journal
https://read.qxmd.com/read/39028299/chloroplast-cell-free-systems-from-different-plant-species-as-a-rapid-prototyping-platform
#1
JOURNAL ARTICLE
Clemens V Böhm, René Inckemann, Michael Burgis, Jessica Baumann, Cedric K Brinkmann, Katarzyna E Lipinska, Sara Gilles, Jonas Freudigmann, Vinca N Seiler, Lauren G Clark, Michael C Jewett, Lars M Voll, Henrike Niederholtmeyer
Climate change poses a significant threat to global agriculture, necessitating innovative solutions. Plant synthetic biology, particularly chloroplast engineering, holds promise as a viable approach to this challenge. Chloroplasts present a variety of advantageous traits for genetic engineering, but the development of genetic tools and genetic part characterization in these organelles is hindered by the lengthy time scales required to generate transplastomic organisms. To address these challenges, we have established a versatile protocol for generating highly active chloroplast-based cell-free gene expression (CFE) systems derived from a diverse range of plant species, including wheat (monocot), spinach, and poplar trees (dicots)...
July 19, 2024: ACS Synthetic Biology
https://read.qxmd.com/read/39025476/bioprinting-of-synthetic-cell-like-lipid-vesicles-to-augment-the-functionality-of-tissues-after-manufacturing
#2
JOURNAL ARTICLE
Ole Thaden, Nicole Schneider, Tobias Walther, Erin Spiller, Alexandre Taoum, Kerstin Göpfrich, Daniela Duarte Campos
Bioprinting is an automated bioassembly method that enables the formation of human tissue-like constructs to restore or replace damaged tissues. Regardless of the employed bioprinting method, cells undergo mechanical stress that can impact their survival and function postprinting. In this study, we investigate the use of a synthetic cell-like unit, giant unilamellar vesicles (GUVs), as adjuvants of the cellular function of human cells postprinting, or in future as the complete replacement of human cells. We analyzed the impact of two nozzle-based bioprinting methods (drop-on-demand and extrusion bioprinting) on the structure, stability, and function of GUVs...
July 18, 2024: ACS Synthetic Biology
https://read.qxmd.com/read/39023433/establishing-a-high-yield-chloroplast-cell-free-system-for-prototyping-genetic-parts
#3
JOURNAL ARTICLE
Lauren Clark, Christopher A Voigt, Michael C Jewett
Plastid engineering offers the potential to carry multigene traits in plants; however, it requires reliable genetic parts to balance expression. The difficulty of chloroplast transformation and slow plant growth makes it challenging to build plants just to characterize genetic parts. To address these limitations, we developed a high-yield cell-free system from Nicotiana tabacum chloroplast extracts for prototyping genetic parts. Our cell-free system uses combined transcription and translation driven by T7 RNA polymerase and works with plasmid or linear template DNA...
July 18, 2024: ACS Synthetic Biology
https://read.qxmd.com/read/39023319/exploring-the-de-novo-nmn-biosynthesis-as-an-alternative-pathway-to-enhance-nmn-production
#4
JOURNAL ARTICLE
Pengju Wang, Yidan Ma, Ju Li, Junchang Su, Junxi Chi, Xingmiao Zhu, Xinna Zhu, Chunzhi Zhang, Changhao Bi, Xueli Zhang
Nicotinamide mononucleotide (NMN) serves as a precursor for NAD+ synthesis and has been shown to have positive effects on the human body. Previous research has predominantly focused on the nicotinamide phosphoribosyltransferase-mediated route (NadV-mediated route) for NMN biosynthesis. In this study, we have explored the de novo NMN biosynthesis route as an alternative pathway to enhance NMN production. Initially, we systematically engineered Escherichia coli to enhance its capacity for NMN synthesis and accumulation, resulting in a remarkable over 100-fold increase in NMN yield...
July 18, 2024: ACS Synthetic Biology
https://read.qxmd.com/read/39012160/a-genomewide-evolution-based-crispr-cas9-with-donor-free-gebcd-for-developing-robust-and-productive-industrial-yeast
#5
JOURNAL ARTICLE
Jinwei Zhang, Guomiao Zhao, Wenxin Bai, Ying Chen, Yuan Zhang, Fan Li, Manyi Wang, Yue Shen, Yun Wang, Xiaoyan Wang, Chun Li
Developing more robust and productive industrial yeast is crucial for high-efficiency biomanufacturing. However, the challenges posed by the long time required and the low abundance of mutations generated through genomewide evolutionary engineering hinder the development and optimization of desired hosts for industrial applications. To address these issues, we present a novel solution called the Genomewide Evolution-based CRISPR/Cas with Donor-free (GEbCD) system, in which nonhomologous-end-joining (NHEJ) repair can accelerate the acquisition of highly abundant yeast mutants...
