Molecular Biology Tools Research Articles

Conservation of the Polyamines Pathway in Ustilaginomycetes A Genomic and Experimental Approach.

Polyamines are organic and aliphatic molecules essential for the growth, development, and survival of both eukaryotes and prokaryotes. In fungi, polyamines play a crucial role in cellular differentiation and pathogenesis. Since fungi and animals are closely related evolutionarily, and fungi can be easily genetically manipulated in the lab, they serve as excellent models for studying polyamine metabolism and the molecular mechanisms controlled by these biomolecules. Although the metabolism of polyamines has been extensively studied in model fungi such as Saccharomyces cerevisiae and Ustilago maydis, the conservation of the polyamine biosynthesis pathway in other Ustilaginomycetes, a class of fungi that includes phytopathogens, saprophytes, mutualists, and mycorrhizae, has not been thoroughly investigated. In this study, using a genomic and bioinformatics approach, we analyzed the conservation of the polyamine biosynthesis pathway in Ustilaginomycetes. Additionally, we confirmed the functional conservation of ornithine decarboxylase (Odc), which is involved in the synthesis of putrescine, one of the most important polyamines in fungi and complex multicellular eukaryotic organisms, using genetics and molecular biology tools. Moreover, we identified the differentially regulated genes by this polyamine in U. maydis. This research provides insights into the similarities and differences in the conservation of the polyamine biosynthesis pathway in fungi, and it expands our understanding of the role of polyamines and the mechanisms regulated by these molecules in eukaryotes.

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae (and Its Resistance to Ciprofloxacin): Validation of a Molecular Biology Tool for Rapid Diagnosis and Treatment

Background and Objectives: Neisseria gonorrhoeae and Chlamydia trachomatis can cause similar clinical syndromes and may coexist in infections. In emergency medicine, empirical treatment targeting both pathogens is often employed, potentially contributing to antibiotic resistance. Gonococcal resistance has emerged against first-line antimicrobials, necessitating prior testing for fluoroquinolone susceptibility. Certest Biotec developed two molecular diagnostic products for simultaneous detection: VIASURE C. trachomatis & N. gonorrhoeae Real-Time PCR Detection Kit and VIASURE Neisseria gonorrhoeae Ciprofloxacin-Resistant Real-Time PCR Detection Kit. To evaluate these products, clinical performance assessments were conducted at the Clinical Microbiology Laboratory of Miguel Servet University Hospital in Zaragoza, Spain. Results and Conclusions: Both VIASURE assays under study demonstrated high clinical sensitivity and specificity compared to reference molecular assays and Sanger sequencing. These kits offer an accurate diagnosis, facilitating appropriate treatment choices while addressing concerns about emerging antibiotic resistance. Methods: A total of 540 clinical samples from 540 patients already characterized as positive or negative for CT and NG by a molecular assay and by antibiotic susceptibility testing for ciprofloxacin using a concentration gradient diffusion method were used for the clinical evaluation. In cases where sensitivity results were unavailable, conventional PCR and Sanger sequencing were employed.

Intrastrand Photo-Crosslinking of 5-Fluoro-2'-O-methyl-4-thiouridine-Modified Oligonucleotides and Its Implication for Fluorescence-Based Detection of DNA Sequences.

DNA photo-crosslinking reactions occur widely in biological systems and are often used as valuable tools in molecular biology. In this article, we demonstrate the application of an oligonucleotide 5-fluoro-2'-O-methyl-4-thiouridine (FSU)-containing probe for the fluorescent detection of specific DNA sequences. The design of the probe was predicated on studies of agents that could adversely affect its efficiency. The most important of these is the intrastrand photo-crosslinking of single-stranded oligodeoxynucleotides bearing FSU. Our research findings indicate that FSU after photoexcitation can react with nonadjacent bases; specifically, it can react with distant thymine and cytosine residues in the chain, forming fluorescent and nonfluorescent intrastrand crosslinks, respectively. In addition, partial photooxidation of the FSU residue to 5-fluorouridine was also observed. The results of the study are significant in terms of the use of FSU-labeled oligonucleotide probes in the fluorescence-based detection of specific DNA sequences because the creation of a fluorescent intrastrand crosslink could produce a false signal. To overcome this problem, replacing thymidine with deoxyuridine in the FSU-labeled oligonucleotide probe is proposed and tested.

Chem(Pro)2: the atlas of chemoproteomic probes labelling human proteins.

