Bioactive Compounds from Cyanobacteria and Microalgae: An Overview

ABSTRACT

Cyanobacteria (blue-green algae) are photosynthetic prokaryotes used as food by humans. They have also been recognized as an excellent source of vitamins and proteins and as such are found in health food stores throughout the world. They are also reported to be a source of fine chemicals, renewable fuel and bioactive compounds. This potential is being realized as data from research in the areas of the physiology and chemistry of these organisms are gathered and the knowledge of cyanobacterial genetics and genetic engineering increased. Their role as antiviral, anti-tumour, antibacterial, anti-HIV and a food additive have been well established. The production of cyanobacteria in artificial and natural environments has been fully exploited. In this review the use of cyanobacteria and microalgae, production processes and biosynthesis of pigments, colorants and certain bioactive compounds are discussed in detail. The genetic manipulation of cyanobacteria and microalgae to improve their quality are also described at length.     

Methods for enhancing cyanobacterial stress tolerance to enable improved production of biofuels and industrially relevant chemicals

Cyanobacteria are photosynthetic prokaryotes that can fix atmospheric CO₂ and can be engineered to produce industrially important compounds such as alcohols, free fatty acids, alkanes used in next-generation biofuels, and commodity chemicals such as ethylene or farnesene. They can be easily genetically manipulated, have minimal nutrient requirements, and are quite tolerant to abiotic stress making them an appealing alternative to other biofuel-producing microbes which require additional carbon sources and plants which compete with food crops for arable land. Many of the compounds produced in cyanobacteria are toxic as titers increase which can slow growth, reduce production, and decrease overall biomass. Additionally, many factors associated with outdoor culturing of cyanobacteria such as UV exposure and fluctuations in temperature can also limit the production potential of cyanobacteria. For cyanobacteria to be utilized successfully as biofactories, tolerance to these stressors must be increased and ameliorating stress responses must be enhanced. Genetic manipulation, directed evolution, and supplementation of culture media with antioxidants are all viable strategies for designing more robust cyanobacterial strains that have the potential to meet industrial production goals.

     

Cyanobacteria: photosynthetic factories combining biodiversity,radiation resistance,and genetics to facilitate drug discovery

Cyanobacteria are ancient, abundant, and widely diverse photosynthetic prokaryotes, which are viewed as promising cell factories for the ecologically responsible production of chemicals. Natural cyanobacteria synthesize a vast array of biologically active (secondary) metabolites with great potential for human health, while a few genetic models can be engineered for the (low level) production of biofuels. Recently, genome sequencing and mining has revealed that natural cyanobacteria have the capacity to produce many more secondary metabolites than have been characterized. The corresponding panoply of enzymes (polyketide synthases and non-ribosomal peptide synthases) of interest for synthetic biology can still be increased through gene manipulations with the tools available for the few genetically manipulable strains. In this review, we propose to exploit the metabolic diversity and radiation resistance of cyanobacteria, and when required the genetics of model strains, for the production and radioactive (¹⁴C) labeling of bioactive products, in order to facilitate the screening for new drugs.

     

Sustainability and cyanobacteria (blue-green algae): facts and challenges

Cyanobacteria (blue-green algae) are widely distributed Gram-negative oxygenic photosynthetic prokaryotes with a long evolutionary history. They have potential applications such as nutrition (food supplements and fine chemicals), in agriculture (as biofertilizer and in reclamation of saline USAR soils) and in wastewater treatment (production of exopolysaccharides and flocculants). In addition, they also produce wide variety of chemicals not needed for their normal growth (secondary metabolites) which show powerful biological activities such as strong antiviral, antibacterial, antifungal, antimalarial, antitumoral and anti-inflammatory activities useful for therapeutic purposes. In recent years, cyanobacteria have gained interest for producing biofuels (both biomass and H₂ production). Because of their simple growth needs, it is potentially cost-effective to exploit cyanobacteria for the production of recombinant compounds of medicinal and commercial value. Recent advances in culture, screening and genetic engineering techniques have opened new ways to exploit the potential of cyanobacteria. This review analyses the sustainability of cyanobacteria to solve global problems such as food, energy and environmental degradation. It emphasizes the need to adopt multidisciplinary approaches and a multi-product production (biorefinery) strategy to harness the maximum benefit of cyanobacteria.     

Bioactive compounds from cyanobacteria and microalgae: an overview

Cyanobacteria (blue-green algae) are photosynthetic prokaryotes used as food by humans. They have also been recognized as an excellent source of vitamins and proteins and as such are found in health food stores throughout the world. They are also reported to be a source of fine chemicals, renewable fuel and bioactive compounds. This potential is being realized as data from research in the areas of the physiology and chemistry of these organisms are gathered and the knowledge of cyanobacterial genetics and genetic engineering increased. Their role as antiviral, anti-tumour, antibacterial, anti-HIV and a food additive have been well established. The production of cyanobacteria in artificial and natural environments has been fully exploited. In this review the use of cyanobacteria and microalgae, production processes and biosynthesis of pigments, colorants and certain bioactive compounds are discussed in detail. The genetic manipulation of cyanobacteria and microalgae to improve their quality are also described at length.     

