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Bioresources and Bioprocessing Jun 2023Cell-free protein synthesis (CFPS) system is an ideal platform for fast and convenient protein research and has been used for macromolecular assembly, unnatural amino...
Cell-free protein synthesis (CFPS) system is an ideal platform for fast and convenient protein research and has been used for macromolecular assembly, unnatural amino acid embedding, glycoprotein production, and more. To realize the construction of an efficient eukaryotic CFPS platform with the advantages of low cost and short time, a CFPS system based on the yeast Pichia pastoris was built in this study. The internal ribosomal entry site (IRES) can independently initiate translation and thus promote protein synthesis. The Kozak sequences can facilitate translation initiation. Therefore, the screening of IRES and its combination with Kozak was performed, in which cricket paralysis virus (CRPV) exhibited as the best translation initiation element from 14 different IRESs. Furthermore, the system components and reaction environment were explored. The protein yield was nearly doubled by the addition of RNase inhibitor. The cell extract amount, energy regeneration system (phosphocreatine and phosphocreatine kinase), and metal ions (K and Mg) were optimized to achieve the best protein synthesis yield. This P. pastoris CFPS system can extend the eukaryotic CFPS platform, providing an enabling technology for fast prototyping design and functional protein synthesis.
PubMed: 38647944
DOI: 10.1186/s40643-023-00653-4 -
Journal of Fungi (Basel, Switzerland) Oct 2023is the most widely used microorganism for the production of secreted industrial proteins and therapeutic proteins. Recently, this yeast has been repurposed as a cell... (Review)
Review
is the most widely used microorganism for the production of secreted industrial proteins and therapeutic proteins. Recently, this yeast has been repurposed as a cell factory for the production of chemicals and natural products. In this review, the general physiological properties of are summarized and the readily available genetic tools and elements are described, including strains, expression vectors, promoters, gene editing technology mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, and adaptive laboratory evolution. Moreover, the recent achievements in -based biosynthesis of proteins, natural products, and other compounds are highlighted. The existing issues and possible solutions are also discussed for the construction of efficient cell factories.
PubMed: 37888283
DOI: 10.3390/jof9101027 -
Pathogens (Basel, Switzerland) Mar 2024has been previously classified as , , and and was recently reclassified in the genus after phylogenetic analysis of its genetic sequence. An increasing number of... (Review)
Review
has been previously classified as , , and and was recently reclassified in the genus after phylogenetic analysis of its genetic sequence. An increasing number of reports of human infections by have emerged, suggesting that this microorganism is an emerging pathogen. The present review aimed to provide data on the epidemiology, antifungal resistance, clinical characteristics, treatment, and outcomes of fungemia by by extracting all the available information from published original reports in the literature. PubMed/Medline, Cochrane Library, and Scopus databases were searched for eligible articles reporting data on patients with this disease. In total, 36 studies involving 170 patients were included. The age of patients with fungemia by ranged from 0 to 89 years; the mean age was 22.8 years, the median age was 2.2 years, with more than 37 patients being less than one month old, and 54% (88 out of 163 patients) were male. Regarding patients' history, 70.4% had a central venous catheter use (CVC), 28.7% were on total parenteral nutrition (TPN), 97% of neonates were hospitalized in the neonatal ICU (NICU), and 39.4% of the rest of the patients were hospitalized in the intensive care unit (ICU). Previous antimicrobial use was noted in 65.9% of patients. The most common identification method was the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in 34.1%, VITEK and VITEK 2 in 20.6%, and ID32 C in 15.3%. had minimal antifungal resistance to fluconazole, echinocandins, and amphotericin B, the most commonly used antifungals for treatment. Fever and sepsis were the most common clinical presentation noted in 95.8% and 86%, respectively. Overall mortality was 20% and was slightly higher in patients older than one year. Due to the rarity of this disease, future multicenter studies should be performed to adequately characterize patients' characteristics, treatment, and outcomes, which will increase our understanding and allow drawing safer conclusions regarding optimal management.
PubMed: 38535612
DOI: 10.3390/pathogens13030269 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Jun 2023Non-conventional yeasts such as , , , and have proven to be efficient cell factories in producing a variety of natural products due to their wide substrate utilization... (Review)
Review
Non-conventional yeasts such as , , , and have proven to be efficient cell factories in producing a variety of natural products due to their wide substrate utilization spectrum, strong tolerance to environmental stresses and other merits. With the development of synthetic biology and gene editing technology, metabolic engineering tools and strategies for non-conventional yeasts are expanding. This review introduces the physiological characteristics, tool development and current application of several representative non-conventional yeasts, and summarizes the metabolic engineering strategies commonly used in the improvement of natural products biosynthesis. We also discuss the strengths and weaknesses of non-conventional yeasts as natural products cell factories at current stage, and prospects future research and development trends.
