Cytokinin synthase enzymes, constructs, and related methods

Abstract

The present disclosure relates to a new class of cytokinin biosynthetic enzymes, cytokinin synthases, which have two domains: an isopentenyl transfer (IPT)-like domain and a cytokinin nucleotide phosphoribohydrolase (PRH)-like domain. The invention provides compositions and methods for the recombinant production of cytokinin synthase, host cells and transformants that include the cytokinin synthases, as well as compositions and formulations that include the disclosed cytokinin synthase.

What is claimed is: 
     
         1 . A recombinant polynucleotide comprising
 a. a cytokinin synthase coding sequence having at least 57% amino acid sequence identity to any one of SEQ ID NO:3 (EfCKS), SEQ ID NO:9 (AtCKS), SEQ ID NO:13 (BoCKS), SEQ ID NO:17 (IrCKS), SEQ ID NO:21 (AhCKS), or SEQ ID NO:25 (FfCKS), wherein the encoded cytokinin synthase comprises a sequence that, when aligned with a consensus sequence of SEQ ID NO:3 (EfCKS), SEQ ID NO:9 (AtCKS), SEQ ID NO:13 (BoCKS), SEQ ID NO:17 (IrCKS), SEQ ID NO:21 (AhCKS), or SEQ ID NO:25 (FfCKS), comprises (i) a first motif GPTXaa1Xaa 2 GKT (SEQ ID NO:30), wherein Xaa 1  is G or A and Xaa 2  is V, S, A, or T, at consensus sequence amino acid positions 13-20; (ii) a second motif PXaa 3 Xaa 4 Xaa 5 GGSXaa 6 S (SEQ ID NO:31), wherein Xaa 3  is I or V, Xaa 4  is L or V, Xaa 5  is V or C, and Xaa 6  is T or I, at consensus sequence amino acid positions 96-104, (iii) a third motif Xaa 7 Xaa 8 YGGG (SEQ ID NO:32), wherein Xaa 7  is L or I, and Xaa 8  is V or I, at consensus sequence amino acid positions 333-338, and (iv) Xaa 9 GGYGT Xaa 10 EEL (SEQ ID NO:33), where Xaa 9  is S or P and Xaa 10  is L or M, at consensus sequence amino acid positions 426-438, and   b. a heterologous sequence.   
     
     
         2 . The polynucleotide of  claim 1 , wherein the cytokinin synthase coding sequence (i) comprises a sequence having at least 70% nucleotide sequence identity to one of SEQ ID NO:1 (EfCKS), SEQ ID NO:8 (AtCKS), SEQ ID NO:12 (BoCKS), SEQ ID NO:16 (IrCKS), SEQ ID NO:21 (AhCKS), or SEQ ID NO:24 (FfCKS) or (ii) encodes a cytokinin synthase having at least 80% amino acid sequence identity to any one of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54. 
     
     
         3 . The polynucleotide of  claim 1 , wherein the cytokinin synthase coding sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NO:1 (EfCKS), SEQ ID NO:8 (AtCKS), SEQ ID NO:12 (BoCKS), SEQ ID NO:16 (IrCKS), SEQ ID NO:21 (AhCKS), or SEQ ID NO:24 (FfCKS). 
     
     
         4 . The polynucleotide of  claim 1 , wherein the cytokinin synthase coding sequence comprises a sequence having at least 95% sequence identity to any one of SEQ ID NO:1 (EfCKS), SEQ ID NO:8 (AtCKS), SEQ ID NO:12 (BoCKS), SEQ ID NO:16 (IrCKS), SEQ ID NO:21 (AhCKS), or SEQ ID NO:24 (FfCKS). 
     
     
         5 . The polynucleotide of  claim 1 , wherein the cytokinin synthase coding sequence comprises one of SEQ ID NO:1 (EfCKS), SEQ ID NO:8 (AtCKS), SEQ ID NO:12 (BoCKS), SEQ ID NO:16 (IrCKS), SEQ ID NO:20 (AhCKS), or SEQ ID NO:24 (FfCKS). 
     
     
         6 . The polynucleotide of  claim 1 , wherein the cytokinin synthase coding sequence is optimized for expression in a host cell. 
     
