Article
Biotechnology & Applied Microbiology
Hongguan Xing, Pingping Wang, Xing Yan, Yi Yang, Xinliang Li, Rui Liu, Zhihua Zhou
Summary: Directed evolution was used to enhance the thermostability of phytase, resulting in the generation of five mutants with higher residual activities and catalytic efficiencies. The substitution of C75 residue was identified as the main reason for the improved thermostability, indicating the crucial role of this amino acid in the stability and catalytic efficiency of phytase.
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
(2023)
Article
Biotechnology & Applied Microbiology
Mengmeng Liu, Qiu Li, Xiaoxiao Liu, Pengpai Zhang, Haiyan Zhang
Summary: Through two rounds of random mutagenesis PCR, an improved starch debranching enzyme with higher catalytic activity and thermostability was screened from Bacillus thermoliquefaciens. The mutant enzyme G250P/T252S/G253T/N255K showed a 1.9-fold increase in specific activity, 22.7% decrease in Km, 28.7% increase in kcat, and 68.4% increase in kcat/Km compared to the parent enzyme. The thermostability of the mutant enzyme was significantly enhanced, with a half-life extended to 7.5 hours at 60 degrees C, which was 87.5% higher than that of the parent enzyme. The mutation sites were concentrated in the 250-255 region, indicating its importance in affecting catalytic activity and thermostability. The reasons for the change in enzymatic properties were preliminarily analyzed through three-dimensional simulation.
ENZYME AND MICROBIAL TECHNOLOGY
(2023)
Article
Biotechnology & Applied Microbiology
Saeed Ebrahimi Fana, Aliakbar Fazaeli, Mahdi Aminian
Summary: An improved variant of cholesterol oxidase with enhanced thermostability was obtained through error-prone PCR. This mutant showed 30% increased thermostability at 50 degrees C for 5 h compared to the wild type, without changing the optimum temperature and pH. Circular dichroism analysis revealed no significant structural changes in the mutant enzymes. These findings indicate that error-prone PCR is an effective method for enhancing enzyme characteristics and provide a platform for the practical use of cholesterol oxidase in industrial and clinical settings.
BIOTECHNOLOGY LETTERS
(2023)
Article
Biotechnology & Applied Microbiology
Yan-Ni Xing, Jie Tan, Yonghua Wang, Jiaqi Wang
Summary: A lipase from Malassizia globose, named SMG1, was mutated to improve thermostability, with mutations on the protein surface resulting in significant enhancements. Specific mutations, such as D245N and L270P, showed the most significant increase in T50, with the double mutant exhibiting a 5-degree Celsius increase. The double mutant displayed a 100-fold increase in half-life at 45 degrees Celsius and higher enzymatic activities at temperatures above 30 degrees Celsius compared to the wild-type.
ENZYME AND MICROBIAL TECHNOLOGY
(2021)
Article
Biotechnology & Applied Microbiology
Ru Jia, Senmiao Tian, Zhaofeng Yang, Faizan Ahmed Sadiq, Lan Wang, Simeng Lu, Guohua Zhang, Jianhui Li
Summary: An improved variant of recombinant aflatoxin-degrading enzyme (rMADE) with enhanced thermostability and catalytic activity was obtained using error-prone PCR. A mutant library containing over 5000 individual mutants was constructed, and three mutants with higher T-50 values compared to the wild-type rMADE were screened. The catalytic activity of two mutants, rMADE-1795 and rMADE-2848, was significantly improved compared to the wild-type.
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
(2023)
Article
Biochemical Research Methods
Sea On Lee, Stephen D. Fried
Summary: An improved error-prone PCR protocol using a Touchdown approach and commercially available components is reported, which can efficiently concentrate mutations into a small region of plasmid DNA and achieve very high mutational loads on a standard-sized amplicon.
ANALYTICAL BIOCHEMISTRY
(2021)
Article
Biochemistry & Molecular Biology
Gheorghita Menghiu, Vasile Ostafe, Radivoje Prodanovic, Rainer Fischer, Raluca Ostafe
Summary: Chitinases catalyze the degradation of chitin, a polymer found in various organisms. Developing improved chitinases is important for addressing environmental concerns and potential applications of chitosan products. This study focused on creating a high-throughput screening system and selecting enhanced chitinase variants by utilizing fluorescence-activated cell sorting and mutagenesis techniques.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2021)
Article
Chemistry, Applied
Yingqi Ruan, Rongzhen Zhang, Yan Xu
Summary: Mutated maltogenic amylase from Bacillus licheniformis R-53 showed higher activity and thermostability. Mutant V296F/K418I had 2.16 times the specific activity of the wild-type enzyme and its optimum activity temperature increased to 65°C. Additionally, the mutant enzyme was more effective in improving bread texture and sensory properties during bread storage.
