A wave of specific transcript and protein accumulation accompanies pollen dehydration.

In flowering plants, male gametes are immotile and carried by dry pollen grains to the female organ. Dehydrated pollen is thought to withstand abiotic stress when grains are dispersed from the anther to the pistil, after which sperm cells are delivered via pollen tube growth for fertilization and seed set. Yet, the underlying molecular changes accompanying dehydration and the impact on pollen development are poorly understood. To gain a systems perspective, we analyzed published transcriptomes and proteomes of developing Arabidopsis thaliana pollen. Waves of transcripts are evident as microspores develop to bicellular, tricellular, and mature pollen. Between the "early"- and "late"-pollen-expressed genes, an unrecognized cluster of transcripts accumulated, including those encoding late-embryogenesis abundant (LEA), desiccation-related protein, transporters, lipid-droplet associated proteins, pectin modifiers, cysteine-rich proteins, and mRNA-binding proteins. Results suggest dehydration onset initiates after bicellular pollen is formed. Proteins accumulating in mature pollen like ribosomal proteins, initiation factors, and chaperones are likely components of mRNA-protein condensates resembling "stress" granules. Our analysis has revealed many new transcripts and proteins that accompany dehydration in developing pollen. Together with published functional studies, our results point to multiple processes, including (1) protect developing pollen from hyperosmotic stress, (2) remodel the endomembrane system and walls, (3) maintain energy metabolism, (4) stabilize presynthesized mRNA and proteins in condensates of dry pollen, and (5) equip pollen for compatibility determination at the stigma and for recovery at rehydration. These findings offer novel models and molecular candidates to further determine the mechanistic basis of dehydration and desiccation tolerance in plants.

Broadening The Substrate Scope of Aldolases Through Metagenomic Enzyme Discovery.

Bio-processes based on enzymatic catalysis play a major role in the development of green, sustainable processes, and the discovery of new enzymes is key to this approach. In this work, we analysed ten metagenomes and retrieved 48 genes coding for deoxyribose-5-phosphate aldolases (DERAs, EC 4.1.2.4) using a sequence-based approach. These sequences were recombinantly expressed in Escherichia coli and screened for activity towards a range of aldol additions. Among these, one enzyme, DERA-61, proved to be particularly interesting and catalysed the aldol addition of furfural or benzaldehyde with acetone, butanone and cyclobutanone with unprecedented activity. The product of these reactions, aldols, can find applications as building blocks in the synthesis of biologically active compounds. Screening was carried out to identify optimized reaction conditions targeting temperature, pH, and salt concentrations. Lastly, the kinetics and the stereochemistry of the products were investigated, revealing that DERA-61 and other metagenomic DERAs have superior activity and stereoselectivity when they are provided with non-natural substrates, compared to well-known DERAs.

Posttranslational regulation of transporters important for symbiotic interactions.

Coordinated sharing of nutritional resources is a central feature of symbiotic interactions, and, despite the importance of this topic, many questions remain concerning the identification, activity, and regulation of transporter proteins involved. Recent progress in obtaining genome and transcriptome sequences for symbiotic organisms provides a wealth of information on plant, fungal, and bacterial transporters that can be applied to these questions. In this update, we focus on legume-rhizobia and mycorrhizal symbioses and how transporters at the symbiotic interfaces can be regulated at the protein level. We point out areas where more research is needed and ways that an understanding of transporter mechanism and energetics can focus hypotheses. Protein phosphorylation is a predominant mechanism of posttranslational regulation of transporters in general and at the symbiotic interface specifically. Other mechanisms of transporter regulation, such as protein-protein interaction, including transporter multimerization, polar localization, and regulation by pH and membrane potential are also important at the symbiotic interface. Most of the transporters that function in the symbiotic interface are members of transporter families; we bring in relevant information on posttranslational regulation within transporter families to help generate hypotheses for transporter regulation at the symbiotic interface.

Methyltransferases: Functions and Applications.

In this review the current state-of-the-art of S-adenosylmethionine (SAM)-dependent methyltransferases and SAM are evaluated. Their structural classification and diversity is introduced and key mechanistic aspects presented which are then detailed further. Then, catalytic SAM as a target for drugs, and approaches to utilise SAM as a cofactor in synthesis are introduced with different supply and regeneration approaches evaluated. The use of SAM analogues are also described. Finally O-, N-, C- and S-MTs, their synthetic applications and potential for compound diversification is given.

Segregationally stabilised plasmids improve production of commodity chemicals in glucose-limited continuous fermentation.