Chemoproteomic probes (CPPs) have been widely considered as powerful molecular biological tools that enable the highly efficient discovery of both binding proteins and modes of action for the studied compounds. They have been successfully used to validate targets and identify binders. The design of CPP has been considered extremely challenging, which asks for the generalization using a large number of probe data. However, none of the existing databases gives such valuable data of CPPs. Herein, a database entitled 'Chem(Pro)2' was therefore developed to systematically describe the atlas of diverse types of CPPs labelling human protein in living cell/lysate. With the booming application of chemoproteomic technique and artificial intelligence in current chemical biology study, Chem(Pro)2 was expected to facilitate the AI-based learning of interacting pattern among molecules for discovering innovative targets and new drugs. Till now, Chem(Pro)2 has been open to all users without any login requirement at: https://idrblab.org/chemprosquare/.

Arterial-Lymphatic-Like Endothelial Cells Appear in Hereditary Hemorrhagic Telangiectasia 2 and Contribute to Vascular Leakage and Arteriovenous Malformations.

Arteriovenous malformations (AVMs) are characteristic of hereditary hemorrhagic telangiectasia. Loss-of-function mutations in the activin receptor-like kinase 1 (Alk1) are linked to hemorrhagic telangiectasia type 2. Endothelial-specific deletion of Alk1, endothelial lineage tracing, transcriptomics of single-cell analysis, and electron microscopy were performed to examine the vascular phenotype and characteristics of ALK1-deficient endothelial cells (ECs) after EC-specific Alk1 deletion. Ischemia assays were used to examine the cell capacity for vascular malformation. Connectivity Map with transcriptomic analysis was applied to identify chemical compounds. Specific methods for arteriovenous malformations, such as micro-computed tomography, with other molecular and cell biological tools were also performed. We performed endothelial-specific deletion of Alk1 in mice and found severe arteriovenous malformations and vascular leakage. The transcriptomics of single-cell analysis revealed a new distinctive cell cluster formed after Alk1 deletion where the cells coexpressed arterial and lymphatic endothelial markers. The analysis projected that these cells potentially originated from arterial ECs after Alk1 deletion. This new population was referred to as arterial-lymphatic-like ECs according to its cellular markers, and its appearance was validated in the pulmonary small arteries after Alk1 deletion. Transplantation of these cells caused vascular malformations. Endothelial lineage tracing confirmed that these new arterial-lymphatic-like ECs were derived from ALK1 depleted ECs, potentially arterial ECs. We discovered that SOX17 (SRY-box transcription factor 17) induction was responsible for the derivation of these arterial-lymphatic-like ECs. We showed that direct binding of MDM2 (mouse double minute 2) was required for Sox17 to execute this activity. Inhibition of MDM2 reduced the arteriovenous malformations in the mouse model. Together, our studies revealed the mechanistic underpinnings of ALK1 signaling in regulating the endothelial phenotype and provided possibilities for new therapeutic strategies in hemorrhagic telangiectasia type 2.

An undergraduate research experience in CRISPR-Cas9 mediated eukaryotic genome editing to teach fundamental biochemistry techniques.

CRISPR-Cas9 technology is an established, powerful tool for genome editing through the ability to target specific DNA sequences of interest for introduction of desired genetic modifications. CRISPR-Cas9 is utilized for a variety of purposes, ranging from a research molecular biology tool to treatment for human diseases. Due to its prominence across a variety of applications, it is critical that undergraduates in the life sciences are educated on CRISPR-Cas9 technology. To this end, we created an intensive eight-week long course-based undergraduate research experience (CURE) designed for students to understand CRISPR-Cas9 genome editing and perform it in Saccharomyces cerevisiae. Students enrolled in the CURE participate in 2, 3-h sessions a week and are engaged in the entire process of CRISPR-Cas9 genome editing, from preparation of genome editing reagents to characterization of mutant yeast strains. During the process, students master fundamental techniques in the life sciences, including sterile technique, Polymerase Chain Reaction (PCR), primer design, sequencing requirements, and data analysis. The course is developed with flexibility in the schedule for repetition of techniques in the event of a failed experiment, providing an authentic research experience for the students. Additionally, we have developed the course to be easily modified for the editing of any yeast gene, offering the potential to expand the course in research-driven classroom or laboratory settings.

From Knallgas Bacterium to Promising Biomanufacturing Host: The Evolution of Cupriavidus necator.

