Metabolic modelling and flux analysis of microorganisms from the Atacama Desert used in biotechnological processes

Metabolic modelling is a useful tool that enables the rational design of metabolic engineering experiments and the study of the unique capabilities of biotechnologically important microorganisms. The extreme abiotic conditions of the Atacama Desert have selected microbial diversity with exceptional characteristics that can be applied in the mining industry for bioleaching processes and for production of specialised metabolites with antimicrobial, antifungal, antiviral, antitumoral, among other activities. In this review we summarise the scientific data available of the use of metabolic modelling and flux analysis to improve the performance of Atacama Desert microorganisms in biotechnological applications.     

Designing synthetic microbial communities for effectual bioremediation: A review

Abstract

Bioremediation is a better alternative and widely accepted approach used for efficient degradation of environmental pollutants released from industries, urban and agricultural activities due to its eco-friendly nature. Systems biology helps in the identification of new genes, proteins, metabolites, and metabolic pathways involved in bioremediation. Such information can be used for designing synthetic microbial communities that can degrade multiple recalcitrant pollutants simultaneously. This review gives a brief insight into various systems biology tools towards providing a greater understanding of microbial behaviour and improving the way of bioremediation. These techniques alone or in combination, provide a way to understand and improve the genetic potential of microorganisms to remediate various environmental contaminants efficiently. Further, this review also describes the successful employment of synthetic microbial consortium in the bioremediation. Moreover, In-silico tools are also described to analyse the data obtained through different laboratory experiments as well to predict the behaviour of microbial consortium towards the pollutants using different databases.     

Interspecific interactions in mixed microbial cultures in a biodegradation perspective

In recent works, microbial consortia consisting of various bacteria and fungi exhibited a biodegradation performance superior to single microbial strains. A highly efficient biodegradation of synthetic dyes, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and other organic pollutants can be achieved by mixed microbial cultures that combine degradative enzyme activities inherent to individual consortium members. This review summarizes biodegradation results obtained with defined microbial cocultures and real microbial consortia. The necessity of using a proper strategy for the microbial consortium development and optimization was clearly demonstrated. Molecular genetic and proteomic techniques have revolutionized the study of microbial communities, and techniques such as the denaturing gradient gel electrophoresis, rRNA sequencing, and metaproteomics have been used to identify consortium members and to study microbial population dynamics. These analyses could help to further enhance and optimize the natural activities of mixed microbial cultures.     

Dealing with water stress and microbial preservation

The relevance of preserving microorganisms has been well accepted for several decades. Interest is now shifting towards investigating adequate preservation methods to improve microbial survival rates and to preserve new taxa of previously considered unculturable microorganisms. In addition, a growing interest in preserving fragile microbial consortia or communities with biotechnological interest motivates the improvement of preservation methods. In the present study, we reviewed the effect of water availability in microbial diversity shift. We describe the effect of drought on microorganisms at the molecular level and their molecular responses to this life-threatening challenge focusing on the production of xeroprotectants. We also review the interspecies interactions of those drought-tolerant microorganisms with other sensitive organisms including neighbouring prokaryotes and eukaryotes such as plants, and the potential role of these microorganisms at determining the ecological composition of stressed environments. We emphasize the importance of applying the knowledge derived from the molecular mechanisms used by desiccation-tolerant microorganisms for the improvement of the preservation techniques. An overview of the current and newer techniques for preserving microorganisms and microbial communities is provided. The biotechnological interest in preserving pure cultures, microbial consortia and communities is also discussed.     

Comparative metagenomics of three Dehalococcoides-containing enrichment cultures: the role of the non-dechlorinating community

