A comprehensive study on Mecanum wheel-based mobility and suspension solutions for intelligent nursing wheelchairs.

Intelligent nursing wheelchairs significantly enhance mobility and independence for elderly individuals with disabilities. However, traditional designs often suffer from large turning radii that restrict their functionality in confined spaces. Addressing this critical challenge, this study introduces an innovative design utilizing a Mecanum wheel chassis that allows for omnidirectional movement, significantly improving maneuverability and stability. Our design incorporates independently controlled Mecanum wheels, overcoming traditional constraints and enhancing user autonomy. To address issues such as wheel spacing variations and hub center tilting, which can lead to slipping and inaccurate motion control, we developed a novel suspension system that stabilizes the chassis, minimizes slipping risks, and boosts motion control accuracy. Experimental validations, including shock absorption and positioning tests, demonstrate that our suspension system markedly enhances the wheelchair's control performance and stability, thereby providing users with enhanced precision and potentially improving their quality of life.

Pangenomic landscapes shape performances of a synthetic genetic circuit across Stutzerimonas species.

Engineering identical genetic circuits into different species typically results in large differences in performance due to the unique cellular environmental context of each host, a phenomenon known as the "chassis-effect" or "context-dependency". A better understanding of how genomic and physiological contexts underpin the chassis-effect will improve biodesign strategies across diverse microorganisms. Here, we combined a pangenomic-based gene expression analysis with quantitative measurements of performance from an engineered genetic inverter device to uncover how genome structure and function relate to the observed chassis-effect across six closely related Stutzerimonas hosts. Our results reveal that genome architecture underpins divergent responses between our chosen non-model bacterial hosts to the engineered device. Specifically, differential expression of the core genome, gene clusters shared between all hosts, was found to be the main source of significant concordance to the observed differential genetic device performance, whereas specialty genes from respective accessory genomes were not significant. A data-driven investigation revealed that genes involved in denitrification and components of trans-membrane transporter proteins were among the most differentially expressed gene clusters between hosts in response to the genetic device. Our results show that the chassis-effect can be traced along differences among the most conserved genome-encoded functions and that these differences create a unique biodesign space among closely related species.IMPORTANCEContemporary synthetic biology endeavors often default to a handful of model organisms to host their engineered systems. Model organisms such as Escherichia coli serve as attractive hosts due to their tractability but do not necessarily provide the ideal environment to optimize performance. As more novel microbes are domesticated for use as biotechnology platforms, synthetic biologists are urged to explore the chassis-design space to optimize their systems and deliver on the promises of synthetic biology. The consequences of the chassis-effect will therefore only become more relevant as the field of biodesign grows. In our work, we demonstrate that the performance of a genetic device is highly dependent on the host environment it operates within, promoting the notion that the chassis can be considered a design variable to tune circuit function. Importantly, our results unveil that the chassis-effect can be traced along similarities in genome architecture, specifically the shared core genome. Our study advocates for the exploration of the chassis-design space and is a step forward to empowering synthetic biologists with knowledge for more efficient exploration of the chassis-design space to enable the next generation of broad-host-range synthetic biology.

Recent advances in genome mining and synthetic biology for discovery and biosynthesis of natural products.

Natural products have long served as critical raw materials in chemical and pharmaceutical manufacturing, primarily which can provide superior scaffolds or intermediates for drug discovery and development. Over the last century, natural products have contributed to more than a third of therapeutic drug production. However, traditional methods of producing drugs from natural products have become less efficient and more expensive over the past few decades. The combined utilization of genome mining and synthetic biology based on genome sequencing, bioinformatics tools, big data analytics, genetic engineering, metabolic engineering, and systems biology promises to counter this trend. Here, we reviewed recent (2020-2023) examples of genome mining and synthetic biology used to resolve challenges in the production of natural products, such as less variety, poor efficiency, and low yield. Additionally, the emerging efficient tools, design principles, and building strategies of synthetic biology and its application prospects in NPs synthesis have also been discussed.

Promising non-model microbial cell factories obtained by genome reduction.

The development of sustainable processes is the most important basis to realize the shift from the fossil-fuel based industry to bio-based production. Non-model microbes represent a great resource due to their advantageous traits and unique repertoire of bioproducts. However, most of these microbes require modifications to improve their growth and production capacities as well as robustness in terms of genetic stability. For this, genome reduction is a valuable and powerful approach to meet industry requirements and to design highly efficient production strains. Here, we provide an overview of various genome reduction approaches in prokaryotic microorganisms, with a focus on non-model organisms, and highlight the example of a successful genome-reduced model organism chassis. Furthermore, we discuss the advances and challenges of promising non-model microbial chassis.

