V2: Integrated management of rainwater for crop-livestock agroecosystems

With mixed crop-livestock systems projected to remain the main providers of food in the coming decades, opportunities exist for smallholders to participate and benefit from emerging crop and livestock markets in the Volta Basin. This project intends to identify, evaluate, adapt, and disseminate best-fit integrated rainwater management strategies (RMS), targeted to different biophysical and socio-economic domains. The integrated RMS are comprised of technological solutions, directed at different components of the agroecosystems, underpinned by enabling institutional and policy environments and linked to market incentives that can drive adoptio

Summary of the growth characteristics of the WΔ<i>treR</i> double mutants generated in this study.

<p>Strains plated on M9 minimal media supplemented with various carbon sources as indicated in the table. Phenotype, +++ fast growth (sizable colonies in 15 h); ++, growth (small colonies after 15 h); −, no growth after 24 h; ND, not determined. Statistical significance was determined using the Kruskal-Wallis test combined with Dunn's Multiple Comparison test. ρ value¹, comparison between each sample and W; ρ value², comparison between each sample and WΔ<i>treR</i>.</p><p>**, ρ value≤0.01;</p><p>ns, not significant; n/a, not applicable.</p

Tn<i>5</i> insertion sites in selected non-<i>csc</i> insertion/mutation strains.

<p>Strains were grown on M9S2 and M9S20. Sucrose phenotype (Suc) is listed as positive if the strain can grow on low sucrose (0.2%) and negative if it cannot grow on sucrose at all. COG (Clusters of Orthologous Genes) groupings are listed.</p

An Expanded Heterologous <i>GAL</i> Promoter Collection for Diauxie-Inducible Expression in <i>Saccharomyces cerevisiae</i>

The <i>GAL</i> promoters are applied in metabolic engineering and synthetic biology to control gene expression in the budding yeast <i>Saccharomyces cerevisiae</i>. In <i>gal80Δ</i> background strains, they show diauxie-inducible expression, a feature beneficial in metabolic pathway optimization. However, only a limited number of <i>GAL</i> promoters have been characterized and are available for engineering purposes. Multiple uses of the same promoters can result in genetic instability in engineered strains due to homologous recombination. Here, 11 <i>GAL1</i>/2 promoters from other <i>Saccharomyces</i> species were isolated and characterized in <i>S. cerevisiae</i>. They exhibited diauxie-inducible expression patterns with low strength in exponential growth phase and induction in the ethanol growth phase. These promoters represent an expansion to the collection of <i>GAL</i> promoters available for genetic engineering in <i>S. cerevisiae</i>, including an increased diversity of expression levels. This provides the capacity for increased numbers of genetic manipulations with a lower risk of genetic instability

Primers used in this study.

<p>* Used for the direct sequencing of Tn<i>5</i> insertion sites from purified genomic DNA.</p

Models for disaccharide transport and utilisation by EIIC^Tre.

