Review
Biotechnology & Applied Microbiology
Haoran Yu, Shuang Ma, Yiwen Li, Paul A. Dalby
Summary: Directed evolution is a powerful strategy to engineer protein properties, and hot spot prediction is crucial for producing smart libraries. Selection of hot spots based on sequence and structure allows for efficient generation of proteins with desired properties.
BIOTECHNOLOGY ADVANCES
(2022)
Article
Acoustics
Yunliang Li, Siyu Ruan, Feng Lu, Pengfei Xie, Xiaoshuang Liu, Haile Ma
Summary: Low-lethality ultrasound technology is receiving increasing attention in regulating microorganisms in the fermentation industry. In this study, two representative Ames tester strains TA97a and TA98 were used to investigate the effects of ultrasound on insertion-deletion (InDel) polymorphisms of microbial DNA and its underlying mechanisms. The results showed an increase in reversion mutation of TA98 under sonication. Sequencing of 1752 TA98 revertants revealed a total of 127 InDels, with 36 more InDels unique to ultrasound compared to the control. Ultrasound-mediated InDels of DNA exhibited additional 29 bp deletions and +7-+43 bp insertions of direct repeat sequences.
ULTRASONICS SONOCHEMISTRY
(2023)
Article
Chemistry, Physical
Yutaka Saito, Misaki Oikawa, Takumi Sato, Hikaru Nakazawa, Tomoyuki Ito, Tomoshi Kameda, Koji Tsuda, Mitsuo Umetsu
Summary: The study shows that machine learning is a useful tool in designing proteins with desired functions in protein engineering. Depending on the presence or absence of highly positive variants in the training data, machine learning-guided directed evolution can lead to improved variants in different regions of sequence space.
Review
Chemistry, Organic
Houchao Xu, Jeroen S. Dickschat
Summary: In the past three decades, numerous terpene synthases have been characterized from various life kingdoms. The mechanisms of terpene synthases have been investigated through site-directed mutagenesis, providing valuable insights into their catalysis and resulting in mutants with improved yields.
SYNTHESIS-STUTTGART
(2022)
Review
Biochemistry & Molecular Biology
Serena Gargiulo, Patrice Soumillion
Summary: Biocatalysis requires the continuous development of enzymes with tailored activity, selectivity, stability, or tolerance, achieved through directed evolution. Key steps include designing "smarter" libraries, library assembly and expression, and selecting different screening strategies based on the final objective.
CURRENT OPINION IN CHEMICAL BIOLOGY
(2021)
Article
Multidisciplinary Sciences
Femila Manoj, Laura W. Tai, Katelyn Sun Mi Wang, Thomas E. Kuhlman
Summary: The GENEWRITE system combines the site-specific targetable activity of Cas endonucleases with the reverse transcriptase activity of the ORF2p protein, enabling the insertion of large genetic constructs at precise locations. Despite off-target effects, successful insertion of large genetic payloads has been achieved in E. coli through proper payload design and co-expression of helper proteins.
SCIENTIFIC REPORTS
(2021)
Letter
Evolutionary Biology
Ward C. Wheeler
Summary: The treatment of inapplicable characters has been a challenging problem for systematists. Researchers have tried different coding scenarios to capture the presence and absence of a feature, as well as its variation. The approach of treating presence/absence as insertion/deletion of a character removes three problems in a simple and straightforward manner.
Article
Computer Science, Information Systems
Yubo Sun, Yuanxiao Xi, Gennian Ge
Summary: Motivated by applications in modern storage devices, this paper studies the sequence reconstruction problem by determining the maximum intersection size between two error balls and designing reconstruction codes. It focuses on channels with single-burst-insertion/deletion errors and fully determines the maximum intersection size for both fixed-length and variable-length models. Characterizations of sequence pairs with a certain intersection size are used to design reconstruction codes and analyze their lower bounds. The results are extended to the burst-edit channel.