Article
Pharmacology & Pharmacy
Christin Pohl, Sujata Mahapatra, Alina Kulakova, Werner Streicher, Gunther H. J. Peters, Allan Norgaard, Pernille Harris
Summary: This study applies small angle X-ray scattering (SAXS), which is not typically used in industrial protein screening, in a high throughput screening workflow to address problems of contradicting results and reproducibility among different methods. The results demonstrate that SAXS can provide valuable information on interparticle interactions, even without time-consuming modeling, and can be used as a tool for in-depth knowledge highly useful for protein formulation development.
EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS
(2022)
Article
Medicine, Research & Experimental
Rit Pratik Mishra, Surbhi Gupta, Anurag Singh Rathore, Gaurav Goel
Summary: A multi-level virtual screening protocol is developed to identify lead molecules that can inhibit insulin aggregation. The protocol involves multiple steps, including screening the inactives database, docking and molecular dynamics simulations, and experimental validation. Riboflavin is identified as the most effective lead molecule for inhibiting insulin aggregation.
MOLECULAR PHARMACEUTICS
(2022)
Article
Microbiology
Zhaokun Zhang, Mingyue Ge, Qi Guo, Yi Jiang, Wendi Jia, Le Gao, Jianhua Hu
Summary: A droplet-based microfluidic ultrahigh-throughput screening technology was developed to select high-beta-xylosidase-producing Penicillium piceum W6 from a mutated library. A novel beta-D-xylosidase with bifunctional activities was purified and identified from this strain. The addition of this enzyme significantly enhanced the yields of glucose and xylose from delignified biomass.
Article
Chemistry, Analytical
Jinpeng Liu, Mashooq Khan, Yong Wei, Anna Liu, Tao Wang, Qiongzheng Hu, Li Yu
Summary: A robust and versatile sensing platform based on liquid crystal-infused porous surfaces (LCIPS) is developed for high-throughput screening of lipase inhibitors. The sliding time of droplets on LCIPS is found to be extended due to the adsorption of oleate at the solution/liquid crystal (LC) interface. However, the presence of lipase inhibitors inhibits the enzymatic hydrolysis of glycerol trioleate (GT), resulting in a shortened sliding time. The study demonstrates the screening of three popular anti-obesity drugs, orlistat, cetilistat, and epigallocatechin gallate (EGCG), with respective IC50 values of 17.45 +/- 3.15 nM, 72.15 +/- 6.32 nM, and 2.19 +/- 0.24 mu M. This research provides a simple, inexpensive, portable, user-friendly, and instrument-free method for rapid, accurate, and high-throughput screening of lipase inhibitors. The LCIPS-based sensing platform shows great potential in developing advanced bioanalytical sensors.
SENSORS AND ACTUATORS B-CHEMICAL
(2023)
Article
Biochemical Research Methods
Amani Alma'abadi, Hayedeh Behzad, Mohammed Alarawi, David Conchouso, Yoshimoto Saito, Masahito Hosokawa, Yohei Nishikawa, Masato Kogawa, Haruko Takeyama, Katsuhiko Mineta, Takashi Gojobori
Summary: The demand for novel microbial biocatalysts in industrial and pharmaceutical applications is increasing rapidly. Advanced tools and technologies are needed to explore the metabolic potential of unculturable microorganisms. Single-cell and functional metagenomics studies have contributed to the discovery of unique microbial genes. In this study, a high-throughput single-cell microfluidic platform combined with fluorescent screening was used to discover new genes encoding lipolytic enzymes from the Red Sea.
Article
Multidisciplinary Sciences
Shuyi Lv, Chao Wang, Ke Xue, Jiaxin Wang, Minghui Xiao, Zhencheng Sun, Lei Han, Linqi Shi, Chunlei Zhu
Summary: This article introduces an activated alkyne-based cascade signal amplification strategy for ultrafast and high-throughput antibiotic screening. By synthesizing a novel water-soluble aggregation-induced emission (AIE) luminogen with an activated alkyne group, fluorescence can be turned on and metal-free click bioconjugation can be achieved under physiological conditions. By utilizing the method of bacterial lysis, clickable biological substances are released, significantly increasing the quantity of analytes. The system fluorescence signal is greatly amplified through activated alkyne-mediated turn-on click bioconjugation. This cascade signal amplification strategy effectively improves the detection sensitivity and enables ultrafast antimicrobial susceptibility assessment. Integrated with a microplate reader, this approach can be applied to high-throughput antibiotic screening.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
(2023)
Article
Biotechnology & Applied Microbiology
Han Byeol Jeong, Hyung Kwoun Kim
Summary: Using error-prone PCR, the lipase activity of the LipCA lipase from Antarctic Croceibacter atlanticus was significantly increased in a mutant strain (M3-1), producing three times more enzyme than the wild-type strain. A single base change at position 73 was identified to greatly impact protein expression level, demonstrating the potential for mRNA sequence modifications to increase protein production in E. coli.
JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY
(2021)
Article
Microbiology
Macarena Larroude, Djamila Onesime, Olivier Rue, Jean-Marc Nicaud, Tristan Rossignol
Summary: In this study, the yeast Yarrowia lipolytica was engineered to overproduce pyomelanin by optimizing a chassis strain to increase homogentisic acid synthesis. The overexpression of the 4HPPD gene led to significantly higher pyomelanin yields, with the pathway being strongly induced by phenylalanine supplementation.
Article
Biochemistry & Molecular Biology
Piotr Hapeta, Patrycja Szczepanska, Tadeusz Witkowski, Jean-Marc Nicaud, Anne-Marie Crutz-Le Coq, Zbigniew Lazar
Summary: The translation explores the use of molasses as a substrate for bioprocesses, focusing on the role of hexose transporters and hexokinase in glucose preference in Yarrowia lipolytica. Overexpression of hexokinase with hexose transporters accelerated citric acid biosynthesis and enhanced pentose phosphate pathway, leading to increased production of polyols. Additionally, overexpression of fructophilic hexokinase resulted in a shorter processing time and higher polyol yield compared to the glycerol medium.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2021)
Article
Food Science & Technology
Sara Mitri, Mohamed Koubaa, Richard G. Maroun, Tristan Rossignol, Jean-Marc Nicaud, Nicolas Louka
Summary: Due to the pleasant rosy scent, there is a huge market demand for the aromatic alcohol 2-phenylethanol (2-PE). The most promising method for its production is through microbial fermentation, particularly using yeasts, which have the ability to produce 2-PE using l-Phe as precursor. Some agro-industrial waste and by-products with high nutritional value can be utilized as suitable media for microbial growth, including the production of 2-PE through yeast fermentation.
Article
Biochemistry & Molecular Biology
Sonia Abreu, Young-Kyoung Park, Camilla Pires de Souza, Lea Vidal, Pierre Chaminade, Jean-Marc Nicaud
Summary: Yarrowia lipolytica is a promising yeast for producing unusual lipids. Recent studies have shown that optimizing substrate combination and metabolic pathways can improve lipid production. In this study, the lipid composition of obese LP and obese L strains was compared, revealing differences in the fatty acid composition and molecular species distributions of various lipids.
Article
Biotechnology & Applied Microbiology
Lea Vidal, Esteban Lebrun, Young-Kyoung Park, Guillaume Mottet, Jean-Marc Nicaud
Summary: In this study, novel bidirectional and hybrid bidirectional promoters were developed, whose expression can be fine-tuned to meet the demand for versatile promoters in the yeast Y. lipolytica. This study provides effective tools for smoothly adjusting the erythritol-inducible co-expression of two target genes in biotechnology applications. BDPs developed in this study have potential applications in the fields of heterologous protein production, metabolic engineering, and synthetic biology.
MICROBIAL CELL FACTORIES
(2023)
Article
Microbiology
Jaroslav Hambalko, Peter Gajdos, Jean-Marc Nicaud, Rodrigo Ledesma-Amaro, Michal Tupec, Iva Pichova, Milan Certik
Summary: Research has found that sex pheromone mixtures of certain insect species can produce unique C16 long conjugated fatty acids with two or three conjugated double bonds. The supplementation of fatty acid methyl esters into the medium resulted in the identification of 20 new fatty acids with two or three double bonds. The study proves that Y. lipolytica is capable of synthesizing C16-conjugated fatty acids, and further optimization could lead to increased production of novel fatty acid derivatives with biotechnologically interesting properties.
Article
Biotechnology & Applied Microbiology
Maria Gorczyca, Jean-Marc Nicaud, Ewelina Celinska
Summary: Resistance to environmental stress and synthesis of recombinant proteins (r-Prots) are complex biological traits that rely on multiple genes. Modifying the operation of transcription factors (TFs) associated with these traits can be a strategy to engineer them. This study examined the implications of five selected TFs in stress resistance and r-Prot synthesis in Yarrowia lipolytica. Over-expression or deletion of the TFs in a host strain resulted in significant changes in growth and r-Prot yields under specific conditions. Mathematical modeling assisted in understanding the contribution of individual TFs and identifying new functions of the studied TFs.
