Biotechnology Journal最新文献

{"title":"Human platelet lysate supports SH-SY5Y neuroblastoma cell proliferation and differentiation into a dopaminergic-like neuronal phenotype under xenogeneic-free culture conditions","authors":"Miguel Ribeiro,&nbsp;Jonas Campos,&nbsp;Tiffany S. Pinho,&nbsp;Belém Sampaio-Marques,&nbsp;Sandra Barata-Antunes,&nbsp;Jorge Ribeiro Cibrão,&nbsp;Ricardo Araújo,&nbsp;Sara Duarte-Silva,&nbsp;Elsa Moreira,&nbsp;Rui A. Sousa,&nbsp;Pedro M. Costa,&nbsp;António J. Salgado","doi":"10.1002/biot.202400068","DOIUrl":"10.1002/biot.202400068","url":null,"abstract":"<p>SH-SY5Y is a human neuroblastoma cell line that can be differentiated into several neuronal phenotypes, depending on culture conditions. For this reason, this cell line has been widely used as an in vitro model of neurodegenerative conditions, such as Parkinson's disease (PD). However, most studies published to date used fetal bovine serum (FBS) as culture medium supplement for SH-SY5Y cell differentiation. We report on the testing of human platelet lysate (hPL) as a culture medium supplement to support SH-SY5Y cell culture. Both standard hPL and a fibrinogen-depleted hPL (FD-hPL) formulation, which does not require the addition of anticoagulants to culture media, promoted an increase in SH-SY5Y cell proliferation in comparison to FBS, without compromising metabolic activity. SH-SY5Y cells cultured in hPL or FD-hPL also displayed a higher number of neurite extensions and stained positive for MAP2 and synaptophysin, in the absence of differentiation stimuli; reducing hPL or FD-hPL concentration to 1% v/v did not affect cell proliferation or metabolic activity. Furthermore, following treatment with retinoic acid (RA) and further stimulation with brain-derived neurotrophic factor (BDNF) and nerve growth factor beta (NGF-β), the percentage of SH-SY5Y cells stained positive for dopaminergic neuronal differentiation markers (tyrosine hydroxylase [TH] and Dopamine Transporter [DAT]) was higher in hPL or FD-hPL than in FBS, and gene expression of dopaminergic markers TH, DAT, and DR2 was also detected.</p><p>Overall, the data herein presented supports the use of hPL to differentiate SH-SY5Y cells into a neuronal phenotype with dopaminergic features, and the adoption of FD-hPL as a fully xenogeneic free alternative to FBS to support the use of SH-SY5Y cells as a neurodegeneration model.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141578353","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Toehold region triggered CRISPR/Cas12a trans-cleavage for detection of uracil-DNA glycosylase activity","authors":"Chenyu Cui,&nbsp;Guihuan Guo,&nbsp;Ting-Hsuan Chen","doi":"10.1002/biot.202400097","DOIUrl":"10.1002/biot.202400097","url":null,"abstract":"<p>DNA glycosylases are a group of enzymes that play a crucial role in the DNA repair process by recognizing and removing damaged or incorrect bases from DNA molecules, which maintains the integrity of the genetic information. The abnormal expression of uracil-DNA glycosylase (UDG), one of significant DNA glycosylases in the base-excision repair pathway, is linked to numerous diseases. Here, we proposed a simple UDG activity detection method based on toehold region triggered CRISPR/Cas12a <i>trans</i>-cleavage. The toehold region on hairpin DNA probe (HP) produced by UDG could induce the <i>trans</i>-cleavage of ssDNA with fluorophore and quencher, generating an obvious fluorescence signal. This protospacer adjacent motif (PAM)-free approach achieves remarkable sensitivity and specificity in detecting UDG, with a detection limit as low as 0.000368 U mL⁻¹. Moreover, this method is able to screen inhibitors and measure UDG in complex biological samples. These advantages render it highly promising for applications in clinical diagnosis and drug discovery.