Synthetic Biology Toolkits for Metabolic Engineering of Cyanobacteria

Cyanobacteria are of great importance to Earth's ecology. Due to their capability in photosynthesis and C1 metabolism, they are ideal microbial chassis that can be engineered for direct conversion of carbon dioxide and solar energy into biofuels and biochemicals. Facilitated by the elucidation of the basic biology of the photoautotrophic microbes and rapid advances in synthetic biology, genetic toolkits have been developed to enable implementation of nonnatural functionalities in engineered cyanobacteria. Hence, cyanobacteria are fast becoming an emerging platform in synthetic biology and metabolic engineering. Herein, the progress made in the synthetic biology toolkits for cyanobacteria and their utilization for transforming cyanobacteria into microbial cell factories for sustainable production of biofuels and biochemicals is outlined. Current techniques in heterologous gene expression, strategies in genome editing, and development of programmable regulatory parts and modules for engineering cyanobacteria towards biochemical production are discussed and prospected. As cyanobacteria synthetic biology is still in its infancy, apart from the achievements made, the difficulties and challenges in applying and developing genetic toolkits in cyanobacteria for biochemical production are also evaluated.     

Introduction of NADH-dependent nitrate assimilation in Synechococcus sp. PCC 7002 improves photosynthetic production of 2-methyl-1-butanol and isobutanol

Cyanobacteria hold promise for renewable chemical production due to their photosynthetic nature, but engineered strains frequently display poor production characteristics. These difficulties likely arise in part due to the distinctive photoautotrophic metabolism of cyanobacteria. In this work, we apply a genome-scale metabolic model of the cyanobacteria Synechococus sp. PCC 7002 to identify strain designs accounting for this unique metabolism that are predicted to improve the production of various biofuel alcohols (e.g. 2-methyl-1-butanol, isobutanol, and 1-butanol) synthesized via an engineered biosynthesis pathway. Using the model, we identify that the introduction of a large, non-native NADH-demand into PCC 7002's metabolic network is predicted to enhance production of these alcohols by promoting NADH-generating reactions upstream of the production pathways. To test this, we construct strains of PCC 7002 that utilize a heterologous, NADH-dependent nitrite reductase in place of the native, ferredoxin-dependent enzyme to create an NADH-demand in the cells when grown on nitrate-containing media. We find that photosynthetic production of both isobutanol and 2-methyl-1-butanol is significantly improved in the engineered strain background relative to that in a wild-type background. We additionally identify that the use of high-nutrient media leads to a substantial prolongment of the production curve in our alcohol production strains. The metabolic engineering strategy identified and tested in this work presents a novel approach to engineer cyanobacterial production strains that takes advantage of a unique aspect of their metabolism and serves as a basis on which to further develop strains with improved production of these alcohols and related products.     

Towards systems metabolic engineering of microorganisms for amino acid production

Microorganisms capable of efficient production of amino acids have traditionally been developed by random mutation and selection method, which might cause unwanted physiological changes in cellular metabolism. Rational genome-wide metabolic engineering based on systems and synthetic biology tools, which is termed 'systems metabolic engineering', is rising as an alternative to overcome these problems. Recently, several amino acid producers have been successfully developed by systems metabolic engineering, where the metabolic engineering procedures were performed within a systems biology framework, and entire metabolic networks, including complex regulatory circuits, were engineered in an integrated manner. Here we review the current status of systems metabolic engineering successfully applied for developing amino acid producing strains and discuss future prospects.     

Cyanobacteria as an eco-friendly resource for biofuel production: A critical review

Cyanobacteria are photosynthetic microorganisms which can be found in various environmental habitats. These photosynthetic bacteria are considered as promising feedstock for the production of the third- and the fourth-generation biofuels. The main subject of this review is highlighting the significant aspects of the biofuel production from cyanobacteria. The most recent investigations about the extraction or separation of the bio-oil from cyanobacteria are also adduced in the present review. Moreover, the genetic engineering of cyanobacteria for improving biofuel production and the impact of bioinformatics studies on the designing better-engineered strains are mentioned. The large-scale biofuel production is challenging, so the economic considerations to provide inexpensive biofuels are also cited. It seems that the future of biofuels is strongly dependent to the following items; understanding the metabolic pathways of the cyanobacterial species, progression in the construction of the engineered cyanobacteria, and inexpensive large-scale cultivation of them.     

