Engineering in Life Sciences最新文献

{"title":"Online deployment of an O-PLS model for dielectric spectroscopy-based inline monitoring of viable cell concentrations in Chinese hamster ovary cell perfusion cultivations","authors":"Johannes Lemke,&nbsp;Robert Söldner,&nbsp;Jonas Austerjost","doi":"10.1002/elsc.202200053","DOIUrl":"10.1002/elsc.202200053","url":null,"abstract":"<p>Viable cell concentration (VCC) is an essential parameter that is required to support the efficient cultivation of mammalian cells. Although commonly determined using at-line or off-line analytics, in-line capacitance measurements represent a suitable alternative method for the determination of VCC. In addition, these latter efforts are complimentary with the Food and Drug Administration's initiative for process analytical technologies (PATs). However, current applications for online determination of the VCC often rely on single frequency measurements and corresponding linear regression models. It has been reported that this may be insufficient for application at all stages of a mammalian cell culture processes due to changes in multiple cell parameters over time. Alternatively, dielectric spectroscopy, measuring capacitance at multiple frequencies, in combination with multivariate mathematical models, has proven to be more robust. However, this has only been applied for retrospective data analysis. Here, we present the implementation of an O-PLS model for the online processing of multifrequency capacitance signals and the on-the-fly integration of the models’ VCC results into a supervisory control and data acquisition (SCADA) system commonly used for cultivation observation and control. This system was evaluated using a Chinese hamster ovary (CHO) cell perfusion process.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"23 6","pages":""},"PeriodicalIF":2.7,"publicationDate":"2023-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.202200053","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9956079","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Salting-out extraction of recombinant κ-carrageenase and phage T7 released from Escherichia coli cells","authors":"Da Chen,&nbsp;Yue-Sheng Dong,&nbsp;Yong-Ming Bao,&nbsp;Zhi-Long Xiu","doi":"10.1002/elsc.202200125","DOIUrl":"https://doi.org/10.1002/elsc.202200125","url":null,"abstract":"Traditional technology of cell disruption has become one of the bottlenecks restricting the industrialization of genetic engineering products due to its high cost and low efficiency. In this study, a novel bioprocess of phage lysis coupled with salting‐out extraction (SOE) was evaluated. The lysis effect of T7 phage on genetically engineered Escherichia coli expressing κ‐carrageenase was investigated at different multiplicity of infection (MOI), meanwhile the phage and enzyme released into the lysate were separated by SOE. It was found that T7 phage could lyse 99.9% of host cells at MOI = 1 and release more than 90.0% of enzyme within 90 min. After phage lysis, 87.1% of T7 phage and 71.2% of κ‐carrageenase could be distributed at the middle phase and the bottom phase, respectively, in the SOE system composed of 16% ammonium sulfate and 20% ethyl acetate (w/w). Furthermore, κ‐carrageenase in the bottom phase could be salted out by ammonium sulfate with a yield of 40.1%. Phage lysis exhibits some advantages, such as mild operation conditions and low cost. While SOE can efficiently separate phage and intracellular products. Therefore, phage lysis coupled with SOE is expected to become a viable alternative to the classical cell disruption and intracellular product recovery.","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"23 6","pages":""},"PeriodicalIF":2.7,"publicationDate":"2023-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.202200125","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50136094","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluating microbial contaminations of alternative heating oils","authors":"Maximilian J. Surger,&nbsp;Katharina Mayer,&nbsp;Karthik Shivaram,&nbsp;Felix Stibany,&nbsp;Wilfried Plum,&nbsp;Andreas Schäffer,&nbsp;Simon Eiden,&nbsp;Lars M. Blank","doi":"10.1002/elsc.202300010","DOIUrl":"10.1002/elsc.202300010","url":null,"abstract":"<p>Since 2008, European and German legislative initiatives for climate protection and reduced dependency on fossil resources led to the introduction of biofuels as CO₂-reduced alternatives in the heating oil sector. In the case of biodiesel, customers were confronted with accelerated microbial contaminations during storage. Since then, other fuel alternatives, like hydrogenated vegetable oils (HVOs), gas-to-liquid (GtL) products, or oxymethylene ether (OME) have been developed. In this study, we use online monitoring of microbial CO₂ production and the simulation of onset of microbial contamination to investigate the contamination potential of fuel alternatives during storage. As references, fossil heating oil of German refineries are used. Biodiesel blends with fossil heating oils confirmed the promotion of microbial activity. In stark contrast, OMEs have an antimicrobial effect. The paraffinic Fischer–Tropsch products and biogenic hydrogenation products demonstrate to be at least as resistant to microbial contamination as fossil heating oils despite allowing a diversity of representative microbes. Through mass spectrometry, elemental analysis, and microbial sequencing, we can discuss fuel properties that affect microbial contaminations. In summary, novel, non-fossil heating oils show clear differences in microbial resistance during long-term storage. Designing blends with an intrinsic resistance against microbial contamination and hence reduced activity might be an option.