scholarly journals In vitroprototyping and rapid optimization of biosynthetic enzymes for cellular design

2019 ◽  
Author(s):  
Ashty S. Karim ◽  
Quentin M. Dudley ◽  
Alex Juminaga ◽  
Yongbo Yuan ◽  
Samantha A. Crowe ◽  
...  
Keyword(s):  
Model Organisms ◽  
Industrial Biotechnology ◽  
Biosynthetic Enzymes ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Design Build ◽  
Mix And Match ◽  
Cell Free Protein Synthesis

AbstractMicrobial cell factories offer an attractive approach for production of biobased products. Unfortunately, designing, building, and optimizing biosynthetic pathways remains a complex challenge, especially for industrially-relevant, non-model organisms. To address this challenge, we describe a platform forin vitroPrototyping andRapidOptimization ofBiosyntheticEnzymes (iPROBE). In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE with two examples. First, we tested and ranked 54 different pathways for 3-hydroxybutyrate production, improvingin vivoproduction inClostridiumby 20-fold to 14.63 ± 0.48 g/L and identifying a new biosynthetic route to(S)-(+)-1,3-butanediol. Second, we used iPROBE and data-driven design to optimize a 6-stepn-butanol pathway, increasing titers 4-fold across 205 pathways, and showed strong correlation between cell-free and cellular performance. We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.

scholarly journals Cell-free prototyping of limonene biosynthesis using cell-free protein synthesis

2020 ◽  
Author(s):  
Quentin M. Dudley ◽  
Ashty S. Karim ◽  
Connor J. Nash ◽  
Michael C. Jewett
Keyword(s):  
Protein Synthesis ◽  
Metabolic Engineering ◽  
Biosynthetic Enzymes ◽  
List Type ◽  
Free Protein ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
The Impact ◽  
Cell Free Protein Synthesis

AbstractMetabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-built-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 hours from 0.2 to 4.5 mM (23 to 610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. We anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design.TOC FigureHighlightsApplied the iPROBE framework to build the nine-enzyme pathway to produce limoneneAssessed the impact of cofactors and 54 enzyme homologs on cell-free enzyme performanceIteratively optimized the cell-free production of limonene by exploring more than 580 unique reactionsExtended pathway to biofuel precursors pinene and bisabolene

scholarly journals A multifaceted review on dihydromyricetin resources, extraction, bioavailability, biotransformation, bioactivities, and food applications with future perspectives to maximize its value

eFood ◽  
2021 ◽  
Author(s):  
Haolin Zhang ◽  
Giovanni Caprioli ◽  
Hidayat Hussain ◽  
Nguyen Phan Khoi Le ◽  
Mohamed A. Farag ◽  
...  
Keyword(s):  
Comprehensive Overview ◽  
In Vivo Experiments ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Action Mechanisms ◽  
Biotechnological Approach ◽  
Food Applications ◽  
Low Solubility

Natural bioactive compounds present a better alternative to prevent and treat chronic diseases owing to their lower toxicity and abundant resources. (+)-Dihydromyricetin (DMY) is a flavanonol, possessing numerous interesting bioactivities with abundant resources. This review provides a comprehensive overview of the recent advances in DMY natural resources, stereoisomerism, physicochemical properties, extraction, biosynthesis, pharmacokinetics, and biotransformation. Stereoisomerism of DMY should be considered for better indication of its efficacy. Biotechnological approach presents a potential tool for the production of DMY using microbial cell factories. DMY high instability is related to its powerful antioxidant capacity due to pyrogallol moiety in ring B, and whether preparation of other analogues could demonstrate improved properties. DMY demonstrates poor bioavailability based on its low solubility and permeability with several attempts to improve its pharmacokinetics and efficacy. DMY possesses various pharmacological effects, which have been proven by many in vitro and in vivo experiments, while clinical trials are rather scarce, with underlying action mechanisms remaining unclear. Consequently, to maximize the usefulness of DMY in nutraceuticals, improvement in bioavailability, and better understanding of its actions mechanisms and drug interactions ought to be examined in the future along with more clinical evidence.