Combinatorial assembly platform enabling engineering of genetically stable metabolic pathways in cyanobacteria

Cyanobacteria are simple, efficient, genetically-tractable photosynthetic microorganisms which in principle represent ideal biocatalysts for CO₂ capture and conversion. However, in practice, genetic instability and low productivity are key, linked problems in engineered cyanobacteria. We took a massively parallel approach, generating and characterising libraries of synthetic promoters and RBSs for the cyanobacterium Synechocystis sp. PCC 6803, and assembling a sparse combinatorial library of millions of metabolic pathway-encoding construct variants. Genetic instability was observed for some variants, which is expected when variants cause metabolic burden. Surprisingly however, in a single combinatorial round without iterative optimisation, 80% of variants chosen at random and cultured photoautotrophically over many generations accumulated the target terpenoid lycopene from atmospheric CO₂, apparently overcoming genetic instability. This large-scale parallel metabolic engineering of cyanobacteria provides a new platform for development of genetically stable cyanobacterial biocatalysts for sustainable light-driven production of valuable products directly from CO₂, avoiding fossil carbon or competition with food production.     

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     

Regulation of Gene Expression in Diverse Cyanobacterial Species by Using Theophylline-Responsive Riboswitches

Cyanobacteria are photosynthetic bacteria that are currently being developed as biological production platforms. They derive energy from light and carbon from atmospheric carbon dioxide, and some species can fix atmospheric nitrogen. One advantage of developing cyanobacteria for renewable production of biofuels and other biological products is that they are amenable to genetic manipulation, facilitating bioengineering and synthetic biology. To expand the currently available genetic toolkit, we have demonstrated the utility of synthetic theophylline-responsive riboswitches for effective regulation of gene expression in four diverse species of cyanobacteria, including two recent isolates. We evaluated a set of six riboswitches driving the expression of a yellow fluorescent protein reporter in Synechococcus elongatus PCC 7942, Leptolyngbya sp. strain BL0902, Anabaena sp. strain PCC 7120, and Synechocystis sp. strain WHSyn. We demonstrated that riboswitches can offer regulation of gene expression superior to that of the commonly used isopropyl-β-d-thiogalactopyranoside induction of a lacI^q-P_trc promoter system. We also showed that expression of the toxic protein SacB can be effectively regulated, demonstrating utility for riboswitch regulation of proteins that are detrimental to biomass accumulation. Taken together, the results of this work demonstrate the utility and ease of use of riboswitches in the context of genetic engineering and synthetic biology in diverse cyanobacteria, which will facilitate the development of algal biotechnology.     

Engineered cyanobacteria: teaching an old bug new tricks

Cyanobacteria have played an important role in the development of the Earth and have long been studied as model organisms for photosynthesis and the circadian rhythm. Recent developments have led to increased interest in the use of engineered cyanobacteria for the production of protein and chemical products. This review highlights the genetic tools and strategies for manipulation of cyanobacteria as well as previous accomplishments in the development of engineered cyanobacteria for applied use. Particular attention is given to the engineering of cyanobacteria for biofuel production, including both hydrocarbon and hydrogen fuels. Genetic engineering efforts to enhance cyanobacterial fitness are reviewed with an emphasis on physiological improvements for large-scale production. Lastly, a future outlook on engineered cyanobacteria is presented, highlighting the future areas of focus and technical challenges in this field. With the uncertainty of future energy security, it is an exciting time in applied cyanobacterial research, but we must take the time to learn from these past accomplishments before we can capitalize on the potential of these photosynthetic microorganisms.     

Estimation of photosynthesis in cyanobacteria by pulse-amplitude modulation chlorophyll fluorescence: problems and solutions

Cyanobacteria are photosynthetic prokaryotes and widely used for photosynthetic research as model organisms. Partly due to their prokaryotic nature, however, estimation of photosynthesis by chlorophyll fluorescence measurements is sometimes problematic in cyanobacteria. For example, plastoquinone pool is reduced in the dark-acclimated samples in many cyanobacterial species so that conventional protocol developed for land plants cannot be directly applied for cyanobacteria. Even for the estimation of the simplest chlorophyll fluorescence parameter, F _v/F _m, some additional protocol such as addition of DCMU or illumination of weak blue light is necessary. In this review, those problems in the measurements of chlorophyll fluorescence in cyanobacteria are introduced, and solutions to those problems are given.     

