Neutral Sites Research Articles

Antisera-Neutralizing Capacity of a Highly Evolved Type 2 Vaccine-Derived Poliovirus from an Immunodeficient Patient

Background: The serotype 2 oral poliovirus vaccine (OPV2) can revert to regain wild-type neurovirulence and spread, causing the emergence of vaccine-derived poliovirus (VDPV2) and immunodeficiency-related vaccine-derived polioviruses (iVDPVs). In the United States, testing carried out by the CDC of type II iVDPV (iVDPV2) with human immune serum from the vaccine has shown that the presence of the virus poses a threat to eradication efforts. Methods: We analyzed the major neutralization sites of VP1, VP2, and VP3 of the iVDPV using bioinformatics techniques and homology modeling (SWISS-MODEL). The three amino acid residues 679, 680, and 141 of the P1 region changed, which had an impact on the spatial conformation of the viral-neutralizing site. We tested polio-vaccinated human sera and rabbit anti-Sabin II polyantibodies against a panel of iVDPV pseudoviruses. Results: The results demonstrated that the serum’s capacity to neutralize mutant pseudoviruses diminished when amino acid substitutions were introduced into the P1 encapsidated protein, particularly when 141 and 679 were mutated together. This study emphasizes the significance of continually monitoring individuals who are known to be immunocompromised and maintaining high vaccination rates in OPV-using communities.

Highly Active Immobilized Catalyst for Ethylene Polymerization: Neutral Single Site Y(III) Complex Bearing Bulky Silylallyl Ligand.

Exploring the surface organometallic chemistry on silica of highly electrophilic yttrium complexes is a relatively uncommon endeavor, particularly when focusing on tris-alkyl complexes characterized by Y-C σ-alkyl bonds. A drawback with this class of complexes once grafted on silica, is the frequent occurrence of alkyl transfer by ring opening of siloxane groups, resulting in a mixture of species. Herein, we employed a more stable homoleptic yttrium allyl complex bearing bulky η3-1,3-bis(trimethylsilyl)allyl ligand to limit this transfer reaction. This strategy has been validated by comparing the reactivity between [Y{ η3-1,3-C3H3(SiMe3)2}3] and [Y(o-CH2PhNMe2)3] with SiO2-700, where the undesired alkyl transfer reaction occurred for [Y(o-CH2PhNMe2)3] leading to a bipodal [(≡SiO)2Y(o-CH2PhNMe2)] as major surface species, 2, while [Y{ η 3-1,3-C3H3(SiMe3)2}3] resulted selectively in a monopodal species, [(≡SiO)Y{η3-1,3-C3H3(SiMe3)2}2], 1. The materials obtained were characterized by DRIFT, solid state NMR, mass balance analysis and EXAFS. Catalyst 1 showed high activity compared to 2 in ethylene polymerization. The catalytic performance of this neutral catalyst 1 was extended to pre-industrial scale in the presence of hydrogen and 1-hexene. An unprecedented activity, up to 7400 gPE gcat -1 h-1 was obtained even with very low concentration of scavenger AliBu3 (TIBA/Y=1.2). The obtained HDPE exhibited desired spherical particle morphology with broad molar mass distribution.

Optimized CRISPR Interference System for Investigating Pseudomonas alloputida Genes Involved in Rhizosphere Microbiome Assembly.

Pseudomonas alloputida KT2440 (formerly P. putida) has become both a well-known chassis organism for synthetic biology and a model organism for rhizosphere colonization. Here, we describe a CRISPR interference (CRISPRi) system in KT2440 for exploring microbe-microbe interactions in the rhizosphere and for use in industrial systems. Our CRISPRi system features three different promoter systems (XylS/Pm, LacI/Plac, and AraC/PBAD) and a dCas9 codon-optimized for Pseudomonads, all located on a mini-Tn7-based transposon that inserts into a neutral site in the genome. It also includes a suite of pSEVA-derived sgRNA expression vectors, where the expression is driven by synthetic promoters varying in strength. We compare the three promoter systems in terms of how well they can precisely modulate gene expression, and we discuss the impact of environmental factors, such as media choice, on the success of CRISPRi. We demonstrate that CRISPRi is functional in bacteria colonizing the rhizosphere, with repression of essential genes leading to a 10-100-fold reduction in P. alloputida cells per root. Finally, we show that CRISPRi can be used to modulate microbe-microbe interactions. When the gene pvdH is repressed and P. alloputida is unable to produce pyoverdine, it loses its ability to inhibit other microbes in vitro. Moreover, our design is amendable for future CRISPRi-seq studies and in multispecies microbial communities, with the different promoter systems providing a means to control the level of gene expression in many different environments.

