Search

The Lipomyces clade contains oleaginous yeast species with advantageous metabolic features for biochemical and biofuel production. Limited knowledge about the metabolic networks of the species and limited tools for genetic engineering have led to a relatively small amount of research on the microbes. Here, a genome-scale metabolic model (GSM) of Lipomyces starkeyi NRRL Y-11557 was built using orthologous protein mappings to model yeast species. Phenotypic growth assays were used to validate the GSM (66% accuracy) and indicated that NRRL Y-11557 utilized diverse carbohydrates but had more limited catabolism of organic acids. The final GSM contained 2,193 reactions, 1,909 metabolites, and 996 genes and was thus named iLst996. The model contained 96 of the annotated carbohydrate-active enzymes. iLst996 predicted a flux distribution in line with oleaginous yeast measurements and was utilized to predict theoretical lipid yields. Twenty-five other yeasts in the Lipomyces clade were then genome sequenced and annotated. Sixteen of the Lipomyces species had orthologs for more than 97% of the iLst996 genes, demonstrating the usefulness of iLst996 as a broad GSM for Lipomyces metabolism. Pathways that diverged from iLst996 mainly revolved around alternate carbon metabolism, with ortholog groups excluding NRRL Y-11557 annotated to be involved in transport, glycerolipid, and starch metabolism, among others. Overall, this study provides a useful modeling tool and data for analyzing and understanding Lipomyces species metabolism and will assist further engineering efforts in Lipomyces.

In 1970, the Southern Corn Leaf Blight epidemic ravaged U.S. fields to great economic loss. The outbreak was caused by never-before-seen, supervirulent, Race T of the fungus Cochliobolus heterostrophus. The functional difference between Race T and O, the previously known, far less aggressive strain, is production of T-toxin, a host-selective polyketide. Supervirulence is associated with ~1 Mb of Race T-specific DNA; only a fraction encodes T-toxin biosynthetic genes (Tox1). Tox1 is genetically and physically complex, with unlinked loci (Tox1A, Tox1B) genetically inseparable from breakpoints of a Race O reciprocal translocation that generated hybrid Race T chromosomes. Previously, we identified 10 genes for T-toxin biosynthesis. Unfortunately, high-depth, short-read sequencing placed these genes on four small, unconnected scaffolds surrounded by repeated A+T rich sequence, concealing context. To sort out Tox1 topology and pinpoint the hypothetical Race O translocation breakpoints corresponding to Race T-specific insertions, we undertook PacBio long-read sequencing which revealed Tox1 gene arrangement and the breakpoints. Six Tox1A genes are arranged as three small islands in a Race T-specific sea (~634 kb) of repeats. Four Tox1B genes are linked, on a large loop of Race T-specific DNA (~210 kb). The race O breakpoints are short sequences of race O-specific DNA; corresponding positions in race T are large insertions of race T-specific, A+T rich DNA, often with similarity to transposable (predominantly Gypsy) elements. Nearby, are 'Voyager Starship' elements and DUF proteins. These elements may have facilitated Tox1 integration into progenitor Race O and promoted large scale recombination resulting in race T. IMPORTANCE In 1970 a corn disease epidemic ravaged fields in the United States to great economic loss. The outbreak was caused by a never-before seen, supervirulent strain of the fungal pathogen Cochliobolus heterostrophus. This was a plant disease epidemic, however, the current COVID-19 pandemic of humans is a stark reminder that novel, highly virulent, pathogens evolve with devastating consequences, no matter what the host-animal, plant, or other organism. Long read DNA sequencing technology allowed in depth structural comparisons between the sole, previously known, much less aggressive, version of the pathogen and the supervirulent version and revealed, in meticulous detail, the structure of the unique virulence-causing DNA. These data are foundational for future analysis of mechanisms of DNA acquisition from a foreign source.