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400097","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141578355","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fast-track adaptive laboratory evolution of Cupriavidus necator H16 with divalent metal cations","authors":"Sepwin Nosten Sitompul,&nbsp;Laura Andrea Diaz Garcia,&nbsp;Joseph Price,&nbsp;Kang Lan Tee,&nbsp;Tuck Seng Wong","doi":"10.1002/biot.202300577","DOIUrl":"10.1002/biot.202300577","url":null,"abstract":"<p>Microbial strain improvement through adaptive laboratory evolution (ALE) has been a key strategy in biotechnology for enhancing desired phenotypic traits. In this Biotech Method paper, we present an accelerated ALE (aALE) workflow and its successful implementation in evolving <i>Cupriavidus necator</i> H16 for enhanced tolerance toward elevated glycerol concentrations. The method involves the deliberate induction of genetic diversity through controlled exposure to divalent metal cations, enabling the rapid identification of improved variants. Through this approach, we observed the emergence of robust variants capable of growing in high glycerol concentration environments, demonstrating the efficacy of our aALE workflow. When cultivated in 10% v/v glycerol, the adapted variant Mn-C2-B11, selected through aALE, achieved a final OD₆₀₀ value of 56.0 and a dry cell weight of 15.2 g L⁻¹, compared to the wild type (WT) strain's final OD₆₀₀ of 39.1 and dry cell weight of 8.4 g L⁻¹. At an even higher glycerol concentration of 15% v/v, Mn-C2-B11 reached a final OD₆₀₀ of 48.9 and a dry cell weight of 12.7 g L⁻¹, in contrast to the WT strain's final OD₆₀₀ of 9.0 and dry cell weight of 3.1 g L⁻¹. Higher glycerol consumption by Mn-C2-B11 was also confirmed by high-performance liquid chromatography (HPLC) analysis. This adapted variant consumed 34.5 times more glycerol compared to the WT strain at 10% v/v glycerol. Our method offers several advantages over other reported ALE approaches, including its independence from genetically modified strains, specialized genetic tools, and potentially carcinogenic DNA-modifying agents. By utilizing divalent metal cations as mutagens, we offer a safer, more efficient, and cost-effective alternative for expansion of genetic diversity. With its ability to foster rapid microbial evolution, aALE serves as a valuable addition to the ALE toolbox, holding significant promise for the advancement of microbial strain engineering and bioprocess optimization.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202300577","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141578352","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biomass specific perfusion rate as a control lever for the continuous manufacturing of biosimilar monoclonal antibodies from CHO cell cultures","authors":"Jiayu Leong,&nbsp;Wen Qin Tang,&nbsp;Jake Chng,&nbsp;Wei Xuan Ler,&nbsp;Norhaizat Abdul Manan,&nbsp;Lyn Chiin Sim,&nbsp;Zi Ying Zheng,&nbsp;Wei Zhang,&nbsp;Ian Walsh,&nbsp;Gerben Zijlstra,&nbsp;Maarten Pennings,&nbsp;Say Kong Ng","doi":"10.1002/biot.202400092","DOIUrl":"10.1002/biot.202400092","url":null,"abstract":"<p>Continuous manufacturing enables high volumetric productivities of biologics such as monoclonal antibodies. However, it is challenging to maintain both high viable cell densities and productivities at the same time for long culture durations. One of the key controls in a perfusion process is the perfusion rate which determines the nutrient availability and potentially controls the cell metabolism. Cell Specific Perfusion Rate (CSPR) is a feed rate proportional to the viable cell density while Biomass Specific Perfusion Rate (BSPR) is a feed rate proportional to the biomass (cell volume multiply by cell density). In this study, perfusion cultures were run at three BSPRs in the production phase. Low BSPR favored a growth arresting state that led to gradual increase in cell volume, which in turn led to an increase in net perfusion rate proportional to the increase in cell volume. Consequently, at low BSPR, while the cell viability and cell density decreased, high specific productivity of 55 pg per cell per day was achieved. In contrast, the specific productivity was lower in bioreactors operating at a high BSPR. The ability to modulate the cell metabolism by using BSPR was confirmed when the specific productivity increased after lowering the BSPR in one of the bioreactors that was initially operating at a high BSPR. This study demonstrated that BSPR significantly influenced cell growth, metabolism, and productivity in cultures with variable cell volumes.