The F-box protein gene exo-1 is a target for reverse engineering enzyme hypersecretion in filamentous fungi.

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.

Online Hydrophilic Interaction Chromatography (HILIC) Enhanced Top-Down Mass Spectrometry Characterization of the SARS-CoV-2 Spike Receptor-Binding Domain.

SARS-CoV-2 cellular infection is mediated by the heavily glycosylated spike protein. Recombinant versions of the spike protein and the receptor-binding domain (RBD) are necessary for seropositivity assays and can potentially serve as vaccines against viral infection. RBD plays key roles in the spike protein's structure and function, and thus, comprehensive characterization of recombinant RBD is critically important for biopharmaceutical applications. Liquid chromatography coupled to mass spectrometry has been widely used to characterize post-translational modifications in proteins, including glycosylation. Most studies of RBDs were performed at the proteolytic peptide (bottom-up proteomics) or released glycan level because of the technical challenges in resolving highly heterogeneous glycans at the intact protein level. Herein, we evaluated several online separation techniques: (1) C2 reverse-phase liquid chromatography (RPLC), (2) capillary zone electrophoresis (CZE), and (3) acrylamide-based monolithic hydrophilic interaction chromatography (HILIC) to separate intact recombinant RBDs with varying combinations of glycosylations (glycoforms) for top-down mass spectrometry (MS). Within the conditions we explored, the HILIC method was superior to RPLC and CZE at separating RBD glycoforms, which differ significantly in neutral glycan groups. In addition, our top-down analysis readily captured unexpected modifications (e.g., cysteinylation and N-terminal sequence variation) and low abundance, heavily glycosylated proteoforms that may be missed by using glycopeptide data alone. The HILIC top-down MS platform holds great potential in resolving heterogeneous glycoproteins for facile comparison of biosimilars in quality control applications.

The role of FSH in body composition in hematopoietic cell transplant recipients.

BACKGROUND: Childhood cancer survivors who received a hematopoietic cell transplantation (HCT) are at increased risk for follicle-stimulating hormone (FSH) abnormalities, which may have a significant negative impact on bone health and body composition. This study's purpose was to examine FSH and body composition in HCT recipients, non-HCT recipients and healthy controls. METHODS: The study included HCT recipients (n = 24), non-HCT recipients (n = 309), and a control group of healthy siblings (n = 211) all aged 9-18 years. A fasting blood sample was collected to measure FSH. All participants underwent a dual X-ray absorptiometry scan to assess total and regional percent fat, lean mass (LM), fat mass (FM), bone mineral content (BMC), bone mineral density (BMD), and visceral adipose tissue (VAT) mass. RESULTS: FSH was significantly higher in HCT recipients compared to non-HCT recipients and healthy controls. HCT recipients had significantly lower total body weight, total LM, arm and leg LM, BMC and BMD compared to non-HCT recipients and healthy controls (p < .05). Non-HCT recipients had significantly higher total, trunk, android, gynoid, arm and leg FM compared to healthy controls. Also, healthy controls had significantly lower VAT mass compared to non-HCT recipients. CONCLUSIONS: This study's results show that HCT recipients have significant reductions in BMD, worse body composition, and abnormal FSH levels compared to non-HCT recipients and healthy controls.

Major involvement of two laccase genes in conidial pigment biosynthesis in Aspergillus oryzae.

Wild-type strains of Aspergillus oryzae develop yellow, yellow-green, green, or brown conidia. Previous reports suggested that the conidiation initiates with the biosynthesis of a yellow pigment YWA1 from acetyl-CoA by a polyketide synthase encoded by wA (AO090102000545). This is followed by the conversion to other pigment by a laccase encoded by yA (AO090011000755). Based on orthologous pathways in other Aspergilli, it is reasonable to hypothesize that in addition to yA, AO090102000546 encoding laccase and AO090005000332 encoding Ayg1-like hydrolase play a role in A. oryzae conidial pigment biosynthesis. However, the involvement of these two genes in conidial pigmentation remains unclear. In this study, we tested this hypothesis by assessing the conidial colors of both disruption and overexpression mutants to verify whether AO090102000546 and AO090005000332 were associated with the conidial pigmentation. Observation of single, double, and triple disruptants of these three genes suggested that conidial pigments were synthesized by two laccase genes, AO090011000755 and AO090102000546, whereas Ayg1-like hydrolase gene AO090005000332 was proven to have no obvious association with the synthesis. This was corroborated by observing the phenotype of each overexpression mutant. Interestingly, AO090005000332 overexpression mutant produced smoky yellow-green conidia, different from the wild-type strain. Thus, the AO090005000332-encoded protein is likely to maintain the enzymatic activity. However, the expression level was observed to be one-third of that of AO090102000546 and one-seventh of that of AO090011000755. Consequently, apparent lack of obvious contribution of AO090005000332 to conidial pigmentation could be attributed to its low expression level. Expression analysis indicated similar profiles in several wild-type strains. KEY POINTS: • Conidial pigment biosynthesis after YWA1 mainly involves two laccases in A. oryzae. • Ayg1-like hydrolase in A. oryzae is not obviously involved in conidial pigmentation. • Conidial color is deemed dependent on expression level of two laccases and hydrolase.