BACKGROUND: The production of chemicals via bio-based routes is held back by limited easy-to-use stabilisation systems. A wide range of plasmid stabilisation mechanisms can be found in the literature, however, how these mechanisms effect genetic stability and how host strains still revert to non-productive variants is poorly understood at the single-cell level. This phenomenon can generate difficulties in production-scale bioreactors as different populations of productive and non-productive cells can arise. To understand how to prevent non-productive strains from arising, it is vital to understand strain behaviour at a single-cell level. The persistence of genes located on plasmid vectors is dependent on numerous factors but can be broadly separated into structural stability and segregational stability. While structural stability refers to the capability of a cell to resist genetic mutations that bring about a loss of gene function in a production pathway, segregational stability refers to the capability of a cell to correctly distribute plasmids into daughter cells to maintain copy number. A lack of segregational stability can rapidly generate plasmid-free variants during replication, which compromises productivity. RESULTS: Citramalate synthase expression was linked in an operon to the expression of a fluorescent reporter to enable rapid screening of the retention of a model chemical synthesis pathway in a continuous fermentation of E. coli. Cells without additional plasmid stabilisation started to lose productivity immediately after entering the continuous phase. Inclusion of a multimer resolution site, cer, enabled a steady-state production period of 58 h before a drop in productivity was detected. Single-cell fluorescence measurements showed that plasmid-free variants arose rapidly without cer stabilisation and that this was likely due to unequal distribution of plasmid into daughter cells during cell division. The addition of cer increased total chemical yield by more than 50%. CONCLUSIONS: This study shows the potential remains high for plasmids to be used as pathway vectors in industrial bio-based chemicals production, providing they are correctly stabilised. We demonstrate the need for accessible bacterial 'toolkits' to enable rapid production of known, stabilised bacterial production strains to enable continuous fermentation at scale for the chemicals industry.

Discovery of New Carbonyl Reductases Using Functional Metagenomics and Applications in Biocatalysis.

Enzyme discovery for use in the manufacture of chemicals, requiring high stereoselectivities, continues to be an important avenue of research. Here, a sequence directed metagenomics approach is described to identify short chain carbonyl reductases. PCR from a metagenomic template generated 37 enzymes, with an average 25% sequence identity, twelve of which showed interesting activities in initial screens. Six of the most productive enzymes were then tested against a panel of 21 substrates, including bulkier substrates that have been noted as challenging in biocatalytic reductions. Two enzymes were selected for further studies with the Wieland Miescher ketone. Notably, enzyme SDR-17, when co-expressed with a co-factor recycling system produced the anti-(4aR,5S) isomer in excellent isolated yields of 89% and 99% e.e. These results demonstrate the viability of a sequence directed metagenomics approach for the identification of multiple homologous sequences with low similarity, that can yield highly stereoselective enzymes with applicability in industrial biocatalysis.

A cell engineering approach to enzyme-based fed-batch fermentation.

BACKGROUND: A fundamental problem associated with E. coli fermentations is the difficulty in achieving high cell densities in batch cultures, attributed in large part to the production and accumulation of acetate through a phenomenon known as overflow metabolism when supplying enough glucose for the cell density desired. Although a fed-batch configuration is the standard method for reducing such issues, traditional fed-batch systems require components which become problematic when applying them at smaller scale. One alternative has been the development of a system whereby the enzymatic degradation of starch is used to release glucose at a controlled rate. However, to date, amylolytic enzymes have only been applied to the culture exogenously, whereas our goal is to design and construct a self-secreting amylolytic chassis capable of self-regulated enzyme-based fed-batch fermentation. RESULTS: A putative glucoamylase from C. violaceum has been cloned and expressed in E. coli BL21(DE3) and W3110, which exhibits significant glucose releasing amylolytic activity. Extracellular amylolytic activity was enhanced following a replacement of the enzymes native signal peptide with the DsbA signal sequence, contributing to a glucoamylase secreting strain capable of utilising starch as a sole carbon source in defined media. Introduction of PcstA, a glucose sensitive K12 compatible promoter, and the incorporation of this alongside C. violaceum glucoamylase in E. coli W3110, gave rise to increased cell densities in cultures grown on starch (OD600 ∼ 30) compared to those grown on an equivalent amount of glucose (OD600 ∼ 15). Lastly, a novel self-secreting enzyme-based fed-batch fermentation system was demonstrated via the simultaneous expression of the C. violaceum glucoamylase and a recombinant protein of interest (eGFP), resulting in a fourfold increase in yield when grown in media containing starch compared with the glucose equivalent. CONCLUSIONS: This study has developed, through the secretion of a previously uncharacterised bacterial glucoamylase, a novel amylolytic E. coli strain capable of direct starch to glucose conversion. The ability of this strain to achieve increased cell densities as well as an associated increase in recombinant protein yield when grown on starch compared with an equivalent amount of glucose, demonstrates for the first time a cell engineering approach to enzyme-based fed-batch fermentation.