IEEE TRANSACTIONS ON INFORMATION THEORY
(2023)
Article
Agriculture, Multidisciplinary
Yangyang Li, Cen Li, Hao Huang, Shengqi Rao, Quan Zhang, Jingwen Zhou, Jianghua Li, Guocheng Du, Song Liu
Summary: This study enhanced the thermostability of an acid-resistant GH11 xylanase from Aspergillus niger AG11 through systematic modification, resulting in the variant xynAm1 with increased half-life and specific activity. XynAm1 exhibited the highest thermostability among reported xylanases and could be a robust candidate for various industrial applications.
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
(2022)
Article
Biochemistry & Molecular Biology
Michal Gala, Peter Pristas, Gabriel Zoldak
Summary: This paper examined the impact of opening and formation of subdomain interfaces during the evolution of Heat shock proteins 70 (Hsp70). It found that indel events, such as insertions and deletions, in specific regions disrupt the mechanical balance of the protein and cause dysfunction. The study highlights the importance of considering the balance between structural elements in the rational design of functional allosteric machines.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2022)
Article
Computer Science, Hardware & Architecture
Yi-Han Lien, Yi-Hua Chen, Po-Chun Huang
Summary: The file model based on character streams is widely used in file systems today but lacks efficient data insertion and deletion. This study proposes data compaction strategies and a chunk anonymization scheme to enhance the performance and space utilization of file systems.
IEEE TRANSACTIONS ON COMPUTERS
(2022)
Article
Clinical Neurology
Cindy W. Yoon, Jonguk Kim, Young Ju Suh, Byeong C. Kim, Young Chul Youn, Jee Hyang Jeong, Hyun Jeong Han, Seong Hye Choi
Summary: This study examined the relationship between the ACE insertion/deletion polymorphism and changes in cerebral microbleeds (CMBs) over a 2-year period. The results indicated that individuals with the ACE DD genotype experienced a greater progression of CMBs compared to those with the combined II/ID genotypes, particularly in lobar CMBs. Further studies with larger sample sizes are necessary to confirm this association.
FRONTIERS IN NEUROLOGY
(2023)
Article
Microbiology
Maria Masoura, Mathew T. Milner, Tim W. Overton, Konstantinos Gkatzionis, Peter A. Lund
Summary: This study used high-throughput techniques to investigate the antibacterial mechanism of honey, identifying genes related to fitness loss when cells were exposed to honey, as well as genes that confer enhanced resistance to honey treatment when mutated.
FRONTIERS IN MICROBIOLOGY
(2022)
Review
Chemistry, Applied
Manfred T. Reetz
Summary: The concept of directed evolution of stereoselective enzymes was born and implemented at the Max-Planck-Institut fur Kohlenforschung in Mulheim, Germany. Andreas Pfaltz witnessed the birth and development of this field during his time at the institute.
ADVANCED SYNTHESIS & CATALYSIS
(2022)
Article
Virology
Lili Chu, Shengqiang Ye, Jiaying Wang, Duo Peng, Xiaoyan Wang, Yunguo Qian, Dabing Zhang
Summary: Duck adenovirus 3 (DuAdV-3; strain HB) isolated in this study is a mutant with several insertion and deletion events in its genome, demonstrating genetic variation among DuAdV-3 isolates.
ARCHIVES OF VIROLOGY
(2022)
Editorial Material
Biochemistry & Molecular Biology
Maximilian Gantz, Godwin A. Aleku, Florian Hollfelder
TRENDS IN BIOCHEMICAL SCIENCES
(2022)
Article
Multidisciplinary Sciences
Remkes A. Scheele, Laurens H. Lindenburg, Maya Petek, Markus Schober, Kevin N. Dalby, Florian Hollfelder
Summary: In this study, a droplet-based screen was used to test variants of the human protein kinase MKK1 for its ability to activate its downstream target ERK2. The results reveal a flexible motif in the MKK1 docking domain that promotes efficient activation of ERK2, suggesting synergy between the residues within that sequence. The combination of ultrahigh-throughput screening and sequencing provides insights into the function and intragenic epistasis within protein mutant libraries, and sheds light on the importance of co-dependent residues in mammalian protein kinase-substrate interactions and the evolution of diverse human signaling networks.