Topics: Yeasts; Yarrowia; Gene Editing; Metabolic Engineering
PubMed: 37401595
DOI: 10.13345/j.cjb.220914 -
Human Vaccines & Immunotherapeutics Dec 2024Current estimates of the HPV infection rate in China vary by geographic region (9.6-23.6%), with two age peaks in prevalence in women ≤20-25 years of age and... (Review)
Review
Current estimates of the HPV infection rate in China vary by geographic region (9.6-23.6%), with two age peaks in prevalence in women ≤20-25 years of age and 50-60 years of age. HPV-16, 52 and 58 are the most commonly-detected HPV genotypes in the Chinese population. In China, five HPV vaccines are licensed and several others are undergoing clinical trials. Multiple RCTs have shown the efficacy and safety of the bvHPV (Cervarix), -produced bvHPV (Cecolin), -produced bvHPV (Walrinvax), qvHPV (Gardasil) and 9vHPV (Gardasil-9) vaccines in Chinese populations, including two studies showing long-term efficacy (≥8 years) for the bvHPV and qvHPV vaccines. Real-world data from China are scarce. Although modeling studies in China show HPV vaccination is cost-effective, uptake and population coverage are relatively low. Various policies have been implemented to raise awareness and increase vaccine coverage, with the long-term aim of eliminating cervical cancer in China.
Topics: Humans; Female; Young Adult; Adult; Human Papillomavirus Recombinant Vaccine Quadrivalent, Types 6, 11, 16, 18; Papillomavirus Vaccines; Papillomavirus Infections; Uterine Cervical Neoplasms; Vaccination; Human papillomavirus 16; China
PubMed: 38575524
DOI: 10.1080/21645515.2024.2329450 -
Microbial Cell Factories Jul 2023Ablynx NV, a subsidiary of Sanofi, has a long-standing focus on the development of Nanobody® molecules as biopharmaceuticals (Nanobody® is a registered trademark of...
BACKGROUND
Ablynx NV, a subsidiary of Sanofi, has a long-standing focus on the development of Nanobody® molecules as biopharmaceuticals (Nanobody® is a registered trademark of Ablynx NV). Nanobody molecules are single variable domains, and they have been met with great success part due to their favorable expression properties in several microbial systems. Nevertheless, the search for the host of the future is an ongoing and challenging process. Komagataella phaffi (Pichia pastoris) is one of the most suitable organisms to produce Nanobody molecules. In addition, genetic engineering of Pichia is easy and an effective approach to improve titers.
RESULTS
Here we report that P. pastoris engineered to co-express genes encoding four auxiliary proteins (HAC1, KAR2, PDI and RPP0), leads to a marked improvement in the expression of Nanobody molecules using the AOX1 methanol induction system. Titer improvement is mainly attributed to HAC1, and its beneficial effect was also observed in a methanol-free expression system.
CONCLUSION
Our findings are based on over a thousand fed-batch fermentations and offer a valuable guide to produce Nanobody molecules in P. pastoris. The presented differences in expressability between types of Nanobody molecules will be helpful for researchers to select both the type of Nanobody molecule and Pichia strain and may stimulate further the development of a more ecological methanol-free expression platform.
Topics: Saccharomycetales; Pichia; Biological Products; Fermentation; Methanol
PubMed: 37481525
DOI: 10.1186/s12934-023-02132-z -
Engineering and Expression Strategies for Optimization of L-Asparaginase Development and Production.International Journal of Molecular... Oct 2023Genetic engineering for heterologous expression has advanced in recent years. Model systems such as , and are often used as host microorganisms for the enzymatic... (Review)
Review
Genetic engineering for heterologous expression has advanced in recent years. Model systems such as , and are often used as host microorganisms for the enzymatic production of L-asparaginase, an enzyme widely used in the clinic for the treatment of leukemia and in bakeries for the reduction of acrylamide. Newly developed recombinant L-asparaginase (L-ASNase) may have a low affinity for asparagine, reduced catalytic activity, low stability, and increased glutaminase activity or immunogenicity. Some successful commercial preparations of L-ASNase are now available. Therefore, obtaining novel L-ASNases with improved properties suitable for food or clinical applications remains a challenge. The combination of rational design and/or directed evolution and heterologous expression has been used to create enzymes with desired characteristics. Computer design, combined with other methods, could make it possible to generate mutant libraries of novel L-ASNases without costly and time-consuming efforts. In this review, we summarize the strategies and approaches for obtaining and developing L-ASNase with improved properties.