The expanding field of synthetic biology requires diversification of microbial chassis to expedite the transition from a fossil fuel-dependent economy to a sustainable bioeconomy. Relying exclusively on established model organisms such as Escherichia coli and Saccharomyces cerevisiae may not suffice to drive the profound advancements needed in biotechnology. In this context, Cupriavidus necator, an extraordinarily versatile microorganism, has emerged as a potential catalyst for transformative breakthroughs in industrial biomanufacturing. This comprehensive book chapter offers an in-depth review of the remarkable technological progress achieved by C. necator in the past decade, with a specific focus on the fields of molecular biology tools, metabolic engineering, and innovative fermentation strategies. Through this exploration, we aim to shed light on the pivotal role of C. necator in shaping the future of sustainable bioprocessing and bioproduct development.

Epidemiology of Hepatitis C virus among thalassemia patients in Al-Muthanna Province

One of the main causes of blood transfusion problem including thalassemia, is the hepatitis C virus (HCV). In total, 1125 patients with thalassemia (635 men and 490 women, ages 2-23 years) took part in the trial from January 1, 2019, to December 31, 2021. Prior to PCR assay, the clinical symptoms, complete blood count (CBC) and particular hemoglobin tests were used to diagnose the thalassemia patients. Three blood transfusions every month were on an average. 1125 patients in total underwent ELISA testing to identify anti-HCV antibody (IgG) in serum. There were 111 (9.8%) Hepatitis C virus infections. In comparison to 2019 and 2021, the year 2020 had the highest numbers of thalassemia patients 400 (36%) and hepatitis C virus infections 41 (36.9%). Hepatitis C virus infected male individuals with thalassemia are more prevalent than female individuals. Compared to married, widowed and divorced states, the single status had the highest rate of hepatitis C virus infection. In comparison to urban residence, the number and percentage of Hepatitis C virus infections were highest in rural areas. To lessen the spread of the virus and eradicate it, we recommend using routine laboratory testing of blood donors and Thalassemia patients and the use of molecular biology tools like the PCR assay in the early diagnosis of the virus.

Distinct Communities of Bacteria and Unicellular Eukaryotes in the Different Water Masses of Cretan Passage Water Column (Eastern Mediterranean Sea).

Elucidating marine microbiota diversity and dynamics holds significant importance due to their role in maintaining vital ecosystem functions and services including climate regulation. This work aims to contribute in the understanding of microbial ecology and networking in one of the world's most understudied marine regions, the Eastern Mediterranean Sea. High-throughput 16S and 18S rRNA gene sequencing analysis was applied to study the diversity of bacteria and unicellular eukaryotes in the different water masses of the Cretan Passage during two seasonally-different sampling expeditions. We assumed that microbial associations differ between the surface and deepwater masses and created co-occurrence networks to evaluate this hypothesis. Our results unveiled vertical variations in both bacterial and unicellular eukaryotic diversity with species fluctuations indicative of seasonality being recorded in the surface water mass. Heterotrophic taxa and grazers related to organic matter degradation and nutrient cycling were enriched in the deepest water layers. Moreover, surface waters presented a higher number of microbial associations indicating abundant ecological niches compared to the deepest layer, possibly related to the lack of bottom-up resources in the oligotrophic deep ocean. Overall, our data provide insight in a heavily stressed, yet underexplored, marine area that requires further research to unravel the ecological roles of marine microbes. To our knowledge, this is the first study that combines molecular biology tools to provide data on both planktic prokaryotes and unicellular eukaryotes across the different water masses in this marine region of the Eastern Mediterranean basin.

Synthesis and Properties of α-Phosphate-Modified Nucleoside Triphosphates.

This review article is focused on the progress made in the synthesis of 5'-α-P-modified nucleoside triphosphates (α-phosphate mimetics). A variety of α-P-modified nucleoside triphosphates (NTPαXYs, Y = O, S; X = S, Se, BH3, alkyl, amine, N-alkyl, imido, or others) have been developed. There is a unique class of nucleoside triphosphate analogs with different properties. The main chemical approaches to the synthesis of NTPαXYs are analyzed and systematized here. Using the data presented here on the diversity of NTPαXYs and their synthesis protocols, it is possible to select an appropriate method for obtaining a desired α-phosphate mimetic. Triphosphates' substrate properties toward nucleic acid metabolism enzymes are highlighted too. We reviewed some of the most prominent applications of NTPαXYs including the use of modified dNTPs in studies on mechanisms of action of polymerases or in systematic evolution of ligands by exponential enrichment (SELEX). The presence of heteroatoms such as sulfur, selenium, or boron in α-phosphate makes modified triphosphates nuclease resistant. The most distinctive feature of NTPαXYs is that they can be recognized by polymerases. As a result, S-, Se-, or BH3-modified phosphate residues can be incorporated into DNA or RNA. This property has made NTPαXYs a multifunctional tool in molecular biology. This review will be of interest to synthetic chemists, biochemists, biotechnologists, or biologists engaged in basic or applied research.

