The Cierva "Autogiro" Mark III (British) : Armstrong-Siddeley "Genet Major" Engine
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The Westland "Widgeon III" "Cirrus II: or "Genet II" engine
The Widgeon III is a parasol type monoplane. One of it's strongest features was the high degree of visibility available to the pilot.
AIRPLANES - WESTLAND WIDGEON 3 (BRIT)↗
Genome-Scale 13 C Fluxomics Modeling for Metabolic Engineering of Saccharomyces cerevisiae .
Synthetic biology is a rapidly developing field that pursues the application of engineering principles and development approaches to biological engineering. Synthetic biology is poised to change the way biology is practiced, and has important practical applications: for example, building genetically engineered organisms to produce biofuels, medicines, and other chemicals. Traditionally, synthetic biology has focused on manipulating a few genes (e.g., in a single pathway or genetic circuit), but its combination with systems biology holds the promise of creating new cellular architectures and constructing complex biological systems from the ground up. Enabling this merge of synthetic and systems biology will require greater predictive capability for modeling the behavior of cellular systems, and more comprehensive data sets for building and calibrating these models. The so-called "-omics" data sets can now be generated via high throughput techniques in the form of genomic, proteomic, transcriptomic, and metabolomic information on the engineered biological system. Of particular interest with respect to the engineering of microbes capable of producing biofuels and other chemicals economically and at scale are metabolomic datasets, and their insights into intracellular metabolic fluxes. Metabolic fluxes provide a rapid and easy to understand picture of how carbon and energy flow throughout the cell. Furthermore, we present a detailed guide to performing metabolic flux analysis and modeling using the open source JBEI Quantitative Metabolic Modeling (jQMM) library. This library allows the user to transform metabolomics data in the form of isotope labeling data from a 13 C labeling experiment into a determination of cellular fluxes that can be used to develop genetic engineering strategies for metabolic engineering.The jQMM library presents a complete toolbox for performing a range of different tasks of interest in metabolic engineering. Various different types of flux analysis and modeling can be performed such as flux balance analysis, 13 C metabolic flux analysis, and two-scale 13 C metabolic flux analysis (2S- 13 C MFA). 2S- 13 C MFA is a novel method that determines genome-scale fluxes without the need of every single carbon transition in the metabolic network. In addition to several other capabilities, the jQMM library can make model based predictions for how various genetic engineering strategies can be incorporated toward bioengineering goals: it can predict the effects of reaction knockouts on metabolism using both the MoMA and ROOM methodologies. In this chapter, we will illustrate the use of the jQMM library through a step-by-step demonstration of flux determination and knockout prediction in a complex eukaryotic model organism: Saccharomyces cerevisiae ( S. cerevisiae ). Included with this chapter is a digital Jupyter Notebook file that provides a computable appendix showing a self-contained example of jQMM usage, which can be changed to fit the user's specific needs. As an open source software project, users can modify and extend the code base to make improvements at will, allowing them to share their development work and contribute back to the jQMM modeling community.
-Omics data↗
Improving the Transformation Efficiency of Synechococcus sp. PCC 7002 via Methylome-Guided Premethylation of DNA
Cyanobacteria are promising microbial platforms for a diverse set of biotechnology applications, from living materials to photosynthetic chemical production, but are less well characterized than commonly engineered microbes such as Escherichia coli. This study facilitates genetic engineering in Synechococcus sp. PCC 7002, a fast-growing, halotolerant, and naturally competent strain, by identifying ten native methylation motifs and designing shuttle strains that mimic the native methylation state by expressing a subset of heterologous methyltransferases. DNA methylation in E. coli with as few as two active methyltransferases increased transformation efficiency up to 30-fold across four distinct integration sites in PCC 7002. This work provides an experimental framework to bypass native restriction-modification systems for efficient genome editing and metabolic engineering in nonmodel bacteria.