Haloarchaea as Promising Chassis to Green Chemistry.

Climate change and the scarcity of primary resources are driving the development of new, more renewable and environmentally friendly industrial processes. As part of this green chemistry approach, extremozymes (extreme microbial enzymes) can be used to replace all or part of the chemical synthesis stages of traditional industrial processes. At present, the production of these enzymes is limited by the cellular chassis available. The production of a large number of extremozymes requires extremophilic cellular chassis, which are not available. This is particularly true of halophilic extremozymes. The aim of this review is to present the current potential and challenges associated with the development of a haloarchaea-based cellular chassis. By overcoming the major obstacle of the limited number of genetic tools, it will be possible to propose a robust cellular chassis for the production of functional halophilic enzymes that can participate in the industrial transition of many sectors.

Engineering oleaginous red yeasts as versatile chassis for the production of oleochemicals and valuable compounds: Current advances and perspectives.

Enabling the transition towards a future circular bioeconomy based on industrial biomanufacturing necessitates the development of efficient and versatile microbial platforms for sustainable chemical and fuel production. Recently, there has been growing interest in engineering non-model microbes as superior biomanufacturing platforms due to their broad substrate range and high resistance to stress conditions. Among these non-conventional microbes, red yeasts belonging to the genus Rhodotorula have emerged as promising industrial chassis for the production of specialty chemicals such as oleochemicals, organic acids, fatty acid derivatives, terpenoids, and other valuable compounds. Advancements in genetic and metabolic engineering techniques, coupled with systems biology analysis, have significantly enhanced the production capacity of red yeasts. These developments have also expanded the range of substrates and products that can be utilized or synthesized by these yeast species. This review comprehensively examines the current efforts and recent progress made in red yeast research. It encompasses the exploration of available substrates, systems analysis using multi-omics data, establishment of genome-scale models, development of efficient molecular tools, identification of genetic elements, and engineering approaches for the production of various industrially relevant bioproducts. Furthermore, strategies to improve substrate conversion and product formation both with systematic and synthetic biology approaches are discussed, along with future directions and perspectives in improving red yeasts as more versatile biotechnological chassis in contributing to a circular bioeconomy. The review aims to provide insights and directions for further research in this rapidly evolving field. Ultimately, harnessing the capabilities of red yeasts will play a crucial role in paving the way towards next-generation sustainable bioeconomy.

An efficient cre-based workflow for genomic integration and expression of large biosynthetic pathways in Eubacterium limosum.

Acetogenic Clostridia are obligate anaerobes that have emerged as promising microbes for the renewable production of biochemicals owing to their ability to efficiently metabolize sustainable single-carbon feedstocks. Additionally, Clostridia are increasingly recognized for their biosynthetic potential, with recent discoveries of diverse secondary metabolites ranging from antibiotics to pigments to modulators of the human gut microbiota. Lack of efficient methods for genomic integration and expression of large heterologous DNA constructs remains a major challenge in studying biosynthesis in Clostridia and using them for metabolic engineering applications. To overcome this problem, we harnessed chassis-independent recombinase-assisted genome engineering (CRAGE) to develop a workflow for facile integration of large gene clusters (>10 kb) into the human gut acetogen Eubacterium limosum. We then integrated a non-ribosomal peptide synthetase gene cluster from the gut anaerobe Clostridium leptum, which previously produced no detectable product in traditional heterologous hosts. Chromosomal expression in E. limosum without further optimization led to production of phevalin at 2.4 mg/L. These results further expand the molecular toolkit for a highly tractable member of the Clostridia, paving the way for sophisticated pathway engineering efforts, and highlighting the potential of E. limosum as a Clostridial chassis for exploration of anaerobic natural product biosynthesis.

Chassis engineering for high light tolerance in microalgae and cyanobacteria.

Oxygenic photosynthesis in microalgae and cyanobacteria is considered an important chassis to accelerate energy transition and mitigate global warming. Currently, cultivation systems for photosynthetic microbes for large-scale applications encountered excessive light exposure stress. High light stress can: affect photosynthetic efficiency, reduce productivity, limit cell growth, and even cause cell death. Deciphering photoprotection mechanisms and constructing high-light tolerant chassis have been recent research focuses. In this review, we first briefly introduce the self-protection mechanisms of common microalgae and cyanobacteria in response to high light stress. These mechanisms mainly include: avoiding excess light absorption, dissipating excess excitation energy, quenching excessive high-energy electrons, ROS detoxification, and PSII repair. We focus on the species-specific differences in these mechanisms as well as recent advancements. Then, we review engineering strategies for creating high-light tolerant chassis, such as: reducing the size of the light-harvesting antenna, optimizing non-photochemical quenching, optimizing photosynthetic electron transport, and enhancing PSII repair. Finally, we propose a comprehensive exploration of mechanisms: underlying identified high light tolerant chassis, identification of new genes pertinent to high light tolerance using innovative methodologies, harnessing CRISPR systems and artificial intelligence for chassis engineering modification, and introducing plant photoprotection mechanisms as future research directions.