High throughput mutational scanning of a protein via alchemistry on a high-performance computing resource.

Antibiotic resistance presents a significant challenge to public health, as bacteria can develop resistance to antibiotics through random mutations during their life cycles, making the drugs ineffective. Understanding how these mutations contribute to drug resistance at the molecular level is crucial for designing new treatment approaches. Recent advancements in molecular biology tools have made it possible to conduct comprehensive analyses of protein mutations. Computational methods for assessing molecular fitness, such as binding energies, are not as precise as experimental techniques like deep mutational scanning. Although full atomistic alchemical free energy calculations offer the necessary precision, they are seldom used to assess high throughput data as they require significantly more computational resources. We generated a computational library using deep mutational scanning for dihydrofolate reductase (DHFR), a protein commonly studied in antibiotic resistance research. Due to resource limitations, we analyzed 33 out of 159 positions, identifying 16 single amino acid replacements. Calculations were conducted for DHFR in its drug-free state and in the presence of two different inhibitors. We demonstrate the feasibility of such calculations, made possible due to the enhancements in computational resources and their optimized use.

Use of isotopic (C, Cl) and molecular biology tools to assess biodegradation in a source area of chlorinated ethenes after biostimulation with Emulsified Vegetable Oil (EVO)

Enhanced In Situ Bioremediation (EISB) using Emulsified Vegetable Oil (EVO) as a long-term electron donor has gained prominence for the treatment of groundwater contaminated with chlorinated ethenes (CEs). This study explores the potential of isotopic and molecular biology tools (MBT) to investigate the CEs (PCE, TCE and cis-DCE) bioremediation using EVO in a contaminated site. A multiple approach using C and Cl-CSIA, quantification of Dehalococcoides (Dhc) and specific reductive dechlorination (RD) gene population, and hydrochemical data in microcosm experiments and field samples was applied. Despite the high partitioning of CEs into the EVO phase, the carbon isotopic values of the remaining CEs fraction in the aqueous phase did not exhibit significant changes caused by phase partitioning in laboratory experiments. Both microcosm experiments and field data revealed a rapid RD of PCE and TCE, resulting in the transient accumulation of cis-DCE, which was slowly degraded to vinyl chloride (VC). These results agreed with the presence of Dhc populations and a shift to stronger reducing conditions in the field: i) RD functional genes (tceA, vcrA and bvcA) exhibited a trend to higher values and ii) a substantial increase in Dhc populations (up to 30% of the total bacterial populations) was observed over time. The dual-element isotope slope ΛC-Cl for RD of cis-DCE obtained from field data (ΛC – Cl = 5 ± 3) was similar to the one determined from the microcosm experiments under controlled anoxic conditions (ΛC – Cl = 4.9 ± 0.8). However, ΛC-Cl values differ from those reported so far for laboratory studies with Dhc strains and mixed cultures containing Dhc, i.e., between 8.3 and 17.8. This observation underscores the potential variety of reductive dehalogenases involved during cis-DCE RD and the importance of determining site-specific Λ and ɛ values in order to improve the identification and quantification of transformation processes in the field.

Characterization of Ligilactobacillus salivarius CRISPR-Cas systems.

Ligilactobacillus salivarius is a diverse bacterial species widely used in the food and dietary supplement industries. In this study, we investigate the occurrence and diversity of their adaptive immune systems, CRISPR-Cas, in over 500 genomes. We establish their function and provide insights into their role in the interplay between the bacterial host and the predatory phages that infect them. Such findings expand our knowledge about these important CRISPR-Cas immune systems widespread across the bacterial tree of life and also provide a technical basis for the repurposing of these molecular machines for the development of molecular biology tools and the manipulation and engineering of bacteria and other life forms.

Developing a microbial community structure index (MCSI) as an approach to evaluate and optimize bioremediation performance.

