Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently

Ness, Jon E.; Kim, Seran; Gottman, Andrea; Pak, Rob; Krebber, Anke; Borchert, Torben V.; Govindarajan, Sridhar; Mundorff, Emily C.; Minshull, Jeremy

doi:10.1038/nbt754

Cited by 186 publications

(111 citation statements)

References 28 publications

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“…In the first six rounds, shuffled gene variant libraries were made based on the maize HPPD protein template using techniques including family shuffling, single-gene shuffling, back-crossing, and semisynthetic and synthetic shuffling (Zhang et al, 1997;Crameri et al, 1998;Ness et al, 2002). The amino acid diversity for those libraries originated from phylogenetic sequence diversity, random mutagenesis, site-directed mutagenesis, and structural features based on crystal structures of proteins in the Protein Data Bank (PDB; www.pdb.org/pdb/home/ home.do).…”

Section: Directed Evolution Of the Maize Hppd Protein For Increased Imentioning

confidence: 99%

Broad 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor Herbicide Tolerance in Soybean with an Optimized Enzyme and Expression Cassette

et al. 2014

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With an optimized expression cassette consisting of the soybean (Glycine max) native promoter modified for enhanced expression driving a chimeric gene coding for the soybean native amino-terminal 86 amino acids fused to an insensitive shuffled variant of maize (Zea mays) 4-hydroxyphenylpyruvate dioxygenase (HPPD), we achieved field tolerance in transgenic soybean plants to the HPPD-inhibiting herbicides mesotrione, isoxaflutole, and tembotrione. Directed evolution of maize HPPD was accomplished by progressively incorporating amino acids from naturally occurring diversity and novel substitutions identified by saturation mutagenesis, combined at random through shuffling. Localization of heterologously expressed HPPD mimicked that of the native enzyme, which was shown to be dually targeted to chloroplasts and the cytosol. Analysis of the native soybean HPPD gene revealed two transcription start sites, leading to transcripts encoding two HPPD polypeptides. The N-terminal region of the longer encoded peptide directs proteins to the chloroplast, while the short form remains in the cytosol. In contrast, maize HPPD was found almost exclusively in chloroplasts. Evolved HPPD enzymes showed insensitivity to five inhibitor herbicides. In 2013 field trials, transgenic soybean events made with optimized promoter and HPPD variant expression cassettes were tested with three herbicides and showed tolerance to four times the labeled rates of mesotrione and isoxaflutole and two times the labeled rates of tembotrione.

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Section: Directed Evolution Of the Maize Hppd Protein For Increased Imentioning

confidence: 99%

Broad 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor Herbicide Tolerance in Soybean with an Optimized Enzyme and Expression Cassette

et al. 2014

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“…Because neutral changes at correlated positions will occur only rarely in a randomly mutagenized pool, a large number of sequences would be required to establish the significance of correlations, especially at pairs of positions that are only weakly correlated. To enhance our ability to identify correlated positions in this ribozyme, a pool was synthesized using a method of in vitro recombination called synthetic shuffling (Ness et al 2002). Variable positions in this pool corresponded to positions that varied among previously isolated ribozyme variants (Curtis and Bartel 2005).…”

Section: In Vitro Recombination By Synthetic Shufflingmentioning

confidence: 99%

“…In this study, we used a method of in vitro recombination called synthetic shuffling (Ness et al 2002), in combination with in vitro selection, comparative sequence analysis, and site-directed mutagenesis, to investigate two related questions about the adaptive fitness landscape of a previously isolated kinase ribozyme (Curtis and Bartel 2005). First, to what extent are mutational effects in this ribozyme independent of one another ( Fig.…”

Section: Introductionmentioning

confidence: 99%

Synthetic shuffling and in vitro selection reveal the rugged adaptive fitness landscape of a kinase ribozyme