NATURE COMMUNICATIONS
(2022)
Article
Chemistry, Multidisciplinary
Godwin A. Aleku, Gabriel R. Titchiner, George W. Roberts, Sasha R. Derrington, James R. Marshall, Florian Hollfelder, Nicholas J. Turner, David Leys
Summary: Allylic amines are versatile precursors for the synthesis of valuable nitrogen-containing organic compounds. We have developed a biocatalytic system that utilizes renewable cinnamic acids to achieve reductive allylation of primary and secondary amines. This system offers high selectivity and conversion rates, while avoiding side reactions.
ACS SUSTAINABLE CHEMISTRY & ENGINEERING
(2022)
Article
Biochemistry & Molecular Biology
Charlotte M. Miton, Nobuhiko Tokuriki
Summary: Protein engineers have studied and borrowed from nature's tricks to accelerate protein evolution. However, our ability to generate new proteins in the laboratory seems limited. This Perspective explores the role of insertions and deletions (indels) in the functional diversification of proteins in nature and discusses their untapped potential for protein engineering.
Article
Developmental Biology
Clara Munger, Timo N. Kohler, Erin Slatery, Anna L. Ellermann, Sophie Bergmann, Christopher A. Penfold, Ioakeim Ampartzidis, Yutong Chen, Florian Hollfelder, Thorsten E. Boroviak
Summary: The early specification and rapid growth of extraembryonic membranes are distinctive characteristics of primate embryogenesis. This study investigates the signals involved in the formation of embryonic and extraembryonic lineages in primates by using microgel culture, single-cell profiling and spatial identity mapping. The findings reveal the crucial roles of activin/nodal signaling in embryonic lineage identity and BMP4 in amnion formation and maturation, counteracted by FGF signaling.
Article
Chemistry, Multidisciplinary
Thomas Fryer, Joel David Rogers, Christopher Mellor, Timo N. Kohler, Ralph Minter, Florian Hollfelder
Summary: The assembly of robust, modular biological components into complex functional systems is crucial in synthetic biology. This study presents a solid-phase protein display system that utilizes modular plug and play design principles. The system enables protein purification, functional assays, and controlled release of protein modules. The use of microfluidic droplet generators and polyacrylamide hydrogel display beads allows for high-density protein binding and efficient valency engineering.
ACS CENTRAL SCIENCE
(2022)
Article
Cell Biology
Min Bao, Jake Cornwall-Scoones, Estefania Sanchez-Vasquez, Dong-Yuan Chen, Joachim De Jonghe, Shahriar Shadkhoo, Florian Hollfelder, Matt Thomson, David M. Glover, Magdalena Zernicka-Goetz
Summary: This study reveals that a cadherin code in stem cells regulates the assembly and sorting of synthetic embryos. By manipulating the expression of cadherin codes in different stem cell lines, the efficiency of correctly forming synthetic embryos was tripled.
NATURE CELL BIOLOGY
(2022)
Article
Chemistry, Multidisciplinary
Morito Sakuma, Shingo Honda, Hiroshi Ueno, Kazuhito V. Tabata, Kentaro Miyazaki, Nobuhiko Tokuriki, Hiroyuki Noji
Summary: Enzymes display heterogeneity in their conformational and functional states, which is crucial for the evolution of new functions. A single-molecule enzyme assay was used to analyze functional substates in wild-type and mutant alkaline phosphatase. Mutant enzymes exhibited significantly heterogeneous functional substates, while the wild-type enzyme showed a highly homogeneous substate. The degree of functional substates correlated with the improvement in promiscuous activities. The findings highlight the importance of functional substates in enzyme evolution.
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
(2023)
Article
Chemistry, Analytical
Elliot J. Medcalf, Maximilian Gantz, Tomasz S. Kaminski, Florian Hollfelder
Summary: Droplet microfluidics is valuable for high throughput screening, but absorbance-activated droplet sorting is slower than fluorescence-activated droplet sorting. In this study, the sorting speed of absorbance-activated droplet sorting is increased through the use of refractive index matching oil, a sorting algorithm, and a chip design, achieving better sensitivity and matching the speed of fluorescence-activated sorting devices.