Chronic Recurrent Multifocal Osteomyelitis (CRMO): A Study of 12 Cases from One Institution and Literature Review.

BACKGROUND: CRMO is a sterile auto inflammatory bone disease that affects the pediatric population. Recently, single gene mutations in LPIN2, DIRA, and IL1RN have been reported in murine models of CRMO. MATERIALS AND METHODS: The medical records and histopathological slides of twelve patients were reviewed. RESULTS: The diagnosis was determined by multiple lesions, imaging, negative cultures, bone biopsy, and lack of antibiotic response. Biopsy showed early neutrophilic infiltrates, and older lesions showed lymphoplasmacytic infiltrates and fibrosis. Patients were treated with anti-inflammatory medication with some lesions completely resolving. CONCLUSION: Bone biopsy aids the diagnosis of CRMO in correlation with clinical presentation, imaging, and culture findings. Our findings indicate the kinetics of CRMO is not well defined and the fibrosis may be reached after months, in contrast to the previously reported several years. We hope that these genetic mutations can be further studied in human models to describe the genetics behind CRMO.

Linking secondary metabolites to gene clusters through genome sequencing of six diverse Aspergillus species.

The fungal genus of Aspergillus is highly interesting, containing everything from industrial cell factories, model organisms, and human pathogens. In particular, this group has a prolific production of bioactive secondary metabolites (SMs). In this work, four diverse Aspergillus species (A. campestris, A. novofumigatus, A. ochraceoroseus, and A. steynii) have been whole-genome PacBio sequenced to provide genetic references in three Aspergillus sections. A. taichungensis and A. candidus also were sequenced for SM elucidation. Thirteen Aspergillus genomes were analyzed with comparative genomics to determine phylogeny and genetic diversity, showing that each presented genome contains 15-27% genes not found in other sequenced Aspergilli. In particular, A. novofumigatus was compared with the pathogenic species A. fumigatus This suggests that A. novofumigatus can produce most of the same allergens, virulence, and pathogenicity factors as A. fumigatus, suggesting that A. novofumigatus could be as pathogenic as A. fumigatus Furthermore, SMs were linked to gene clusters based on biological and chemical knowledge and analysis, genome sequences, and predictive algorithms. We thus identify putative SM clusters for aflatoxin, chlorflavonin, and ochrindol in A. ochraceoroseus, A. campestris, and A. steynii, respectively, and novofumigatonin, ent-cycloechinulin, and epi-aszonalenins in A. novofumigatus Our study delivers six fungal genomes, showing the large diversity found in the Aspergillus genus; highlights the potential for discovery of beneficial or harmful SMs; and supports reports of A. novofumigatus pathogenicity. It also shows how biological, biochemical, and genomic information can be combined to identify genes involved in the biosynthesis of specific SMs.

Pharmacist-Driven Implementation of Fast Identification and Antimicrobial Susceptibility Testing Improves Outcomes for Patients with Gram-Negative Bacteremia and Candidemia.