Engineering microbes for isoprene production

Isoprene is facing a growing global market due to its wide industrial applications. Current industrial production of isoprene is almost entirely petroleum-based, which is influenced by the shrinking C5 supply, while the natural emission of isoprene is predominantly contributed by plants. To bridge the need gap, a highly efficient fermentation-based process for isoprene production might be a suitable and sustainable solution, and extensive research works have been performed to achieve this goal. Here we review the accomplishments in this field by summarizing the history and prospects of microbial isoprene production. The natural producers and biosynthesis pathways of isoprene, the key enzyme isoprene synthase and the metabolic engineering strategies adopted for developing isoprene-producing microorganisms are introduced. In particular, strategies employed for achieving engineered strains with improved performance indices are discussed based on the published papers and patents. The perspectives on further performance improvements and potential future strategies are presented as well.     

Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products

Metabolic engineering of cyanobacteria has the advantage that sunlight and CO₂ are the sole source of energy and carbon for these organisms. However, as photoautotrophs, cyanobacteria generally lack transporters to move hydrophilic primary metabolites across membranes. To address whether cyanobacteria could be engineered to produce and secrete organic primary metabolites, Synechococcus elongatus PCC7942 was engineered to express genes encoding an invertase and a glucose facilitator, which mediated secretion of glucose and fructose. Similarly, expression of lactate dehydrogenase- and lactate transporter-encoding genes allowed lactate accumulation in the extracellular medium. Expression of the relevant transporter was essential for secretion. Production of these molecules was further improved by expression of additional heterologous enzymes. Sugars secreted by the engineered cyanobacteria could be used to support Escherichia coli growth in the absence of additional nutrient sources. These results indicate that cyanobacteria can be engineered to produce and secrete high-value hydrophilic products.Metabolic engineering of photosynthetic microbes is attractive because of the efficient use of light energy by these organisms and the potential for CO₂ mitigation during production (21). Conventional terrestrial plants capture solar energy at low efficiencies (about 0.1 to 0.25% for corn and up to 1% for switchgrass), while fast-growing prokaryotic and eukaryotic microalgal species are about 1 order of magnitude more productive and their photosynthetic efficiencies can be >10% (12, 13). Genetic tools for engineering cyanobacterial species, including Synechococcus elongatus PCC7942 (Synechococcus), can be applied to metabolic engineering (7). For example, Deng and Coleman (8) expressed pyruvate decarboxylase and alcohol dehydrogenase in cyanobacteria to produce small amounts of ethanol, and Atsumi et al. recently described efficient synthesis of isobutanol using a four-step pathway established in Escherichia coli (2).Much attention has been focused on metabolic engineering to produce fuels. However, fuel molecules are generally toxic to microbes even at moderate concentrations. In addition, on a per-photon basis, the actual market value of fuels is at best comparable to, and generally lower than, the market value of other commodity organic compounds, such as sugars, lactic acid, and amino acids. Engineering cyanobacteria to produce and secrete hydrophilic or charged molecules would thus be economically desirable.Commonly used metabolic engineering organisms, such as E. coli and yeast (e.g., Saccharomyces cerevisiae), express a variety of transport systems for exporting waste products as well as importing nutrients. As photoautotrophs, cyanobacteria lack many of the transporters found in these organisms. In addition, while most microbes store energy by pumping protons across the plasma membrane, cyanobacteria store energy by transporting protons across the thylakoid membrane. In fact, cyanobacteria tend to alkalinize their growth medium in both laboratory and natural conditions (4), and thus how effective heterologous transporters can be in metabolic engineering of cyanobacteria is an open question. Here, we investigated whether heterologous transporters belonging to the major facilitator superfamily, in combination with relevant enzymes, could be introduced into cyanobacteria for production and secretion of useful products.     

蓝细菌光驱固碳合成蔗糖技术的发展与展望

生物炼制技术体系是缓解能源和环境危机,推动社会可持续发展的重要选择,而充足的糖原料供应是生物炼制的基础。蓝细菌光驱固碳合成蔗糖是一种潜力巨大的新型糖原料供应路线。基于高效的蓝细菌光驱固碳细胞工厂,可以在单平台上以太阳能为驱动将二氧化碳和水直接转化为蔗糖,过程简单、产品明确、易于提取,而且可以同时达到固碳减排和供应糖原料的效果,具有重要的研究和应用价值。本文回顾了蓝细菌光驱固碳合成蔗糖技术的发展现状,从合成机制、代谢工程策略、技术延伸应用等层面对其最新进展和所遇到的问题进行了总结介绍,并对该技术未来发展方向进行了展望。     

Three-dimensional structures of enzymes useful for beta-lactam antibiotic production

Significant advances have been made in the structure-based engineering of enzymes useful for beta-lactam antibiotic production. Structure-based engineering of penicillin G acylase and cephalosporin acylase has resulted in improved enzymes for use in enzymatic production processes. The structures of many other enzymes that could be used in the production of beta-lactam antibiotics, such as enzymes from the beta-lactam biosynthetic pathway and beta-lactam antibiotic-converting enzymes, have been determined. The interest in these structures suggests that the future may see an even more extensive use of rationally engineered biocatalysts in antibiotic production than today.     