scholarly journals Modular cell-free expression plasmids to accelerate biological design in cells

2020 ◽  
Author(s):  
Ashty S. Karim ◽  
Fungmin Eric Liew ◽  
Shivani Garg ◽  
Bastian Vögeli ◽  
Blake J. Rasor ◽  
...  
Keyword(s):  
Vector System ◽  
Free Expression ◽  
Expression Vectors ◽  
Model Organisms ◽  
Industrial Biotechnology ◽  
Biological Design ◽  
Cellular Expression ◽  
Community Science

AbstractIndustrial biotechnology aims to produce high-value products from renewable resources. This can be challenging because model microorganisms—organisms that are easy to use like Escherichia coli—often lack the machinery required to utilize desired feedstocks like lignocellulosic biomass or syngas. Non-model organisms, such as Clostridium, are industrially proven and have the desired metabolic features but have several hurdles to mainstream use. Namely, these species grow more slowly than conventional laboratory microbes and genetic tools for engineering them are far less prevalent. To address these hurdles for accelerating cellular design, cell-free synthetic biology has emerged as an approach for characterizing non-model organisms and rapidly testing metabolic pathways in vitro. Unfortunately, cell-free systems can require specialized DNA architectures with minimal regulation that are not compatible with cellular expression. In this work, we develop a modular vector system that allows for T7 expression of desired enzymes for cell-free expression and direct Golden Gate assembly into Clostridium expression vectors. Utilizing the Joint Genome Institute’s DNA Synthesis Community Science Program, we designed and synthesized these plasmids and genes required for our projects allowing us to shuttle DNA easily between our in vitro and in vivo experiments. We next validated that these vectors were sufficient for cell-free expression of enzymes, performing on par with the previous state-of-the-art. Lastly, we demonstrated automated six-part DNA assemblies for C. autoethanogenum expression with efficiencies ranging from 68-90%. We anticipate this system of plasmids will enable a framework for facile testing of biosynthetic pathways in vitro and in vivo by shortening development cycles.

scholarly journals Modular cell-free expression plasmids to accelerate biological design in cells

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Ashty S Karim ◽  
Fungmin (Eric) Liew ◽  
Shivani Garg ◽  
Bastian Vögeli ◽  
Blake J Rasor ◽  
...  
Keyword(s):  
Vector System ◽  
Free Expression ◽  
Expression Vectors ◽  
Model Organisms ◽  
Industrial Biotechnology ◽  
Biological Design ◽  
In Vivo Experiments ◽  
Cellular Expression

Abstract Industrial biotechnology aims to produce high-value products from renewable resources. This can be challenging because model microorganisms—organisms that are easy to use like Escherichia coli—often lack the machinery required to utilize desired feedstocks like lignocellulosic biomass or syngas. Non-model organisms, such as Clostridium, are industrially proven and have desirable metabolic features but have several hurdles to mainstream use. Namely, these species grow more slowly than conventional laboratory microbes, and genetic tools for engineering them are far less prevalent. To address these hurdles for accelerating cellular design, cell-free synthetic biology has matured as an approach for characterizing non-model organisms and rapidly testing metabolic pathways in vitro. Unfortunately, cell-free systems can require specialized DNA architectures with minimal regulation that are not compatible with cellular expression. In this work, we develop a modular vector system that allows for T7 expression of desired enzymes for cell-free expression and direct Golden Gate assembly into Clostridium expression vectors. Utilizing the Joint Genome Institute’s DNA Synthesis Community Science Program, we designed and synthesized these plasmids and genes required for our projects allowing us to shuttle DNA easily between our in vitro and in vivo experiments. We next validated that these vectors were sufficient for cell-free expression of functional enzymes, performing on par with the previous state-of-the-art. Lastly, we demonstrated automated six-part DNA assemblies for Clostridium autoethanogenum expression with efficiencies ranging from 68% to 90%. We anticipate this system of plasmids will enable a framework for facile testing of biosynthetic pathways in vitro and in vivo by shortening development cycles.