Characterisation of a hyperthermophilic transketolase from Thermotoga maritima DSM3109 as a biocatalyst for 7-keto-octuronic acid synthesis.

Transketolase (TK) is a fundamentally important enzyme in industrial biocatalysis which carries out a stereospecific carbon-carbon bond formation, and is widely used in the synthesis of prochiral ketones. This study describes the biochemical and molecular characterisation of a novel and unusual hyperthermophilic TK from Thermotoga maritima DSM3109 (TKtmar). TKtmar has a low protein sequence homology compared to the already described TKs, with key amino acid residues in the active site highly conserved. TKtmar has a very high optimum temperature (>90 °C) and shows pronounced stability at high temperature (e.g. t1/2 99 and 9.3 h at 50 and 80 °C, respectively) and in presence of organic solvents commonly used in industry (DMSO, acetonitrile and methanol). Substrate screening showed activity towards several monosaccharides and aliphatic aldehydes. In addition, for the first time, TK specificity towards uronic acids was achieved with TKtmar catalysing the efficient conversion of d-galacturonic acid and lithium hydroxypyruvate into 7-keto-octuronic acid, a very rare C8 uronic acid, in high yields (98%, 49 mM).

A photoelectron imaging study of the deprotonated GFP chromophore anion and RNA fluorescent tags.

Green fluorescent protein (GFP), together with its family of variants, is the most widely used fluorescent protein for in vivo imaging. Numerous spectroscopic studies of the isolated GFP chromophore have been aimed at understanding the electronic properties of GFP. Here, we build on earlier work [A. V. Bochenkova, C. Mooney, M. A. Parkes, J. Woodhouse, L. Zhang, R. Lewin, J. M. Ward, H. Hailes, L. H. Andersen and H. H. Fielding, Chem. Sci., 2017, 8, 3154] investigating the impact of fluorine and methoxy substituents that have been employed to tune the electronic structure of the GFP chromophore for use as fluorescent RNA tags. We present photoelectron spectra following photoexcitation over a broad range of wavelengths (364-230 nm) together with photoelectron angular distributions following photoexcitation at 364 nm, which are interpreted with the aid of quantum chemistry calculations. The results support the earlier high-level quantum chemistry calculations that predicted how fluorine and methoxy substituents tune the electronic structure and we find evidence to suggest that the methoxy substituents enhance internal conversion, most likely from the 2ππ* state which has predominantly Feshbach resonance character, to the 1ππ* state.

Multienzyme One-Pot Cascades Incorporating Methyltransferases for the Strategic Diversification of Tetrahydroisoquinoline Alkaloids.

The tetrahydroisoquinoline (THIQ) ring system is present in a large variety of structurally diverse natural products exhibiting a wide range of biological activities. Routes to mimic the biosynthetic pathways to such alkaloids, by building cascade reactions in vitro, represents a successful strategy and can offer better stereoselectivities than traditional synthetic methods. S-Adenosylmethionine (SAM)-dependent methyltransferases are crucial in the biosynthesis and diversification of THIQs; however, their application is often limited in vitro by the high cost of SAM and low substrate scope. In this study, we describe the use of methyltransferases in vitro in multi-enzyme cascades, including for the generation of SAM in situ. Up to seven enzymes were used for the regioselective diversification of natural and non-natural THIQs on an enzymatic preparative scale. Regioselectivites of the methyltransferases were dependent on the group at C-1 and presence of fluorine in the THIQs. An interesting dual activity was also discovered for the catechol methyltransferases used, which were found to be able to regioselectively methylate two different catechols in a single molecule.

A Palette of Minimally Tagged Sucrose Analogues for Real-Time Raman Imaging of Intracellular Plant Metabolism.

Sucrose is the main saccharide used for long-distance transport in plants and plays an essential role in energy metabolism; however, there are no analogues for real-time imaging in live cells. We have optimised a synthetic approach to prepare sucrose analogues including very small (≈50 Da or less) Raman tags in the fructose moiety. Spectroscopic analysis identified the alkyne-tagged compound 6 as a sucrose analogue recognised by endogenous transporters in live cells and with higher Raman intensity than other sucrose derivatives. Herein, we demonstrate the application of compound 6 as the first optical probe to visualise real-time uptake and intracellular localisation of sucrose in live plant cells using Raman microscopy.