Stepwise increase of fidaxomicin in an engineered heterologous host Streptomyces albus through multi-level metabolic engineering.

The anti-Clostridium difficile infection (CDI) drug fidaxomicin is a natural polyketide metabolite mainly produced by Micromonosporaceae, such as Actinoplanes deccanensis, Dactylosporangium aurantiacum, and Micromonospora echinospora. In the present study, we employed a stepwise strategy by combining heterologous expression, chassis construction, promoter engineering, activator and transporters overexpression, and optimization of fermentation media for high-level production of fidaxomicin. The maximum yield of 384 mg/L fidaxomicin was achieved with engineered Streptomyces albus D7-VHb in 5 L-tank bioreactor, and it was approximately 15-fold higher than the native strain Actinoplanes deccanensis YP-1 with higher strain stability and growth rate. This study developed an enhanced chassis strain, and for the first time, achieved the heterologous synthesis of fidaxomicin through a combinatorial metabolic engineering strategy.

Corrigendum: Genome reduction in Paenibacillus polymyxa DSM 365 for chassis development.

[This corrects the article DOI: 10.3389/fbioe.2024.1378873.].

Engineering xylose induction in Vibrio natriegens for biomanufacturing applications.

Xylose is an abundant, inexpensive and readily available carbohydrate common in minimally processed feedstocks such as seaweed and algae. While a wide variety of marine microbes have evolved to utilize seaweed and algae, only a few currently have the requisite characteristics and genetic engineering tools necessary to entertain the use of these underutilized feedstocks. The rapidly growing Gram-negative halophilic bacterium Vibrio natriegens is one such chassis. In this study, we engineered and tested xylose induction in V. natriegens as a tool for scalable bioproduction applications. First, we created a sensing construct based on the xylose operon from Escherichia coli MG1665 and measured its activity using a fluorescent reporter and identified that cellular import plays a key role in induction strength and that expression required the XylR transcription factor. Next, we identified that select deletions of the promoter region enhance gene expression, limiting the effect of carbohydrate repression when xylose is used as an inducer in the presence of industrially relevant carbon sources. Lastly, we used the optimized constructs to produce the biopolymer melanin using seawater mimetic media. One of these formulations utilized a nori-based seaweed extract as an inducer and demonstrated melanin yields comparable to previously optimized methods using a more traditional and costly inducer. Together, the results demonstrate that engineering xylose induction in V. natriegens can provide an effective and lower cost option for timed biosynthesis in scalable biomanufacturing applications using renewable feedstocks.

Reprogramming naturally evolved switches for Streptomyces chassis development.

The Streptomyces chassis serves as an important platform for efficient biomanufacture of diverse secondary metabolite (SM) compounds, but the current chassis lacks compatibility for integration of these SM biosynthetic pathways reliably and consistently. This forum discusses harnessing naturally evolved multifaceted switches to reprogram the Streptomyces chassis for biomanufacturing applications.

Application of Cyanobacteria as Chassis Cells in Synthetic Biology.

Synthetic biology is an exciting new area of research that combines science and engineering to design and build new biological functions and systems. Predictably, with the development of synthetic biology, more efficient and economical photosynthetic microalgae chassis will be successfully constructed, making it possible to break through laboratory research into large-scale industrial applications. The synthesis of a range of biochemicals has been demonstrated in cyanobacteria; however, low product titers are the biggest barrier to the commercialization of cyanobacterial biotechnology. This review summarizes the applied improvement strategies from the perspectives of cyanobacteria chassis cells and synthetic biology. The harvest advantages of cyanobacterial products and the latest progress in improving production strategies are discussed according to the product status. As cyanobacteria synthetic biology is still in its infancy, apart from the achievements made, the difficulties and challenges in the application and development of cyanobacteria genetic tool kits in biochemical synthesis, environmental monitoring, and remediation were assessed.