ANALYTICAL CHEMISTRY
(2023)
Review
Chemistry, Multidisciplinary
Maximilian Gantz, Stefanie Neun, Elliot J. Medcalf, Liisa D. van Vliet, Florian Hollfelder
Summary: Novel and improved biocatalysts are increasingly derived from libraries through experimental screening, and the success of such campaigns hinges on the number of candidates tested. Water-in-oil emulsion droplets can serve as an alternative screening format, offering in vitro compartments containing genotype and phenotype, and enabling functional readout. Droplets made in automated microfluidic devices can be integrated into modular workflows to set up multistep screening protocols, allowing for the sorting of a large number of variants in a high-throughput manner. The adaptability of droplet screening for enzyme discovery and detailed kinetic characterization is also discussed.
Correction
Cell Biology
Min Bao, Jake Cornwall-Scoones, Estefania Sanchez-Vasquez, Andy L. Cox, Dong-Yuan Chen, Joachim De Jonghe, Shahriar Shadkhoo, Florian Hollfelder, Matt Thomson, David M. Glover, Magdalena Zernicka-Goetz
NATURE CELL BIOLOGY
(2023)
Article
Ecology
Brennen Heames, Filip Buchel, Margaux Aubel, Vyacheslav Tretyachenko, Dmitry Loginov, Petr Novak, Andreas Lange, Erich Bornberg-Bauer, Klara Hlouchova
Summary: A comparison between de novo genes and synthetic random sequences reveals no difference in biophysical properties, but in vitro expression demonstrates de novo proteins' higher solubility, indicating their better integration into the cellular system. The emergence of de novo genes provides a pathway for the formation of new proteins from previously non-coding DNA. While the likelihood of assuming a soluble and stable tertiary structure in de novo proteins remains unclear, evidence suggests that native-like biophysical properties are abundant in sequence space. Experimental characterization of putative de novo proteins in human and fly shows that their solubility and structure propensity are moderately higher compared to synthetic random proteins. The DnaK chaperone system further enhances the solubility of de novo proteins. Therefore, de novo proteins may be better integrated into the cellular system than random sequences due to their higher solubility.
NATURE ECOLOGY & EVOLUTION
(2023)
Article
Chemistry, Physical
Simon Ladeveze, Paul J. Zurek, Tomasz S. Kaminski, Stephane Emond, Florian Hollfelder
Summary: Enzyme discovery and directed evolution are two contemporary approaches for improving biocatalysis in various fields. Screening campaigns for evolving enzymes rely on the sampled fraction of sequence space, while ultrahigh-throughput screening (uHTS) based on microfluidic systems allows for screening rates >1 kHz. However, screening for carbohydrate-active enzymes (CAZymes) that catalyze valuable reactions is challenging due to difficulties in monitoring released carbohydrates. To overcome these issues, microdroplet assays were designed to make optically inactive carbohydrate products visible by specific cascades. Implementing these assays at the picoliter droplet scale enabled the detection of specific complex carbohydrate degradation products. This advancement in screening technology will facilitate the development of specific and efficient biocatalysts for biovalorization.
Article
Chemistry, Multidisciplinary
Morito Sakuma, Shingo Honda, Hiroshi Ueno, Kazuhito V. Tabata, Kentaro Miyazaki, Nobuhiko Tokuriki, Hiroyuki Noji
Summary: Enzymes have inherent heterogeneity in their conformational and functional states, which is key to the evolution of new functions. Single-molecule enzyme assays allow direct observation of these multiple functional substates. We analyzed functional substates in wild-type and mutant Escherichia coli alkaline phosphatase and found that mutant enzymes exhibited significantly heterogeneous functional substates, while the wild-type enzyme showed a highly homogeneous substate. Our study provides comprehensive evidence that functional substates can be easily altered by mutations and that the evolution towards new catalytic activities may involve modulation of these substates.
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
(2023)