Development of a CRISPR/Cas9 system for high efficiency multiplexed gene deletion in Rhodosporidium toruloides

The oleaginous yeast Rhodosporidium toruloides is considered a promising candidate for production of chemicals and biofuels thanks to its ability to grow on lignocellulosic biomass, and its high production of lipids and carotenoids. However, efforts to engineer this organism are hindered by a lack of suitable genetic tools. Here we report the development of a CRISPR/Cas9 system for genome editing in R. toruloides based on a fusion 5S rRNA–tRNA promoter for guide RNA (gRNA) expression, capable of greater than 95% gene knockout for various genetic targets. Additionally, multiplexed double-gene knockout mutants were obtained using this method with an efficiency of 78%. This tool can be used to accelerate future metabolic engineering work in this yeast.     

Microbial interventions are an easier alternative to engineer higher organisms

Advances in synthetic biology have made microbes easier to engineer than ever before. However, synthetic biology in animals and plants has lagged behind. Since it is now known that the phenotype of higher organisms depends largely on their microbiota, we propose that this is an easier route to achieving synthetic biology applications in these organisms.

A transition from reading to writing biology has blurred the lines between basic science and engineering creating the field of synthetic biology. With an ever‐expanding genetic toolbox, we now manipulate natural biological systems to optimize our anthropocentric activities. From the synthesis of complex aromatic compounds, to the production of safer vaccines, a problem identified may find its solution lying in the metabolism of a single cell. Initially, synthetic biology was largely focused on the production of such commodities at the industrial scale, not only to maximize profitability, but also to minimize energy and resource consumption. Consequently, this paradigm shift has come to alter the notion of a factory by many orders of magnitude and to create a new bridge between the built and natural world, as we employ nature’s evolutionary machinery to address our modern endeavours.Growth of the genetic toolbox and maturation of synthetic biology as a field has led to speculation about increasingly ambitious applications of writing biology with implications beyond biosynthesis. To date, most applications have been developed using microbes because they are less complex, more well understood and easier to manipulate. Single‐celled organisms can be optimized for production of complicated organic molecules; however, other exploits of genetic engineering will target more ambitious feats and thus require engineering of more than a large monoculture of microbes. Applications of synthetic biology outside of the bioreactor can address such issues as health and longevity, challenges in industrial agriculture and farming, the degradation of natural habitats and the reclamation of limited natural resources.Scope and scale of these applications provide obvious obstacles to the development of effective biotechnologies, but a more immediate limitation to realizing these technologies is the relative lack of genetic tools and insights which would allow the tinkering and rewiring of more complex organisms such as animals and plants. However, because of the natural intimate interactions between higher eukaryotes and microbes and the effect of these on phenotype, it is our vision that a faster, more tractable route to the engineering animal and plant phenotypes is via engineering their microbiomes.     

Current state of coenzyme Q10 production and its applications

Coenzyme Q₁₀ (CoQ₁₀), an obligatory cofactor in the aerobic respiratory electron transfer for energy generation, is formed from the conjugation of a benzoquinone ring with a hydrophobic isoprenoid chain. CoQ₁₀ is now used as a nutritional supplement because of its antioxidant properties and is beneficial in the treatment of several human diseases when administered orally. Bioprocesses have been developed for the commercial production of CoQ₁₀ because of its increased demand, and these bioprocesses depend on microbes that produce high levels of CoQ₁₀ naturally. However, as knowledge of the biosynthetic enzymes and the regulatory mechanisms modulating CoQ₁₀ production increases, approaches arise for the genetic engineering of CoQ₁₀ production in Escherichia coli and Agrobacterium tumefaciens. This review focused on approaches for CoQ₁₀ production, strategies used to engineer CoQ₁₀ production in microbes, and potential applications of CoQ₁₀.     

Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways

Gas fermentation by autotrophic bacteria, such as clostridia, offers a sustainable path to numerous bioproducts from a range of local, highly abundant, waste and low-cost feedstocks, such as industrial flue gases or syngas generated from biomass or municipal waste. Unfortunately, designing and engineering clostridia remains laborious and slow. The ability to prototype individual genetic part function, gene expression patterns, and biosynthetic pathway performance in vitro before implementing designs in cells could help address these bottlenecks by speeding up design. Unfortunately, a high-yielding cell-free gene expression (CFE) system from clostridia has yet to be developed. Here, we report the development and optimization of a high-yielding (236 ± 24 μg/mL) batch CFE platform from the industrially relevant anaerobe, Clostridium autoethanogenum. A key feature of the platform is that both circular and linear DNA templates can be applied directly to the CFE reaction to program protein synthesis. We demonstrate the ability to prototype gene expression, and quantitatively map aerobic cell-free metabolism in lysates from this system. We anticipate that the C. autoethanogenum CFE platform will not only expand the protein synthesis toolkit for synthetic biology, but also serve as a platform in expediting the screening and prototyping of gene regulatory elements in non-model, industrially relevant microbes.     