Metabolic engineering for compartmentalized biosynthesis of the valuable compounds in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is commonly used as a microbial cell factory to produce high-value compounds or bulk chemicals due to its genetic operability and suitable intracellular physiological environment. The current biosynthesis pathway for targeted products is primarily rewired in the cytosolic compartment. However, the related precursors, enzymes, and cofactors are frequently distributed in various subcellular compartments, which may limit targeted compounds biosynthesis. To overcome above mentioned limitations, the biosynthesis pathways are localized in different subcellular organelles for product biosynthesis. Subcellular compartmentalization in the production of targeted compounds offers several advantages, mainly relieving competition for precursors from side pathways, improving biosynthesis efficiency in confined spaces, and alleviating the cytotoxicity of certain hydrophobic products. In recent years, subcellular compartmentalization in targeted compound biosynthesis has received extensive attention and has met satisfactory expectations. In this review, we summarize the recent advances in the compartmentalized biosynthesis of the valuable compounds in S. cerevisiae, including terpenoids, sterols, alkaloids, organic acids, and fatty alcohols, etc. Additionally, we describe the characteristics and suitability of different organelles for specific compounds, based on the optimization of pathway reconstruction, cofactor supplementation, and the synthesis of key precursors (metabolites). Finally, we discuss the current challenges and strategies in the field of compartmentalized biosynthesis through subcellular engineering, which will facilitate the production of the complex valuable compounds and offer potential solutions to improve product specificity and productivity in industrial processes.

The bioproduction of astaxanthin: A comprehensive review on the microbial synthesis and downstream extraction.

Astaxanthin is a valuable orange-red carotenoid with wide applications in agriculture, food, cosmetics, pharmaceuticals and nutraceuticals areas. At present, the biological synthesis of astaxanthin mainly relies on Haematococcus pluvialis and Xanthophyllomyces dendrorhous. With the rapid development of synthetic biology, more recombinant microbial hosts have been genetically constructed for astaxanthin production including Escherichia coli, Saccharomyces cerevisiae and Yarrowia lipolytica. As multiple genes (15) were involved in the astaxanthin synthesis, it is particularly important to adopt different strategies to balance the metabolic flow towards the astaxanthin synthesis. Furthermore, astaxanthin is a fat-soluble compound stored intracellularly, hence efficient extraction methods are also essential for the economical production of astaxanthin. Several efficient and green extraction methods of astaxanthin have been reported in recent years, including the superfluid extraction, ionic liquid extraction and microwave-assisted extraction. Accordingly, this review will comprehensively introduce the advances on the astaxanthin production and extraction by using different microbial hosts and strategies to improve the astaxanthin synthesis and extraction efficiency.

[Development and characterization of tobacco suspension cell chassis NBS-1].

Plant synthetic biology has significant theoretical advantages in exploration and production of plant natural products. However, its contribution to the field of biosynthesis is currently limited due to the lack of efficient chassis systems and related enabling technologies. Synthetic biologists often avoid tobacco as a chassis system because of its long operation cycle, difficulties in genetic and metabolic modification, complex metabolism and purification background, nicotine toxicity, and challenges in accurately controlling for agricultural production. Nevertheless, the tobacco suspension cell chassis system offers a viable solution to these challenges. The objective of this research was to develop a tobacco suspension cell chassis with high scientific and industrial potential. This chassis should exhibit rapid growth, high biomass, excellent dispersion, high transformation efficiency, and minimal nicotine content. Nicotiana benthamiana, which has high applicability in molecular technology, was used to induce suspension cells. The induced suspension cells, named NBS-1, exhibited rapid growth, excellent dispersion, and high biomass, reaching a maximum biomass of 476.39 g/L (fresh weight), which was significantly higher than that of BY-2. The transformation efficiency of the widely utilized pEAQ-HT transient expression system in NBS-1 reached 81%, which was substantially elevated compared to BY-2. The metabolic characteristics and bias of BY-2 and NBS-1 were analyzed using transcriptome data. It was found that the gene expression of pathways related to biosynthesis of flavonoids and their derivatives in NBS-1 was significantly higher, while the pathways related to alkaloid biosynthesis were significantly lower compared to BY-2. These findings were further validated by the total content of flavonoid and alkaloid. In summary, our research demonstrates NBS-1 possesses minimal nicotine content and provides valuable guidance for selecting appropriate chassis for specific products. In conclusion, this study developed NBS-1, a tobacco suspension cell chassis with excellent growth and transformation, high flavonoid content and minimal nicotine content, which has important guiding significance for the development of tobacco suspension cell chassis.

The elimination of two restriction enzyme genes allows for electroporation-based transformation and CRISPR-Cas9-based base editing in the non-competent Gram-negative bacterium Acinetobacter sp. Tol 5.