Progress and challenges in producing polyhydroxyalkanoate biopolymers from cyanobacteria

Growing concerns over conventional plastic materials and their detrimental effects on the environment have paved the way for exploring alternative sources for the production of bioplastics/biodegradable polymers. Polyhydroxyalkanoates (PHAs), being eco-friendly, biodegradable and renewable, with material properties comparable to conventional plastics, have gained significant attention for research and commercial ventures. Bacteria are reported to be the most efficient microbes in accumulating PHAs, where productivity up to 3.2 g L^?1 h^?1 can be attained. PHA production from a bacterial system, however, is found to be expensive. Cyanobacteria are now considered as prospective photoautotrophic systems with many advantages over higher plants for low-cost production of PHAs. Cyanobacteria have the potential to synthesize polyhydroxybutyrate (PHB) under photoautotrophic and chemoheterotrophic conditions using carbon substrates like glucose, acetate, and maltose, individually or in combination. Several studies have shown improvement in PHA yield in cyanobacteria by limiting nutrients and/or addition of various precursors. Under optimized conditions, PHB and P(3HB-co-3HV) co-polymer accumulation can reach up to 85 and 77% of dry cell weight (dcw) with a productivity of 13.3 and 1.6 mg L^?1 h^?1, respectively. Despite the strategic increase in the potential of PHA accumulation in cyanobacteria, the productivity does not suffice for economic production. Therefore, economically feasible production of PHA in cyanobacteria might be attained by technological improvements in various aspects like improvement in mass cultivation techniques, alternate low-cost organic substrates, use of various metabolic inhibitors to stimulate intracellular accumulation, and by suppression and overexpression of specific biosynthetic pathways by genetic engineering approaches.     

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.     

Biosynthesis of platform chemical 3-hydroxypropionic acid (3-HP) directly from CO2 in cyanobacterium Synechocystis sp. PCC 6803

3-hydroxypropionic acid (3-HP) is an important platform chemical with a wide range of applications. So far large-scale production of 3-HP has been mainly through petroleum-based chemical processes, whose sustainability and environmental issues have attracted widespread attention. With the ability to fix CO₂ directly, cyanobacteria have been engineered as an autotrophic microbial cell factory to produce fuels and chemicals. In this study, we constructed the biosynthetic pathway of 3-HP in cyanobacterium Synechocystis sp. PCC 6803, and then optimized the system through the following approaches: i) increasing expression of malonyl-CoA reductase (MCR) gene using different promoters and cultivation conditions; ii) enhancing supply of the precursor malonyl-CoA by overexpressing acetyl-CoA carboxylase and biotinilase; iii) improving NADPH supply by overexpressing the NAD(P) transhydrogenase gene; iv) directing more carbon flux into 3-HP by inactivating the competing pathways of PHA and acetate biosynthesis. Together, the efforts led to a production of 837.18 mg L⁻¹ (348.8 mg/g dry cell weight) 3-HP directly from CO₂ in Synechocystis after 6 days cultivation, demonstrating the feasibility photosynthetic production of 3-HP directly from sunlight and CO₂ in cyanobacteria. In addition, the results showed that overexpression of the ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) gene from Anabaena sp. PCC 7120 and Synechococcus sp. PCC 7942 led to no increase of 3-HP production, suggesting CO₂ fixation may not be a rate-limiting step for 3-HP biosynthesis in Synechocystis.     

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 of a modular and synthetic phosphoketolase pathway for photosynthetic production of acetone from CO2 in Synechococcus elongatus PCC 7942 under light and aerobic condition

Capture and conversion of CO₂ to valuable chemicals is intended to answer global challenges on environmental issues, climate change and energy security. Engineered cyanobacteria have been enabled to produce industry‐relevant chemicals from CO₂. However, the final products from cyanobacteria have often been mixed with fermented metabolites during dark fermentation. In this study, our engineering of Synechococcus elongatus PCC 7942 enabled continuous conversion of CO₂ to volatile acetone as sole product. This process occurred during lighted, aerobic culture via both ATP‐driven malonyl‐CoA synthesis pathway and heterologous phosphoketolase (PHK)‐phosphotransacetylase (Pta) pathway. Because of strong correlations between the metabolic pathways of acetate and acetone, supplying the acetyl‐CoA directly from CO₂ in the engineered strain, led to sole production of acetone (22.48 mg/L ± 1.00) without changing nutritional constraints, and without an anaerobic shift. Our engineered S. elongatus strains, designed for acetone production, could be modified to create biosolar cell factories for sustainable photosynthetic production of acetyl‐CoA‐derived biochemicals.     