59 BASIC BIOLOGICAL SCIENCES↗
Metabolic Engineering of Oleaginous Yeasts for Production of Fuels and Chemicals
Oleaginous yeasts have been increasingly explored for production of chemicals and fuels via metabolic engineering. Particularly, there is a growing interest in using oleaginous yeasts for the synthesis of lipid-related products due to their high lipogenesis capability, robustness, and ability to utilize a variety of substrates. Most of the metabolic engineering studies in oleaginous yeasts focused on Yarrowia that already has plenty of genetic engineering tools. However, recent advances in systems biology and synthetic biology have provided new strategies and tools to engineer those oleaginous yeasts that have naturally high lipid accumulation but lack genetic tools, such as Rhodosporidium, Trichosporon, and Lipomyces. This review highlights recent accomplishments in metabolic engineering of oleaginous yeasts and recent advances in the development of genetic engineering tools in oleaginous yeasts within the last 3 years.
09 BIOMASS FUELS↗
Chapter 12: Gene Editing Technologies for Biofuel Production in Thermophilic Microbes
Thermophilic microbes are an attractive bioproduction platform due to their inherently lower contamination risk and their ability to perform thermostable enzymatic processes which may be required for biomass processing and other industrial applications. The engineering of microbes for industrial scale processes requires a suite of genetic engineering tools to optimize existing biological systems as well as to design and incorporate new metabolic pathways within strains. Yet, such tools are often lacking and/or inadequate for novel microbes, especially thermophiles. This chapter focuses on genetic tool development and engineering strategies, in addition to challenges, for thermophilic microbes. We provide detailed instructions and techniques for tool development for an anaerobic thermophile, Caldanaerobacter subterraneus subsp. tengcongensis, including culturing, plasmid construction, transformation, and selection. This establishes a foundation for advanced genetic tool development necessary for the metabolic engineering of this microbe and potentially other thermophilic organisms.
BASIC BIOLOGICAL SCIENCES,BIOMASS FUELS↗
Automated model-predictive design of synthetic promoters to control transcriptional profiles in bacteria
Abstract Transcription rates are regulated by the interactions between RNA polymerase, sigma factor, and promoter DNA sequences in bacteria. However, it remains unclear how non-canonical sequence motifs collectively control transcription rates. Here, we combine massively parallel assays, biophysics, and machine learning to develop a 346-parameter model that predicts site-specific transcription initiation rates for any σ 70 promoter sequence, validated across 22132 bacterial promoters with diverse sequences. We apply the model to predict genetic context effects, design σ 70 promoters with desired transcription rates, and identify undesired promoters inside engineered genetic systems. The model provides a biophysical basis for understanding gene regulation in natural genetic systems and precise transcriptional control for engineering synthetic genetic systems.
42 ENGINEERING↗
Space Synthetic Biology Project
Synthetic biology is an effort to make genetic engineering more useful by standardizing sections of genetic code. By standardizing genetic components, biological engineering will become much more similar to traditional fields of engineering, in which well-defined components and subsystems are readily available in markets. Specifications of the behavior of those components and subsystems can be used to model a system which incorporates them. Then, the behavior of the novel system can be simulated and optimized. Finally, the components and subsystems can be purchased and assembled to create the optimized system, which most often will exhibit behavior similar to that indicated by the model. The Space Synthetic Biology project began in 2012 as a multi-Center effort. The purpose of this project was to harness Synthetic Biology principals to enable NASA's missions. A central target for application was to Environmental Control & Life Support (ECLS). Engineers from NASA Marshall Space Flight Center's (MSFC's) ECLS Systems Development Branch (ES62) were brought into the project to contribute expertise in operational ECLS systems. Project lead scientists chose to pursue the development of bioelectrochemical technologies to spacecraft life support. Therefore, the ECLS element of the project became essentially an effort to develop a bioelectrochemical ECLS subsystem. Bioelectrochemical systems exploit the ability of many microorganisms to drive their metabolisms by direct or indirect utilization of electrical potential gradients. Whereas many microorganisms are capable of deriving the energy required for the processes of interest (such as carbon dioxide (CO2) fixation) from sunlight, it is believed that subsystems utilizing electrotrophs will exhibit smaller mass, volume, and power requirements than those that derive their energy from sunlight. In the first 2 years of the project, MSFC personnel conducted modeling, simulation, and conceptual design efforts to assist the project in selecting the best approaches to the application of bioelectrochemical technologies to ECLS. Figure 1 shows results of simulation of charge transport in an experimental system. Figure 2 shows one of five conceptual designs for ECLS subsystems based on bioelectrochemical reactors. Also during the first 2 years, some work was undertaken to gather fundamental data (conductivities, overpotentials) relevant to the modeling efforts.