Molecular genetic improvements of cyanobacteria to enhance the industrial potential of the microbe: A review

The rapid increase in worldwide population coupled with the increasing demand for fossil fuels has led to an increased urgency to develop sustainable sources of energy and chemicals from renewable resources. Using microorganisms to produce high‐value chemicals and next‐generation biofuels is one sustainable option and is the focus of much current research. Cyanobacteria are ideal platform organisms for chemical and biofuel production because they can be genetically engineered to produce a broad range of products directly from CO₂, H₂O, and sunlight, and require minimal nutrient inputs. The purpose of this review is to provide an overview on advances that have been or could be made to improve strains of cyanobacteria for industrial purposes. First, the benefits of using cyanobacteria as a platform for chemical and biofuel production are discussed. Next, an overview of cyanobacterial strain improvements by genetic engineering is provided. Finally, mutagenesis techniques to improve the industrial potential of cyanobacteria are described. Along with providing an overview on various areas of research that are currently being investigated to improve the industrial potential of cyanobacteria, this review aims to elucidate potential targets for future research involving cyanobacteria as an industrial microorganism. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1357–1371, 2016     

蓝细菌乙醇光合细胞工厂的发展与展望

生物乙醇是极具应用潜力和代表性的生物能源产品之一。以蓝细菌为光合平台,利用二氧化碳和太阳能直接进行乙醇合成可以同时起到降低二氧化碳排放和提供可再生能源的效果,具有重要的研究与应用价值。本文回顾了蓝细菌乙醇光合细胞工厂相关技术的发展历程和现状,从途径优化、底盘选择和代谢工程策略等层面对其最新进展和所遇到的问题进行了总结介绍,并对该技术未来发展方向进行了展望。     

Toolboxes for cyanobacteria: Recent advances and future direction

Photosynthetic cyanobacteria are important primary producers and model organisms for studying photosynthesis and elements cycling on earth. Due to the ability to absorb sunlight and utilize carbon dioxide, cyanobacteria have also been proposed as renewable chassis for carbon-neutral “microbial cell factories”. Recent progresses on cyanobacterial synthetic biology have led to the successful production of more than two dozen of fuels and fine chemicals directly from CO₂, demonstrating their potential for scale-up application in the future. However, compared with popular heterotrophic chassis like Escherichia coli and Saccharomyces cerevisiae, where abundant genetic tools are available for manipulations at levels from single gene, pathway to whole genome, limited genetic tools are accessible to cyanobacteria. Consequently, this significant technical hurdle restricts both the basic biological researches and further development and application of these renewable systems. Though still lagging the heterotrophic chassis, the vital roles of genetic tools in tuning of gene expression, carbon flux re-direction as well as genome-wide manipulations have been increasingly recognized in cyanobacteria. In recent years, significant progresses on developing and introducing new and efficient genetic tools have been made for cyanobacteria, including promoters, riboswitches, ribosome binding site engineering, clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease (CRISPR/Cas) systems, small RNA regulatory tools and genome-scale modeling strategies. In this review, we critically summarize recent advances on development and applications as well as technical limitations and future directions of the genetic tools in cyanobacteria. In addition, toolboxes feasible for using in large-scale cultivation are also briefly discussed.     

Engineering cyanobacteria as cell factories for direct trehalose production from CO2

Trehalose is a non-reducing disaccharide with a wide range of applications in food, cosmetic, and pharmaceutical industries. Cyanobacteria are promising cell factories to produce biochemicals by using solar energy and CO₂. Trehalose is biosynthesized at low intracellular concentrations as a salt-inducible compatible solute in some cyanobacteria. In the current study, we demonstrated the efficient trehalose production without salt induction in cyanobacteria by metabolic engineering. The trehalose transporter 1 (TRET1) from an anhydrobiotic insect (Polypedilum vanderplanki) was successfully expressed in the engineered strains and the intracellular trehalose was efficiently secreted to the medium. As the results, the engineered strain co-expressing maltooligosyl trehalose synthase (MTS), maltooligosyl trehalose trehalohydrolase (MTH) and TRET1 secreted 97% of trehalose to the medium, and the titer was up to 2.7 g/L in 15 days. In addition, 5.7 g/L trehalose was produced by semi-continuous cultivation in 34 days. Taken together, this work demonstrates cyanobacteria can be applied as cell factories for direct sunlight-driven conversion of CO₂ into excreted trehalose.     