     
         7 . The polynucleotide of  claim 1 , wherein the cytokinin synthase coding sequence is optimized for expression in a host cell selected from the group consisting of a bacteria, yeast, plant, dicot plant, monocot plant, maize, soybean, canola, cotton, wheat,  Arabidopsis thaliana,  rice ( Oryza sativa ), sunflower, grass, creeping bentgrass ( Agrostis stolonifera ), tall fescue ( Festuca arundinacea ), tobacco ( Nicotiana tabacum ), and poplar hybrid. 
     
     
         8 . The polynucleotide of  claim 1 , wherein the heterologous sequence is a heterologous promoter and the promoter is operably linked to the cytokinin synthase coding sequence. 
     
     
         9 . The polynucleotide of  claim 8 , wherein the promoter is a bacteria promoter, heterologous fungal promoter, yeast promoter, or plant promoter. 
     
     
         10 . The polynucleotide of  claim 8 , wherein the promoter is a dicot promoter or a monocot promoter. 
     
     
         11 . The polynucleotide of  claim 8 , wherein the promoter is a maize promoter. 
     
     
         12 . The polynucleotide of  claim 8 , wherein the promoter is a 35S CaMV, 34S FMV, Napin, 7S alpha, 7S alpha', Glob, Lec, ZmGS2, ZmSTP13, or ZmGSTU6 promoter. 
     
     
         13 . The polynucleotide of  claim 1 , wherein the cytokinin synthase coding sequence is flanked by a first upstream heterologous sequence and a second downstream heterologous sequence, and the first and second heterologous sequences are (i) more than 80% identical genomic sequence from a heterologous host and (ii) suitable for catalyzing integration by homologous recombination into the host. 
     
     
         14 . A gene expression cassette comprising the polynucleotide of  claim 1 . 
     
     
         15 . A recombinant vector comprising
 a. the gene expression cassette of claim  144 ; and   b. a selectable marker.   
     
     
         16 . The recombinant vector of  claim 15 , wherein
 a. the gene expression cassette comprises the polynucleotide of  claim 9 ; and   b. the vector comprises an origin of replication that is functional in bacteria or yeast.   
     
     
         17 . The polynucleotide of  claim 1 , wherein the heterologous sequence encodes a protein fusion tag. 
     
     
         18 . The polynucleotide of claim  177 , wherein the encoded protein fusion tag is a poly-histidine, poly-arginine, haloalkane dehalogenase, streptavidin-binding, glutathione s-transferase (GST), maltose-binding protein (MBP), thioredoxin, small ubiquitin-like modifier (SUMO), N-utilization substance A (NusA), protein disulfide isomerase I (DsbA), Mistic, Ketosteroid isomerase (KSI), or TrpE, c-myc, hemaglutinin antigen (HA), FLAG, 1D4, calmodulin-binding peptide, chitin-binding domain, cellulose-binding domain, S-tag, or Softag3 protein fusion tag. 
     
     
         19 . A host cell comprising the polynucleotide of  claim 1 . 
     
     
         20 . The host cell of  claim 19 , wherein the host cell is a transformed bacteria cell, a transformed fungus cell, a transformed yeast cell, or a transgenic plant cell. 
     
     
         21 . The host cell of  claim 20 , wherein the host cell is a transgenic monocot plant cell or a transgenic dicot plant cell. 
     
     
         22 . The host cell of  claim 21 , wherein the host cell is a soybean host cell or a maize host cell. 
     
     
         23 . A transgenic plant comprising the host cell of  claim 21 . 
     