Howard, David↗
eYGFPuv-Assisted Transgenic Selection in Populus deltoides WV94 and Multiplex Genome Editing in Protoplasts of P. trichocarpa × P. deltoides Clone ‘52-225’
Although CRISPR/Cas-based genome editing has been widely used for plant genetic engineering, its application in the genetic improvement of trees has been limited, partly because of challenges in Agrobacterium-mediated transformation. As an important model for poplar genomics and biotechnology research, eastern cottonwood (Populus deltoides) clone WV94 can be transformed by A. tumefaciens, but several challenges remain unresolved, including the relatively low transformation efficiency and the relatively high rate of false positives from antibiotic-based selection of transgenic events. Moreover, the efficacy of CRISPR-Cas system has not been explored in P. deltoides yet. Here, we first optimized the protocol for Agrobacterium-mediated stable transformation in P. deltoides WV94 and applied a UV-visible reporter called eYGFPuv in transformation. Our results showed that the transgenic events in the early stage of transformation could be easily recognized and counted in a non-invasive manner to narrow down the number of regenerated shoots for further molecular characterization (at the DNA or mRNA level) using PCR. We found that approximately 8.7% of explants regenerated transgenic shoots with green fluorescence within two months. Next, we examined the efficacy of multiplex CRISPR-based genome editing in the protoplasts derived from P. deltoides WV94 and hybrid poplar clone ‘52-225’ (P. trichocarpa × P. deltoides clone ‘52-225’). The two constructs expressing the Trex2-Cas9 system resulted in mutation efficiency ranging from 31% to 57% in hybrid poplar clone 52-225, but no editing events were observed in P. deltoides WV94 transient assay. The eYGFPuv-assisted plant transformation and genome editing approach demonstrated in this study has great potential for accelerating the genome editing-based breeding process in poplar and other non-model plants species and point to the need for additional CRISPR work in P. deltoides.
59 BASIC BIOLOGICAL SCIENCES↗
Recent Advances in Algal Genetic Tool Development
The goal of achieving cost-effective biofuels and bioproducts derived from algal biomass will require improvements along the entire value chain, including identification of robust, high-productivity strains and development of advanced genetic tools. Though there have been modest advances in development of genetic systems for the model alga Chlamydomonas reinhardtii, progress in development of algal genetic tools, especially as applied to non-model algae, has generally lagged behind that of more commonly utilized laboratory and industrial microbes. This is in part due to the complex organellar structure of algae, including robust cell walls and intricate compartmentalization of target loci, as well as prevalent gene silencing mechanisms, which hinder facile utilization of conventional genetic engineering tools and methodologies. However, recent progress in global tool development has opened the door for implementation of strain-engineering strategies in industrially-relevant algal strains. Here, we review recent advances in algal genetic tool development and applications in eukaryotic microalgae.
09 BIOMASS FUELS↗
Restoration and Modification of Magnetosome Biosynthesis by Internal Gene Acquisition in a Magnetotactic Bacterium
We report that the Integration of a large-sized DNA fragment into a chromosome is an important strategy for characterization of cellular functions in microorganisms. Magnetotactic bacteria synthesize intracellular organelles comprising membrane-bound single crystalline magnetite, also referred to as magnetosomes. Magnetosomes have gained interest in both scientific and engineering sectors as they can be utilized as a material for biomedical and nanotechnological applications. Although genetic engineering of magnetosome biosynthesis mechanism has been investigated, the current method requires cumbersome gene preparation processes. Here, the chromosomal integration of a plasmid containing ≈27 magnetosome genes (≈26 kbp region) in a non-magnetic mutant of Magnetospirillum magneticum AMB-1 using a broad-host-range plasmid is shown. The genome sequencing of gene-complemented strains reveals the chromosomal integration of the plasmid with magnetosome genes at a specific site, most likely by catalysis of an endogenous transposase. Magnetosome production is successfully enhanced by integrating a variation of magnetosome gene operons in the chromosome. This chromosomal integration mechanism will allow the design of functional magnetosomes de novo and M. magneticum AMB-1 may be used as a chassis for the designed magnetosome production.