ABSTRACT: BACKGROUND: The Dehalococcoides are strictly anaerobic bacteria that gain metabolic energy via the oxidation of H2 coupled to the reduction of halogenated organic compounds. Dehalococcoides spp. grow best in mixed microbial consortia, relying on non-dechlorinating members to provide essential nutrients and maintain anaerobic conditions. A metagenome sequence was generated for the dechlorinating mixed microbial consortium KB-1. A comparative metagenomic study utilizing two additional metagenome sequences for Dehalococcoides-containing dechlorinating microbial consortia was undertaken to identify common features that are provided by the non-dechlorinating community and are potentially essential to Dehalococcoides growth. RESULTS: The KB-1 metagenome contained eighteen novel homologs to reductive dehalogenase genes. The metagenomes obtained from the three consortia were automatically annotated using the MG-RAST server, from which statistically significant differences in community composition and metabolic profiles were determined. Examination of specific metabolic pathways, including corrinoid synthesis, methionine synthesis, oxygen scavenging, and electron-donor metabolism identified the Firmicutes, methanogenic Archaea, and the delta-Proteobacteria as key organisms encoding these pathways, and thus potentially producing metabolites required for Dehalococcoides growth. CONCLUSIONS: Comparative metagenomics of the three Dehalococcoides-containing consortia identified that similarities across the three consortia are more apparent at the functional level than at the taxonomic level, indicating the non-dechlorinating organisms' identities can vary provided they fill the same niche within a consortium. Functional redundancy was identified in each metabolic pathway of interest, with key processes encoded by multiple taxonomic groups. This redundancy likely contributes to the robust growth and dechlorination rates in dechlorinating enrichment cultures.     

Bacterial degradation of aromatic pollutants: a paradigm of metabolic versatility.

Although most organisms have detoxification abilities (i.e mineralization, transformation and/or immobilization of pollutants), microorganisms, particularly bacteria, play a crucial role in biogeochemical cycles and in sustainable development of the biosphere. Next to glucosyl residues, the benzene ring is the most widely distributed unit of chemical structure in nature, and many of the aromatic compounds are major environmental pollutants. Bacteria have developed strategies for obtaining energy from virtually every compound under oxic or anoxic conditions (using alternative final electron acceptors such as nitrate, sulfate, and ferric ions). Clusters of genes coding for the catabolism of aromatic compounds are usually found in mobile genetic elements, such as transposons and plasmids, which facilitate their horizontal gene transfer and, therefore, the rapid adaptation of microorganisms to new pollutants. A successful strategy for in situ bioremediation has been the combination, in a single bacterial strain or in a syntrophic bacterial consortium, of different degrading abilities with genetic traits that provide selective advantages in a given environment. The advent of high-throughput methods for DNA sequencing and analysis of gene expression (genomics) and function (proteomics), as well as advances in modelling microbial metabolism in silico, provide a global, rational approach to unravel the largely unexplored potentials of microorganisms in biotechnological processes thereby facilitating sustainable development.     

石油烃生物降解过程的研究进展

石油烃污染物属于难降解混合物,生物修复已经成为石油烃污染环境的主要修复方法。文中简述了微生物对石油烃的间期适应过程和转运过程,并通过对部分典型石油烃成分的微生物降解机理和代谢路径的梳理和综述,阐释了石油烃生物降解过程中的菌株、基因、代谢路径等研究进展。此外,利用基因工程和代谢工程等手段,可对野生型石油烃降解菌进行改造,进一步提升其对石油烃污染环境的生物修复能力。最后,从石油烃降解菌的代谢途径改造、人工混菌体系的设计构建等角度,结合合成生物学和代谢工程的手段,提出了对石油烃降解的研究展望,以期提升对石油烃污染物的生物修复效果。     

Cyanide detoxification by microbial consortia of natural-industrial complexes of gold heap leaching

Microorganisms that have adapted not only to high concentrations of pollutants but also to environmental conditions develop in autochthonous microbial communities of natural-industrial complexes of gold heap leaching. The biotechnological potential and diversity of autochthonous microbial communities involved in cyanide detoxification was evaluated by the example of a deposit situated in the Sakha (Yakutia) Republic. Under the zoning conditions of the ore heap, the biological component had a greater impact on cyanide destruction than chemical transformation. Metabolically active representatives of a microbial consortium are capable of surviving developed under these conditions. Phylotypes of the genus Serratia and family Alcaligenaceae that are capable of cyanide destruction and are potentially promising for the detoxification of wastes of gold heap leaching were revealed.     