Biofuel production in Escherichia coli: the role of metabolic engineering and synthetic biology

The microbial production of biofuels is a promising avenue for the development of viable processes for the generation of fuels from sustainable resources. In order to become cost and energy effective, these processes must utilize organisms that can be optimized to efficiently produce candidate fuels from a variety of feedstocks. Escherichia coli has become a promising host organism for the microbial production of biofuels in part due to the ease at which this organism can be manipulated. Advancements in metabolic engineering and synthetic biology have led to the ability to efficiently engineer E. coli as a biocatalyst for the production of a wide variety of potential biofuels from several biomass constituents. This review focuses on recent efforts devoted to engineering E. coli for the production of biofuels, with emphasis on the key aspects of both the utilization of a variety of substrates as well as the synthesis of several promising biofuels. Strategies for the efficient utilization of carbohydrates, carbohydrate mixtures, and noncarbohydrate carbon sources will be discussed along with engineering efforts for the exploitation of both fermentative and nonfermentative pathways for the production of candidate biofuels such as alcohols and higher carbon biofuels derived from fatty acid and isoprenoid pathways. Continued advancements in metabolic engineering and synthetic biology will help improve not only the titers, yields, and productivities of biofuels discussed herein, but also increase the potential range of compounds that can be produced.     

Systems metabolic engineering design: Fatty acid production as an emerging case study

Increasing demand for petroleum has stimulated industry to develop sustainable production of chemicals and biofuels using microbial cell factories. Fatty acids of chain lengths from C₆ to C₁₆ are propitious intermediates for the catalytic synthesis of industrial chemicals and diesel‐like biofuels. The abundance of genetic information available for Escherichia coli and specifically, fatty acid metabolism in E. coli, supports this bacterium as a promising host for engineering a biocatalyst for the microbial production of fatty acids. Recent successes rooted in different features of systems metabolic engineering in the strain design of high‐yielding medium chain fatty acid producing E. coli strains provide an emerging case study of design methods for effective strain design. Classical metabolic engineering and synthetic biology approaches enabled different and distinct design paths towards a high‐yielding strain. Here we highlight a rational strain design process in systems biology, an integrated computational and experimental approach for carboxylic acid production, as an alternative method. Additional challenges inherent in achieving an optimal strain for commercialization of medium chain‐length fatty acids will likely require a collection of strategies from systems metabolic engineering. Not only will the continued advancement in systems metabolic engineering result in these highly productive strains more quickly, this knowledge will extend more rapidly the carboxylic acid platform to the microbial production of carboxylic acids with alternate chain‐lengths and functionalities. Biotechnol. Biotechnol. Bioeng. 2014;111: 849–857. © 2014 Wiley Periodicals, Inc.     

Engineering biosynthesis of high-value compounds in photosynthetic organisms

The photosynthetic, autotrophic lifestyle of plants and algae position them as ideal platform organisms for sustainable production of biomolecules. However, their use in industrial biotechnology is limited in comparison to heterotrophic organisms, such as bacteria and yeast. This usage gap is in part due to the challenges in generating genetically modified plants and algae and in part due to the difficulty in the development of synthetic biology tools for manipulating gene expression in these systems. Plant and algal metabolism, pre-installed with multiple biosynthetic modules for precursor compounds, bypasses the requirement to install these pathways in conventional production organisms, and creates new opportunities for the industrial production of complex molecules. This review provides a broad overview of the successes, challenges and future prospects for genetic engineering in plants and algae for enhanced or de novo production of biomolecules. The toolbox of technologies and strategies that have been used to engineer metabolism are discussed, and the potential use of engineered plants for industrial manufacturing of large quantities of high-value compounds is explored. This review also discusses the routes that have been taken to modify the profiles of primary metabolites for increasing the nutritional quality of foods as well as the production of specialized metabolites, cosmetics, pharmaceuticals and industrial chemicals. As the universe of high-value biosynthetic pathways continues to expand, and the tools to engineer these pathways continue to develop, it is likely plants and algae will become increasingly valuable for the biomanufacturing of high-value compounds.     