scholarly journals Heavily Armed Ancestors: CRISPR Immunity and Applications in Archaea with a Comparative Analysis of CRISPR Types in Sulfolobales

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1523
Author(s):  
Isabelle Anna Zink ◽  
Erika Wimmer ◽  
Christa Schleper
Keyword(s):  
Comparative Analysis ◽  
Molecular Mechanisms ◽  
Model Organisms ◽  
Special Focus ◽  
Natural Environments ◽  
Type I ◽  
Accessory Proteins ◽  
Type Iii

Prokaryotes are constantly coping with attacks by viruses in their natural environments and therefore have evolved an impressive array of defense systems. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is an adaptive immune system found in the majority of archaea and about half of bacteria which stores pieces of infecting viral DNA as spacers in genomic CRISPR arrays to reuse them for specific virus destruction upon a second wave of infection. In detail, small CRISPR RNAs (crRNAs) are transcribed from CRISPR arrays and incorporated into type-specific CRISPR effector complexes which further degrade foreign nucleic acids complementary to the crRNA. This review gives an overview of CRISPR immunity to newcomers in the field and an update on CRISPR literature in archaea by comparing the functional mechanisms and abundances of the diverse CRISPR types. A bigger fraction is dedicated to the versatile and prevalent CRISPR type III systems, as tremendous progress has been made recently using archaeal models in discerning the controlled molecular mechanisms of their unique tripartite mode of action including RNA interference, DNA interference and the unique cyclic-oligoadenylate signaling that induces promiscuous RNA shredding by CARF-domain ribonucleases. The second half of the review spotlights CRISPR in archaea outlining seminal in vivo and in vitro studies in model organisms of the euryarchaeal and crenarchaeal phyla, including the application of CRISPR-Cas for genome editing and gene silencing. In the last section, a special focus is laid on members of the crenarchaeal hyperthermophilic order Sulfolobales by presenting a thorough comparative analysis about the distribution and abundance of CRISPR-Cas systems, including arrays and spacers as well as CRISPR-accessory proteins in all 53 genomes available to date. Interestingly, we find that CRISPR type III and the DNA-degrading CRISPR type I complexes co-exist in more than two thirds of these genomes. Furthermore, we identified ring nuclease candidates in all but two genomes and found that they generally co-exist with the above-mentioned CARF domain ribonucleases Csx1/Csm6. These observations, together with published literature allowed us to draft a working model of how CRISPR-Cas systems and accessory proteins cross talk to establish native CRISPR anti-virus immunity in a Sulfolobales cell.

scholarly journals Synergic Interaction between Permethrin and Bt toxins discovered using RNA-seq

2019 ◽  
Author(s):  
Takuma Sakamoto ◽  
Toshinori Kozaki ◽  
Norichika Ogata
Keyword(s):  
Differentially Expressed Gene ◽  
Model Organisms ◽  
Cross Resistance ◽  
Agricultural Pests ◽  
Bt Toxin ◽  
Development Of Resistance ◽  
Bt Toxins ◽  
Increased Susceptibility

AbstractActing against the development of resistance to antibiotics and insecticides, involving negatively correlated cross-resistance (NCR) is an alternative to use- and-discard approach. It is termed NCR that toxic chemicals interact with each other and resistance of target organisms to one chemical is sometimes associated with increased susceptibility to a second chemical when; an allele confers resistance to one toxic chemical and hyper-susceptibility to another, NCR occurs. However, only 11 toxin pairs have been revealed to cause NCR in insects. Finding novel NCRs is needed for integrated pest management. We analyzed permethrin, an insecticide, induced transcriptomes of cultured fat bodies of the silkworm Bombyx mori, a lepidopteran model insect. Differentially expressed gene analyses suggested Bacillus thuringiensis (Bt) toxin was an NCR toxin of permethrin. NCR to permethrin and Bt toxins in Thysanoplusia intermixta, the agricultural pest moth, was examined; the children of permethrin survivor T. intermixta had increased susceptibility to Bt toxin. A novel NCR toxin pair, permethrin and Bt toxin, was discovered. The screening and developmental method for negatively correlated cross-resistance toxins established in this study was effective, in vitro screening using model organisms and in vivo verification using agricultural pests.