July 16, 2024: ACS Synthetic Biology
https://read.qxmd.com/read/39002162/toward-practical-applications-of-engineered-living-materials-with-advanced-fabrication-techniques
#6
REVIEW
Chenjing Lu, Yaying Huang, Jian Cui, Junhua Wu, Chunping Jiang, Xiaosong Gu, Yi Cao, Sheng Yin
Engineered Living Materials (ELMs) are materials composed of or incorporating living cells as essential functional units. These materials can be created using bottom-up approaches, where engineered cells spontaneously form well-defined aggregates. Alternatively, top-down methods employ advanced materials science techniques to integrate cells with various kinds of materials, creating hybrids where cells and materials are intricately combined. ELMs blend synthetic biology with materials science, allowing for dynamic responses to environmental stimuli such as stress, pH, humidity, temperature, and light...
July 13, 2024: ACS Synthetic Biology
https://read.qxmd.com/read/38991546/degron-based-bioprotacs-for-controlling-signaling-in-car-t-cells
#7
JOURNAL ARTICLE
Matthew S Kim, Hersh K Bhargava, Gavin E Shavey, Wendell A Lim, Hana El-Samad, Andrew H Ng
Chimeric antigen receptor (CAR) T cells have made a tremendous impact in the clinic, but potent signaling through the CAR can be detrimental to treatment safety and efficacy. The use of protein degradation to control CAR signaling can address these issues in preclinical models. Existing strategies for regulating CAR stability rely on small molecules to induce systemic degradation. In contrast to small molecule regulation, genetic circuits offer a more precise method to control CAR signaling in an autonomous cell-by-cell fashion...
July 11, 2024: ACS Synthetic Biology
https://read.qxmd.com/read/38991172/shuffleanalyzer-a-comprehensive-tool-to-visualize-dna-shuffling
#8
JOURNAL ARTICLE
Franz Schweiggert, Gregor Habeck, Patrick Most, Martin Busch, Jörg Schweiggert
DNA shuffling is a powerful technique for generating synthetic DNA via recombination of homologous parental sequences. Resulting chimeras are often incorporated into complex libraries for functionality screenings that identify novel variants with improved characteristics. To survey shuffling efficiency, subsequences of chimeras can be computationally assigned to their corresponding parental counterpart, yielding insight into frequency of recombination events, diversity of shuffling libraries and actual composition of final variants...
July 11, 2024: ACS Synthetic Biology
https://read.qxmd.com/read/38986010/bioinformatic-prediction-and-high-throughput-in-vivo-screening-to-identify-cis-regulatory-elements-for-the-development-of-algal-synthetic-promoters
#9
JOURNAL ARTICLE
Y Torres-Tiji, H Sethuram, A Gupta, J McCauley, J-V Dutra-Molino, R Pathania, L Saxton, K Kang, N J Hillson, S P Mayfield
Algae biotechnology holds immense promise for revolutionizing the bioeconomy through the sustainable and scalable production of various bioproducts. However, their development has been hindered by the lack of advanced genetic tools. This study introduces a synthetic biology approach to develop such tools, focusing on the construction and testing of synthetic promoters. By analyzing conserved DNA motifs within the promoter regions of highly expressed genes across six different algal species, we identified cis-regulatory elements (CREs) associated with high transcriptional activity...
July 10, 2024: ACS Synthetic Biology
https://read.qxmd.com/read/38981096/directed-evolution-of-acoustic-reporter-genes-using-high-throughput-acoustic-screening
#10
JOURNAL ARTICLE
Robert C Hurt, Zhiyang Jin, Mohamed Soufi, Katie K Wong, Daniel P Sawyer, Hao K Shen, Przemysław Dutka, Ramya Deshpande, Ruby Zhang, David R Mittelstein, Mikhail G Shapiro
A major challenge in the fields of biological imaging and synthetic biology is noninvasively visualizing the functions of natural and engineered cells inside opaque samples such as living animals. One promising technology that addresses this limitation is ultrasound (US), with its penetration depth of several cm and spatial resolution on the order of 100 μm. Within the past decade, reporter genes for US have been introduced and engineered to link cellular functions to US signals via heterologous expression in commensal bacteria and mammalian cells...