Plant artificial chromosome technology and its potential application in genetic engineering

Genetic engineering with just a few genes has changed agriculture in the last 20 years. The most frequently used transgenes are the herbicide resistance genes for efficient weed control and the Bt toxin genes for insect resistance. The adoption of the first‐generation genetically engineered crops has been very successful in improving farming practices, reducing the application of pesticides that are harmful to both human health and the environment, and producing more profit for farmers. However, there is more potential for genetic engineering to be realized by technical advances. The recent development of plant artificial chromosome technology provides a super vector platform, which allows the management of a large number of genes for the next generation of genetic engineering. With the development of other tools such as gene assembly, genome editing, gene targeting and chromosome delivery systems, it should become possible to engineer crops with multiple genes to produce more agricultural products with less input of natural resources to meet future demands.     

Use of designer nucleases for targeted gene and genome editing in plants

The ability to efficiently inactivate or replace genes in model organisms allowed a rapid expansion of our understanding of many of the genetic, biochemical, molecular and cellular mechanisms that support life. With the advent of new techniques for manipulating genes and genomes that are applicable not only to single‐celled organisms, but also to more complex organisms such as animals and plants, the speed with which scientists and biotechnologists can expand fundamental knowledge and apply that knowledge to improvements in medicine, industry and agriculture is set to expand in an exponential fashion. At the heart of these advancements will be the use of gene editing tools such as zinc finger nucleases, modified meganucleases, hybrid DNA/RNA oligonucleotides, TAL effector nucleases and modified CRISPR/Cas9. Each of these tools has the ability to precisely target one specific DNA sequence within a genome and (except for DNA/RNA oligonucleotides) to create a double‐stranded DNA break. DNA repair to such breaks sometimes leads to gene knockouts or gene replacement by homologous recombination if exogenously supplied homologous DNA fragments are made available. Genome rearrangements are also possible to engineer. Creation and use of such genome rearrangements, gene knockouts and gene replacements by the plant science community is gaining significant momentum. To document some of this progress and to explore the technology's longer term potential, this review highlights present and future uses of designer nucleases to greatly expedite research with model plant systems and to engineer genes and genomes in major and minor crop species for enhanced food production.     

Establishing microbial co‐cultures for 3‐hydroxybenzoic acid biosynthesis on glycerol

Converting renewable feedstocks to aromatic compounds using engineered microbes offers a robust approach for sustainable, environment‐friendly, and cost‐effective production of these value‐added products without the reliance on petroleum. In this study, rationally designed E. coli–E. coli co‐culture systems were established for converting glycerol to 3‐hydroxybenzoic acid (3HB). Specifically, the 3HB pathway was modularized and accommodated by two metabolically engineered E. coli strains. The co‐culture biosynthesis was optimized by using different cultivation temperatures, varying the inoculum ratio between the co‐culture strains, recruitment of a key pathway intermediate transporter, strengthening the critical pathway enzyme expression, and adjusting the timing for inducing pathway gene expression. Compared with the E. coli mono‐culture, the optimized co‐culture showed 5.3‐fold improvement for 3HB biosynthesis. This study demonstrated the applicability of modular co‐culture engineering for addressing the challenges of aromatic compound biosynthesis.     

Synthetic conversion of a graded receptor signal into a tunable,reversible switch

The ability to engineer an all‐or‐none cellular response to a given signaling ligand is important in applications ranging from biosensing to tissue engineering. However, synthetic gene network ‘switches’ have been limited in their applicability and tunability due to their reliance on specific components to function. Here, we present a strategy for reversible switch design that instead relies only on a robust, easily constructed network topology with two positive feedback loops and we apply the method to create highly ultrasensitive (n_H>20), bistable cellular responses to a synthetic ligand/receptor complex. Independent modulation of the two feedback strengths enables rational tuning and some decoupling of steady‐state (ultrasensitivity, signal amplitude, switching threshold, and bistability) and kinetic (rates of system activation and deactivation) response properties. Our integrated computational and synthetic biology approach elucidates design rules for building cellular switches with desired properties, which may be of utility in engineering signal‐transduction pathways.     