Genetic diversity during selective sweeps in non-recombining populations.

Selective sweeps, resulting from the spread of beneficial, neutral, or deleterious mutations through a population, shape patterns of genetic variation at linked neutral sites. While many theoretical, computational, and statistical advances have been made in understanding the genomic signatures of selective sweeps in recombining populations, substantially less is understood in populations with little/no recombination. We present a mathematical framework based on diffusion theory for obtaining the site frequency spectrum (SFS) at linked neutral sites immediately post and during the fixation of moderately or strongly beneficial mutations. We find that when a single hard sweep occurs, the SFS decays as 1/ x for low derived allele frequencies ( x ), similar to the neutral SFS at equilibrium, whereas at higher derived allele frequencies, it follows a 1/ x 2 power law. These power laws are universal in the sense that they are independent of the dominance and inbreeding coefficient, and also characterize the SFS during the sweep. Additionally, we find that the derived allele frequency where the SFS shifts from the 1/ x to 1/ x 2 law, is inversely proportional to the selection strength: thus under strong selection, the SFS follows the 1/ x 2 dependence for most allele frequencies, resembling a rapidly expanding neutral population. When clonal interference is pervasive, the SFS immediately post-fixation becomes U-shaped and is better explained by the equilibrium SFS of selected sites. Our results will be important in developing statistical methods to infer the timing and strength of recent selective sweeps in asexual populations, genomic regions that lack recombination, and clonally propagating tumor populations.

Investigation of radioactivity and heavy metal levels in soil samples from neutral and vegetation land of Punjab, India.

In this work, radioactivity investigations of soil samples from neutral and agricultural sites in Punjab (India) have been carried out to study the impact of land use patterns. Analyzing soil samples radiological, mineralogical, and physicochemical attributes has employed state-of-the-art techniques. The mean activity concentration of 238U/226Ra, 232Th, 40K, 235U, and 137Cs, measured using a carbon fiber endcap p-type HPGe detector, in neutral land was observed as 58.03, 83.95, 445.18, 2.83, and 1.16Bqkg-1, respectively. However, in vegetation land, it was found to be 40.07, 64.68, 596.74, 2.26, and 1.90Bqkg-1, respectively. In the detailed activity analysis, radium equivalent (Raeq) radioactivity is in the safe prescribed limit of 370Bqkg-1 for all investigated soil samples. However, the dosimetric investigations revealed that the outdoor absorbed gamma dose rate (96.08 nGy h-1) and consequent annual effective dose rate (0.12mSv y-1) for neutral land and the gamma dose rate (82.46 nGy h-1) and subsequent annual effective dose rate (0.10mSv y-1) for vegetation land marginally exceeded the global average. The soil's physicochemical parameters (pH, EC, and porosity) from both sites were measured, and their correlations with radionuclides were analyzed. Various heavy metals of health concern, namely, chromium (Cr), arsenic (As), copper (Cu), cobalt (Co), cadmium (Cd), lead (Pb), mercury (Hg), selenium (Se), and zinc (Zn), were also evaluated in soil samples using Inductively Coupled Plasma-Mass Spectroscopy (ICP-MS). Pollution Load Index (PLI) and Ecological Risk Index (RI) revealed that vegetation land was more anthropogenically contaminated than neutral land, with maximum contamination from Hg and As.

Continuously fluctuating selection reveals fine granularity of adaptation.

Temporally fluctuating environmental conditions are a ubiquitous feature of natural habitats. Yet, how finely natural populations adaptively track fluctuating selection pressures via shifts in standing genetic variation is unknown1,2. Here we generated genome-wide allele frequency data every 1-2 generations from a genetically diverse population of Drosophila melanogaster in extensively replicated field mesocosms from late June to mid-December (a period of approximately 12 total generations). Adaptation throughout the fundamental ecological phases of population expansion, peak density and collapse was underpinned by extremely rapid, parallel changes in genomic variation across replicates. Yet, the dominant direction of selection fluctuated repeatedly, even within each of these ecological phases. Comparing patterns of change in allele frequency to an independent dataset procured from the same experimental system demonstrated that the targets of selection are predictable across years. In concert, our results reveala fitness relevance of standing variation that is likely to be masked by inference approaches based on static population sampling or insufficiently resolved time-series data. We propose that such fine-scaled, temporally fluctuating selection may be an important force contributing to the maintenance of functional genetic variation in natural populations and an important stochastic force impacting genome-wide patterns of diversity at linked neutral sites, akin to genetic draft.