July 9, 2024: ACS Synthetic Biology
https://read.qxmd.com/read/38981062/metabolic-profile-of-the-genome-reduced-bacillus-subtilis-strain-iig-bs-27-39-an-attractive-chassis-for-recombinant-protein-production
#11
JOURNAL ARTICLE
Rocío Aguilar Suárez, Michael Kohlstedt, Ayşegül Öktem, Jolanda Neef, Yuzheng Wu, Kaiya Ikeda, Ken-Ichi Yoshida, Josef Altenbuchner, Christoph Wittmann, Jan Maarten van Dijl
The Gram-positive bacterium Bacillus subtilis is extensively used in the industry for the secretory production of proteins with commercial value. To further improve its performance, this microbe has been the subject of extensive genome engineering efforts, especially the removal of large genomic regions that are dispensable or even counterproductive. Here, we present the genome-reduced B. subtilis strain IIG-Bs-27-39, which was obtained through systematic deletion of mobile genetic elements, as well as genes for extracellular proteases, sporulation, flagella formation, and antibiotic production...
July 9, 2024: ACS Synthetic Biology
https://read.qxmd.com/read/38979618/cell-free-translation-quantification-via-a-fluorescent-minihelix
#12
JOURNAL ARTICLE
Jessica A Willi, Ashty S Karim, Michael C Jewett
Cell-free gene expression systems are used in numerous applications, including medicine making, diagnostics, and educational kits. Accurate quantification of nonfluorescent proteins in these systems remains a challenge. To address this challenge, we report the adaptation and use of an optimized tetra-cysteine minihelix both as a fusion protein and as a standalone reporter with the FlAsH dye. The fluorescent reporter helix is short enough to be encoded on a primer pair to tag any protein of interest via PCR...
July 9, 2024: ACS Synthetic Biology
https://read.qxmd.com/read/38975718/autobiotech%C3%A2-a-versatile-biofoundry-for-automated-strain-engineering
#13
JOURNAL ARTICLE
Tobias Michael Rosch, Julia Tenhaef, Tim Stoltmann, Till Redeker, Dominic Kösters, Niels Hollmann, Karin Krumbach, Wolfgang Wiechert, Michael Bott, Susana Matamouros, Jan Marienhagen, Stephan Noack
The inevitable transition from petrochemical production processes to renewable alternatives has sparked the emergence of biofoundries in recent years. Manual engineering of microbes will not be sufficient to meet the ever-increasing demand for novel producer strains. Here we describe the AutoBioTech platform, a fully automated laboratory system with 14 devices to perform operations for strain construction without human interaction. Using modular workflows, this platform enables automated transformations of Escherichia coli with plasmids assembled via modular cloning...
July 8, 2024: ACS Synthetic Biology
https://read.qxmd.com/read/38968698/investigating-and-engineering-an-1-2-propanediol-responsive-transcription-factor-based-biosensor
#14
JOURNAL ARTICLE
Yuxi Teng, Xinyu Gong, Jianli Zhang, Ziad Obideen, Yajun Yan
Transcription factor (TF)-based biosensors have arisen as powerful tools in the advancement of metabolic engineering. However, with the emergence of numerous bioproduction targets, the variety of applicable TF-based biosensors remains severely limited. In this study, we investigated and engineered an 1,2-propanediol (1,2-PD)-responsive transcription activator, PocR, from Salmonella typhimurium to enrich the current biosensor repertoire. Heterologous characterization of PocR in E. coli revealed a significantly limited operational range and dynamic range, primarily attributed to the leaky binding between PocR and its corresponding promoters in the absence of the 1,2-PD inducer...
July 5, 2024: ACS Synthetic Biology
https://read.qxmd.com/read/38968167/reliable-genomic-integration-sites-in-pseudomonas-putida-identified-by-two-dimensional-transcriptome-analysis
#15
JOURNAL ARTICLE
Sebastian Köbbing, Thorsten Lechtenberg, Benedikt Wynands, Lars M Blank, Nick Wierckx
Genomic integration is commonly used to engineer stable production hosts. However, so far, for many microbial workhorses, only a few integration sites have been characterized, thereby restraining advanced strain engineering that requires multiple insertions. Here, we report on the identification of novel genomic integration sites, so-called landing pads, for Pseudomonas putida KT2440. We identified genomic regions with constant expression patterns under diverse experimental conditions by using RNA-Seq data...