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400092","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141578403","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Streamlined screening of extracellularly expressed PETase libraries for improved polyethylene terephthalate degradation","authors":"Guy Orr,&nbsp;Yoav Niv,&nbsp;Maya Barakat,&nbsp;Alexandra Boginya,&nbsp;Moshe Dessau,&nbsp;Livnat Afriat-Jurnou","doi":"10.1002/biot.202400021","DOIUrl":"10.1002/biot.202400021","url":null,"abstract":"<p>Enzyme-mediated polyethylene terephthalate (PET) depolymerization has recently emerged as a sustainable solution for PET recycling. Towards an industrial-scale implementation of this technology, various strategies are being explored to enhance PET depolymerization (PETase) activity and improve enzyme stability, expression, and purification processes. Recently, rational engineering of a known PET hydrolase (LCC—leaf compost cutinase) has resulted in the isolation of a variant harboring four-point mutations (LCC-ICCG), presenting increased PETase activity and thermal stability. Here, we revealed the enzyme's natural extracellular expression and used it to efficiently screen error-prone genetic libraries based on LCC-ICCG for enhanced activity toward consumer-grade PET. Following multiple rounds of mutagenesis and screening, we successfully isolated variants that exhibited up to a 60% increase in PETase activity. Among other mutations, the improved variants showed a histidine to tyrosine substitution at position 218, a residue known to be involved in substrate binding and stabilization. Introducing H218Y mutation on the background of LCC-ICCG (named here LCC-ICCG/H218Y) resulted in a similar level of activity improvement. Analysis of the solved structure of LCC-ICCG/H218Y compared to other known PETases featuring different amino acids at the equivalent position suggests that H218Y substitution promotes enhanced PETase activity. The expression and screening processes developed in this study can be further used to optimize additional enzymatic parameters crucial for efficient enzymatic degradation of consumer-grade PET.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400021","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141578354","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Efficient CRISPR-mediated C-to-T base editing in Komagataella phaffii","authors":"Ling-Yu Wu,&nbsp;Yan Xu,&nbsp;Xiao-Wei Yu","doi":"10.1002/biot.202400115","DOIUrl":"10.1002/biot.202400115","url":null,"abstract":"<p>The nonconventional methylotrophic yeast <i>Komagataella phaffii</i> is widely applied in the production of industrial enzymes, pharmaceutical proteins, and various high-value chemicals. The development of robust and versatile genome editing tools for <i>K. phaffii</i> is crucial for the design of increasingly advanced cell factories. Here, we first developed a base editing method for <i>K. phaffii</i> based on the CRISPR-nCas9 system. We engineered 24 different base editor constructs, using a variety of promoters and cytidine deaminases (CDAs). The optimal base editor (P<i>_AOX2*</i>-KpA3A-nCas9-KpUGI-<i>DAS1</i>TT) comprised a truncated <i>AOX2</i> promoter (P<i>_AOX2*</i>), a <i>K. phaffii</i> codon-optimized human APOBEC3A CDA (KpA3A), human codon-optimized nCas9 (D10A), and a <i>K. phaffii</i> codon-optimized uracil glycosylase inhibitor (KpUGI). This optimal base editor efficiently performed C-to-T editing in <i>K. phaffii</i>, with single-, double-, and triple-locus editing efficiencies of up to 96.0%, 65.0%, and 5.0%, respectively, within a 7-nucleotide window from C_-18 to C_-12. To expand the targetable genomic region, we also replaced nCas9 in the optimal base editor with nSpG and nSpRy, and achieved 50.0%–60.0% C-to-T editing efficiency for NGN-protospacer adjacent motif (PAM) sites and 20.0%–93.2% C-to-T editing efficiency for NRN-PAM sites, respectively. Therefore, these constructed base editors have emerged as powerful tools for gene function research, metabolic engineering, genetic improvement, and functional genomics research in <i>K. phaffii</i>.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141578404","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Issue Information: Biotechnology Journal 7/2024","authors":"","doi":"10.1002/biot.202470071","DOIUrl":"10.1002/biot.202470071","url":null,"abstract":"","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202470071","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141549899","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Outside Front Cover: (Biotechnology Journal 4/2024)","authors":"","doi":"10.1002/biot.202470042","DOIUrl":"10.1002/biot.202470042","url":null,"abstract":"<p>The cover image is based on the Rapid Publication <i>Accelerated generation of gene-engineered monoclonal CHO cell lines using FluidFM nanoinjection and CRISPR/Cas9</i> by Justin S. Antony et al., https://doi.org/10.1002/biot.202300505.