Much attention is placed on organohalide-respiring bacteria (OHRB), such as Dehalococcoides, during the design and performance monitoring of chlorinated solvent bioremediation systems. However, many OHRB cannot function effectively without the support of a diverse group of other microbial community members (MCMs), who play key roles fermenting organic matter into more readily useable electron donors, producing corrinoids such as vitamin B12, or facilitating other important metabolic processes or biochemical reactions. While it is known that certain MCMs support dechlorination, a metric considering their contribution to bioremediation performance has yet to be proposed. Advances in molecular biology tools offer an opportunity to better understand the presence and activity of specific microbes, and their relation to bioremediation performance. In this paper, we test the hypothesis that a specific microbial consortium identified within 16S ribosomal ribonucleic acid (rRNA) gene next generation sequencing (NGS) data can be predictive of contaminant degradation rates. Field-based data from multiple contaminated sites indicate that increasing relative abundance of specific MCMs correlates with increasing first-order degradation rates. Based on these results, we present a framework for computing a simplified metric using NGS data, the Microbial Community Structure Index, to evaluate the adequacy of the microbial ecosystem during assessment of bioremediation performance.

A project‐based learning mode for science students: Fluorescent protein expression in trypanosomes

AbstractAn effective, practical approach for incorporating molecular biology tools to biology and biochemistry students is described. For 6 weeks, students are exposed to bacterial transformation, plasmid DNA purification and analysis, parasite transfection, ectopic expression of fluorescent proteins, fluorescence microscopy, and flow cytometry analysis. This research‐oriented laboratory course delves into theoretical and practical concepts of basic techniques in molecular biology to emphasize the logical connections between the obtained results and the resolution of problems that arise daily in a research laboratory, training the students in planning, conducting, discussing, and presenting their own experiments and results.

Metataxonomic Studies to Evaluate the Beneficial Effect of Enzymatic Pretreatment on the Anaerobic Digestion of Waste Generated in Turkey Farming.

Turkey litter waste is lignocellulosic and keratinous, requiring prior enzymatic treatment to facilitate fiber hydrolysis and utilization by microorganisms in anaerobic digestion (AD) process. The understanding of the performance of microorganisms in AD can be facilitated through molecular biology and bioinformatics tools. This study aimed to determine the taxonomic profile and functional prediction of microbial communities in the AD of turkey litter waste subjected to enzymatic pretreatment and correlate it with operational parameters. The tests involved the use of turkey litter (T) at 25gL-1 of volatile solids, a granular inoculum (S) (10% m/v), and the addition of cellulase (C), and pectinase (P) enzymes at four concentrations. The use of enzymes increased methane production by 19% (turkey litter, inoculum, and cellulase-TSC4) and 15% (turkey litter, inoculum, and enzymatic pectinase-TSP4) compared to the control (turkey litter and inoculum-TS), being more effective in TSC4 (667.52 mLCH4), where there was consumption of acetic, butyric, and propionic acids. The pectinase assay (TSP4) showed a methane production of 648 mLCH4 and there was the accumulation of metabolites. Cellulolytic microorganisms Bacteroides, Ruminofilibacter, Lachnospiraceae, Ruminococcaceae, and Methanosaeta were favored in TSC4. In TSP4, the predominant genus was Macellibacteroides and Methanosarcina, and genes involved in methylotrophic methanogenesis were also found (mtaB, mtmB, and mtbB). Enzymes involved in hydrogenotrophic methanogenesis were identified in both assays (TSC4 and TSP4). Molecular tools helped to understand the metabolic routes involved in AD with enzymatic treatment, allowing the elaboration of strategies to improve the sustainable degradation of turkey litter waste.

The potential of genome editing to create novel alleles of resistance genes in rice.

Rice, a staple food for a significant portion of the global population, faces persistent threats from various pathogens and pests, necessitating the development of resilient crop varieties. Deployment of resistance genes in rice is the best practice to manage diseases and reduce environmental damage by reducing the application of agro-chemicals. Genome editing technologies, such as CRISPR-Cas, have revolutionized the field of molecular biology, offering precise and efficient tools for targeted modifications within the rice genome. This study delves into the application of these tools to engineer novel alleles of resistance genes in rice, aiming to enhance the plant's innate ability to combat evolving threats. By harnessing the power of genome editing, researchers can introduce tailored genetic modifications that bolster the plant's defense mechanisms without compromising its essential characteristics. In this study, we synthesize recent advancements in genome editing methodologies applicable to rice and discuss the ethical considerations and regulatory frameworks surrounding the creation of genetically modified crops. Additionally, it explores potential challenges and future prospects for deploying edited rice varieties in agricultural landscapes. In summary, this study highlights the promise of genome editing in reshaping the genetic landscape of rice to confront emerging challenges, contributing to global food security and sustainable agriculture practices.