July 5, 2024: ACS Synthetic Biology
https://read.qxmd.com/read/38954490/non-native-pathway-engineering-with-crispri-for-carbon-dioxide-assimilation-and-valued-5-aminolevulinic-acid-synthesis-in-escherichia-coli-nissle
#16
JOURNAL ARTICLE
Sefli Sri Wahyu Effendi, I-Son Ng
Carbon dioxide emission and acidification during chemical biosynthesis are critical challenges toward microbial cell factories' sustainability and efficiency. Due to its acidophilic traits among workhorse lineages, the probiotic Escherichia coli Nissle (EcN) has emerged as a promising chemical bioproducer. However, EcN lacks a CO2 -fixing system. Herein, EcN was equipped with a simultaneous CO2 fixation system and subsequently utilized to produce low-emission 5-aminolevulinic acid (5-ALA). Two different artificial CO2 -assimilating pathways were reconstructed: the novel ribose-1,5-bisphosphate (R15P) route and the conventional ribulose-5-phosphate (Ru5P) route...
July 2, 2024: ACS Synthetic Biology
https://read.qxmd.com/read/38946081/metabolic-engineering-of-corynebacterium-glutamicum-for-the-high-level-production-of-l-valine-under-aerobic-conditions
#17
JOURNAL ARTICLE
Feiao Wang, Ningyun Cai, Yanlin Leng, Chen Wu, Yanan Wang, Siyu Tian, Chenglin Zhang, Qingyang Xu, Huadong Peng, Ning Chen, Yanjun Li
l-Valine, an essential amino acid, serves as a valuable compound in various industries. However, engineering strains with both high yield and purity are yet to be delivered for microbial l-valine production. We engineered a Corynebacterium glutamicum strain capable of highly efficient production of l-valine. We initially introduced an acetohydroxy acid synthase mutant from an industrial l-valine producer and optimized a cofactor-balanced pathway, followed by the activation of the nonphosphoenolpyruvate-dependent carbohydrate phosphotransferase system and the introduction of an exogenous Entner-Doudoroff pathway...
June 30, 2024: ACS Synthetic Biology
https://read.qxmd.com/read/38941613/characterization-and-engineering-of-a-novel-miniature-eubacterium-siraeum-crispr-cas12f-system
#18
JOURNAL ARTICLE
Yannan Wang, Yujue Wang, Na Tang, Zhipeng Wang, Deng Pan, Quanjiang Ji
Cas12f nucleases are one of the most compact genome editors, exhibiting promising potential for in vivo therapeutic applications. However, the availability of active Cas12f genome editors remains relatively limited in the field. Here, we report the characterization and engineering of a novel miniature Cas12f endonuclease from Eubacterium siraeum (EsCas12f1, 433 amino acids). We elucidate the specific Protospacer Adjacent Motifs preference and the detailed biochemical properties for DNA targeting and cleavage...
June 28, 2024: ACS Synthetic Biology
https://read.qxmd.com/read/38934464/meta-analysis-driven-strain-design-for-mitigating-oxidative-stresses-important-in-biomanufacturing
#19
JOURNAL ARTICLE
P V Phaneuf, S H Kim, K Rychel, C Rode, F Beulig, B O Palsson, L Yang
As the availability of data sets increases, meta-analysis leveraging aggregated and interoperable data types is proving valuable. This study leveraged a meta-analysis workflow to identify mutations that could improve robustness to reactive oxygen species (ROS) stresses using an industrially important melatonin production strain as an example. ROS stresses often occur during cultivation and negatively affect strain performance. Cellular response to ROS is also linked to the SOS response and resistance to pH fluctuations, which is important to strain robustness in large-scale biomanufacturing...
June 27, 2024: ACS Synthetic Biology
https://read.qxmd.com/read/38925629/expression-and-subcellular-localization-of-lanthipeptides-in-human-cells
#20
JOURNAL ARTICLE
Sara M Eslami, Chandrashekhar Padhi, Imran R Rahman, Wilfred A van der Donk
Cyclic peptides, such as most ribosomally synthesized and post-translationally modified peptides (RiPPs), represent a burgeoning area of interest in therapeutic and biotechnological research because of their conformational constraints and reduced susceptibility to proteolytic degradation compared to their linear counterparts. Herein, an expression system is reported that enables the production of structurally diverse lanthipeptides and derivatives in mammalian cells. Successful targeting of lanthipeptides to the nucleus, the endoplasmic reticulum, and the plasma membrane is demonstrated...
June 26, 2024: ACS Synthetic Biology
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