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
(2023)
Article
Biochemistry & Molecular Biology
Veronika Urbanikova, Young-Kyoung Park, Daniela Krajciova, Mehdi Tachekort, Milan Certik, Ioana Grigoras, Roman Holic, Jean-Marc Nicaud, Peter Gajdos
Summary: In this study, the oleaginous yeast Yarrowia lipolytica was used as a host for the production of Punicic acid (PuA). By supplementing the medium with pomegranate seed oil, lipids containing PuA esters were accumulated up to 31.2% in Y. lipolytica. Strategies involving the expression of the bifunctional fatty acid conjugase/desaturase gene from Punica granatum (PgFADX) allowed for de novo production of PuA in Y. lipolytica. The optimized promoter for PgFADX expression resulted in a PuA production of 36.6 mg/L. These findings highlight the potential of Y. lipolytica as a promising host for PuA production.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2023)
Article
Biochemistry & Molecular Biology
Georgio Nemer, Nicolas Louka, Paul Rabiller Blandin, Richard G. Maroun, Eugene Vorobiev, Tristan Rossignol, Jean-Marc Nicaud, Erwann Guenin, Mohamed Koubaa
Summary: This study focuses on the biosynthesis of a mixture of violacein and deoxyviolacein using a genetically modified Y. lipolytica strain as a production chassis. The intracellular pigments were extracted and purified using column chromatography. The optimal separation between the pigments was achieved using an ethyl acetate/cyclohexane mixture with different ratios, followed by analysis using thin-layer chromatography and nuclear magnetic resonance.
Article
Biotechnology & Applied Microbiology
Lea Vidal, Zehui Dong, Kim Olofsson, Eva Nordberg Karlsson, Jean-Marc Nicaud
Summary: Yarrowia lipolytica is a suitable yeast for producing foreign proteins. A set of 18 chromogenic cloning vectors were developed, each containing an excisable auxotrophic selective marker and a different promoter. An improved recipient strain was developed by eliminating filamentation and introducing an auxotrophy for lysine. The optimal targeting sequence for Rhizopus oryzae ROL lipase secretion was determined and higher lipase activity was achieved with certain promoters.
FEMS YEAST RESEARCH
(2023)
Article
Biochemical Research Methods
Esteban Lebrun, Vasily Shenshin, Cecile Plaire, Vincent Vigneres, Theo Pizette, Bruno Dumas, Jean-Marc Nicaud, Guillaume Mottet
Summary: The search for new antibodies is time-consuming and expensive due to repeated library screenings. A new antibody screening platform based on the encapsulation of antibody-secreting yeast cells into picoreactor droplets is proposed in this study. This platform enables high-throughput screening of millions of antibodies per day and is useful in various contexts.
Article
Biotechnology & Applied Microbiology
Wendy Al Sahyouni, Sally El Kantar, Anissa Khelfa, Young-Kyoung Park, Jean-Marc Nicaud, Nicolas Louka, Mohamed Koubaa
Summary: This study investigated the production of odd-chain fatty acid-enriched lipids through fermentation using a genetically engineered strain. The optimal conditions for maximizing cell density and fatty acid content were determined, and the results show potential for industrial production.
FERMENTATION-BASEL
(2022)
Review
Biotechnology & Applied Microbiology
Young-Kyoung Park, Cristina Gonzalez-Fernandez, Raul Robles-Iglesias, Lea Vidal, Pierre Fontanille, Christian Kennes, Elia Tomas Pejo, Jean-Marc Nicaud, Patrick Fickers
Summary: This review discusses the processes of creating valuable molecules from organic wastes-derived VFAs, the pathways used by oleaginous yeast Yarrowia lipolytica to metabolize such molecules, and the relationship between organic waste composition, anaerobic digestion, and VFA profiles. It also summarizes the current knowledge about VFA toxicity, the metabolic pathways of VFAs, and the metabolic engineering strategies in Y. lipolytica for producing value-added biobased compounds from VFAs.
FEMS YEAST RESEARCH
(2021)
Article
Biotechnology & Applied Microbiology
Young-Kyoung Park, Florence Bordes, Fabien Letisse, Jean-Marc Nicaud
Summary: Microbial production of odd-chain fatty acids (OCFAs) has gained significant interest for its diverse applications, and this study successfully increased OCFA accumulation through strain engineering and optimizing the precursors balance. The highest recombinant OCFAs titer reported in yeast was achieved, showing the potential of microbial production in various applications.
METABOLIC ENGINEERING COMMUNICATIONS
(2021)
Review
Biotechnology & Applied Microbiology
Georgio Nemer, Nicolas Louka, Eugene Vorobiev, Dominique Salameh, Jean-Marc Nicaud, Richard G. Maroun, Mohamed Koubaa
Summary: The production of pigments using single cell microorganisms is emerging as a sustainable alternative to conventional synthesis methods heavily reliant on petrochemicals. Research is focused on producing pigments coveted for their unique qualities beyond just dyeing abilities. Efforts are also directed towards developing effective cell disruption methods to ensure successful recovery of biosynthetic pigments.
FERMENTATION-BASEL
(2021)