<p>In phospho<i>enol</i>pyruvate∶carbohydrate transport systems, sugars (in this case, trehalose) are transported with concomitant phosphorylation <i>via</i> a PTS-associated phosphorylation cascade (A). For the trehalose PTS, the transporter protein EIIBC^Tre consists of a permease (EIIC^Tre) and a kinase (EIIB^Tre) domain. EIIB^Tre accepts a phosphate group from EIIA^Glc in the presence of trehalose transport. EIIC^Glc accepts a phosphate from HPr, which in turn accepts a phosphate from the PEP-dependent histidine-protein kinase EI^Glc, which accepts its phosphate group from phospho<i>enol</i>pyruvate. Phosphorylated trehalose is cleaved by TreC to yield 1× phosphorylated (G6P) and 1× unphosphorylated glucose; both of these feed into central carbon metabolism (CCM) (the unphosphorylated glucose is phosphorylated by glucokinase, Glk). In the presence of PTS-mediated sugar transport, the concentration of unphosphorylated EIIA^Glc increases; unphosphorylated EIIA^Glc transcriptionally inhibits non-PTS permeases and catabolic enzymes that generate internal inducers of the various catabolic regulons (a mechanism known as inducer exclusion) <i>via</i> a variety of transcription factors <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088688#pone.0088688-Postma1" target="_blank">[51]</a>. The trehalose PTS has also been shown to transport maltose <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088688#pone.0088688-Decker1" target="_blank">[50]</a> (B). In this case, transport is thought to be achieved through facilitated diffusion by EIIC^Tre and the maltose not phosphorylated by EIIB^Tre. In the absence of PTS-mediated phosphorylation, carbon catabolite repression is released: the concentration of phosphorylated EIIA^Glc remains high; this activates adenylate cyclase (AC), resulting in an increase in intracellular cAMP concentration; cAMP complexes with the transcription factor CrpA; and cAMP-CrpA transcriptionally activates a wide variety of non-PTS transport systems, including the ABC transporter for maltose. Sucrose transport by TreB (C) may occur with or without concomitant phosphorylation, but most likely occurs without phosphorylation (see text). Transport results in sufficient intracellular sucrose to facilitate induction of the <i>csc</i> regulon; phosphorylated EIIA^Glc remains high, and the <i>csc</i> regulon is activated through cAMP-CrpA. Transported unphosphorylated sucrose may be metabolised <i>via csc</i> gene products; phosphorylated sucrose is most likely not metabolised. Once the <i>csc</i> genes are induced, sucrose can be imported through the CscB permease and cleaved by the CscA invertase into fructose and sucrose. Fructose is phosphorylated by the CscK fructokinase, and glucose is phosphorylated by Glk; both phosphorylated sugars feed into CCM.</p

<i>E. coli</i> strains and plasmids used in this study.

<p><i>csc</i>^LS+: positive growth on low sucrose (2 g/l).</p><p>Amp^R, ampillicin resistance (100 µg/mL).</p><p>Kan^R, kanamycin resistance (50 µg/mL).</p><p>Chl^R, chloramphenicol resistance (40 µg/mL).</p

Relative expression of <i>cscA</i> in the WΔ<i>treR</i> mutants.

<p>The expression level of <i>cscA</i> was determined by qRT-PCR and log normalized to the level of <i>dld</i> (D-lactate dehydrogenase). As a control, W was cultured in minimal media containing 1% glycerol and 2% sucrose. The test strains W, WΔ<i>treR</i>, WΔ<i>treR</i>Δ<i>treB</i> and WΔ<i>treR</i>Δ<i>cscB</i> were cultured in minimal media containing 1% glycerol and 0.2% sucrose. Statistical significance was determined using one way ANOVA followed by Tukey's HSD test. The average relative expression was determined from three independent mutants (or three biological replicates of W). Error bars are SEM; * p≤0.05, ** p≤0.01, *** p≤0.001.</p

Process Proteomics of Beer Reveals a Dynamic Proteome with Extensive Modifications

Modern beer production is a complex industrial process. However, some of its biochemical details remain unclear. Using mass spectrometry proteomics, we have performed a global untargeted analysis of the proteins present across time during nanoscale beer production. Samples included sweet wort produced by a high temperature infusion mash, hopped wort, and bright beer. This analysis identified over 200 unique proteins from barley and yeast, emphasizing the complexity of the process and product. We then used data independent SWATH-MS to quantitatively compare the relative abundance of these proteins throughout the process. This identified large and significant changes in the proteome at each process step. These changes described enrichment of proteins by their biophysical properties, and identified the appearance of dominant yeast proteins during fermentation. Altered levels of malt modification also quantitatively changed the proteomes throughout the process. Detailed inspection of the proteomic data revealed that many proteins were modified by protease digestion, glycation, or oxidation during the processing steps. This work demonstrates the opportunities offered by modern mass spectrometry proteomics in understanding the ancient process of beer production