Arabidopsis Sucrose Transporter AtSuc1 introns act as strong enhancers of expression.

The expression of AtSUC1 is controlled by the promoter and intragenic sequences. AtSUC1 is expressed in roots, pollen and trichomes. However, AtSUC1 promoter-GUS transgenics only show expression in trichomes and pollen. Here, we show that the root expression of AtSUC1 is controlled by an interaction between the AtSUC1 promoter and two short introns. The deletion of either intron from whole-gene-GUS constructs results in no root expression, showing that both introns are required. The two introns in tandem, fused to GUS, produce high constitutive expression throughout the vegetative parts of the plant. When combined with the promoter, the expression driven by the introns is reduced and localized to the roots. In Arabidopsis seedlings, exogenously applied sucrose induces the expression of AtSUC1 in roots and causes anthocyanin accumulation. atsuc1 loss-of-function mutants are defective in sucrose-induced anthocyanin accumulation. We show that an AtSUC1 whole-gene-GUS construct expressing a nonfunctional AtSUC1 (D152N) mutant, that is transport inactive, is defective in sucrose-induced AtSUC1 expression when expressed in an atsuc1-null background. We also show that the transport-defective allele does not complement the loss of sucrose-induced anthocyanin accumulation in null atsuc1 mutants. The results indicate that sucrose uptake via AtSUC1 is required for sucrose-induced AtSUC1 expression and sucrose-induced anthocyanin accumulation and that the site for sucrose detection is intracellular.

The Molecular Dialog between Flowering Plant Reproductive Partners Defined by SNP-Informed RNA-Sequencing.

The molecular interactions between reproductive cells are critical for determining whether sexual reproduction between individuals results in fertilization and can result in barriers to interspecific hybridization. However, it is a challenge to define the complete molecular exchange between reproductive partners because parents contribute to a complex mixture of cells during reproduction. We unambiguously defined male- and female-specific patterns of gene expression during Arabidopsis thaliana reproduction using single nucleotide polymorphism-informed RNA-sequencing analysis. Importantly, we defined the repertoire of pollen tube-secreted proteins controlled by a group of MYB transcription factors that are required for sperm release from the pollen tube to the female gametes, a critical barrier to interspecific hybridization. Our work defines the pollen tube gene products that respond to the pistil and are required for reproductive success; moreover, we find that these genes are highly evolutionarily plastic both at the level of coding sequence and expression across A. thaliana accessions.

Biomimetic Phosphate-Catalyzed Pictet-Spengler Reaction for the Synthesis of 1,1'-Disubstituted and Spiro-Tetrahydroisoquinoline Alkaloids.

Tetrahydroisoquinoline (THIQ) alkaloids are an important group of compounds that exhibit a range of bioactivities. Here, a phosphate buffer-catalyzed Pictet-Spengler reaction (PSR) using unreactive ketone substrates is described. A variety of 1,1'-disubstituted and spiro-tetrahydroisoquinoline alkaloids were readily prepared in one-step and high yields, highlighting the general applicability of this approach. This study features the role of phosphate in the aqueous-based PSR and provides an atom-efficient, sustainable route to new THIQs.

Aminopolyols from Carbohydrates: Amination of Sugars and Sugar-Derived Tetrahydrofurans with Transaminases.

Carbohydrates are the major component of biomass and have unique potential as a sustainable source of building blocks for chemicals, materials, and biofuels because of their low cost, ready availability, and stereochemical diversity. With a view to upgrading carbohydrates to access valuable nitrogen-containing sugar-like compounds such as aminopolyols, biocatalytic aminations using transaminase enzymes (TAms) have been investigated as a sustainable alternative to traditional synthetic strategies. Demonstrated here is the reaction of TAms with sugar-derived tetrahydrofuran (THF) aldehydes, obtained from the regioselective dehydration of biomass-derived sugars, to provide access to cyclic aminodiols in high yields. In a preliminary study we have also established the direct transamination of sugars to give acyclic aminopolyols. Notably, the reaction of the ketose d-fructose proceeds with complete stereoselectivity to yield valuable aminosugars in high purity.

Design and Use of de novo Cascades for the Biosynthesis of New Benzylisoquinoline Alkaloids.