Transposon tagging in maize

Through recent government- and industry-sponsored efforts, several forward and reverse genetic screening programs have emerged over the past few years to aid in the genetic dissection of gene function in maize. Despite a US maize crop valued at $18.4 billion last year (http://www.ncga.com/03world/main/US_crop_value_2000.html) and rich genetic history, maize has taken a back seat to Arabidopsis thaliana as the model genetic system for plants over the past decade. With a fully sequenced genome, short generation time and small size, studies of Arabidopsis have provided plant scientists with a molecular framework for hormonal, developmental and environmental signaling pathways in plants. As investigations into Arabidopsis continue, our capacity to engineer biochemical pathways and alter plant physiological responses will become increasingly sophisticated. Nevertheless, approximately 130 million years have passed since monocot and higher eudicot lineages diverged. Thus, our ability to engineer agronomically important monocot grasses such as maize, rice and wheat will become increasingly limited by our lack of understanding of the physiological and morphological differences that have evolved in the monocots and higher eudicots. The sophisticated transposon collections now being generated for maize are but one of several recent projects (http://www.nsf.gov/bio/pubs/awards/genome01.htm) to provide grass researchers with essential tools for genome analysis. Because grain crops are such a closely related group, it is hoped that many of the findings made in one grass will be directly applicable to understanding the biology of another. The goal of this review is to highlight the recent developments in maize transposon-based gene characterization programs and provide a critical examination of the advantages and disadvantages each system offers. Electronic Publication     

An integrated approach: advances in the use of Clostridium for biofuel

Almost 90% of our energy comes from fossil fuels, which are both limited and polluting, hence the need to find alternative sources. Biofuels can provide a sustainable and renewable source of energy for the future. Recent significant advances in genetic engineering and fermentation technology have made microbial bio-based production of chemicals from renewable resources more viable. Clostridium species are considered as promising micro-organisms for the production of a wide range of chemicals for industrial use. However, a number of scientific challenges still need to be overcome to facilitate an economically viable production system. These include the use of cheap non-food-based substrates, a better understanding of the metabolic processes involved, improvement of strains through genetic engineering and innovation in process technology. This paper reviews recent developments in these areas, advancing the use of Clostridium within an industrial context especially for the production of biofuels.     

Metabolic design for cyanobacterial chemical synthesis

Photosynthetic chemical production in cyanobacteria is a promising technology for renewable energy, CO₂ mitigation, and fossil fuel replacement. Metabolic engineering has enabled a direct biosynthetic process from CO₂ fixation to chemical feedstocks and biofuels, without requiring costly production, storage, and breakdown of cellulose or sugars. However, direct production technology is challenged by a need to achieve high-carbon partitioning to products in order to be competitive. This review discusses principles for the design of biosynthetic pathways in cyanobacteria and describes recent advances in relevant genetic tools. This field is at a critical juncture in assessing the strength and feasibility of carbon partitioning. To address this, we have included the stoichiometry of reducing equivalents and carbon conservation for heterologous pathways, and a method for calculating product yields against a theoretical maximum.     

Recombineering facilitates the discovery of natural product biosynthetic pathways in Pseudomonas parafulva

Microbial natural products among other functions they play a vital role in the disease prevention in humans, animals and plants. Pseudomonas parafulva CRS01-1 is a broad-spectrum antagonistic bacterium present in plants. However, no natural products have been isolated from this strain till date. Corresponding biosynthetic gene clusters to natural products is an effective method for bioprospecting, for which, genome manipulation tools are essential. We previously developed Pseudomonas-specific phage-derived homologous recombination systems for genetic engineering in four Pseudomonas species. Herein, we report the application of these recombineering systems in Pseudomonas parafulva CRS01-1, along with structural elucidation and bioactivity evaluation of natural products. The Pseudomonas recombineering toolbox established before in different four species is efficient for genome mining and bioactive metabolite discovery from more distant species.     

Production of fuels and chemicals from renewable resources using engineered Escherichia coli

Biotechnological production of fuels and chemicals from renewable resources is an appealing way to move from the current petroleum-based economy to a biomass-based green economy. Recently, the feedstocks that can be used for bioconversion or fermentation have been expanded to plant biomass, microbial biomass, and industrial waste. Several microbes have been engineered to produce chemicals from renewable resources, among which Escherichia coli is one of the best studied. Much effort has been made to engineer E. coli to produce fuels and chemicals from different renewable resources. In this paper, we focused on E. coli and systematically reviewed a range of fuels and chemicals that can be produced from renewable resources by engineered E. coli. Moreover, we proposed how can we further improve the efficiency for utilizing renewable resources by engineered E. coli, and how can we engineer E. coli for utilizing alternative renewable feedstocks. e.g. C1 gases and methanol. This review will help the readers better understand the current progress in this field and provide insights for further metabolic engineering efforts in E. coli.     