Using Orthodromic Distance to Determine Homefield Advantage in Professional Bass Fishing Tournaments

The home team is widely viewed as having an advantage in most team sports. Individual sports are less inclined to have homefield advantage, since they are contested on neutral sites with contestants from a wide variety of locales. Bass fishing differs from most individual sports as the venue fished is governed by local biological and climatological variation. Thus, there may be a homefield advantage that accrues to anglers living close to the venue where the tournament is held. In this paper we examine angler performance in the two premier bass fishing tournaments in the U.S.: the Bassmaster Elite Series and Major League Fishing's Bass Pro Tour. We find that angler performance in both tournaments is related to characteristics that measure experience, aptitude, and past success. In addition, we find evidence that angler performance suffers as distance to the lake increases, and this result holds after controlling for other angler characteristics.

A toolbox to engineer the highly productive cyanobacterium Synechococcus sp. PCC 11901.

Synechococcus sp. PCC 11901 (PCC 11901) is a fast-growing marine cyanobacterial strain that has a capacity for sustained biomass accumulation to very high cell densities, comparable to that achieved by commercially relevant heterotrophic organisms. However, genetic tools to engineer PCC 11901 for biotechnology applications are limited. Here we describe a suite of tools based on the CyanoGate MoClo system to unlock the engineering potential of PCC 11901. First, we characterized neutral sites suitable for stable genomic integration that do not affect growth even at high cell densities. Second, we tested a suite of constitutive promoters, terminators, and inducible promoters including a 2,4-diacetylphloroglucinol (DAPG)-inducible PhlF repressor system, which has not previously been demonstrated in cyanobacteria and showed tight regulation and a 228-fold dynamic range of induction. Lastly, we developed a DAPG-inducible dCas9-based CRISPR interference (CRISPRi) system and a modular method to generate markerless mutants using CRISPR-Cas12a. Based on our findings, PCC 11901 is highly responsive to CRISPRi-based repression and showed high efficiencies for single insertion (31% to 81%) and multiplex double insertion (25%) genome editing with Cas12a. We envision that these tools will lay the foundations for the adoption of PCC 11901 as a robust model strain for engineering biology and green biotechnology.

Light Induced Proton Coupled Charge Transfer Triggers Counterion Directional Translocation

AbstractWe demonstrate directed translocation of ClO4− anions from cationic to neutral binding site along the synthetized BPym‐OH dye molecule that exhibits coupled excited‐state intramolecular proton‐transfer (ESIPT) and charge‐transfer (CT) reaction (PCCT). The results of steady‐state and time‐resolved spectroscopy together with computer simulation and modeling show that in low polar toluene the excited‐state redistribution of electronic charge enhanced by ESIPT generates the driving force, which is much stronger than by CT reaction itself and provides more informative gigantic shifts of fluorescence spectra signaling on ultrafast ion motion. The associated with ion translocation red‐shifted fluorescence band (at 750 nm, extending to near‐IR region) appears at the time ~83 ps as a result of electrochromic modulation of PCCT reaction. It occurs at substantial delay to PCCT that displayed fluorescence band at 640 nm and risetime of <200 fs. Thus, it becomes possible to visualize the manifestations of light‐triggered ion translocation and of its driving force by fluorescence techniques and to separate them in time and energy domains.

Light Induced Proton Coupled Charge Transfer Triggers Counterion Directional Translocation.

We demonstrate directed translocation of ClO4 - anions from cationic to neutral binding site along the synthetized BPym-OH dye molecule that exhibits coupled excited-state intramolecular proton-transfer (ESIPT) and charge-transfer (CT) reaction (PCCT). The results of steady-state and time-resolved spectroscopy together with computer simulation and modeling show that in low polar toluene the excited-state redistribution of electronic charge enhanced by ESIPT generates the driving force, which is much stronger than by CT reaction itself and provides more informative gigantic shifts of fluorescence spectra signaling on ultrafast ion motion. The associated with ion translocation red-shifted fluorescence band (at 750 nm, extending to near-IR region) appears at the time ~83 ps as a result of electrochromic modulation of PCCT reaction. It occurs at substantial delay to PCCT that displayed fluorescence band at 640 nm and risetime of <200 fs. Thus, it becomes possible to visualize the manifestations of light-triggered ion translocation and of its driving force by fluorescence techniques and to separate them in time and energy domains.