Curtis

Bartel

2013

RNA

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The relationship between genotype and phenotype is often described as an adaptive fitness landscape. In this study, we used a combination of recombination, in vitro selection, and comparative sequence analysis to characterize the fitness landscape of a previously isolated kinase ribozyme. Point mutations present in improved variants of this ribozyme were recombined in vitro in more than 10 14 different arrangements using synthetic shuffling, and active variants were isolated by in vitro selection. Mutual information analysis of 65 recombinant ribozymes isolated in the selection revealed a rugged fitness landscape in which approximately one-third of the 91 pairs of positions analyzed showed evidence of correlation. Pairs of correlated positions overlapped to form densely connected networks, and groups of maximally connected nucleotides occurred significantly more often in these networks than they did in randomized control networks with the same number of links. The activity of the most efficient recombinant ribozyme isolated from the synthetically shuffled pool was 30-fold greater than that of any of the ribozymes used to build it, which indicates that synthetic shuffling can be a rich source of ribozyme variants with improved properties.

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“…Designed libraries can be synthesized for roughly the same cost as a designed sequence by recognizing the opportunities in gene synthesis for the combinatorial shuffling of sequence diversity (14)(15)(16)(17). Although many algorithms have now been proposed to design such combinatorial libraries (7-9, 11, 12), few computationally designed libraries have been characterized experimentally (9,18,19), and, to our knowledge, there have been no controlled experiments comparing these methods with each other or with libraries of randomly generated sequence diversity.…”

mentioning

confidence: 99%

Computationally designed libraries of fluorescent proteins evaluated by preservation and diversity of function

Treynor

Vizcarra²,

Nedelcu

et al. 2007

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

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To determine which of seven library design algorithms best introduces new protein function without destroying it altogether, seven combinatorial libraries of green fluorescent protein variants were designed and synthesized. Each was evaluated by distributions of emission intensity and color compiled from measurements made in vivo. Additional comparisons were made with a library constructed by error-prone PCR. Among the designed libraries, fluorescent function was preserved for the greatest fraction of samples in a library designed by using a structure-based computational method developed and described here. A trend was observed toward greater diversity of color in designed libraries that better preserved fluorescence. Contrary to trends observed among libraries constructed by error-prone PCR, preservation of function was observed to increase with a library's average mutation level among the four libraries designed with structure-based computational methods.GFP ͉ library design ͉ protein design ͉ protein engineering ͉ high-throughput screening P rotein sequence space is so vast that one can easily imagine the optimal sequence for a particular application will never be sampled by random mutation and recombination. Structure-based computational protein design tools seek to screen that sequence space more thoroughly than can be screened in the laboratory, but are currently based on approximate representations of candidate sequences and an incomplete understanding of the relationships between structure and function. Although many algorithms used to screen sequences in silico aim to identify a single optimal sequence (1-5), others aim instead to optimize the composition of a library of sequences (6-13). Provided that resources exist to synthesize and screen such libraries, library design algorithms compensate for the approximations built into them by increasing the number of attempts at designing the desired function. Viewed from a complementary perspective, such algorithms aim to sample sequence space more effectively than methods that randomly generate sequence diversity.Designed libraries can be synthesized for roughly the same cost as a designed sequence by recognizing the opportunities in gene synthesis for the combinatorial shuffling of sequence diversity (14-17). Although many algorithms have now been proposed to design such combinatorial libraries (7-9, 11, 12), few computationally designed libraries have been characterized experimentally (9,18,19), and, to our knowledge, there have been no controlled experiments comparing these methods with each other or with libraries of randomly generated sequence diversity. The results of such a comparison would be hard to predict, especially because none of these methods models protein function explicitly. Instead, these algorithms attempt to model protein stability as a surrogate for protein function on the assumption that libraries with a greater fraction of well folded proteins are more likely to contain variants with the desired function.Here, we evaluate seven design...

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Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently

Cited by 186 publications

References 28 publications

Broad 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor Herbicide Tolerance in Soybean with an Optimized Enzyme and Expression Cassette

Broad 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor Herbicide Tolerance in Soybean with an Optimized Enzyme and Expression Cassette

Synthetic shuffling and in vitro selection reveal the rugged adaptive fitness landscape of a kinase ribozyme

Computationally designed libraries of fluorescent proteins evaluated by preservation and diversity of function

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