Bloodstream infections (BSI) are associated with increased morbidity and mortality, especially when caused by Gram-negative or fungal pathogens. The objective of this study was to assess the impact of fast identification-antimicrobial susceptibility testing (ID/AST) with the Accelerate Pheno system (AXDX) from May 2018 to December 2018 on antibiotic therapy and patient outcomes. A pre-post quasiexperimental study of 200 patients (100 pre-AXDX implementation and 100 post-AXDX implementation) was conducted. The primary endpoints measured were time to first antibiotic intervention, time to most targeted antibiotic therapy, and 14-day hospital mortality. Secondary endpoints included hospital and intensive care unit (ICU) length of stay (LOS), antibiotic intensity score at 96 h, and 30-day readmission rates. Of 100 patients with Gram-negative bacteremia or candidemia in each cohort, 84 in the preimplementation group and 89 in the AXDX group met all inclusion criteria. The AXDX group had a decreased time to first antibiotic intervention (26.3 versus 8.0, P = 0.003), hours to most targeted therapy (14.4 versus 9, P = 0.03), hospital LOS (6 versus 8, P = 0.002), and average antibiotic intensity score at 96 h (16 versus 12, P = 0.002). Both groups had a comparable 14-day mortality (0% versus 3.6%, P = 0.11). In this analysis of patients with Gram-negative bacteremia or candidemia, fast ID/AST implementation was associated with decreased hospital LOS, decreased use of broad-spectrum antibiotics, shortened time to targeted therapy, and an improved utilization of antibiotics within the first 96 h of therapy.

Colonies of the fungus Aspergillus niger are highly differentiated to adapt to local carbon source variation.

Saprobic fungi, such as Aspergillus niger, grow as colonies consisting of a network of branching and fusing hyphae that are often considered to be relatively uniform entities in which nutrients can freely move through the hyphae. In nature, different parts of a colony are often exposed to different nutrients. We have investigated, using a multi-omics approach, adaptation of A. niger colonies to spatially separated and compositionally different plant biomass substrates. This demonstrated a high level of intra-colony differentiation, which closely matched the locally available substrate. The part of the colony exposed to pectin-rich sugar beet pulp and to xylan-rich wheat bran showed high pectinolytic and high xylanolytic transcript and protein levels respectively. This study therefore exemplifies the high ability of fungal colonies to differentiate and adapt to local conditions, ensuring efficient use of the available nutrients, rather than maintaining a uniform physiology throughout the colony.

Deletion analysis of the itaconic acid biosynthesis gene cluster components in Aspergillus pseudoterreus ATCC32359.

The filamentous fungus Aspergillus terreus has been successfully used for industrial production of itaconic acid (IA) for many years. The IA biosynthesis pathway has recently been characterized at a molecular genetic level as an IA gene cluster by a clone-based transcriptomic approach. The cluster consists of four genes, including genes for cis-aconitic acid decarboxylase (cadA), a predicted transcription factor (tf), a mitochondrial organic acid transporter (mttA) and an MFS (major facilitator superfamily) type transporter (mfsA). In this research, we performed expressed sequence tag (EST) analysis and systematic gene deletions to further investigate the role of those genes during IA biosynthesis in A. pseudoterreus ATCC32359. EST analysis showed a similar expression pattern among those four genes that were distinct from neighboring genes and further confirmed that they belong to the same biosynthesis cluster. Systematic gene deletion analysis demonstrated that tf, cadA, mttA and mfsA genes in the cluster are essential for IA production; deletion of any of them will either completely abolish the IA production or dramatically decrease the amount of IA produced. The tf gene plays a regulatory role in this cluster. Deletion of tf led to decreased expression levels of cadA, mttA and mfsA. More importantly, a significant amount of aconitic acid was detected in the cadA deletion strain but not in the other deletion strains. Therefore, by deleting only one gene, the cadA, we established a novel microbial host for the production of aconitic acid and other value-added chemicals from sugars in lignocellulosic biomass.

Economic uncertainty before and during the COVID-19 pandemic.

We consider several economic uncertainty indicators for the US and UK before and during the COVID-19 pandemic: implied stock market volatility, newspaper-based policy uncertainty, Twitter chatter about economic uncertainty, subjective uncertainty about business growth, forecaster disagreement about future GDP growth, and a model-based measure of macro uncertainty. Four results emerge. First, all indicators show huge uncertainty jumps in reaction to the pandemic and its economic fallout. Indeed, most indicators reach their highest values on record. Second, peak amplitudes differ greatly - from a 35% rise for the model-based measure of US economic uncertainty (relative to January 2020) to a 20-fold rise in forecaster disagreement about UK growth. Third, time paths also differ: Implied volatility rose rapidly from late February, peaked in mid-March, and fell back by late March as stock prices began to recover. In contrast, broader measures of uncertainty peaked later and then plateaued, as job losses mounted, highlighting differences between Wall Street and Main Street uncertainty measures. Fourth, in Cholesky-identified VAR models fit to monthly U.S. data, a COVID-size uncertainty shock foreshadows peak drops in industrial production of 12-19%.