The global regulator LaeA controls secondary metabolism in diverse Aspergillus species. Here we explored its role in regulation of itaconic acid production in Aspergillus pseudoterreus. To understand its role in regulating metabolism, we deleted and overexpressed laeA, and assessed the transcriptome, proteome, and secreted metabolome prior to and during initiation of phosphate limitation induced itaconic acid production. We found that secondary metabolite clusters, including the itaconic acid biosynthetic gene cluster, are regulated by laeA and that laeA is required for high yield production of itaconic acid. Overexpression of LaeA improves itaconic acid yield at the expense of biomass by increasing the expression of key biosynthetic pathway enzymes and attenuating the expression of genes involved in phosphate acquisition and scavenging. Increased yield was observed in optimized conditions as well as conditions containing excess nutrients that may be present in inexpensive sugar containing feedstocks such as excess phosphate or complex nutrient sources. This suggests that global regulators of metabolism may be useful targets for engineering metabolic flux that is robust to environmental heterogeneity.

In this study, a 14-gene edited Pseudomonas putida KT2440 strain for heterologous indigoidine production was examined using three distinct omic datasets. Transcriptomic data indicated that CRISPR/dCpf1-interference (CRISPRi) mediated multiplex repression caused global gene expression changes, implying potential undesirable changes in metabolic flux. 13C-metabolic flux analysis (13C-MFA) revealed that the core P. putida flux network after CRISPRi repression was conserved, with moderate reduction of TCA cycle and pyruvate shunt activity along with glyoxylate shunt activation during glucose catabolism. Metabolomic results identified a change in intracellular TCA metabolites and extracellular metabolite secretion profiles (sugars and succinate overflow) in the engineered strains. These omic analyses guided further strain engineering, with a random mutagenesis screen first identifying an optimal ribosome binding site (RBS) for Cpf1 that enabled stronger product-substrate pairing (1.6-fold increase). Then, deletion strains were constructed with excision of the PHA operon (ΔphaAZC-IID) resulting in a 2.2-fold increase in indigoidine titer over the optimized Cpf1-RBS construct at the end of the growth phase (∼6 h). The maximum indigoidine titer (at 72 h) in the ΔphaAZC-IID strain had a 1.5-fold and 1.8-fold increase compared to the optimized Cpf1-RBS construct and the original strain, respectively. Overall, this study demonstrated that integration of omic data types is essential for understanding responses to complex metabolic engineering designs and directly quantified the effect of such modifications on central metabolism.

Instructional interactions that occur between teachers and students around foundational mathematics topics are critical for supporting mathematical proficiency among students with mathematics learning disabilities (MLD). This study investigated whether the initial mathematics skill of 880 kindergarten students at risk of MLD predicted the quantity and quality of explicit instructional interactions (i.e., overt teacher modeling, student practice opportunities, and academic feedback) experienced during an empirically validated Tier 2 kindergarten mathematics intervention. It also examined whether the quantity and quality of such instructional interactions predicted gains in student mathematics achievement. Researchers conducted 740 direct observations of 255 intervention groups within a multiyear, randomized controlled trial. Results suggested that intervention groups with lower initial mathematics skill received higher rates of academic feedback and made more frequent errors. In addition, more frequent and higher quality academic feedback and group practice opportunities predicted increased mathematics achievement. Implications for investigating the active ingredients of mathematics interventions are discussed.

A concerning number of students enter kindergarten facing an intractable variation of mathematics difficulties (MD). This study investigated the impact of an explicit, core kindergarten mathematics program on the mathematical outcomes of kindergartners who demonstrated risk for severe MD at kindergarten entry and examined whether these students improved from a category of high MD risk (i.e., <10th percentile) to a lower risk of MD (i.e., norm-referenced performance at or above the 10th, 20th, 30th, and 40th percentiles) between the fall and spring of kindergarten. Differential response to the program based on the classroom-level proportion of students with severe MD was also explored. A total of 795 kindergarteners with severe MD from 122 classrooms were included in the analyses. Results suggested students with severe MD in treatment classrooms improved from fall to spring at a greater rate than their control peers. Treatment students also demonstrated higher rates of improvement from below the 10th percentile to a performance at or above the 20th, 30th, and 40th percentiles across the school year. No evidence of differential efficacy of the program by the classroom-level proportion of students with severe MD was found. Implications for using explicit mathematics programs to thwart the onset of severe MD among academically vulnerable students are discussed.

