evolution, identifying new mutants of the enzyme horseradish peroxidase exhibiting catalytic rates more than 10 times faster than their parent, which is already a very efficient enzyme. We exploit the ultrahigh throughput to use an initial purifying selection that removes inactive mutants; we identify ∼100 variants comparable in activity to the parent from an initial population of ∼10 7 . After a second generation of mutagenesis and high-stringency screening, we identify several significantly improved mutants, some approaching diffusion-limited efficiency. In total, we screen ∼10 8 individual enzyme reactions in only 10 h, using < 150 μL of total reagent volume; compared to state-of-the-art robotic screening systems, we perform the entire assay with a 1,000-fold increase in speed and a 1-million-fold reduction in cost.

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“…In some cases enzymes cannot be expressed efficiently in the usual workhorses of molecular biology such as E. coli, which means that alternative expression systems have to be developed, with examples being horse radish peroxidase (yeast surface display) [57] and oxynitrilases (Pichia pastoris). [129] Finally, ISM offers perspectives which go far beyond the control of substrate acceptance, stereoselectivity, and thermostability required in organic chemistry and white biotechnology.…”

Section: Discussionmentioning

confidence: 99%

“…These advances include the use of enzymes of the type esterase, [29,43] lipase, [44a-b] hydantoinase, [45] epoxide hydrolase, [46] nitrilase, [47] aldolase, [48] monoamine oxidase, [49] Baeyer-Villiger monooxygenase, [50] transaminase, [51] benzoylformate decarboxylase, [52] phosphotriesterase, [53] P450, [54] reductase, [55] oxynitrilase, [56] and horse radish peroxidase. [57] Circular permutation, that is, the intramolecular relocation of the C and N termini of a protein, has been applied to the evolution of enantioselectivity with the lipase Candida antarctica B (CALB).…”

Section: Early Examples Of Directed Evolution Of Enantioselective Enzmentioning

confidence: 99%

Laboratory Evolution of Stereoselective Enzymes: A Prolific Source of Catalysts for Asymmetric Reactions

2010

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Asymmetric catalysis plays a key role in modern synthetic organic chemistry, with synthetic catalysts and enzymes being the two available options. During the latter part of the last century the use of enzymes in organic chemistry and biotechnology experienced a period of rapid growth. However, these biocatalysts have traditionally suffered from several limitations, including in many cases limited substrate scope, poor enantioselectivity, insufficient stability, and sometimes product inhibition. During the last 15 years, the genetic technique of directed evolution has been developed to such an extent that all of these long-standing problems can be addressed and solved. It is based on repeated cycles of gene mutagenesis, expression, and screening (or selection). This Review focuses on the directed evolution of enantioselective enzymes, which constitutes a fundamentally new approach to asymmetric catalysis. Emphasis is placed on the development of methods to make laboratory evolution faster and more efficient, thus providing chemists and biotechnologists with a rich and non-ending source of robust and selective catalysts for a variety of useful applications.

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

Cited by 47 publications

References 32 publications

A general strategy for the evolution of bond-forming enzymes using yeast display

A general strategy for the evolution of bond-forming enzymes using yeast display

Ultrahigh-throughput screening in drop-based microfluidics for directed evolution

Laboratory Evolution of Stereoselective Enzymes: A Prolific Source of Catalysts for Asymmetric Reactions

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