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Protein engineering can enhance desirable features and improve performance outside of the natural context. Several strategies have been adopted over the years for gene diversification, and engineering of modular proteins in particular is most effective when a high-throughput, library-based approach is employed. Nondegenerate saturation mutagenesis plays a dynamic role in engineering proteins by targeting multiple codons to generate massively diverse gene libraries. Herein, we describe the nondegenerate saturation mutagenesis techniques that we have developed for contiguous (ProxiMAX) and noncontiguous (MAX) randomized codon generation to create precisely defined, diverse gene libraries, in the context of other fully nondegenerate strategies. ProxiMAX randomization comprises saturation cycling with repeated cycles of blunt-ended ligation, type IIS restriction, and PCR amplification, and is now a commercially automated process predominantly used for antibody library generation. MAX randomization encompasses a manual process of selective hybridisation between individual custom oligonucleotide mixes and a conventionally randomized template and is principally employed in the research laboratory setting, to engineer alpha helical proteins and active sites of enzymes. DNA libraries generated using either technology create high-throughput amino acid substitutions via codon randomization, to generate genetically diverse clones.

Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation., Mep2 proteins are tightly regulated fungal ammonium transporters. Here, the authors report the crystal structures of closed states of Mep2 proteins and propose a model for their regulation by comparing them with the open ammonium transporters of bacteria.

Summary Synchronization of circadian clocks to the day-night cycle ensures the correct timing of biological events. This entrainment process is essential to ensure that the phase of the circadian oscillator is synchronized with daily events within the environment [1], to permit accurate anticipation of environmental changes [2, 3]. Entrainment in plants requires phase changes in the circadian oscillator, through unidentified pathways, which alter circadian oscillator gene expression in response to light, temperature, and sugars [4, 5, 6]. To determine how circadian clocks respond to metabolic rhythms, we investigated the mechanisms by which sugars adjust the circadian phase in Arabidopsis [5]. We focused upon metabolic regulation because interactions occur between circadian oscillators and metabolism in several experimental systems [5, 7, 8, 9], but the molecular mechanisms are unidentified. Here, we demonstrate that the transcription factor BASIC LEUCINE ZIPPER63 (bZIP63) regulates the circadian oscillator gene PSEUDO RESPONSE REGULATOR7 (PRR7) to change the circadian phase in response to sugars. We find that SnRK1, a sugar-sensing kinase that regulates bZIP63 activity and circadian period [10, 11, 12, 13, 14] is required for sucrose-induced changes in circadian phase. Furthermore, TREHALOSE-6-PHOSPHATE SYNTHASE1 (TPS1), which synthesizes the signaling sugar trehalose-6-phosphate, is required for circadian phase adjustment in response to sucrose. We demonstrate that daily rhythms of energy availability can entrain the circadian oscillator through the function of bZIP63, TPS1, and the KIN10 subunit of the SnRK1 energy sensor. This identifies a molecular mechanism that adjusts the circadian phase in response to sugars., Graphical Abstract, Highlights • The transcription factor bZIP63 binds and regulates the circadian clock gene PRR7 • bZIP63 is required for adjustment of circadian period by sugars • Trehalose-6-phosphate metabolism and KIN10 signaling regulate circadian period • Sugar signals establish the correct circadian phase in light and dark cycles, Frank et al. identify mechanisms by which the Arabidopsis circadian clock entrains to sugars. Metabolic adjustment of circadian phase involves trehalose-6-phosphate, SnRK1 subunit KIN10 and the transcription factor bZIP63. bZIP63 regulates the circadian clock gene PSEUDORESPONSE REGULATOR7. This sets the circadian phase in light and dark cycles.

Emerging seedlings respond to environmental conditions such as light and temperature to optimize their establishment. Seedlings grow initially through elongation of the hypocotyl, which is regulated by signalling pathways that integrate environmental information to regulate seedling development. The hypocotyls of Arabidopsis thaliana also elongate in response to sucrose. Here, we investigated the role of cellular sugar-sensing mechanisms in the elongation of hypocotyls in response to sucrose. We focused upon the role of SnRK1, which is a sugar-signalling hub that regulates metabolism and transcription in response to cellular energy status. We also investigated the role of TPS1, an enzyme that synthesizes the signalling sugar trehalose-6-phosphate (Tre6P) that is proposed to regulate SnRK1 activity. Under light/dark cycles, we found that sucrose-induced hypocotyl elongation did not occur in tps1 mutants and overexpressors of KIN10 (AKIN10/SnRK1.1), a catalytic subunit of SnRK1. We demonstrate that the magnitude of sucrose-induced hypocotyl elongation depends on the day length and light intensity. We identified roles for auxin and gibberellin signalling in sucrose-induced hypocotyl elongation under short photoperiods. We found that sucrose-induced hypocotyl elongation under light/dark cycles does not involve another proposed sugar sensor, HEXOKINASE1, or the circadian oscillator. Our study identifies novel roles for KIN10 and TPS1 in mediating a signal that underlies sucrose-induced hypocotyl elongation in light/dark cycles.

The East Indian sandalwood, Santalum album, valued for its fragrant oil yielding heartwood is a major ingre- dient in indigenous medicines and perfumes. Scarcity of sandal has led to illegal felling of sandal trees, and adulteration of sandalwood and oil. This study represents the first molecular phylogeny of S. album and its adulterant species Osyris wightiana, Erythroxylum monogynum, Buxus sempervirens, Ximenia americana, Osyris lanceolata, and Chukrasia tabularis through 18S and 26S rDNA sequencing. In the Maximum Parsimony (MP) tree for 18S and 26S rDNA data sets, moderate to high bootstrap support was obtained for the nodes. For 18S rDNA data sets, the tree had B. sempervirens and X. Americana as the upper branch, with E. monogynum branched separately to the cluster. The lower branch had S. album and O. wightiana with O. lanceolata joining separately to both clades of the tree. In the MP tree for 26S rDNA datasets, S. album and O. wightiana formed the major cluster with X. americana clustering separate and B. sempervirens and O. wightiana as the lower branch with C. tabularis clustering separate to the tree. The molecular data presented here provided useful information for resolving the phylogenetic relationship of these plants. Inferences from this study are in accordance with Cronquist's system of classification of flowering plants where all the species originate from a single phylogenetic tree of Rosidae.