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.

1-Aminocyclopropane-1-carboxylate deaminase producers associated to maize and other Poaceae species

“…In the root zone, the plant may be colonized by different types of acdS + microorganisms [ 23 , 24 ], which are likely to contribute jointly to degradation of ACC produced by roots, and the overall significance of ACC deamination for the plant is expected to result from the combined functioning of its acdS + microbial partners [ 2 ]. However, even though various kinds of acdS + microorganisms can be readily isolated from the rhizosphere, there is no direct PCR tool available to assess the entire functional group of root-associated acdS + microorganisms, i.e., including non-cultured taxa and strains.…”

A Glucuronic Acid-Producing Endophyte Pseudomonas sp. MCS15 Reduces Cadmium Uptake in Rice by Inhibition of Ethylene Biosynthesis

“…It is well documented that ACC deaminase-producing bacteria can change plant physiology, which is similar to the mutants defective in ethylene signal transduction pathways ( Santoyo et al, 2016 ). ACC, the precursor of ethylene biosynthesis can be converted into α-ketobutyrate and ammonia by bacterial ACC deaminase, thereby mediating the levels of endogenous ethylene in plants under adverse stresses ( Glick, 2005 ; Jin et al, 2015 ; Bouffaud et al, 2018 ; Naing et al, 2021 ). The ACC deaminase-producing bacteria can enhance the tolerance of plants to various stresses, such as drought, salt, and waterlogging, by inhibiting overproduction of ethylene ( Ali and Kim, 2018 ; Gupta and Pandey, 2019 ; Orozco-Mosqueda et al, 2019 ; Danish et al, 2021 ).…”

Effects of Increased 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Activity in <i>Bradyrhizobium</i> sp. SUTN9-2 on Mung Bean Symbiosis under Water Deficit Conditions

“…and Pseudomonas sp., and amino acid residues were also important for enzymatic activity. Based on the alignments of the three-dimensional structures, a small domain of ACC deaminase-containing bacteria (amino acid residues 58 to 169) may be the region responsible for the diversity or establishment of enzymatic activity by ACC deaminase-containing microorganisms ( Jin et al , 2016 ). Yao et al (2000) proposed that amino acid residues 55 to 91, located on the small domain (helix 6) in the extra loop as a binding site of the ACC deaminase enzyme, were fixed after growth in medium broth supplemented with ACC for several selection rounds.…”