PAM forms an atypical SCF ubiquitin ligase complex that ubiquitinates and degrades NMNAT2.

PHR (PAM/Highwire/RPM-1) proteins are conserved RING E3 ubiquitin ligases that function in developmental processes, such as axon termination and synapse formation, as well as axon degeneration. At present, our understanding of how PHR proteins form ubiquitin ligase complexes remains incomplete. Although genetic studies indicate NMNAT2 is an important mediator of PHR protein function in axon degeneration, it remains unknown how PHR proteins inhibit NMNAT2. Here, we decipher the biochemical basis for how the human PHR protein PAM, also called MYCBP2, forms a noncanonical Skp/Cullin/F-box (SCF) complex that contains the F-box protein FBXO45 and SKP1 but lacks CUL1. We show FBXO45 does not simply function in substrate recognition but is important for assembly of the PAM/FBXO45/SKP1 complex. Interestingly, we demonstrate a novel role for SKP1 as an auxiliary component of the target recognition module that enhances binding of FBXO45 to NMNAT2. Finally, we provide biochemical evidence that PAM polyubiquitinates NMNAT2 and regulates NMNAT2 protein stability and degradation by the proteasome.

Gaps in Chlamydia Treatment Within California Family Planning Clinics: Are Patients Filling Prescriptions?

BACKGROUND: Compared with receiving medication dispensed in a health center, patients receiving prescriptions must take additional steps for treatment. Few clinics have protocols for ensuring prescriptions are filled. This study evaluated prescription fill rates for chlamydia treatment based on claims data in California Title X clinics and examined fill rates by patient demographics and clinic type. METHODS: We collected treatment information during Title X site audits for a convenience sample of patients with a positive chlamydia test between January 2008 and March 2013. We categorized patients as receiving treatment on-site versus via prescription and matched prescriptions to pharmacy billing claims within 90 days of test date. We examined treatment rates by patient age, gender, and race/ethnicity, and by clinic type, and assessed the median time to treatment. RESULTS: Among 790 patients diagnosed with chlamydia across 79 clinics, 65% (n = 513) were treated on-site and 33% (n = 260) via prescription; 17 (2%) did not have treatment information. Sixty-seven percent of prescriptions had confirmed receipt of treatment. Prescription fill rates were lower for patients age 18 years and younger (47% vs. 71%, P < 0.01) and for patients attending federally qualified health centers compared with stand-alone family planning clinics (63% vs. 88%, P < 0.01). Median time to treatment was similar for patients treated on-site (5 days) or via prescription (4 days). CONCLUSIONS: Delays in chlamydia treatment increase risk of complications and ongoing transmission. Providing medications on-site can improve treatment rates, especially among younger patients. These insights can inform clinic treatment protocols and efforts to improve quality of chlamydia care.

Contrast-enhanced MRI compared to direct joint visualization at arthroscopy in pediatric patients with suspected temporomandibular joint synovitis.

BACKGROUND: Contrast-enhanced magnetic resonance imaging (MRI) has become the gold standard when assessing the temporomandibular joint (TMJ) in children. To our knowledge, no previous pediatric study has compared findings of TMJ MRI with direct visualization of the joint using arthroscopy. OBJECTIVE: To determine if subjective findings on contrast-enhanced MRI of the temporomandibular joint correlate with arthroscopic findings of acute and chronic inflammation of the TMJ in children. MATERIALS AND METHODS: Patients who had temporally related TMJ arthroscopy and contrast-enhanced TMJ MRI between March 2014 and March 2016 were identified. Imaging was retrospectively reviewed by two radiologists for erosions, condyle shape, bone marrow edema, effusion, severity of synovitis, joint space width measured in the coronal plane and enhancement ratio of the synovium relative to ipsilateral temporal lobe white matter. Joint space width was included because synovial thickening could widen the joint space. TMJ arthroscopy findings assessed included indicators of acute inflammation (active synovitis, number of joint sites affected, presence of retrodiscitis) and markers of chronic inflammation (hyperplastic synovitis, adhesions, disc perforation, chondromalacia). A Total Synovitis Score was given to all patients on arthroscopy, which multiplied the severity of active synovitis (0-4) with the number of joint recesses affected. Data were compared using the Fisher exact test and a P-value <0.05 was considered significant. RESULTS: There was no significant correlation between subjective synovitis on MRI and active synovitis on arthroscopy; however, there was a significant correlation between joint space width and hyperplastic synovitis (P=0.04, 3.7 mm±0.8 vs. 2.9 mm±0.6) and a trend toward significance between subjective synovitis (P=0.08) and enhancement ratio of synovium (P=0.06) on MRI and hyperplastic synovitis on arthroscopy. CONCLUSION: Joint space width and subjective synovitis on TMJ MRI correlate with arthroscopic findings of chronic synovitis. Increased joint space width may be useful when evaluating the TMJ with less time-intensive modalities, such as ultrasound. However, MRI findings did not correlate well with findings of acute inflammation on arthroscopy.