59 BASIC BIOLOGICAL SCIENCES↗
Chapter 2: Genetics of Unstudied Thermophiles for Industry
Thermophilic organisms hold great potential for industry due to their numerous advantages in biotechnological applications such as higher reaction rate, higher substrate loading, decreased susceptibility to reaction contamination, energy savings in industrial fermentations, and ability to express thermostable proteins that can be utilized in many important industrial processes. Bioprospecting for thermophiles will continue to reveal new enzymatic and metabolic paradigms with industrial applicability. In order to translate these paradigms to production scale, routine methods for microbial genetic engineering are needed, yet remain to be developed in many newly isolated thermophiles. Major challenges and recent developments in the establishment of reliable genetic systems in thermophiles are discussed. Here, we use a hyperthermophilic, cellulolytic bacterium, Caldicellulosiruptor bescii, as a case study to demonstrate the development of a genetic system for an industrially useful thermophile, describing in detail methods for transformation, genetic tool utilization, and chromosomal modification using targeted gene deletion and insertion techniques.
93 SC - Biological and Environmental Research (BER↗
XERICO as a target for engineering stress-resilient crops: Mechanisms, applications, and future directions
XERICO's capacity to enhance ABA-driven stress responses across diverse crops, its regulatory crosstalk with other hormonal pathways, and its compatibility with advanced genetic engineering tools highlight its central role in sustainable agriculture. Leveraging XERICO in crop improvement programs aligns with the urgent need to mitigate the impacts of climate-induced stress in agriculture, offering a pathway toward resilient and high-yielding crops. Here, by enabling crops to withstand drought and other environmental stresses, XERICO-based biotechnological approaches hold transformative potential for global food security and environmental sustainability.
09 BIOMASS FUELS↗
Editorial: Multi-Omics Technologies for Optimizing Synthetic Biomanufacturing
Industrial manufacturing endures as an essential human activity yielding a variety of useful products; it plays a significant role in the global economy with huge impacts in everyday life. However, the manufacturing process requires consumption of various raw materials (especially petroleum derivatives), generates a variety of harmful waste products, causes pollution, and is energetically inefficient. Biological manufacturing from sustainable, affordable, and scalable feedstocks potentially enables the displacement of the entire portfolio of currently available products produced by industrial processes, enabling the manufacturing of renewable and eco-friendly products (Clomburg et al., 2017). Thus, successful development of a robust biomanufacturing strategy and technology platform, based on the latest advances in synthetic biology and chemical catalysis, will decrease both the cost and production time compared with previous manufacturing processes. Development of biomanufacturing processes using a synthetic biology platform requires the multidisciplinary efforts of science and engineering fields including molecular biology, microbiology, genetic engineering, informatics, metabolic modeling and chemical or process engineering (El Karoui et al., 2019).
59 BASIC BIOLOGICAL SCIENCES↗
Therapeutic potential of implanted tissue-engineered bioartificial muscles delivering recombinant proteins to the sheep heart
Tissue-engineered primary adult sheep muscle cells genetically engineered to express either rhVEGF or rhIGF-1 secreted the bioactive proteins locally in the sheep heart for at least 30 days.
