Green-Ampt infiltration model parameter determination using SCS curve number (CN) and soil texture class, and application to the SCS runoff model

The U.S. Department of Agriculture (USDA) Soil Conservation Service Curve Number (SCS CN) method is a simple and widely popular technique of estimation of direct runoff volume for design and natural rainfall events in small watersheds. The SCS CN procedure is incorporated into such computer programs as TR-20 and TR-55. Although the method reliably predicts 24-hr runoff volume, the predicted distribution of runoff during a storm event is not realistic. This shortcoming of the SCS CN method is due to weakness in the infiltration concept of the method. Use of a physically realistic and distributed Green-Ampt infiltration model can significantly improve the SCS CN method.;The purposes of the present investigation were to incorporate the Green-Ampt infiltration model into the SCS CN method and to explore its advantages over the Curve Number infiltration model. As a result, a procedure of evaluating the Green-Ampt parameters from the SCS Curve Numbers for various soil texture classes was developed. The comparison of peak discharge estimation by both models for various rainfall depths and time of concentrations showed a divergence in predictions of up to 77 percent for low runoff potential soils

High level secretion of cellobiohydrolases by Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>The main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP). Development of <it>Saccharomyces cerevisiae </it>for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases.</p> <p>Results</p> <p>We expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by <it>S. cerevisiae</it>. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of <it>cbh1 </it>and <it>cbh2 </it>genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel™) to ethanol by CBH-producing <it>S. cerevisiae </it>strains with the addition of beta-glucosidase.</p> <p>Conclusions</p> <p>Gene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome.</p

Improved Gene Targeting through Cell Cycle Synchronization.

Gene targeting is a challenge in organisms where non-homologous end-joining is the predominant form of recombination. We show that cell division cycle synchronization can be applied to significantly increase the rate of homologous recombination during transformation. Using hydroxyurea-mediated cell cycle arrest, we obtained improved gene targeting rates in Yarrowia lipolytica, Arxula adeninivorans, Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris demonstrating the broad applicability of the method. Hydroxyurea treatment enriches for S-phase cells that are active in homologous recombination and enables previously unattainable genomic modifications

Method for increased gene targeting.

<p>Cells are grown in the presence of hydroxyurea to induce cell cycle arrest in S-phase with high HR activity (a). <i>Y</i>. <i>lipolytica</i> YB-392 cells untreated or arrested at the large-budded stage are shown. HU-arrested cells are transformed with an antibiotic resistance cassette bearing the marker flanked by short regions of homology to the promoter and terminator of the target gene (b). Homologous recombination between the cassette and genomic DNA leads to replacement of the target gene with the marker (c). Antibiotic-resistant colonies are screened by PCR to distinguish between random and targeted integration using primer sets specific to each integration outcome (d).</p

Effect of HU treatment on gene targeting efficiency.

<p>Transformants of <i>Y</i>. <i>lipolytica</i>, <i>A</i>. <i>adeninivorans</i>, <i>S</i>. <i>cerevisiae</i>, <i>P</i>. <i>pastoris and K</i>. <i>lactis</i> untreated or pretreated with HU were screened to distinguish random and targeted integration events. The percentage of gene targeting is shown and the number of total transformants screened is included in parentheses. Targeted genes are listed by their systematic names (<i>Y</i>. <i>lipolytica</i>, <i>S</i>. <i>cerevisiae</i>, <i>P</i>. <i>pastoris K</i>. <i>lactis</i>) or GenBank accession numbers (<i>A</i>. <i>adeninivorans</i>). The Fisher’s exact test hypergeometric probability for each individual experiment is tabulated in the last column.</p