Accumulation of metals and polycyclic aromatic hydrocarbons in agricultural soil after additions of street sediment in southern Ontario.

Prior to 2012 street sediment from the Greater Toronto Area was being managed by a local authority and provided to rural landowners under the assumption it was clean fill. The aim of this study was to characterise the chemical and physical composition of that street sediment applied to an agricultural field in southwestern Ontario, Canada and determine if contaminants had migrated to native soil. Soil was sampled from an impact and a background location during the fall of 2016 at four soil depths (0-10, 10-20, 20-30 and 30-40 cm below the surface) to characterise texture, pH, organic content, recoverable metals and total polycyclic aromatic hydrocarbons (PAHs). Textural analysis revealed street sediment was dominated by very coarse and coarse sand which differed from the native silty clay loam and extended to 30 cm below the surface. Some PAHs, including benzo(a)pyrene (1.29 µg g-1) exceeded the typical regional background concentrations. A distinct pattern of high molecular mass PAHs in the native soil below street sediments suggests that PAHs have migrated to native soil. To our knowledge this is the first study to report PAH concentrations in street sediment in Ontario and to show their potential movement and introduction to native soil. Future studies should focus on transport mechanisms and understanding movement of PAHs in native coarse textured soil.

Using propensity score matching to estimate treatment effects of afterschool programs on third-grade reading outcomes.

Evidence on whether afterschool programs enhance academic achievement is limited and mixed. In this study, a group of community providers delivered interventions to students to improve academic achievement. We used propensity score matching to estimate the impact of these afterschool interventions on third-grade reading achievement. A significant effect of moderate magnitude was found favoring students who participated in afterschool programming. Propensity score matching and other methodological considerations are described to support the importance and value of conducting rigorous evaluations of afterschool programs.

Defining Minimal Binding Regions in Regulator of Presynaptic Morphology 1 (RPM-1) Using Caenorhabditis elegans Neurons Reveals Differential Signaling Complexes.

The intracellular signaling protein regulator of presynaptic morphology 1 (RPM-1) is a conserved regulator of synapse formation and axon termination in Caenorhabditis elegans RPM-1 functions in a ubiquitin ligase complex with the F-box protein FSN-1 and functions through the microtubule binding protein RAE-1. Using a structure-function approach and positive selection for transgenic C. elegans, we explored the biochemical relationship between RPM-1, FSN-1, and RAE-1. This led to the identification of two new domains in RPM-1 that are sufficient for binding to FSN-1, called FSN-1 binding domain 2 (FBD2) and FBD3. Furthermore, we map the RAE-1 binding domain to a much smaller region of RPM-1. Point mutations in RPM-1 that reduce binding to RAE-1 did not affect FSN-1 binding, indicating that RPM-1 utilizes different biochemical mechanisms to bind these molecules. Analysis of RPM-1 protein complexes in the neurons of C. elegans elucidated two further discoveries: FSN-1 binds to RAE-1, and this interaction is not mediated by RPM-1, and RPM-1 binding to FSN-1 and RAE-1 reduces FSN-1·RAE-1 complex formation. These results indicate that RPM-1 uses different mechanisms to recruit FSN-1 and RAE-1 into independent signaling complexes in neurons.

Blocking hexose entry into glycolysis activates alternative metabolic conversion of these sugars and upregulates pentose metabolism in Aspergillus nidulans.