A Critical Review of Genome Editing and Synthetic Biology Applications in Metabolic Engineering of Microalgae and Cyanobacteria

Being the green gold of the future, microalgae and cyanobacteria have recently attracted considerable interest worldwide, for their metabolites such as lipids, protein, pigments, and bioactive compounds have immense potential for sustainable energy and pharmaceutical production capabilities. In the last decades, the efforts attended to enhance the usage of microalgae and cyanobacteria by genetic manipulation, synthetic and metabolic engineering. However, the development of photoautotrophic cell factories have rarely compared to the heterotrophic counterparts due to limited tools, bioinformatics, and multi‐omics database. Therefore, recent advances of their genome editing techniques by clustered regularly interspaced short palindromic repeats (CRISPR) technology, and potential applications of their metabolic engineering and regulation approaches are examined in this review. Moreover, the contemporary achievements of synthetic biology approaches of microalgae and cyanobacteria in carbon fixation and sequestration, lipid and triacylglycerol (TAG), and sustainable production of high value‐added chemicals, such as carotenoids and docosahexaenoic acid (DHA), have been also discussed. From recent genomic study to trends in metabolic regulation of microalgae and cyanobacteria and a comprehensive assessment of the current challenges and opportunities for microalgae and cyanobacteria is also conducted.     

Versatility of hydrocarbon production in cyanobacteria

Cyanobacteria are photosynthetic microorganisms using solar energy, H₂O, and CO₂ as the primary inputs. Compared to plants and eukaryotic microalgae, cyanobacteria are easier to be genetically engineered and possess higher growth rate. Extensive genomic information and well-established genetic platform make cyanobacteria good candidates to build efficient biosynthetic pathways for biofuels and chemicals by genetic engineering. Hydrocarbons are a family of compounds consisting entirely of hydrogen and carbon. Structural diversity of the hydrocarbon family is enabled by variation in chain length, degree of saturation, and rearrangements of the carbon skeleton. The diversified hydrocarbons can be used as valuable chemicals in the field of food, fuels, pharmaceuticals, nutrition, and cosmetics. Hydrocarbon biosynthesis is ubiquitous in bacteria, yeasts, fungi, plants, and insects. A wide variety of pathways for the hydrocarbon biosynthesis have been identified in recent years. Cyanobacteria may be superior chassis for hydrocabon production in a photosynthetic manner. A diversity of hydrocarbons including ethylene, alkanes, alkenes, and terpenes can be produced by cyanobacteria. Metabolic engineering and synthetic biology strategies can be employed to improve hydrocarbon production in cyanobacteria. This review mainly summarizes versatility and perspectives of hydrocarbon production in cyanobacteria.

     

Genetic control of hydrogen metabolism in cyanobacteria

The development of methods for the use of phototrophic cyanobacteria as producers of molecular hydrogen via bioconversion of solar energy is a promising filed of hydrogen energetics. Optimization of hydrogen formation and release is based on studying the genetic control of hydrogen metabolism and the use of genetic approaches for obtaining efficient producer strains. Data on genes coding for the hydrogenases that are responsible for hydrogen uptake and production in cyanobacteria are summarized. Bioinformatic methods have been used to construct the scheme of the hydrogen metabolism gene network of nitrogen-fixing heterocystous cyanobacteria. The possible approaches to constructing the cyanobacterium strains producing molecular hydrogen that would be promising for photobiotechnology by mutagenesis and genetic engineering methods are discussed in terms of this model and analysis of the data on hydrogen-producing mutants.     