Policy efforts in mathematics have focused on increasing teachers' mathematics content knowledge (MCK), with the goal of increasing teacher quality and in turn increasing student mathematics learning. An alternative approach to increasing student mathematics achievement is to investigate curricula that can be effectively used by teachers with a range of MCK. Drawing from a large-scale study of kindergarten students (n = 2,598) and their teachers (n = 130), the current study investigated the interaction between teacher MCK and curriculum (Early Learning in Mathematics core kindergarten curriculum vs. business-as-usual curricula) on (a) instructional behaviors and (b) student mathematics achievement gains. Results indicated differential significant interactions across instructional behaviors and a small but negative effect of teacher MCK on student mathematics achievement gains. Implications for future research, policy, and practice are discussed.

A growing body of research suggests that the effects of core mathematics instruction on student mathematics outcomes may not be uniform across different skill levels in mathematics. This study investigated the extent to which observed components of explicit mathematics instruction explained why students' initial mathematics achievement was previously found to moderate the treatment impact of an empirically validated, core kindergarten mathematics program. Instructional components examined were as follows: (a) teacher demonstrations and explanations of mathematical concepts, (b) group and individual student practice opportunities, and (c) teacher-delivered academic feedback. Findings suggest that the rate in which teachers facilitated individual student practice opportunities during core mathematics instruction explained the program's differential effectiveness. Implications in terms of differentiating practice opportunities for at-risk learners and utilizing classroom observation data to test potential mediating variables of academic interventions are discussed.

Opportunities for practice play a critical role in learning complex behaviors. In the context of explicit mathematics instruction, practice facilitates systematic opportunities for students with mathematics difficulties (MD) to learn new mathematics content and apply such knowledge and skills to novel mathematics problems. This study explored whether there is an optimal amount of student practice that teachers should provide in core mathematics instruction to maximize the mathematics achievement of kindergarten students with MD, a so called "Goldilocks effect," as opposed to simply "more is better." Results from observation data collected in a large-scale efficacy trial supported the latter rather than the former. Specifically, we found that three individual practice opportunities for every explicit teacher demonstration of mathematical content was associated with increased mathematics achievement for students with MD relative to fewer practice opportunities. Implications for facilitating frequent student practice opportunities during core mathematics instruction and designing professional development for teachers who work with students with MD are discussed.

Group size and treatment intensity are understudied topics in mathematics intervention research. This study examined whether the treatment intensity and overall intervention effects of an empirically validated Tier 2 mathematics intervention varied between intervention groups with 2:1 and 5:1 student-teacher ratios. Student practice opportunities and the quality of explicit instruction served as treatment intensity metrics. A total of 465 kindergarten students with mathematics difficulties from 136 intervention groups participated. Results suggested comparable performances between the 2:1 and 5:1 intervention groups on six outcome measures. Observation data indicated that student practice differed by group size. Students in the 5:1 groups received more opportunities to practice with their peers, while students in the 2:1 groups participated in more frequent and higher quality individualized practice opportunities. Implications in terms of delivering Tier 2 interventions in small-group formats and engaging at-risk learners in meaningful practice opportunities are discussed.

This study examined the role of initial skill in moderating intervention effects of a 50-lesson mathematics intervention program, ROOTS, for at-risk kindergarten students focused on developing whole-number concepts and skills. The study utilized a randomized block design with at-risk students (n = 592) within classrooms (n = 60) randomly assigned to one of two treatment conditions (a small group of two to five students) or control condition. Proximal and distal measures were collected in the fall (pretest), spring (posttest), and winter of first grade (delayed posttest). Analyses examined the moderating effects of initial student achievement level on mathematics outcomes. Results indicated that initial skill moderated student outcomes but the relationship did not differ by group size. Implications for tiered mathematics instruction are discussed.