Genetically engineering cyanobacteria to convert CO2, water,and light into the long-chain hydrocarbon farnesene

Genetically engineered cyanobacteria offer a shortcut to convert CO₂ and H₂O directly into biofuels and high value chemicals for societal benefits. Farnesene, a long-chained hydrocarbon (C₁₅H₂₄), has many applications in lubricants, cosmetics, fragrances, and biofuels. However, a method for the sustainable, photosynthetic production of farnesene has been lacking. Here, we report the photosynthetic production of farnesene by the filamentous cyanobacterium Anabaena sp. PCC 7120 using only CO₂, mineralized water, and light. A codon-optimized farnesene synthase gene was chemically synthesized and then expressed in the cyanobacterium, enabling it to synthesize farnesene through its endogenous non-mevalonate (MEP) pathway. Farnesene excreted from the engineered cyanobacterium volatilized into the flask head space and was recovered by adsorption in a resin column. The maximum photosynthetic productivity of farnesene was 69.1?±?1.8 μg·L^?1·O.D.^?1·d^?1. Compared to the wild type, the farnesene-producing cyanobacterium also exhibited a 60 % higher PSII activity under high light, suggesting increased farnesene productivity in such conditions. We envision genetically engineered cyanobacteria as a bio-solar factory for photosynthetic production of a wide range of biofuels and commodity chemicals.     

八氢番茄红素脱氢酶的研究进展北大核心CSCD

类胡萝卜素是一类超过700种的萜烯基团类不饱和化合物的总称,根据结构可分为胡萝卜素族和叶黄素族,具有较高的营养价值。八氢番茄红素脱氢酶是类胡萝卜素生物合成途径中的首要限速酶,它参与催化无色的八氢番茄红素转变成有色类胡萝卜素,发挥着中心调控作用。不同生物源的八氢番茄红素脱氢酶在功能上呈现多样性,在大多数蓝细菌,藻类和高等植物的类胡萝卜素生物合成途径中,由Crt P,Crt Q和异构酶Crt H或PDS,ZDS和异构酶Z-ISO、Crt ISO共同参与番茄红素的形成,而在大多数微生物中只有Crt I-type一种酶来完成八氢番茄红素的脱氢反应,且根据脱氢步骤的不同分别可生成链孢红素、番茄红素或脱氢番茄红素。本文阐述了不同生物源八氢番茄红素脱氢酶的基因分离与鉴定,功能多样性及表达调控机制等最新研究进展,并进行了进化分析,为八氢番茄红素脱氢酶的深入研究及利用基因工程策略生产类胡萝卜素的应用提供重要信息。     

Rerouting carbon flux to enhance photosynthetic productivity

The bioindustrial production of fuels, chemicals, and therapeutics typically relies upon carbohydrate inputs derived from agricultural plants, resulting in the entanglement of food and chemical commodity markets. We demonstrate the efficient production of sucrose from a cyanobacterial species, Synechococcus elongatus, heterologously expressing a symporter of protons and sucrose (cscB). cscB-expressing cyanobacteria export sucrose irreversibly to concentrations of >10 mM without culture toxicity. Moreover, sucrose-exporting cyanobacteria exhibit increased biomass production rates relative to wild-type strains, accompanied by enhanced photosystem II activity, carbon fixation, and chlorophyll content. The genetic modification of sucrose biosynthesis pathways to minimize competing glucose- or sucrose-consuming reactions can further improve sucrose production, allowing the export of sucrose at rates of up to 36.1 mg liter(-1) h illumination(-1). This rate of production exceeds that of previous reports of targeted, photobiological production from microbes. Engineered S. elongatus produces sucrose in sufficient quantities (up to ～80% of total biomass) such that it may be a viable alternative to sugar synthesis from terrestrial plants, including sugarcane.     

Production of optically pure d-lactate from CO2 by blocking the PHB and acetate pathways and expressing d-lactate dehydrogenase in cyanobacterium Synechocystis sp. PCC 6803

Lactate is an important industrial material with numerous potential applications, and its production from carbon dioxide is very attractive. d-Lactate is an essential monomer for production of thermostable polylactide. The photoautotrophic prokaryote cyanobacterium Synechocystis sp. PCC 6803 represents a promising host for biosynthesis of d-lactate from CO₂ as it only contains d-lactate dehydrogenase. The production of d-lactate from CO₂ by an engineered strain of Synechocystis sp. PCC 6803 with overexpressing d-lactate dehydrogenase and a soluble transhydrogenase has been reported recently. Here, we report an alternative engineering strategy to produce d-lactate from CO₂. This strategy involves blocking two competitive pathways, the native poly-3-hydroxybutyrate and acetate pathways from the acetyl-CoA node, and introducing a more efficient d-lactate dehydrogenase into Synechocystis sp. PCC 6803. The engineered strain of Synechocystis sp. PCC 6803 was capable of producing 1.06 g/L of d-lactate from CO₂. This alternative strategy for the production of optically pure d-lactate could also be used to produce other acetyl-CoA-derived chemicals from CO₂ by using engineered cyanobacteria.