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"23 6","pages":""},"PeriodicalIF":2.7,"publicationDate":"2023-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.202300010","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9956076","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cover Picture: Engineering in Life Sciences 5'23","authors":"","doi":"10.1002/elsc.202370051","DOIUrl":"https://doi.org/10.1002/elsc.202370051","url":null,"abstract":"","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"23 5","pages":""},"PeriodicalIF":2.7,"publicationDate":"2023-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.202370051","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50121172","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Design of cell expansion processes for adherent-growing cells with mDoE-workflow","authors":"Kim B. Kuchemüller,&nbsp;Ralf Pörtner,&nbsp;Johannes Möller","doi":"10.1002/elsc.202200059","DOIUrl":"10.1002/elsc.202200059","url":null,"abstract":"<p>Adherent cells, mammalian or human, are ubiquitous for production of viral vaccines, in gene therapy and in immuno-oncology. The development of a cell-expansion process with adherent cells is challenging as scale-up requires the expansion of the cell culture surface. Microcarrier (MC)-based cultures are still predominate. However, the development of MC processes from scratch possesses particular challenges due to their complexity. A novel approach for the reduction of development times and costs of cell propagation processes is the combination of mathematical process models with statistical optimization methods, called model-assisted Design of Experiments (mDoE). In this study, an mDoE workflow was evaluated successfully for the design of a MC-based expansion process of adherent L929 cells at a very early stage of development with limited prior knowledge. At the start, the analytical methods and the screening of appropriate MCs were evaluated. Then, cause-effect relationships (e.g., cell growth related to medium conditions) were worked out, and a mathematical process model was set-up and adapted to experimental data for modeling purposes. The model was subsequently used in mDoE to identify optimized process conditions, which were proven experimentally. An eight-fold increase in cell yield was achieved basically by reducing the initial MC concentration.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"23 5","pages":""},"PeriodicalIF":2.7,"publicationDate":"2023-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.202200059","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9424064","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Application of Artificial Neural Network for predicting biomass growth during domestic wastewater treatment through a biological process","authors":"Mpho Muloiwa,&nbsp;Megersa Dinka,&nbsp;Stephen Nyende-Byakika","doi":"10.1002/elsc.202200058","DOIUrl":"10.1002/elsc.202200058","url":null,"abstract":"<p>The biological treatment process is responsible for removing organic and inorganic matter in wastewater. This process relies heavily on microorganisms to successfully remove organic and inorganic matter. The aim of the study was to model biomass growth in the biological treatment process. Multilayer perceptron (MLP) Artificial Neural Network (ANN) algorithm was used to model biomass growth. Three metrics: coefficient of determination (<i>R</i>²), root mean squared error (RMSE), and mean squared error (MSE) were used to evaluate the performance of the model. Sensitivity analysis was applied to confirm variables that have a strong influence on biomass growth. The results of the study showed that MLP ANN algorithm was able to model biomass growth successfully. <i>R</i>² values were 0.844, 0.853, and 0.823 during training, validation, and testing phases, respectively. RMSE values were 0.7476, 1.1641, and 0.7798 during training, validation, and testing phases respectively. MSE values were 0.5589, 1.3551, and 0.6081 during training, validation, and testing phases, respectively. Sensitivity analysis results showed that temperature (47.2%) and dissolved oxygen (DO) concentration (40.2%) were the biggest drivers of biomass growth. Aeration period (4.3%), chemical oxygen demand (COD) concentration (3.2%), and oxygen uptake rate (OUR) (5.1%) contributed minimally. The biomass growth model can be applied at different wastewater treatment plants by different plant managers/operators in order to achieve optimum biomass growth. The optimum biomass growth will improve the removal of organic and inorganic matter in the biological treatment process.