Research Article Received: 27 June 2019 Revised: 24 August 2019 Accepted article published: 10 September 2019 Published online in Wiley Online Library: (wileyonlinelibrary.com) DOI 10.1002/ps.5613 Microbial HemG-type protoporphyrinogen IX oxidase enzymes for biotechnology applications in plant herbicide tolerance traits Clayton T Larue, * Joel E Ream, Xuefeng Zhou, Farhard Moshiri, † Arlene Howe, Michael Goley, Oscar C Sparks, Steven T Voss, Erin Hall, Christine Ellis, Janice Weihe, Qungang Qi, Daniela Ribeiro, Xiaoping Wei, Shirley Guo, Artem G Evdokimov, ‡ Marguerite J Varagona and James K Roberts Abstract BACKGROUND: Protoporphyrinogen IX oxidase (PPO)-inhibiting herbicides act by inhibiting a key enzyme in the heme and chlorophyll biosynthetic pathways in plants. This enzyme, the PPO enzyme, is conserved across plant species. However, some microbes are known to utilize a unique family of PPO enzymes, the HemG family. This enzyme family carries out the same enzymatic step as the plant PPO enzymes, but does not share sequence homology with the plant PPO enzymes. RESULTS: Bioinformatic analysis was used to identify putative HemG PPO enzyme variants from microbial sources. A subset of these variants was cloned and characterized. HemG PPO variants were characterized for functionality and tolerance to PPO-inhibiting herbicides. HemG PPO variants that exhibited insensitivity to PPO-inhibiting herbicides were identified for further characterization. Expression of selected variants in maize, soybean, cotton and canola resulted in plants that displayed tolerance to applications of PPO-inhibiting herbicides. CONCLUSION: Selected microbial-sourced HemG PPO enzyme variants present an opportunity for building new herbicide tolerance biotechnology traits. These traits provide tolerance to PPO-inhibiting herbicides and, therefore, could provide additional tools for farmers to employ in their weed management systems. © 2019 Society of Chemical Industry Supporting information may be found in the online version of this article. Keywords: protoporphyrinogen IX oxidase; PPO-inhibiting herbicides; herbicide tolerance; plant biotechnology; HemG 1 INTRODUCTION The enzyme, protoporphyrinogen IX oxidase (PPO) catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX by molecular oxygen. This enzymatic step is conserved across prokaryotic and eukaryotic life forms in the production of tetrapyrroles such as heme. 1 In plants, the production of chloro- phyll is also dependent on this enzymatic step. In plants, the production of chlorophyll is also dependent on this enzymatic step. In eukaryotic species and many prokaryotic species, the PPO enzyme is roughly 50 kDa and contains a binding domain for flavin adenine dinucleotide (FAD), its cofactor. 2,3 This family of PPO enzymes is often called the HemY family in microbes. Some microbes, especially within proteobacteria, do not possess a HemY PPO, but instead have a HemG PPO enzyme. The HemG PPO enzyme is a smaller enzyme, approximately 20 kDa, with shared domains to flavodoxins. 4,5 This family appears to utilize quinones as cofactors, which likely enables it HemG PPOs to function in oxygen-poor environments where the oxygen-dependent HemY PPOs would be unable to fully function. The HemG PPO enzymes contain a characteristic long-chain insert loop domain which is essential for PPO functionality. In plants, the PPO enzyme is the target of a large and diverse fam- ily of PPO-inhibiting herbicides. 6,7 PPO-inhibiting herbicides have been observed to bind in the active pocket of PPO enzymes when co-crystalized. 8,9 Application of PPO-inhibiting herbicides to sen- sitive plants results in a blockage of heme and chlorophyll biosyn- thetic pathways in the plastids, resulting in the accumulation of pathway intermediates which leak from the plastids and undergo non-specific oxidation to protoporphyrin IX in the cytosol. In the presence of oxygen and light, protoporphyrin IX rapidly generates singlet oxygen, resulting in uncontrolled membrane lipid peroxi- dation and plant death. 6,10,11 PPO-inhibiting herbicides have been used for four decades in agricultural weed control applications. The utility of these ∗ Correspondence to: CT Larue, Bayer Crop Science, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA. E-mail: clayton.larue@bayer.com † Current address: Benson Hill 1100 Corporate Square Drive, St. Louis MO 63132. ‡ Current address: EnkoChem, 19 Presidential Dr, Woburn, MA 01801. Bayer Crop Science, Chesterfield, MO, USA Pest Manag Sci (2019) www.soci.org © 2019 Society of Chemical Industry