Strain engineering for microbial production of value-added chemicals and fuels from glycerol

While the widespread reliance on fossil fuels is driven by their low cost and relative abundance, this fossil-based economy has been deemed unsustainable and, therefore, the adoption of sustainable and environmentally compatible energy sources is on the horizon. Biorefinery is an emerging approach that integrates metabolic engineering, synthetic biology, and systems biology principles for the development of whole-cell catalytic platforms for biomanufacturing. Due to the high degree of reduction and low cost, glycerol, either refined or crude, has been recognized as an ideal feedstock for the production of value-added biologicals, though microbial dissimilation of glycerol sometimes can be difficult particularly under anaerobic conditions. While strain development for glycerol biorefinery is widely reported in the literature, few, if any, commercialized bioprocesses have been developed as a result, such that engineering of glycerol metabolism in microbial hosts remains an untapped opportunity in biomanufacturing. Here we review the recent progress made in engineering microbial hosts for the production of biofuels, diols, organic acids, biopolymers, and specialty chemicals from glycerol. We begin with a broad outline of the major pathways for fermentative and respiratory glycerol dissimilation and key end metabolites, and then focus our analysis on four key genera of bacteria known to naturally dissimilate glycerol, i.e. Klebsiella, Citrobacter, Clostridium, and Lactobacillus, in addition to Escherichia coli, and systematically review the progress made toward engineering these microorganisms for glycerol biorefinery. We also identify the major biotechnological and bioprocessing advantages and disadvantages of each genus, and bottlenecks limiting the production of target metabolites from glycerol in engineered strains. Our analysis culminates in the development of potential strategies to overcome the current technical limitations identified for commonly employed strains, with an outlook on the suitability of different hosts for the production of key metabolites and avenues for their future development into biomanufacturing platforms.     

合成生物学技术在环境保护中的应用

合成生物学是一个基于生物学和工程学原理的科学领域,其目的是重新设计和重组微生物,以优化或创建具有增强功能的新生物系统。该领域利用分子工具、系统生物学和遗传框架的重编程,从而构建合成途径以获得具有替代功能的微生物。传统上,合成生物学方法通常旨在开发具有成本效益的微生物细胞工厂进而从可再生资源中生产化学物质。然而,近年来合成生物学技术开始在环境保护中发挥着更直接的作用。本综述介绍了基因工程中的合成生物学工具,讨论了基于基因工程的微生物修复策略,强调了合成生物学技术可以通过响应特定污染物进行生物修复来保护环境。其中,规律间隔成簇短回文重复序列(Clustered Regularly Interspersed Short Palindromic Repeats, CRISPR)技术在基因工程细菌和古细菌的生物修复中得到了广泛应用,生物修复领域也出现了很多新的先进技术,包括生物膜工程、人工微生物群落的构建、基因驱动、酶和蛋白质工程等。有了这些新的技术和工具,生物修复将成为当今最好和最有效的污染物去除方式之一。     

Reprogramming microorganisms for the biosynthesis of astaxanthin via metabolic engineering

There is an increasing demand for astaxanthin in food, feed, cosmetics and pharmaceutical applications because of its superior anti-oxidative and coloring properties. However, naturally produced astaxanthin is expensive, mainly due to low productivity and limited sources. Reprogramming of microorganisms for astaxanthin production via metabolic engineering is a promising strategy. We primarily focus on the application of synthetic biology, enzyme engineering and metabolic engineering in enhancing the synthesis and accumulation of astaxanthin in microorganisms in this review. We also discuss the biosynthetic pathways of astaxanthin within natural producers, and summarize the achievements and challenges in reprogramming microorganisms for enhancing astaxanthin production. This review illuminates recent biotechnological advances in microbial production of astaxanthin. Future perspectives on utilization of new technologies for boosting microbial astaxanthin production are also discussed.     

Genomic and mechanistic insights into the biodegradation of organic pollutants

Several new methodologies have enabled recent studies on the microbial biodegradation mechanisms of organic pollutants. Culture-independent techniques for analysis of the genetic and metabolic potential of natural and model microbial communities that degrade organic pollutants have identified new metabolic pathways and enzymes for aerobic and anaerobic degradation. Furthermore, structural studies of the enzymes involved have revealed the specificities and activities of key catabolic enzymes, such as dioxygenases. Genome sequencing of several biodegradation-relevant microorganisms have provided the first whole-genome insights into the genetic background of the metabolic capability and biodegradation versatility of these organisms. Systems biology approaches are still in their infancy, but are becoming increasingly helpful to unravel, predict and quantify metabolic abilities within particular organisms or microbial consortia.     