毕赤酵母细胞工厂工程化改造与应用

毕赤酵母作为细胞工厂在小分子代谢产物发酵和蛋白制品生物合成中扮演着重要角色，具有极其重要的工业应用价值。随着CRISPR/Cas9等新型编辑工具的开发和应用，对毕赤酵母细胞工厂进行多基因高效率的工程化改造已成为可能。本文首先对毕赤酵母工程化改造的遗传操作技术和目标方向进行了归纳总结，其次介绍了毕赤酵母作为细胞工厂的应用现状，同时探讨了毕赤酵母细胞工厂的优点及缺陷，并对其发展方向作出展望；以期为未来的毕赤酵母工程化改造研究提供参考和启示，推动毕赤酵母细胞工厂在生物产业中的创新应用。     

L-lactate production in engineered Saccharomyces cerevisiae using a multistage multiobjective automated design framework

The design of alternative biodegradable polymers has the potential of severely reducing the environmental impact, cost and production time currently associated with the petrochemical industry. In fact, growing demand for renewable feedstock has recently brought to the fore synthetic biology and metabolic engineering. These two interdependent research areas focus on the study of microbial conversion of organic acids, with the aim of replacing their petrochemical-derived equivalents with more sustainable and efficient processes. The particular case of Lactic acid (LA) production has been the subject of extensive research because of its role as an essential component for developing an eco-friendly biodegradable plastic—widely used in industrial biotechnological applications. Because of its resistance to acidic environments, among the many LA-producing microbes, Saccharomyces cerevisiae has been the main focus of research into related biocatalysts. In this study, we present an extensive in silico investigation of S. cerevisiae cell metabolism (modeled with Flux Balance Analysis) with the overall aim of maximizing its LA production yield. We focus on the yeast 8.3 steady-state metabolic model and analyze it under the impact of different engineering strategies including: gene knock-in, gene knock-out, gene regulation and medium optimization; as well as a comparison between results in aerobic and anaerobic conditions. We designed ad-hoc constrained multiobjective evolutionary algorithms to automate the engineering process and developed a specific postprocessing methodology to analyze the genetic manipulation results obtained. The in silico results reported in this paper empirically show that our method is able to automatically select a small number of promising genetic and metabolic manipulations, deriving competitive strains that promise to impact microorganisms design in the production of sustainable chemicals.     

Study of biodiversity of <Emphasis Type="Italic">Klebsiella </Emphasis>sp.

Microorganisms provide the largest gamut of genetic and physiological diversity in nature. The determination of the biodiversity of microbes is necessary for the exploitation of their inestimable population, which remains unexplored in the environment. In our study, we have isolated 20 different Klebsiella sp. from contaminated soil and effluent treatment plants and determined their genetic diversity using molecular tools. The phylogenetic result based on 16S rDNA sequencing, shows genetic variation among the strains, which was further confirmed by Randomly Amplified Polymorphic DNA (RAPD) analysis. The occurrence of a broad spectrum of Klebsiella sp. variants in polluted and stress environments portrays the ability of this genus to survive abundantly under stress conditions.     

Rapid and highly efficient genomic engineering with a novel iEditing device for programming versatile extracellular electron transfer of electroactive bacteria

The advances in synthetic biology bring exciting new opportunities to reprogram microorganisms with novel functionalities for environmental applications. For real-world applications, a genetic tool that enables genetic engineering in a stably genomic inherited manner is greatly desired. In this work, we design a novel genetic device for rapid and efficient genome engineering based on the i ntron-encoded homing-endonuclease empowered genome editing (iEditing). The iEditing device enables rapid and efficient genome engineering in Shewanella oneidensis MR-1, the representative strain of the electroactive bacteria group. Moreover, combining with the Red or RecET recombination system, the genome-editing efficiency was greatly improved, up to approximately 100%. Significantly, the iEditing device itself is eliminated simultaneously when genome editing occurs, thereby requiring no follow-up to remove the encoding system. Then, we develop a new extracellular electron transfer (EET) engineering strategy by programming the parallel EET systems to enhance versatile EET. The engineered strains exhibit sufficiently enhanced electron output and pollutant reduction ability. Furthermore, this device has demonstrated its great potential to be extended for genome editing in other important microbes. This work provides a useful and efficient tool for the rapid generation of synthetic microorganisms for various environmental applications.