     
         24 . An isolated or recombinant cytokinin synthase having at least 57% amino acid sequence identity to any one of SEQ ID NO:3 (EfCKS), SEQ ID NO:9 (AtCKS), SEQ ID NO:13 (BoCKS), SEQ ID NO:17 (IrCKS), SEQ ID NO:21 (AhCKS), or SEQ ID NO:25 (FfCKS), wherein the encoded cytokinin synthase comprises a sequence that, when aligned with a consensus sequence of SEQ ID NO:3 (EfCKS), SEQ ID NO:9 (AtCKS), SEQ ID NO:13 (BoCKS), SEQ ID NO:17 (IrCKS), SEQ ID NO:21 (AhCKS), or SEQ ID NO:25 (FfCKS), comprises (i) a first motif GPTXaa 1 Xaa 2 GKT (SEQ ID NO:30), wherein Xaa 1  is G or A and Xaa 2  is V, S, A, or T, at consensus sequence amino acid positions 13-20; (ii) a second motif PXaa 3 Xaa 4 Xaa 5 GGSXaa 6 S (SEQ ID NO:31), wherein Xaa 3  is I or V, Xaa 4  is L or V, Xaa 5  is V or C, and Xaa 6  is T or I, at consensus sequence amino acid positions 96-104, (iii) a third motif Xaa 7 Xaa 8 YGGG (SEQ ID NO:32), wherein Xaa 7  is L or I, and Xaa 8  is V or I, at consensus sequence amino acid positions 333-338, and (iv) Xaa 9 GGYGT Xaa 10 EEL (SEQ ID NO:33), where Xaa 9  is S or P and Xaa 10  is L or M, at consensus sequence amino acid positions 426-438, wherein the cytokinin synthase is substantially free of fungal material from a fungus that comprises native cytokinin synthase. 
     
     
         25 . The isolated or recombinant cytokinin synthase of  claim 24 , wherein the cytokinin synthase comprises a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NO:3 (EfCKS), SEQ ID NO:9 (AtCKS), SEQ ID NO:13 (BoCKS), SEQ ID NO:17 (IrCKS), SEQ ID NO:21 (AhCKS), SEQ ID NO:25 (FfCKS), SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54. 
     
     
         26 . The isolated cytokinin synthase of  claim 24 , wherein the cytokinin synthase is covalently linked to a protein fusion tag. 
     
     
         27 . A composition for application of cytokinin synthase, wherein the composition comprises
 a. cytokinin produced by the cytokinin synthase of  claims 24 ; and   b. one or more inactive ingredients suitable for application to a plant.   
     
     
         28 . The composition of  claim 27 , wherein the composition is a formulation selected from the group consisting of a liquid formulation, water-soluble concentrate, emulsifiable concentrate, flowable suspension, an aqueous suspension, a sprayable formulation, a dry formulation, granule, pellet, wettable powder, soluble powder, water dispersible granule, or dry flowable formulation. 
     
     
         29 . A method for producing recombinant cytokinin synthase, wherein the method comprises,
 a. expressing the recombinant cytokinin synthase in the host cell of  claim 19 ; and   b. isolating the cytokinin synthase from host cell materials.   
     
     
         30 . A method of converting cytokinin precursors to cytokinin, the method comprising contacting cytokinin precursor monophosphate (AMP) and either dimethylallyl diphosphate (DMAPP) or hydroxymethylbutenyl diphosphate (HMBDP) to an isolated or recombinant cytokinin synthase in a host cell, a transgenic plant, or in vitro, wherein the isolated or recombinant cytokinin synthase has at least 57% amino acid sequence identity to any one of SEQ ID NO:3 (EfCKS), SEQ ID NO:9 (AtCKS), SEQ ID NO:13 (BoCKS), SEQ ID NO:17 (IrCKS), SEQ ID NO:21 (AhCKS), or SEQ ID NO:25 (FfCKS), wherein the encoded cytokinin synthase comprises a sequence that, when aligned with a consensus sequence of SEQ ID NO:3 (EfCKS), SEQ ID NO:9 (AtCKS), SEQ ID NO:13 (BoCKS), SEQ ID NO:17 (IrCKS), SEQ ID NO:21 (AhCKS), or SEQ ID NO:25 (FfCKS), comprises (i) a first motif GPTXaa 1 Xaa 2 GKT (SEQ ID NO:30), wherein Xaa 1  is G or A and Xaa 2  is V, S, A, or T, at consensus sequence amino acid positions 13-20; (ii) a second motif PXaa 3 Xaa 4 Xaa 5 GGSXaa 6 S (SEQ ID NO:31), wherein Xaa 3  is I or V, Xaa 4  is L or V, Xaa 5  is V or C, and Xaa6 is T or I, at consensus sequence amino acid positions 96-104, (iii) a third motif Xaa 7 Xaa8YGGG (SEQ ID NO:32), wherein Xaa 7  is L or I, and Xaa 8  is V or I, at consensus sequence amino acid positions 333-338, and (iv) Xaa 9 GGYGT Xaa 10 EEL (SEQ ID NO:33), where Xaa 9  is S or P and Xaa 10  is L or M, at consensus sequence amino acid positions 426-438.