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"23 5","pages":""},"PeriodicalIF":2.7,"publicationDate":"2023-04-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.202200058","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9424062","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cover Picture: Engineering in Life Sciences 4'23","authors":"","doi":"10.1002/elsc.202370041","DOIUrl":"https://doi.org/10.1002/elsc.202370041","url":null,"abstract":"","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"23 4","pages":""},"PeriodicalIF":2.7,"publicationDate":"2023-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.202370041","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50130783","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Implementation of ubiquitous chromatin opening elements as artificial integration sites for CRISPR/Cas9-mediated knock-in in mammalian cells","authors":"Seul Mi Kim,&nbsp;Jaejin Lee,&nbsp;Jae Seong Lee","doi":"10.1002/elsc.202200047","DOIUrl":"10.1002/elsc.202200047","url":null,"abstract":"<p>CRISPR/Cas9-mediated targeted gene integration (TI) has been used to generate recombinant mammalian cell lines with predictable transgene expression. Identifying genomic hot spots that render high and stable transgene expression and knock-in (KI) efficiency is critical for fully implementing TI-mediated cell line development (CLD); however, such identification is cumbersome. In this study, we developed an artificial KI construct that can be used as a hot spot at different genomic loci. The ubiquitous chromatin opening element (UCOE) was employed because of its ability to open chromatin and enable stable and site-independent transgene expression. UCOE KI cassettes were randomly integrated into CHO-K1 and HEK293T cells, followed by TI of enhanced green fluorescent protein (EGFP) onto the artificial UCOE KI site. The CHO-K1 random pool harboring 5′2.2A2UCOE-CMV displayed a significant increase in EGFP expression level and KI efficiency compared with that of the control without UCOE. In addition, 5′2.2A2UCOE-CMV showed improved Cas9 accessibility in the HEK293T genome, leading to an increase in indel frequency and homology-independent KI. Overall, this assessment revealed the potential of UCOE KI constructs as artificial integration sites in streamlining the screening of high-production targeted integrants by mitigating the selection of genomic hot spots.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"23 4","pages":""},"PeriodicalIF":2.7,"publicationDate":"2023-03-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.202200047","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9270239","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Site-directed mutagenesis improves the practical application of L-glutamic acid decarboxylase in Escherichia coli","authors":"Liu Fengmin,&nbsp;Zhang Heng,&nbsp;Zhang Xiangjun,&nbsp;Wei Xiaobo,&nbsp;Liu Huiyan,&nbsp;Fang Haitian","doi":"10.1002/elsc.202200064","DOIUrl":"https://doi.org/10.1002/elsc.202200064","url":null,"abstract":"<p>γ-Aminobutyric acid (GABA) is a kind of non-proteinogenic amino acid which is highly soluble in water and widely used in the food and pharmaceutical industries. Enzymatic conversion is an efficient method to produce GABA, whereby glutamic acid decarboxylase (GAD) is the key enzyme that catalyzes the process. The activity of wild-type GAD is usually limited by temperature, pH or biotin concentration, and hence directional modification is applied to improve its catalytic properties and practical application. GABA was produced using whole cell transformation of the recombinant strains <i>Escherichia coli</i> BL21(DE3)-Gad B, <i>E. coli</i> BL21(DE3)-Gad B-T62S and <i>E. coli</i> BL21(DE3)-Gad B-Q309A. The corresponding GABA concentrations in the fermentation broth were 219.09, 238.42, and 276.66 g/L, and the transformation rates were 78.02%, 85.04%, and 98.58%, respectively. The results showed that Gad B-T62S and Gad B-Q309A are two effective mutation sites. These findings may contribute to ideas for constructing potent recombinant strains for GABA production.</p><p><b><i>Practical Application</i></b>: Enzymatic properties of the GAD from <i>Escherichia coli</i> and GAD site-specific mutants were examined by analyzing their conserved sequences, substrate contacts, contact between GAD amino acid residues and mutation energy (ΔΔG) of the GAD mutants. The enzyme activity and stability of Gad B-T62S and Gad B-Q309A mutants were improved compared to Gad B. The kinetic parameters K_m and V_max of Gad B, Gad B-T62S, and Gad B-Q309A mutants were 11.3 ± 2.1 mM and 32.1 ± 2.4 U/mg, 7.3 ± 2.5 mM and 76.1 ± 3.1 U/mg, and 7.2 ± 3.8 mM and 87.3 ± 1.1 U/mg, respectively. GABA was produced using whole cell transformation of the recombinant strains <i>E. coli</i> BL21(DE3)-Gad B, <i>E. coli</i> BL21(DE3)-Gad B-T62S, and <i>E. coli</i> BL21(DE3)-Gad B-Q309A. The corresponding GABA concentrations in the fermentation broth were 219.09, 238.42, and 276.66 g/L, and the transformation rates were 78.02%, 85.04%, and 98.58%, respectively.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"23 4","pages":""},"PeriodicalIF":2.7,"publicationDate":"2023-03-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.202200064","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50126456","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cover Picture: Engineering in Life Sciences 3'23","authors":"","doi":"10.1002/elsc.202370031","DOIUrl":"https://doi.org/10.1002/elsc.202370031","url":null,"abstract":"","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"23 3","pages":""},"PeriodicalIF":2.7,"publicationDate":"2023-03-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.202370031","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50118147","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}