BACKGROUND: Plant biomass is the most abundant carbon source for many fungal species. In the biobased industry fungi, are used to produce lignocellulolytic enzymes to degrade agricultural waste biomass. Here we evaluated if it would be possible to create an Aspergillus nidulans strain that releases, but does not metabolize hexoses from plant biomass. For this purpose, metabolic mutants were generated that were impaired in glycolysis, by using hexokinase (hxkA) and glucokinase (glkA) negative strains. To prevent repression of enzyme production due to the hexose accumulation, strains were generated that combined these mutations with a deletion in creA, the repressor involved in regulating preferential use of different carbon catabolic pathways. RESULTS: Phenotypic analysis revealed reduced growth for the hxkA1 glkA4 mutant on wheat bran. However, hexoses did not accumulate during growth of the mutants on wheat bran, suggesting that glucose metabolism is re-routed towards alternative carbon catabolic pathways. The creAΔ4 mutation in combination with preventing initial phosphorylation in glycolysis resulted in better growth than the hxkA/glkA mutant and an increased expression of pentose catabolic and pentose phosphate pathway genes. This indicates that the reduced ability to use hexoses as carbon sources created a shift towards the pentose fraction of wheat bran as a major carbon source to support growth. CONCLUSION: Blocking the direct entry of hexoses to glycolysis activates alternative metabolic conversion of these sugars in A. nidulans during growth on plant biomass, but also upregulates conversion of other sugars, such as pentoses.

Dichomitus squalens partially tailors its molecular responses to the composition of solid wood.

White-rot fungi, such as Dichomitus squalens, degrade all wood components and inhabit mixed-wood forests containing both soft- and hardwood species. In this study, we evaluated how D. squalens responded to the compositional differences in softwood [guaiacyl (G) lignin and higher mannan content] and hardwood [syringyl/guaiacyl (S/G) lignin and higher xylan content] using semi-natural solid cultures. Spruce (softwood) and birch (hardwood) sticks were degraded by D. squalens as measured by oxidation of the lignins using 2D-NMR. The fungal response as measured by transcriptomics, proteomics and enzyme activities showed a partial tailoring to wood composition. Mannanolytic transcripts and proteins were more abundant in spruce cultures, while a proportionally higher xylanolytic activity was detected in birch cultures. Both wood types induced manganese peroxidases to a much higher level than laccases, but higher transcript and protein levels of the manganese peroxidases were observed on the G-lignin rich spruce. Overall, the molecular responses demonstrated a stronger adaptation to the spruce rather than birch composition, possibly because D. squalens is mainly found degrading softwoods in nature, which supports the ability of the solid wood cultures to reflect the natural environment.

Protein hyperproduction in fungi by design.

The secretion of enzymes used by fungi to digest their environment has been exploited by humans for centuries for food and beverage production. More than a century after the first biotechnology patent, we know that the enzyme cocktails secreted by these amazing organisms have tremendous use across a number of industrial processes. Secreting the maximum titer of enzymes is critical to the economic feasibility of these processes. Traditional mutagenesis and screening approaches have generated the vast majority of strains used by industry for the production of enzymes. Until the emergence of economical next generation DNA sequencing platforms, the majority of the genes mutated in these screens remained uncharacterized at the sequence level. In addition, mutagenesis comes with a cost to an organism's fitness, making tractable rational strain design approaches an attractive alternative. As an alternative to traditional mutagenesis and screening, controlled manipulation of multiple genes involved in processes that impact the ability of a fungus to sense its environment, regulate transcription of enzyme-encoding genes, and efficiently secrete these proteins will allow for rational design of improved fungal protein production strains.

Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in Aspergillus niger.

Plant biomass, once reduced to its composite sugars, can be converted to fuel substitutes. One means of overcoming the recalcitrance of lignocellulose is pretreatment followed by enzymatic hydrolysis. However, currently available commercial enzyme cocktails are inhibited in the presence of residual pretreatment chemicals. Recent studies have identified a number of cellulolytic enzymes from bacteria that are tolerant to pretreatment chemicals such as ionic liquids. The challenge now is generation of these enzymes in copious amounts, an arena where fungal organisms such as Aspergillus niger have proven efficient. Fungal host strains still need to be engineered to increase production titers of heterologous protein over native enzymes, which has been a difficult task. Here, we developed a forward genetics screen coupled with whole-genome resequencing to identify specific lesions responsible for a protein hyper-production phenotype in A. niger. This strategy successfully identified novel targets, including a low-affinity glucose transporter, MstC, whose deletion significantly improved secretion of recombinant proteins driven by a glucoamylase promoter.