Digest: Does the insertion preference of a transposable element represent a strategy for the mitigation of genetic conflicts with the host?

Transposable elements can exhibit a predilection for specific insertion regions. In a recent study, Munasinghe et al. (2023) consider how variation in where TE families prefer to insert within the genome influences their copy number evolution. The study emphasizes how a preference for neutral insertion sites is only advantageous in conjunction with host restriction mechanisms, which suggests that insertion preference may be a strategy to mitigate genetic conflicts with the host.

De novo genome assembly and pan-genome analysis of the fast-growing Indian isolates of Synechococcus elongatus: Potential chassis for bioproduction

Cyanobacteria are explored as phototrophic cell factories for the conversion of CO2 to chemicals. Although several cyanobacterial platforms have been established, discovering novel cyanobacterial strains with different growth and metabolic traits would be advantageous. Here, we describe de novo genome assembly and pan-genome analysis of eight fast-growing Indian isolates of Synechococcus elongatus, displaying an average doubling time of 3–4 h. The draft genome sequences of S. elongatus PCC 11801 and PCC 11802 were earlier reported by our group. However, here we present circular un-gapped genome sequences of all the eight strains. Their pan-genome analysis showed that these strains form a distinct sub-clade within the S. elongatus clade, and are closely related to a widely used cyanobacterium S. elongatus PCC 7942. Pan-genome and extra-chromosomal DNA sequence analyses further revealed intriguing features, including CRISPR-associated protein genes, circular episomal elements, and other essential genes which were previously not reported for this clade. Further, from bio-engineering perspective, we optimized their transformation using neutral site integrative plasmids. The heterologous protein expression was confirmed using eYFP reporter system, and validated by engineering a mannitol biosynthesis pathway in these strains. Although the strains exhibited comparable eYFP fluorescence, the mannitol production varied significantly, indicating physiological differences among the strains. The maximum mannitol was obtained from S. elongatus IITB6 strain. Overall, this work encompasses comparative growth analysis of the eight Indian isolates of S. elongatus, their genome sequences and pan-genome analysis. We demonstrated their potential as phototrophic cell factories, providing cyanobacterial bio-chassis options.

Deep removal of COS by carbon aerogel in natural gas: A 3D network structure of nitrogen doped and Cu based adsorbent

For the purpose of overcoming the difficulty of low removal rate of COS at low temperatures, N-doped copper oxide-supported carbon aerogels were prepared with sodium alginate as a carbon source for deep removal of COS (about 20 ppm) from natural gas. By exploring the effects of different nitrogen sources (urea, melamine and p-phenylenediamine), carbon–nitrogen ratio and carbonization temperature, p-phenylenediamine-doped copper oxide-loaded carbon aerogels (PPD-Cu/CA) with both excellent catalytic hydrolysis of COS and adsorption were prepared. The COS adsorption capacity of optimized PPD-Cu/CA was 18.6 mg·g−1 with 0.94 %Vol of water content at 40 °C, meanwhile, the COS removal efficiency at 40,000 and 70,000 mL·g−1·h−1 could reach 100 % and 94.6 %, respectively, showing excellent high space velocity resistance. Combined with characterization and experimental results, the introduction of p-phenylenediamine enriched the pore structure of the carbon carrier, increased the neutral base site, and played a positive role in the adsorption and hydrolysis of COS. This study provides a new insight into the removal of carbonyl sulfur from natural gas.

CRISPR-COPIES: an in silico platform for discovery of neutral integration sites for CRISPR/Cas-facilitated gene integration.