The benzylisoquinoline alkaloids (BIAs) are an important group of secondary metabolites from higher plants and have been reported to show significant biological activities. The production of BIAs through synthetic biology approaches provides a higher-yielding strategy than traditional synthetic methods or isolation from plant material. However, the reconstruction of BIA pathways in microorganisms by combining heterologous enzymes can also give access to BIAs through cascade reactions. Most importantly, non-natural BIAs can be generated through such artificial pathways. In the current study, we describe the use of tyrosinases and decarboxylases and combine these with a transaminase enzyme and norcoclaurine synthase for the efficient synthesis of several BIAs, including six non-natural alkaloids, in cascades from l-tyrosine and analogues.

MpAMT1;2 from Marchantia polymorpha is a High-Affinity, Plasma Membrane Ammonium Transporter.

Plant ammonium transporters in the AMT/MEP/Rh (ammonium transporter/methylammonium and ammonium permease/Rhesus factor) superfamily have only been previously characterized in flowering plants (angiosperms). Plant AMT1s are electrogenic, while plant AMT2s are electroneutral, and MEP and Rh transporters in other organisms are electroneutral. We analyzed the transport function of MpAMT1;2 from the basal land plant Marchantia polymorpha, a liverwort. MpAMT1;2 was shown to localize to the plasma membrane in Marchantia gametophyte thallus by stable transformation using a C-terminal citrine fusion. MpAMT1;2 expression was studied using quantitative real-time PCR and shown to be higher when plants were N deficient and lower when plants were grown on media containing ammonium, nitrate or the amino acid glutamine. Expression in Xenopus oocytes and analysis by electrophysiology revealed that MpAMT1;2 is an electrogenic ammonium transporter with a very high affinity for ammonium (7 µM at pH 5.6 and a membrane potential of -137 mV). A conserved inhibitory phosphorylation site identified in angiosperm AMT1s is also present in all AMT1s in Marchantia. Here we show that a phosphomimetic mutation T475D in MpAMT1;2 completely inhibits ammonium transport activity. The results indicate that MpAMT1;2 may be important for ammonium uptake into cells in the Marchantia thallus.

Sucrose Transporter Localization and Function in Phloem Unloading in Developing Stems.

How sucrose transporters (SUTs) regulate phloem unloading in monocot stems is poorly understood and particularly so for species storing high Suc concentrations. To this end, Sorghum bicolor SUTs SbSUT1 and SbSUT5 were characterized by determining their transport properties heterologously expressed in yeast or Xenopus laevis oocytes, and their in planta cellular and subcellular localization. The plasma membrane-localized SbSUT1 and SbSUT5 exhibited a strong selectivity for Suc and high Suc affinities in X. laevis oocytes at pH 5-SbSUT1, 6.3 ± 0.7 mm, and SbSUT5, 2.4 ± 0.5 mm Suc. The Suc affinity of SbSUT1 was dependent on membrane potential and pH. In contrast, SbSUT5 Suc affinity was independent of membrane potential and pH but supported high transport rates at neutral pH. Suc transport by the tonoplast localized SbSUT4 could not be detected using yeast or X. laevis oocytes. Across internode development, SUTs, other than SbSUT4, were immunolocalized to sieve elements, while for elongating and recently elongated internodes, SUTs also were detected in storage parenchyma cells. We conclude that apoplasmic Suc unloading from de-energized protophloem sieve elements in meristematic zones may be mediated by reversal of SbSUT1 and/or by uniporting SWEETs. Storage parenchyma localized SbSUT1 and SbSUT5 may accumulate Suc from the stem apoplasms of elongating and recently elongated internodes, whereas SbSUT4 may function to release Suc from vacuoles. Transiting from an apoplasmic to symplasmic unloading pathway as the stem matures, SbSUT1 and SbSUT5 increasingly function in Suc retrieval into metaphloem sieve elements to maintain a high turgor to drive symplasmic unloading by bulk flow.

Probing binding specificity of the sucrose transporter AtSUC2 with fluorescent coumarin glucosides.

The phloem sucrose transporter, AtSUC2, is promiscuous with respect to substrate recognition, transporting a range of glucosides in addition to sucrose, including naturally occurring coumarin glucosides. We used the inherent fluorescence of coumarin glucosides to probe the specificity of AtSUC2 for its substrates, and determined the structure-activity relationships that confer phloem transport in vivo using Arabidopsis seedlings. In addition to natural coumarin glucosides, we synthesized new compounds to identify key structural features that specify recognition by AtSUC2. Our analysis of the structure-activity relationship revealed that the presence of a free hydroxyl group on the coumarin moiety is essential for binding by AtSUC2 and subsequent phloem mobility. Structural modeling of the AtSUC2 substrate-binding pocket explains some important structural requirements for the interaction of coumarin glucosides with the AtSUC2 transporter.