scholarly journals Developing Clostridia as Cell Factories for Short- and Medium-Chain Ester Production

2021 ◽  
Vol 9 ◽  
Author(s):  
Qingzhuo Wang ◽  
Naief H. Al Makishah ◽  
Qi Li ◽  
Yanan Li ◽  
Wenzheng Liu ◽  
...  
Keyword(s):  
Organic Acids ◽  
Value Added ◽  
Carbon Chain ◽  
Chain Elongation ◽  
Medium Chain ◽  
Cell Factories ◽  
Alcohol Acyltransferase ◽  
Volatile Esters

Short- and medium-chain volatile esters with flavors and fruity fragrances, such as ethyl acetate, butyl acetate, and butyl butyrate, are usually value-added in brewing, food, and pharmacy. The esters can be naturally produced by some microorganisms. As ester-forming reactions are increasingly deeply understood, it is possible to produce esters in non-natural but more potential hosts. Clostridia are a group of important industrial microorganisms since they can produce a variety of volatile organic acids and alcohols with high titers, especially butanol and butyric acid through the CoA-dependent carbon chain elongation pathway. This implies sufficient supplies of acyl-CoA, organic acids, and alcohols in cells, which are precursors for ester production. Besides, some Clostridia could utilize lignocellulosic biomass, industrial off-gas, or crude glycerol to produce other branched or straight-chain alcohols and acids. Therefore, Clostridia offer great potential to be engineered to produce short- and medium-chain volatile esters. In the review, the efforts to produce esters from Clostridia via in vitro lipase-mediated catalysis and in vivo alcohol acyltransferase (AAT)-mediated reaction are comprehensively revisited. Besides, the advantageous characteristics of several Clostridia and clostridial consortia for bio-ester production and the driving force of synthetic biology to clostridial chassis development are also discussed. It is believed that synthetic biotechnology should enable the future development of more effective Clostridia for ester production.

scholarly journals An Integrated Approach to Studying Rare Neuromuscular Diseases Using Animal and Human Cell-Based Models

2022 ◽  
Vol 9 ◽  
Author(s):  
Timothy J. Hines ◽  
Cathleen Lutz ◽  
Stephen A. Murray ◽  
Robert W. Burgess
Keyword(s):  
Human Disease ◽  
Neuromuscular Diseases ◽  
Trna Synthetase ◽  
Genetically Engineered ◽  
Model Systems ◽  
Model Organisms ◽  
Cellular Mechanisms ◽  
Disease Mechanisms

As sequencing technology improves, the identification of new disease-associated genes and new alleles of known genes is rapidly increasing our understanding of the genetic underpinnings of rare diseases, including neuromuscular diseases. However, precisely because these disorders are rare and often heterogeneous, they are difficult to study in patient populations. In parallel, our ability to engineer the genomes of model organisms, such as mice or rats, has gotten increasingly efficient through techniques such as CRISPR/Cas9 genome editing, allowing the creation of precision human disease models. Such in vivo model systems provide an efficient means for exploring disease mechanisms and identifying therapeutic strategies. Furthermore, animal models provide a platform for preclinical studies to test the efficacy of those strategies. Determining whether the same mechanisms are involved in the human disease and confirming relevant parameters for treatment ideally involves a human experimental system. One system currently being used is induced pluripotent stem cells (iPSCs), which can then be differentiated into the relevant cell type(s) for in vitro confirmation of disease mechanisms and variables such as target engagement. Here we provide a demonstration of these approaches using the example of tRNA-synthetase-associated inherited peripheral neuropathies, rare forms of Charcot-Marie-Tooth disease (CMT). Mouse models have led to a better understanding of both the genetic and cellular mechanisms underlying the disease. To determine if the mechanisms are similar in human cells, we will use genetically engineered iPSC-based models. This will allow comparisons of different CMT-associated GARS alleles in the same genetic background, reducing the variability found between patient samples and simplifying the availability of cell-based models for a rare disease. The necessity of integrating mouse and human models, strategies for accomplishing this integration, and the challenges of doing it at scale are discussed using recently published work detailing the cellular mechanisms underlying GARS-associated CMT as a framework.