Environmental isolates are promising candidates for new chassis of synthetic biology because of their inherent capabilities, which include efficiently converting a wide range of substrates into valuable products and resilience to environmental stresses; however, many remain genetically intractable and unamenable to established genetic tools tailored for model bacteria. Acinetobacter sp. Tol 5, an environmentally isolated Gram-negative bacterium, possesses intriguing properties for use in synthetic biology applications. Despite the previous development of genetic tools for the engineering of strain Tol 5, its genetic manipulation has been hindered by low transformation efficiency via electroporation, rendering the process laborious and time-consuming. This study demonstrated the genetic refinement of the Tol 5 strain, achieving efficient transformation via electroporation. We deleted two genes encoding type I and type III restriction enzymes. The resulting mutant strain not only exhibited marked efficiency of electrotransformation but also proved receptive to both in vitro and in vivo DNA assembly technologies, thereby facilitating the construction of recombinant DNA without reliance on intermediate Escherichia coli constructs. In addition, we successfully adapted a CRISPR-Cas9-based base-editing platform developed for other Acinetobacter species. Our findings provide genetic modification strategies that allow for the domestication of environmentally isolated bacteria, streamlining their utilization in synthetic biology applications.IMPORTANCERecent synthetic biology has sought diverse bacterial chassis from environmental sources to circumvent the limitations of laboratory Escherichia coli strains for industrial and environmental applications. One of the critical barriers in cell engineering of bacterial chassis is their inherent resistance to recombinant DNA, propagated either in vitro or within E. coli cells. Environmental bacteria have evolved defense mechanisms against foreign DNA as a response to the constant threat of phage infection. The ubiquity of phages in natural settings accounts for the genetic intractability of environmental isolates. The significance of our research is in demonstrating genetic modification strategies for the cell engineering of such genetically intractable bacteria. This research marks a pivotal step in the domestication of environmentally isolated bacteria, promising candidates for emerging synthetic biology chassis. Our work thus significantly contributes to advancing their applications across industrial, environmental, and biomedical fields.

Rational construction of genome-minimized Streptomyces host for the expression of secondary metabolite gene clusters.

Streptomyces offer a wealth of naturally occurring compounds with diverse structures, many of which possess significant pharmaceutical values. However, new product exploration and increased yield of specific compounds in Streptomyces have been technically challenging due to their slow growth rate, complex culture conditions and intricate genetic backgrounds. In this study, we screened dozens of Streptomyces strains inhabiting in a plant rhizosphere for fast-growing candidates, and further employed CRISPR/Cas-based engineering techniques for stepwise refinement of a particular strain, Streptomyces sp. A-14 that harbors a 7.47 Mb genome. After strategic removal of nonessential genomic regions and most gene clusters, we reduced its genome size to 6.13 Mb, while preserving its growth rate to the greatest extent. We further demonstrated that cleaner metabolic background of this engineered strain was well suited for the expression and characterization of heterologous gene clusters, including the biosynthetic pathways of actinorhodin and polycyclic tetramate macrolactams. Moreover, this streamlined genome is anticipated to facilitate directing the metabolic flux towards the production of desired compounds and increasing their yields.

Genome reduction in Paenibacillus polymyxa DSM 365 for chassis development.

The demand for highly robust and metabolically versatile microbes is of utmost importance for replacing fossil-based processes with biotechnological ones. Such an example is the implementation of Paenibacillus polymyxa DSM 365 as a novel platform organism for the production of value-added products such as 2,3-butanediol or exopolysaccharides. For this, a complete genome sequence is the first requirement towards further developing this host towards a microbial chassis. A genome sequencing project has just been reported for P. polymyxa DSM 365 showing a size of 5,788,318 bp with a total of 47 contigs. Herein, we report the first complete genome sequence of P. polymyxa DSM 365, which consists of 5,889,536 bp with 45 RNAs, 106 tRNAs, 5,370 coding sequences and an average GC content of 45.6%, resulting in a closed genome of P. polymyxa 365. The additional nucleotide data revealed a novel NRPS synthetase that may contribute to the production of tridecaptin. Building on these findings, we initiated the top-down construction of a chassis variant of P. polymyxa. In the first stage, single knock-out mutants of non-essential genomic regions were created and evaluated for their biological fitness. As a result, two out of 18 variants showed impaired growth. The remaining deletion mutants were combined in two genome-reduced P. polymyxa variants which either lack the production of endogenous biosynthetic gene clusters (GR1) or non-essential genomic regions including the insertion sequence ISPap1 (GR2), with a decrease of the native genome of 3.0% and 0.6%, respectively. Both variants, GR1 and GR2, showed identical growth characteristics to the wild-type. Endpoint titers of 2,3-butanediol and EPS production were also unaffected, validating these genome-reduced strains as suitable for further genetic engineering.