Targeting Oral Cancer: In Silico Docking Studies of Phytochemicals on Oncogenic Molecular Markers.

Molecular docking is a key tool in structural molecular biology and computer-assisted drug design. Oral carcinogenesis is a complex, multistep process in which genetic events within signal transduction pathways governing normal cellular physiology are quantitatively or qualitatively altered. There are various molecular targets like Cyclin D and PI3k- alpha Ras Binding Domain receptor protein involved in the pathogenesis of Oral Squamous Cell Carcinoma. The aim of the study is to demonstrate the computer aided drug design to identify a potent natural molecule for targeting cyclin D4 and PI3K RAS binding protein. Target selection (Cyclin D1 and PI3K-alpha Ras Binding Domain receptor) was done and structures were derived from protein data bank. Ligands (Apigenin, Chrysoeriol and Luteolin) selection was done and structure derived. Final docking was performed by Autodock. From the docking results it can be seen that luteolin has the highest binding energy (-5.45) with the Cyclin D receptor molecule followed by Chrysoeriol (-4.99) and Apigenin (-4.96). The binding energies of the ligands against PI3K-alpha Ras Binding Domain receptors were Apigenin (-4.51), Chrysoeriol (-4.6) and Luteolin (-4.56). The study concludes that all the three selected ligands possess high binding energy with both the target proteins involved in carcinogenesis with highest binding energy possessed by Luteolin against the Cyclin D receptor and by Chrysoeriol against PI3K-RAS binding protein. Thus their activity can be utilized to derive potential Anti-cancer therapeutic drugs.

Microinjection of the CRISPR/Cas9 editing system through the germ pore of a wheat microspore induces mutations in the target Ms2 gene.

Microinjection is a direct procedure for delivering various compounds via micropipette into individual cells. Combined with the CRISPR/Cas9 editing technology, it has been used to produce genetically engineered animal cells. However, genetic micromanipulation of intact plant cells has been a relatively unexplored area of research,partly due to the cytological characteristics of these cells. This study aimed to gain insight into the genetic micromanipulation of wheat microspores using microinjection procedures combined with the CRISPR/Cas9 editing system targeting the Ms2 gene. Microspores were first reprogrammed by starvation and heat shock treatment to make them structurally suitable for microinjection. The large central vacuole was fragmented and the nucleus with cytoplasm was positioned in the center of the cell. This step and an additional maltose gradient provided an adequate source of intact single cells in the three wheat genotypes. The microcapillary was inserted into the cell through the germ pore to deliver a working solution with a fluorescent marker. This procedure was much more efficient and less harmful to the microspore than inserting the microcapillary through the cell wall. The CRISPR/Cas9 binary vectors injected into reprogrammed microspores induced mutations in the target Ms2 gene with deletions ranging from 1 to 16bp. This is the first report of successful genome editing in an intact microspore/wheat cell using the microinjection technique and the CRISPR/Cas9 editing system. The study presented offers a range of molecular and cellular biology tools that can aid in genetic micromanipulation and single-cell analysis.

Enhancement of efficient molecular biology tools for Tremella fuciformis spore

Tremella fuciformis, one of the most approved mushrooms worldwide, possesses both edible and medicinal value. However, low transformation efficiency and high false positive rate, long verification period, and low expression of heterologous proteins hinder further genetic research. In this study, a polyethylene glycol-mediated genetic transformation system was established by using the homologous resistance marker carboxin in T. fuciformis. By optimizing the protoplast quality and transformation conditions, the transformation efficiency reached 8.23 CFU/μg, and the positive rate reached 64.95%. Moreover, Driselase-Heating-Freezing screening system was developed for rapid transformants verification, which took only 35–45 min and made the colony PCR 100% accurate. Considering the rationality of tRNA use frequency in heterologous genes, the coordinated relative synonymous codon usage (RSCU) strategy was adopted to recode the heterologous gene sequence, which ensured the protein expression level. This study comprehensively enhances molecular biology tools in T. fuciformis, and lays a foundation for further development.