Carbohydrate active enzymes (CAZymes) are vital for the lignocellulose-based biorefinery. The development of hypersecreting fungal protein production hosts is therefore a major aim for both academia and industry. However, despite advances in our understanding of their regulation, the number of promising candidate genes for targeted strain engineering remains limited. Here, we resequenced the genome of the classical hypersecreting Neurospora crassa mutant exo-1 and identified the causative point of mutation to reside in the F-box protein-encoding gene, NCU09899. The corresponding deletion strain displayed amylase and invertase activities exceeding those of the carbon catabolite derepressed strain Δcre-1, while glucose repression was still mostly functional in Δexo-1 Surprisingly, RNA sequencing revealed that while plant cell wall degradation genes are broadly misexpressed in Δexo-1, only a small fraction of CAZyme genes and sugar transporters are up-regulated, indicating that EXO-1 affects specific regulatory factors. Aiming to elucidate the underlying mechanism of enzyme hypersecretion, we found the high secretion of amylases and invertase in Δexo-1 to be completely dependent on the transcriptional regulator COL-26. Furthermore, misregulation of COL-26, CRE-1, and cellular carbon and nitrogen metabolism was confirmed by proteomics. Finally, we successfully transferred the hypersecretion trait of the exo-1 disruption by reverse engineering into the industrially deployed fungus Myceliophthora thermophila using CRISPR-Cas9. Our identification of an important F-box protein demonstrates the strength of classical mutants combined with next-generation sequencing to uncover unanticipated candidates for engineering. These data contribute to a more complete understanding of CAZyme regulation and will facilitate targeted engineering of hypersecretion in further organisms of interest.

A socioeconomic status (SES)-related achievement gap in mathematics emerges prior to school entry, and increases in elementary school. This gap makes implementation of demanding mathematics standards (e.g., the Common Core State Standards) an ongoing challenge. Early educational intervention is a strategy for addressing this challenge. A randomised controlled trial was conducted in public American preschools to (1) replicate the efficacy of an intervention, "Pre-K Mathematics," for low-SES children, and (2) test the combined impact of this intervention and a Common-Core-aligned kindergarten intervention, "Early Learning in Mathematics." Forty-one clusters of pre-kindergarten and kindergarten classrooms, containing a sample of 389 low-SES children from an agricultural region, were randomly assigned to treatment and control conditions. The original impact findings were replicated: Child mathematics outcomes in pre-kindergarten were positive and significant. Gains were maintained in kindergarten. Thus, the gap can be reduced and gains maintained by sustained early intervention. [For the corresponding grantee submission, see ED618489.]

An oleaginous yeast Rhodosporidium toruloides is a promising host for converting lignocellulosic biomass to bioproducts and biofuels. In this work, we performed multi-omics analysis of lignocellulosic carbon utilization in R. toruloides and reconstructed the genome-scale metabolic network of R. toruloides. High-quality metabolic network models for model organisms and orthologous protein mapping were used to build a draft metabolic network reconstruction. The reconstruction was manually curated to build a metabolic model using functional annotation and multi-omics data including transcriptomics, proteomics, metabolomics, and RB-TDNA sequencing. The multi-omics data and metabolic model were used to investigate R. toruloides metabolism including lipid accumulation and lignocellulosic carbon utilization. The developed metabolic model was validated against high-throughput growth phenotyping and gene fitness data, and further refined to resolve the inconsistencies between prediction and data. We believe that this is the most complete and accurate metabolic network model available for R. toruloides to date.

Biological engineering of microorganisms to produce value-added chemicals is a promising route to sustainable manufacturing. However, overproduction of metabolic intermediates at high titer, rate, and yield from inexpensive substrates is challenging in non-model systems where limited information is available regarding metabolic flux and its control in production conditions. Integrated multi-omic analyses of engineered strains offers an in-depth look at metabolites and proteins directly involved in growth and production of target and non-target bioproducts. Here we applied multi-omic analyses to overproduction of the polymer precursor 3-hydroxypropionic acid (3HP) in the filamentous fungus Aspergillus pseudoterreus. A synthetic pathway consisting of aspartate decarboxylase, beta-alanine pyruvate transaminase, and 3HP dehydrogenase was designed and built for A. pseudoterreus. Strains with single- and multi-copy integration events were isolated and multi-omics analysis consisting of intracellular and extracellular metabolomics and targeted and global proteomics was used to interrogate the strains in shake-flask and bioreactor conditions. Production of a variety of co-products (organic acids and glycerol) and oxidative degradation of 3HP were identified as metabolic pathways competing with 3HP production. Intracellular accumulation of nitrogen as 2,4-diaminobutanoate was identified as an off-target nitrogen sink that may also limit flux through the engineered 3HP pathway. Elimination of the high-expression oxidative 3HP degradation pathway by deletion of a putative malonate semialdehyde dehydrogenase improved the yield of 3HP by 3.4 × after 10 days in shake-flask culture. This is the first report of 3HP production in a filamentous fungus amenable to industrial scale biomanufacturing of organic acids at high titer and low pH.

