title: Novel Rhizosphere Soil Alleles for the Enzyme 1-Aminocyclopropane-1-Carboxylate Deaminase Queried for Function with an In Vivo Competition Assay.
abstract: Metagenomes derived from environmental microbiota encode a vast diversity of protein homologs. How this diversity impacts protein function can be explored through selection assays aimed to optimize function. While artificially generated gene sequence pools are typically used in selection assays, their usage may be limited because of technical or ethical reasons. Here, we investigate an alternative strategy, the use of soil microbial DNA as a starting point. We demonstrate this approach by optimizing the function of a widely occurring soil bacterial enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase. We identified a specific ACC deaminase domain region (ACCD-DR) that, when PCR amplified from the soil, produced a variant pool that we could swap into functional plasmids carrying ACC deaminase-encoding genes. Functional clones of ACC deaminase were selected for in a competition assay based on their capacity to provide nitrogen to Escherichia coli in vitro. The most successful ACCD-DR variants were identified after multiple rounds of selection by sequence analysis. We observed that previously identified essential active-site residues were fixed in the original unselected library and that additional residues went to fixation after selection. We identified a divergent essential residue whose presence hints at the possible use of alternative substrates and a cluster of neutral residues that did not influence ACCD performance. Using an artificial ACCD-DR variant library generated by DNA oligomer synthesis, we validated the same fixation patterns. Our study demonstrates that soil metagenomes are useful starting pools of protein-coding-gene diversity that can be utilized for protein optimization and functional characterization when synthetic libraries are not appropriate.
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA Department of Microbiology, Cornell University, Ithaca, New York, USA.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Jin
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA Department of Microbiology, Cornell University, Ithaca, New York, USA.
authors.forename: Sara C
authors.initials: SC
authors.lastname: Di Rienzi
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA Department of Microbiology, Cornell University, Ithaca, New York, USA.
authors.forename: Anders
authors.initials: A
authors.lastname: Janzon
authors.affiliation: Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York, USA.
authors.forename: Jeff J
authors.initials: JJ
authors.lastname: Werner
authors.affiliation: Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York, USA.
authors.forename: Largus T
authors.initials: LT
authors.lastname: Angenent
authors.affiliation: Howard Hughes Medical Institute, Chevy Chase, Maryland, USA Department of Biology, University of North Carolina, Chapel Hill, North Carolina, USA Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina, USA Carolina Center for Genome Sciences, University of North Carolina, Chapel Hill, North Carolina, USA.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Dangl
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Fowler
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA Department of Microbiology, Cornell University, Ithaca, New York, USA Max Planck Institute for Developmental Biology, Tübingen, Germany rel222@cornell.edu.
authors.forename: Ruth E
authors.initials: RE
authors.lastname: Ley
mesh_terms: D000483:Alleles; D019755:Carbon-Carbon Lyases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism; D005820:Genetic Testing / Q000379:methods*; D056186:Metagenomics / Q000379:methods*; D009584:Nitrogen / Q000378:metabolism; D058441:Rhizosphere; D012988:Soil Microbiology
chemical_list: C033634:1-aminocyclopropane-1-carboxylate deaminase; D019755:Carbon-Carbon Lyases; D009584:Nitrogen