The CRISPR/Cas system has emerged as a powerful tool for genome editing in metabolic engineering and human gene therapy. However, locating the optimal site on the chromosome to integrate heterologous genes using the CRISPR/Cas system remains an open question. Selecting a suitable site for gene integration involves considering multiple complex criteria, including factors related to CRISPR/Cas-mediated integration, genetic stability, and gene expression. Consequently, identifying such sites on specific or different chromosomal locations typically requires extensive characterization efforts. To address these challenges, we have developed CRISPR-COPIES, a COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites. This tool leverages ScaNN, a state-of-the-art model on the embedding-based nearest neighbor search for fast and accurate off-target search, and can identify genome-wide intergenic sites for most bacterial and fungal genomes within minutes. As a proof of concept, we utilized CRISPR-COPIES to characterize neutral integration sites in three diverse species: Saccharomyces cerevisiae, Cupriavidus necator, and HEK293T cells. In addition, we developed a user-friendly web interface for CRISPR-COPIES (https://biofoundry.web.illinois.edu/copies/). We anticipate that CRISPR-COPIES will serve as a valuable tool for targeted DNA integration and aid in the characterization of synthetic biology toolkits, enable rapid strain construction to produce valuable biochemicals, and support human gene and cell therapy applications.

Electrode Montage for Bilateral Cervical Vestibular Evoked Myogenic Potential (cVEMP) Testing.

Cervical vestibular evoked myogenic potentials (cVEMPs) are predominantly ipsilateral, myogenic responses originating from saccular activation. Some individuals have contralateral-crossed cVEMP responses with monaural air-conducted stimulation (ACS) which can contaminate cVEMP responses with bilateral stimulation. While the origin of the contralateral-crossed response is under debate, its presence has implications for cVEMP testing with midline bone conduction vibration (BCV). The purpose of this study was to determine the origin of the contralateral-crossed cVEMP response. It was hypothesized that the crossed response is due to electrode contamination and would disappear with a modified electrode montage. Cross-sectional research study. Fifteen healthy participants (30 ears; mean age: 27.4 19-39; 10 females). Participants completed cVEMP testing using three stimulation methods (monoaural ACS, binaural ACS, and midline BCV) and two electrode montages (sternum reference and Fp reference). In the monoaural ACS with sternum reference condition, 53.3% ears had contralateral-crossed cVEMP responses that were in-phase with the ipsilateral response for all but 3 ears. Whereas in the monoaural ACS with Fp reference condition, 3% had a contralateral-crossed cVEMP response. ACS and BCV cVEMP corrected amplitudes were significantly larger in the sternum reference conditions, which is attributed to artificial enhancement from the in-phase contralateral-crossed responses. The significant reduction of contralateral-crossed responses in the Fp reference condition suggests that the contralateral-crossed cVEMP response is due to reference electrode contamination and may be a more appropriate reference placement when completing cVEMPs with midline BCV. The purpose of this study was to determine the origin of the contralateral-crossed cVEMP response. It was hypothesized that the crossed response is due to electrode contamination and would disappear with a modified electrode montage. Cross-sectional research study. Fifteen healthy participants (30 ears; mean age: 27.4 19-39; 10 females). Participants completed cVEMP testing using three stimulation methods (monoaural ACS, binaural ACS, and midline BCV) and three electrode montages (sternum reference, Fp reference, and active on Fp). In the monoaural ACS with sternum reference condition, 53.3% ears had contralateral-crossed cVEMP responses that were in-phase with the ipsilateral response for all but 3 ears. Whereas in the monoaural ACS with Fp reference condition, 3% had a contralateral-crossed cVEMP response. No participants demonstrated responses using Fp for the active electrode suggesting this is a neutral site. ACS and BCV cVEMP corrected amplitudes were significantly larger in the sternum reference conditions, which is attributed to artificial enhancement from the in-phase contralateral-crossed responses. The significant reduction of contralateral-crossed responses in the Fp reference condition suggests that the contralateral-crossed cVEMP response is due to reference electrode contamination and may be a more appropriate reference placement when completing cVEMPs with midline BCV.

Multiple energy dissipation modes in dynamic polymer networks with neutral and ionic junctions.

Polymer networks with controlled ratios of neutral and ionic dynamic crosslink points were prepared from ethylene glycol, boric acid, and lithium hydroxide. Both neutral and ionic sites led to the emergence of distinct damping modes separate from the glass transition. This work highlights the potential of polymer networks for multimodal damping spectra through dynamic bond selection.

Comparing microbial populations from diverse hydrothermal features in Yellowstone National Park: hot springs and mud volcanoes.