Topics: Humans; Asparaginase; Asparagine; Leukemia; Escherichia coli; Models, Biological; Antineoplastic Agents
PubMed: 37894901
DOI: 10.3390/ijms242015220 -
International Journal of Molecular... Jun 2023Researchers are often interested in proteins that are present in cells in small ratios compared to the total amount of proteins. These proteins include transcription... (Review)
Review
Researchers are often interested in proteins that are present in cells in small ratios compared to the total amount of proteins. These proteins include transcription factors, hormones and specific membrane proteins. However, sufficient amounts of well-purified protein preparations are required for functional and structural studies of these proteins, including the creation of artificial proteoliposomes and the growth of protein 2D and 3D crystals. This aim can be achieved by the expression of the target protein in a heterologous system. This review describes the applications of yeast heterologous expression systems in studies of plant membrane proteins. An initial brief description introduces the widely used heterologous expression systems of the baker's yeast and the methylotrophic yeast . is further considered a convenient model system for functional studies of heterologously expressed proteins, while has the advantage of using these yeast cells as factories for producing large quantities of proteins of interest. The application of both expression systems is described for functional and structural studies of membrane proteins from plants, namely, K- and Na-transporters, various ATPases and anion transporters, and other transport proteins.
Topics: Saccharomyces cerevisiae; Membrane Proteins; Plant Proteins; Pichia; Recombinant Proteins
PubMed: 37445944
DOI: 10.3390/ijms241310768 -
Microorganisms May 2024Because data on the fungal gut community structure of African children are scarce, we aimed to describe it by reanalysing rRNA ITS1 and ITS2 metabarcoding data from a...
Because data on the fungal gut community structure of African children are scarce, we aimed to describe it by reanalysing rRNA ITS1 and ITS2 metabarcoding data from a study designed to assess the influence of microbiota in malaria susceptibility in Malian children from the Dogon country. More specifically, we aimed to establish the core gut mycobiome and compare the gut fungal community structure of breastfed children, aged 0-2 years, with other age groups. Briefly, DNA was extracted from 296 children's stool samples. Both rRNA ITS1 and ITS2 genomic barcodes were amplified and subjected to Illumina MiSeq sequencing. The ITS2 barcode generated 1,975,320 reads and 532 operational taxonomic units (OTUs), while the ITS1 barcode generated 647,816 reads and 532 OTUs. The alpha diversity was significantly higher by using the ITS1 compared to the ITS2 barcode ( < 0.05); but, regardless of the ITS barcode, we found no significant difference between breastfed children, aged 0-2 years, compared to the other age groups. The core gut mycobiome of the Malian children included , , , , and section , which were present in at least 50% of the 296 children. Further studies in other African countries are warranted to reach a global view of African children's core gut mycobiome.
PubMed: 38792756
DOI: 10.3390/microorganisms12050926 -
Synthetic and Systems Biotechnology Sep 2023CRISPR interference (CRISPRi) has been developed and widely used for gene repression in various hosts. Here we report an improved CRISPRi system in by fusing dCas9 with...
CRISPR interference (CRISPRi) has been developed and widely used for gene repression in various hosts. Here we report an improved CRISPRi system in by fusing dCas9 with endogenous transcriptional repressor domains. The CRISPRi system shows strong repression of , with the highest efficiency of 85%. Repression of native genes is demonstrated by targeting promoter. is efficiently repressed and the mutant strains show much slower growth in methanol medium. Effects of gRNA expression and processing on CRISPRi efficiency is also investigated. It is found that gRNA processing by HH/HDV ribozymes or Csy4 endoribonuclease generating clean gRNA is critical to achieve strong repression, and Csy4 cleavage shows higher repression efficiency. However, gRNA expression using native tRNA transcription and processing systems results in relatively weaker repression of . By expression of two gRNAs targeting promoters of and in an array together with Cys4 recognition sites, both genes can be repressed simultaneously. Cys4-mediated gRNA array processing is further applied to repress fatty acyl-CoA synthetase genes ( and ). Both genes are efficiently repressed, demonstrating that Cys4 endoribonuclease has the ability to cleave gRNAs array and can be can be used for multiplexed gene repression in .
PubMed: 37692202
DOI: 10.1016/j.synbio.2023.06.008