Engineering of a high lipid producing Yarrowia lipolytica strain

Background: Microbial lipids are produced by many oleaginous organisms including the well-characterized yeast Yarrowia lipolytica, which can be engineered for increased lipid yield by up-regulation of the lipid biosynthetic pathway and down-regulation or deletion of competing pathways. Results: We describe a strain engineering strategy centered on diacylglycerol acyltransferase (DGA) gene overexpression that applied combinatorial screening of overexpression and deletion genetic targets to construct a high lipid producing yeast biocatalyst. The resulting strain, NS432, combines overexpression of a heterologous DGA1 enzyme from Rhodosporidium toruloides, a heterlogous DGA2 enzyme from Claviceps purpurea, and deletion of the native TGL3 lipase regulator. These three genetic modifications, selected for their effect on lipid production, enabled a 77 % lipid content and 0.21 g lipid per g glucose yield in batch fermentation. In fed-batch glucose fermentation NS432 produced 85 g/L lipid at a productivity of 0.73 g/L/h. Conclusions: The yields, productivities, and titers reported in this study may further support the applied goal of cost effective, large -scale microbial lipid production for use as biofuels and biochemicals. Keywords: Yarrowia lipolytica, Lipid accumulation, Oleaginous yeast, Metabolic engineeringNovogy, Inc

High-oleate yeast oil without polyunsaturated fatty acids

Abstract Background Oleate-enriched triacylglycerides are well-suited for lubricant applications that require high oxidative stability. Fatty acid carbon chain length and degree of desaturation are key determinants of triacylglyceride properties and the ability to manipulate fatty acid composition in living organisms is critical to developing a source of bio-based oil tailored to meet specific application requirements. Results We sought to engineer the oleaginous yeast Yarrowia lipolytica for production of high-oleate triacylglyceride oil. We studied the effect of deletions and overexpressions in the fatty acid and triacylglyceride synthesis pathways to identify modifications that increase oleate levels. Oleic acid accumulation in triacylglycerides was promoted by exchanging the native ∆9 fatty acid desaturase and glycerol-3-phosphate acyltransferase with heterologous enzymes, as well as deletion of the Δ12 fatty acid desaturase and expression of a fatty acid elongase. By combining these engineering steps, we eliminated polyunsaturated fatty acids and created a Y. lipolytica strain that accumulates triglycerides with > 90% oleate content. Conclusions High-oleate content and lack of polyunsaturates distinguish this triacylglyceride oil from plant and algal derived oils. Its composition renders the oil suitable for applications that require high oxidative stability and further demonstrates the potential of Y. lipolytica as a producer of tailored lipid profiles

Characterization of the Pichia pastoris protein-O-mannosyltransferase gene family.

The methylotrophic yeast, Pichiapastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, either N-linked or O-linked, can elicit an immune response or enable the expressed protein to bind to mannose receptors, thus reducing their efficacy. Previously we have reported the elimination of β-linked glycans in this organism. In the current report we have focused on reducing the O-linked mannose content of proteins produced in P. pastoris, thereby reducing the potential to bind to mannose receptors. The initial step in the synthesis of O-linked glycans in P. pastoris is the transfer of mannose from dolichol-phosphomannose to a target protein in the yeast secretory pathway by members of the protein-O-mannosyltransferase (PMT) family. In this report we identify and characterize the members of the P. pastoris PMT family. Like Candida albicans, P. pastoris has five PMT genes. Based on sequence homology, these PMTs can be grouped into three sub-families, with both PMT1 and PMT2 sub-families possessing two members each (PMT1 and PMT5, and PMT2 and PMT6, respectively). The remaining sub-family, PMT4, has only one member (PMT4). Through gene knockouts we show that PMT1 and PMT2 each play a significant role in O-glycosylation. Both, by gene knockouts and the use of Pmt inhibitors we were able to significantly reduce not only the degree of O-mannosylation, but also the chain-length of these glycans. Taken together, this reduction of O-glycosylation represents an important step forward in developing the P. pastoris platform as a suitable system for the production of therapeutic glycoproteins

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Additional file 1. Heterologous gene sequences used in Tsakraklides et al. 2018