\u0000\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture><p></p>\u0000 </div>\u0000 </figure></p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 4","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202470042","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141514263","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Expanding the RNA polymerase biocatalyst solution space for mRNA manufacture","authors":"Edward Curry,&nbsp;Svetlana Sedelnikova,&nbsp;John Rafferty,&nbsp;Martyn Hulley,&nbsp;Melinda Pohle,&nbsp;George Muir,&nbsp;Adam Brown","doi":"10.1002/biot.202400012","DOIUrl":"https://doi.org/10.1002/biot.202400012","url":null,"abstract":"<p>All mRNA products are currently manufactured in in vitro transcription (IVT) reactions that utilize single-subunit RNA polymerase (RNAP) biocatalysts. Although it is known that discrete polymerases exhibit highly variable bioproduction phenotypes, including different relative processivity rates and impurity generation profiles, only a handful of enzymes are generally available for mRNA biosynthesis. This limited RNAP toolbox restricts strategies to design and troubleshoot new mRNA manufacturing processes, which is particularly undesirable given the continuing diversification of mRNA product lines toward larger and more complex molecules. Herein, we describe development of a high-throughput RNAP screening platform, comprising complementary in silico and in vitro testing modules, that enables functional characterization of large enzyme libraries. Utilizing this system, we identified eight novel sequence-diverse RNAPs, with associated active cognate promoters, and subsequently validated their performance as recombinant enzymes in IVT-based mRNA production processes. By increasing the number of available characterized functional RNAPs by more than 130% and providing a platform to rapidly identify further potentially useful enzymes, this work significantly expands the RNAP biocatalyst solution space for mRNA manufacture, thereby enhancing the capability for application-specific and molecule-specific optimization of both product yield and quality.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 6","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400012","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141439617","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Advances in serum-free media for CHO cells: From traditional serum substitutes to microbial-derived substances","authors":"Mingcan Zhang,&nbsp;Xinyu Zhao,&nbsp;Ying Li,&nbsp;Qinghua Ye,&nbsp;Yuwei Wu,&nbsp;Qinya Niu,&nbsp;Ying Zhang,&nbsp;Guanghan Fan,&nbsp;Tianxiang Chen,&nbsp;Jiarui Xia,&nbsp;Qingping Wu","doi":"10.1002/biot.202400251","DOIUrl":"https://doi.org/10.1002/biot.202400251","url":null,"abstract":"<p>The Chinese hamster ovary (CHO) cell is an epithelial-like cell that produces proteins with post-translational modifications similar to human glycosylation. It is widely used in the production of recombinant therapeutic proteins and monoclonal antibodies. Culturing CHO cells typically requires the addition of a certain proportion of fetal bovine serum (FBS) to maintain cell proliferation and passaging. However, serum is characterized by its complex composition, batch-to-batch variability, high cost, and potential risk of exogenous contaminants such as mycoplasma and viruses, which impact the purity and safety of the synthesized proteins. Therefore, search for serum alternatives and development of serum-free media for CHO-based protein biomanufacturing are of great significance. This review systematically summarizes the application advantages of CHO cells and strategies for high-density expression. It highlights the developmental trends of serum substitutes from human platelet lysates to animal-free extracts and microbial-derived substances and elucidates the mechanisms by which these substitutes enhance CHO cell culture performance and recombinant protein production, aiming to provide theoretical guidance for exploring novel serum alternatives and developing serum-free media for CHO cells.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 6","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141439664","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}