Accumulating research has established explicit mathematics instruction as an evidence-based teaching practice. This study utilized observation data from a multi-year efficacy trial to examine the longitudinal effects of a core kindergarten mathematics program on the use of explicit mathematics instruction among two distinct groups of teachers: one group that used standard practices in Year 1 of the efficacy trial and the core program in Year 2, and a second group that used the core program in both years. Targeted teaching practices consisted of teacher models, student practice opportunities, and teacher-provided academic feedback. Implementation of the program in Year 2 was found to increase the mean rates of teaching practices of teachers who used standard teaching practices in Year 1. Effect sizes are also suggestive of a positive impact of a second year of implementation with the core program. Implications for designing explicit mathematics programs and investigating evidence-based practices in future research are discussed.

We report on a practitioner implementation of Fusion, a first-grade mathematics intervention. Studies such as this evaluation of a loose implementation under realistic conditions are important to curriculum developers' understanding of how evidence-based programs and tools work under a variety of implementation scenarios. In this quasi-experimental study, first-grade students (n = 253) in 10 schools were assigned to treatment (n = 154) or control (n = 99). Rather than randomly assigning students, schools assigned those students most at risk to treatment and, typically, those less at risk to control. School staff administered pre- and postassessments and led Fusion sessions approximately 30 min per day, 3 days per week. The intervention resulted in a significant positive effect on a researcher-developed first-grade math measure. The implementation of Fusion and feedback from school staff provided insights to guide the curriculum development process.

Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high genome diversity, especially in sub-telomeric regions. We predict abundant CAZymes (598 per species) and prolific secondary metabolite gene clusters (73 per species) in section Flavi. However, the observed phenotypes (growth characteristics, polysaccharide degradation) do not necessarily correlate with inferences made from the predicted CAZyme content. Our work, including genomic analyses, phenotypic assays, and identification of secondary metabolites, highlights the genetic and metabolic diversity within section Flavi.

An oleaginous yeast Rhodosporidium toruloides is a promising host for converting lignocellulosic biomass to bioproducts and biofuels. In this work, we performed multi-omics analysis of lignocellulosic carbon utilization in R. toruloides and reconstructed the genome-scale metabolic network of R. toruloides. High-quality metabolic network models for model organisms and orthologous protein mapping were used to build a draft metabolic network reconstruction. The reconstruction was manually curated to build a metabolic model using functional annotation and multi-omics data including transcriptomics, proteomics, metabolomics, and RB-TDNA sequencing. The multi-omics data and metabolic model were used to investigate R. toruloides metabolism including lipid accumulation and lignocellulosic carbon utilization. The developed metabolic model was validated against high-throughput growth phenotyping and gene fitness data, and further refined to resolve the inconsistencies between prediction and data. We believe that this is the most complete and accurate metabolic network model available for R. toruloides to date.

We situate education, and the science of reading (SOR) specifically, in the midst of a broad, evidence-based revolution involving an array of disciplines focused on improving the health and well-being of individuals and populations. Low and stagnant levels of reading proficiency, massive reading disparities, and a robust SOR knowledge base suggest that the withholding of evidence-based practices in schools differentially harms students of Color, students from poor families, English learners, and students with disabilities. We acknowledge that simply expecting greater use of evidence-based reading practices in schools will not suffice. We present a framework where practitioners and policymakers would continue to gain better and easier access to the SOR knowledge base and evidence-based reading practices and where much greater emphasis would be placed on fueling the demand for evidence-based practices in schools. How schools are organized to provide reading instruction for students is also a key consideration in efforts to expand the use of evidence-based practices. We make the case that schools engaged in comprehensive use of multi-tiered systems of support approaches in reading are well positioned to increase their use of evidence-based reading practices. Because much is not known about how to scale the use of effective practices, scaling efforts themselves represent opportunities to generate new SOR knowledge on both the supply and demand sides. This work would be consistent with the SOR knowledge base as a dynamic and constantly emerging phenomenon, rather than a static repository waiting to be accessed and used.