Geothermal features, such as hot springs and mud volcanoes, host diverse microbial life, including many extremophile organisms. The physicochemical parameters of the geothermal feature, such as temperature, pH, and heavy metal concentration, can influence the alpha and beta diversity of microbial life in these environments, as can spatiotemporal differences between sites and sampling. In this study, water and sediment samples were collected and analyzed from eight geothermal sites at Yellowstone National Park, including six hot springs, a mud volcano, and an acidic lake within the same week in July 2019, and these geothermal sites varied greatly in their temperature, pH, and chemical composition. All samples were processed and analyzed with the same methodology and taxonomic profiles and alpha and beta diversity metrics determined with 16S rRNA sequencing. These microbial diversity results were then analyzed with respect to pH, temperature, and chemical composition of the geothermal features. Results indicated that predominant microbial species varied greatly depending on the physicochemical composition of the geothermal site, with decreases in pH and increases in dissolved heavy metals in the water corresponding to decreases in alpha diversity, especially in the sediment samples. Similarly, sites with acidic pH values had more similar microbial populations (beta diversity) to one another than to relatively neutral or alkaline pH geothermal sites. This study suggests that pH and/or heavy metal concentration is a more important driver for microbial diversity and population profile than the temperature for these sites and is also the first reported microbial diversity study for multiple geothermal sites in Yellowstone National Park, including the relatively new mud volcano Black Dragon's Caldron, which erupted in 1948.

Nonunion Supervisors’ Humility at Work and Union Intolerance: An Exploratory Study Centered on One Hypothesis

AbstractViewing supervisors’ humility at work as experiential from a self-awareness perspective, we formulated a central hypothesis that more humility would be related to less union intolerance in the work environment. Based on a broad sample of nonunion supervisors (N = 101) surveyed at neutral sites away from work, support was found for the relationship. To explore supervisors’ experience as indicated by their demographics as moderators, our data suggested that the relationship was stronger for supervisors at lower levels of supervision and for supervisors with at least one parent who was a union member. Results were discussed in relation to future studies to verify and extend this first-ever hypothesis, in which supervisors’ humility experienced at work is linked to unions and union employees.

Comprehensive profiling of neutralizing polyclonal sera targeting coxsackievirus B3

Despite their fundamental role in resolving viral infections, our understanding of how polyclonal neutralizing antibody responses target non-enveloped viruses remains limited. To define these responses, we obtained the full antigenic profile of multiple human and mouse polyclonal sera targeting the capsid of a prototypical picornavirus, coxsackievirus B3. Our results uncover significant variation in the breadth and strength of neutralization sites targeted by individual human polyclonal responses, which contrasted with homogenous responses observed in experimentally infected mice. We further use these comprehensive antigenic profiles to define key structural and evolutionary parameters that are predictive of escape, assess epitope dominance at the population level, and reveal a need for at least two mutations to achieve significant escape from multiple sera. Overall, our data provide a comprehensive analysis of how polyclonal sera target a non-enveloped viral capsid and help define both immune dominance and escape at the population level.

Enhancing the Heterologous Expression of a Thermophilic Endoglucanase and Its Cost-Effective Production in Pichia pastoris Using Multiple Strategies.

Although Pichia pastoris was successfully used for heterologous gene expression for more than twenty years, many factors influencing protein expression remain unclear. Here, we optimized the expression of a thermophilic endoglucanase from Thermothielavioides terrestris (TtCel45A) for cost-effective production in Pichia pastoris. To achieve this, we established a multifactorial regulation strategy that involved selecting a genome-editing system, utilizing neutral loci, incorporating multiple copies of the heterologous expression cassette, and optimizing high-density fermentation for the co-production of single-cell protein (SCP). Notably, even though all neutral sites were used, there was still a slight difference in the enzymatic activity of heterologously expressed TtCel45A. Interestingly, the optimal gene copy number for the chromosomal expression of TtCel45A was found to be three, indicating limitations in translational capacity, post-translational processing, and secretion, ultimately impacting protein yields in P. pastoris. We suggest that multiple parameters might influence a kinetic competition between protein elongation and mRNA degradation. During high-density fermentation, the highest protein concentration and endoglucanase activity of TtCel45A with three copies reached 15.8 g/L and 9640 IU/mL, respectively. At the same time, the remaining SCP of P. pastoris exhibited a crude protein and amino acid content of up to 59.32% and 46.98%, respectively. These findings suggested that SCP from P. pastoris holds great promise as a sustainable and cost-effective alternative for meeting the global protein demand, while also enabling the production of thermophilic TtCel45A in a single industrial process.