产乙醇工程菌研究进展

伴随着21世纪的到来,低油价的时代也悄然落幕。简要概述了燃料乙醇产生菌代谢工程的研究进展,包括了利用淀粉、戊糖及纤维素的工程酵母构建,运动发酵单胞菌利用戊糖工程菌的构建,引入外源乙醇合成途径的大肠埃希氏菌和产酸克雷伯氏菌等。对燃料乙醇的重视将促进开发能利用廉价原料和要求粗放的工程菌株用于高产乙醇的生产过程,以降低成本和能耗,其中能利用生淀粉的工程酵母及利用木质纤维素水解物的运动发酵单胞菌工程菌有较大的工业化潜力。     

Genetic control of hydrogen metabolism in cyanobacteria

The development of methods for the use of phototrophic cyanobacteria as producers of molecular hydrogen via bioconversion of solar energy is a promising filed of hydrogen energetics. Artificial optimization of hydrogen formation and release is based on studying the genetic control of hydrogen metabolism and the use of genetic approaches for obtaining efficient producer strains. Data on genes coding for the hydrogenases that are responsible for hydrogen uptake and production in cyanobacteria are summarized. Bioinformatic methods have been used to construct the scheme of the hydrogen metabolism gene network of nitrogen-fixing heterocyst cyanobacteria. The possible approaches to constructing the cyanobacterium strains producing molecular hydrogen that would be promising for photobiotechnology by mutagenesis and genetic engineering methods are discussed in terms of this model and analysis of the data on hydrogen-producing mutants.     

Metabolic engineering of Escherichia coli: A sustainable industrial platform for bio-based chemical production

In order to decrease carbon emissions and negative environmental impacts of various pollutants, more bulk and/or fine chemicals are produced by bioprocesses, replacing the traditional energy and fossil based intensive route. The Gram-negative rod-shaped bacterium, Escherichia coli has been studied extensively on a fundamental and applied level and has become a predominant host microorganism for industrial applications. Furthermore, metabolic engineering of E. coli for the enhanced biochemical production has been significantly promoted by the integrated use of recent developments in systems biology, synthetic biology and evolutionary engineering. In this review, we focus on recent efforts devoted to the use of genetically engineered E. coli as a sustainable platform for the production of industrially important biochemicals such as biofuels, organic acids, amino acids, sugar alcohols and biopolymers. In addition, representative secondary metabolites produced by E. coli will be systematically discussed and the successful strategies for strain improvements will be highlighted. Moreover, this review presents guidelines for future developments in the bio-based chemical production using E. coli as an industrial platform.     

Alternative affinity tools: more attractive than antibodies?

Antibodies are the most successful affinity tools used today, in both fundamental and applied research (diagnostics, purification and therapeutics). Nonetheless, antibodies do have their limitations, including high production costs and low stability. Alternative affinity tools based on nucleic acids (aptamers), polypeptides (engineered binding proteins) and inorganic matrices (molecular imprinted polymers) have received considerable attention. A major advantage of these alternatives concerns the efficient (microbial) production and in vitro selection procedures. The latter approach allows for the high-throughput optimization of aptamers and engineered binding proteins, e.g. aiming at enhanced chemical and physical stability. This has resulted in a rapid development of the fields of nucleic acid- and protein-based affinity tools and, although they are certainly not as widely used as antibodies, the number of their applications has steadily increased in recent years. In the present review, we compare the properties of the more conventional antibodies with these innovative affinity tools. Recent advances of affinity tool developments are described, both in a medical setting (e.g. diagnostics, therapeutics and drug delivery) and in several niche areas for which antibodies appear to be less attractive. Furthermore, an outlook is provided on anticipated future developments.     

A modular approach to creating large engineered cartilage surfaces

Native articular cartilage has limited capacity to repair itself from focal defects or osteoarthritis. Tissue engineering has provided a promising biological treatment strategy that is currently being evaluated in clinical trials. However, current approaches in translating these techniques to developing large engineered tissues remains a significant challenge. In this study, we present a method for developing large-scale engineered cartilage surfaces through modular fabrication. Modular Engineered Tissue Surfaces (METS) uses the well-known, but largely under-utilized self-adhesion properties of de novo tissue to create large scaffolds with nutrient channels. Compressive mechanical properties were evaluated throughout METS specimens, and the tensile mechanical strength of the bonds between attached constructs was evaluated over time. Raman spectroscopy, biochemical assays, and histology were performed to investigate matrix distribution. Results showed that by Day 14, stable connections had formed between the constructs in the METS samples. By Day 21, bonds were robust enough to form a rigid sheet and continued to increase in size and strength over time. Compressive mechanical properties and glycosaminoglycan (GAG) content of METS and individual constructs increased significantly over time. The METS technique builds on established tissue engineering accomplishments of developing constructs with GAG composition and compressive properties approaching native cartilage. This study demonstrated that modular fabrication is a viable technique for creating large-scale engineered cartilage, which can be broadly applied to many tissue engineering applications and construct geometries.