scholarly journals Modular, synthetic chromosomes as new tools for large scale engineering of metabolism

2021 ◽  
Author(s):  
Eline Postma ◽  
Else-Jasmijn Hassing ◽  
Venda Mangkusaputra ◽  
Jordi Geelhoed ◽  
Pilar de la Torre ◽  
...  
Keyword(s):  
Copy Number ◽  
Large Scale ◽  
Metabolic Networks ◽  
De Novo ◽  
Microbial Cell ◽  
Cell Factory ◽  
Microbial Cell Factory ◽  
Microbial Cell Factories ◽  
Cell Factories

The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range pharmacological properties. Turned into exclusive expression platforms for heterologous and essential metabolic routes, the neochromosomes mimic native chromosomes regarding mitotic and genetic stability, copy number, harmlessness for the host and editability by CRISPR/Cas9. This study paves the way for future microbial cell factories with modular genomes in which core metabolic networks, localized on satellite, specialized neochromosomes can be swapped for alternative configurations and serve as landing pads for the addition of functionalities.

scholarly journals Pharmacological and functional similarities of the human neuropeptide Y system in C. elegans challenges phylogenetic views on the FLP/NPR system

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Miron Mikhailowitsch Gershkovich ◽  
Victoria Elisabeth Groß ◽  
Anette Kaiser ◽  
Simone Prömel
Keyword(s):  
Neuropeptide Y ◽  
Pharmacological Study ◽  
Receptor Activation ◽  
Cross Reactivity ◽  
Model Organisms ◽  
Loss Of Function ◽  
C Elegans ◽  
Molecular Studies

Abstract Background The neuropeptide Y system affects various processes, among others food intake, and is frequently discussed in the context of targeting obesity. Studies in model organisms are indispensable to enable molecular studies in a physiological context. Although the NPY system is evolutionarily conserved in all bilaterians, in the widely used model Caenorhabditis elegans there is controversy on the existence of NPY orthologous molecules. While the FMRFamide-like peptide (FLP)/Neuropeptide receptor-Resemblance (NPR) system in the nematode was initially suggested to be orthologous to the mammalian NPY system, later global phylogenetic studies indicate that FLP/NPR is protostome-specific. Methods We performed a comprehensive pharmacological study of the FLP/NPR system in transfected cells in vitro, and tested for functional substitution in C. elegans knockout strains. Further, we phenotypically compared different flp loss-of-function strains. Differences between groups were compared by ANOVA and post-hoc testing (Dunnett, Bonferroni). Results Our pharmacological analysis of the FLP/NPR system including formerly functionally uncharacterized NPY-like peptides from C. elegans demonstrates that G protein-coupling and ligand requirements for receptor activation are similar to the human NPY system. In vitro and in vivo analyses show cross-reactivity of NPY with the FLP/NPR system manifesting in the ability of the human GPCRs to functionally substitute FLP/NPR signaling in vivo. The high pharmacological/functional similarities enabled us to identify C. elegans FLP-14 as a key molecule in avoidance behavior. Conclusions Our data demonstrate the pharmacological and functional similarities of human NPY and C. elegans NPR systems. This adds a novel perspective to current phylogenetic reconstructions of the neuropeptide Y system. NPY and NPR receptors are pharmacologically so similar that the human receptors can functionally compensate for the C. elegans ones, suggesting orthologous relationships. This is also underlined by the presence of NPY-like peptides and parallels in peptide requirements for receptor activation. Further, the results presented here highlight the potential of this knowledge for physiological as well as molecular studies on neuropeptide GPCRs such as the NPY system in the future.

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