NASA Discipline Musculoskeletal↗
Utilizing plant synthetic biology to accelerate plant-microbe interactions research
Plant-microbe interactions are critical to ecosystem resilience and substantially influence crop production. From the perspective of plant science, two important focus areas concerning plant-microbe interactions include: 1) understanding plant molecular mechanisms involved in plant-microbe interfaces and 2) engineering plants for increasing plant disease resistance or enhancing beneficial interactions with microbes to increase their resilience to biotic and abiotic stress conditions. Molecular biology and genetics approaches have been used to investigate the molecular mechanisms underlying plant responses to various beneficial and pathogenic microbes. While these approaches are valuable for elucidating the functions of individual genes and pathways, they fall short of unraveling the complex cross-talk across pathways or systems that plants employ to respond and adapt to environmental stresses. Also, genetic engineering of plants to increase disease resistance or enhance symbiosis with microbes has mainly been attempted or conducted through targeted manipulation of single genes/pathways of plants. Recent advancements in synthetic biology tool development are paving the way for multi-gene characterization and engineering in plants in relation to plant-microbe interactions. Here, we briefly summarize the current understanding of plant molecular pathways involved in plant interactions with beneficial and pathogenic microorganisms. Then, we highlight the progress in applying plant synthetic biology to elucidate the molecular basis of plant responses to microbes, enhance plant disease resistance, engineer synthetic symbiosis, and conduct in situ microbiome engineering. Lastly, we discuss the challenges, opportunities, and future directions for advancing plant-microbe interactions research using the capabilities of plant synthetic biology.
59 BASIC BIOLOGICAL SCIENCES↗
Biological funneling of phenolics from transgenic plants engineered to express the bacterial 3-dehydroshikimate dehydratase (qsuB) gene
The economic and environmental sustainability of lignocellulosic biomass biorefineries is predicated on generating biofuels and bioproducts from cell-wall polysaccharide and lignin polymers. Historical efforts in plant genetic engineering have focused on the development of strategies that facilitate biomass deconstruction, with more recently efforts including the synthesis of high-value chemicals in planta . One such genetic modification is the expression of the bacterial quinate and shikimate utilization B ( qsuB ) gene that increases the accumulation of protocatechuic acid in lignocellulosic biomass. Herein, we evaluated the effectiveness of an alkaline pretreatment process to extract phenolics directly from wild-type and QsuB-transgenic lines of Arabidopsis, poplar, and sorghum, and then upgrade them to the polyester precursor 2-pyrone-4,6-dicarboxylic acid (PDC) with an engineered strain of Novosphingobium aromaticivorans . Protocatechuic acid extracted from all QsuB transgenic lines was found to be mostly in the glycosylated form. Glycosylated protocatechuic acid and other plant-derived phenolics were effectively metabolized by N. aromaticivorans, and PDC production was greatest using extracts from an Arabidopsis QsuB transgenic line (∼5% w/w), followed by QsuB sorghum (∼1.1% w/w), and QsuB poplar (∼0.4% w/w) lines. The comparison of PDC production from wild-type and QsuB transgenic lines of Arabidopsis, poplar, and sorghum demonstrates the utility of a mild alkaline pretreatment to liberate phenolics from plant biomass that are either naturally present or that accumulate as a consequence of genetic engineering strategies. All QsuB transgenic lines outperformed their wild-type counterparts with respect to observed PDC yields. In addition, microbial funneling to PDC was effective even when most of the protocatechuic acid extracted was in glycosylated form, clearly demonstrating that this bacterium can metabolize these aromatic conjugates. These findings illustrate the benefits of combining plant and microbial engineering for bioproduct formation from phenolics in lignocellulosic biorefineries.
Umana, German E.↗
Improved Microalgal Carbon Utilization Efficiency via Integrated CO 2 Electro-Conversion to Formate and Microalgal Sequestration
This project developed a process to convert industrial carbon dioxide (CO 2 ) emissions into high-value, sustainable products through genetically engineered algae cultivation. While traditional microalgae cultivation depends on sparging CO 2 gas through water, this method is often inefficient because much of the gas escapes into the atmosphere before the algae can consume it. To overcome this challenge, the project designed an integrated system that first uses a CO 2 to formic acid electrolyzer to convert CO 2 into water-soluble formic acid/formate, then introduces formic acid/formate into the algae pond for cultivation, which allows the algae to access and utilize nearly all of the provided carbon, greatly increasing the efficiency of carbon utilization. The project team has successfully scaled up the CO 2 to formic acid electrolyzer from lab-scale to 1000 cm² and demonstrated industrially relevant current densities with the scaled-up electrolyzers using a CO 2 source that simulates industrial CO 2 waste.
42 ENGINEERING↗