States are increasingly recommending that districts and schools use multi-tiered systems of support (MTSS) to improve reading outcomes for all students. States have also suggested MTSS is a viable service delivery model in response to new state legislation to screen, identify, and treat students with word-level reading disability (i.e., dyslexia). One model of MTSS that utilizes Enhanced Core Reading Instruction (ECRI MTSS), has demonstrated significant increases in students' early acquisition of foundational reading skills (Smith et al., 2016). The purpose of this study was to conduct a conceptual replication of the Smith's (2016) original impact study. In a cluster-randomized controlled trial, 44 schools were randomly assigned to the ECRI MTSS treatment or a business-as-usual (BAU) MTSS control condition. Across conditions, 754 students were assigned to receive Tier 2 intervention in addition to Tier 1 instruction. Impact data indicate moderate to strong effects on student decoding, word reading, and fluency skills for students in the ECRI MTSS schools. Results suggest that schools can use ECRI MTSS to improve foundational reading skills for struggling early readers, including students with or at risk for word-level reading disabilities (i.e., dyslexia).

This study examines the effect of 30 min of small group explicit instruction on reading outcomes for first-grade Spanish-speaking English learners (ELs) at risk of reading difficulties. Participants were 78 ELs from seven schools who were receiving Spanish only, or Spanish and English, whole group reading instruction in first grade. Students were rank-ordered within schools and then randomly assigned to a treatment condition (n = 39) or a comparison condition (n = 39). Students in the treatment condition received instruction on transition elements that supported their transfer of skills from Spanish to English. Students in the comparison condition received Business as Usual instruction from a variety of commercially available programs. Findings indicated that ELs in both conditions made significant gains from pretest to posttest on all reading outcomes even though instruction in the treatment condition focused significantly more on higher order skills (i.e., vocabulary, comprehension, and transition elements) whereas instruction in the comparison condition focused significantly more on lower order skills (i.e., phonics, word work, and sentence reading). Implications for practice and future research are discussed.

The purpose of this closely aligned conceptual replication study was to investigate the efficacy of a Tier 2 kindergarten mathematics intervention. The replication study differed from the initial randomized controlled trial on three important elements: geographical region, timing of the intervention, and instructional context of the counterfactual. Similar to the original investigation, however, the current study tested the same intervention, used the same outcome measures and statistical analyses, and involved the same population of learners. A total of 319 kindergarten students with mathematics difficulties from 36 kindergarten classrooms participated in the study. Students who were randomly assigned to the treatment condition received the intervention in small-group formats, with 2 or 5 students per group. Control students participated in a no-treatment control condition. Significant effects on proximal and distal measures of mathematics achievement were found. Effect sizes obtained for all measures fell within or exceeded the upper bound of the effects reported in the initial study. Implications for systematically situating replication studies in larger frameworks of intervention research and reporting rates of treatment response across replication studies are discussed. [This paper was published in "Exceptional Children" (EJ1116305).]

Aspergillus niger and other filamentous fungi are widely used in industry, but efficient genetic engineering of these hosts remains nascent. For example, while molecular genetic tools have been developed, including CRISPR/Cas9, facile genome engineering of A. niger remains challenging. To address these challenges, we have developed a simple Cas9-based gene targeting method that provides selectable, iterative, and ultimately marker-free generation of genomic deletions and insertions. This method leverages locus-specific "pop-out" recombination to suppress off-target integrations. We demonstrated the effectiveness of this method by targeting the phenotypic marker albA and validated it by targeting the glaA and mstC loci. After two selection steps, we observed 100% gene editing efficiency across all three loci. This method greatly reduces the effort required to engineer the A. niger genome and overcomes low Cas9 transformations efficiency by eliminating the need for extensive screening. This method represents a significant addition to the A. niger genome engineering toolbox and could be adapted for use in other organisms. It is expected that this method will impact several areas of industrial biotechnology, such as the development of new strains for the secretion of heterologous enzymes and the discovery and optimization of metabolic pathways.