Biotransformation of 2,4-dinitrotoluene in a phototrophic co-culture of engineered Synechococcus elongatus and Pseudomonas putida

In contrast to the current paradigm of using microbial mono-cultures in most biotechnological applications, increasing efforts are being directed towards engineering mixed-species consortia to perform functions that are difficult to programme into individual strains. In this work, we developed a synthetic microbial consortium composed of two genetically engineered microbes, a cyanobacterium (Synechococcus elongatus PCC 7942) and a heterotrophic bacterium (Pseudomonas putida EM173). These microbial species specialize in the co-culture: cyanobacteria fix CO₂ through photosynthetic metabolism and secrete sufficient carbohydrates to support the growth and active metabolism of P. putida, which has been engineered to consume sucrose and to degrade the environmental pollutant 2,4-dinitrotoluene (2,4-DNT). By encapsulating S. elongatus within a barium–alginate hydrogel, cyanobacterial cells were protected from the toxic effects of 2,4-DNT, enhancing the performance of the co-culture. The synthetic consortium was able to convert 2,4-DNT with light and CO₂ as key inputs, and its catalytic performance was stable over time. Furthermore, cycling this synthetic consortium through low nitrogen medium promoted the sucrose-dependent accumulation of polyhydroxyalkanoate, an added-value biopolymer, in the engineered P. putida strain. Altogether, the synthetic consortium displayed the capacity to remediate the industrial pollutant 2,4-DNT while simultaneously synthesizing biopolymers using light and CO₂ as the primary inputs.     

Harnessing plant-bacteria-fungi interactions to improve plant growth and degradation of organic pollutants

Exploiting the potential of bacteria in phytoremediation for the removal of organic and inorganic pollutants from soils and (ground)water holds great promise. Besides bacteria, mycorrhizal fungi and free-living saprotrophs are well known for their strong degradative capacities and plant growth promotion effects, which makes them of high interest for use in different bioremediation strategies. To further increase the efficiency and successes of phytoremediation, interactions between plants and their associated microorganisms, both bacteria and fungi, should be further investigated, in addition to the close interactions between bacteria and fungi. Benefitting from an increased understanding of microbial community structure and assembly allows us to better understand how the holobiont can be modified to improve pollutant degradation and plant growth. In this review, we present an overview of insights in plant-bacteria-fungi interactions and the opportunities of exploiting these tripartite interactions to enhance the effectiveness of phytoremediation of organic pollutants.     

Medicinally important secondary metabolites in recombinant microorganisms or plants: Progress in alkaloid biosynthesis

Plants produce a high diversity of natural products or secondary metabolites which are important for the communication of plants with other organisms. A prominent function is the protection against herbivores and/or microbial pathogens. Some natural products are also involved in defence against abiotic stress, e.g. UV-B exposure. Many of the secondary metabolites have interesting biological properties and quite a number are of medicinal importance. Because the production of the valuable natural products, such as the anticancer drugs paclitaxel, vinblastine or camptothecin in plants is a costly process, biotechnological alternatives to produce these alkaloids more economically become increasingly important. This review provides an overview of the state of art to produce alkaloids in recombinant microorganisms, such as bacteria or yeast. Some progress has been made in metabolic engineering usually employing a single recombinant alkaloid gene. More importantly, for benzylisoquinoline, monoterpene indole and diterpene alkaloids (taxanes) as well as some terpenoids and phenolics the proof of concept for production of complex alkaloids in recombinant Escherichia coli and yeast has already been achieved. In a long-term perspective, it will probably be possible to generate gene cassettes for complete pathways, which could then be used for production of valuable natural products in bioreactors or for metabolic engineering of crop plants. This will improve their resistance against herbivores and/or microbial pathogens.     

Streamlining of a synthetic co-culture towards an individually controllable one-pot process for polyhydroxyalkanoate production from light and CO2

Rationally designed synthetic microbial consortia carry a vast potential for biotechnological applications. The application of such a consortium in a bioprocess, however, requires tight and individual controllability of the involved microbes. Here, we present the streamlining of a co-cultivation process consisting of Synechococcus elongatus cscB and Pseudomonas putida for the production of polyhydroxyalkanoates (PHA) from light and CO₂. First, the process was improved by employing P. putida cscRABY, a strain with a higher metabolic activity towards sucrose. Next, the individual controllability of the co-culture partners was addressed by providing different nitrogen sources, each exclusively available for one strain. By this, the growth rate of the co-culture partners could be regulated individually, and defined conditions could be set. The molC/molN ratio, a key value for PHA accumulation, was estimated from the experimental data, and the necessary feeding rates to obtain a specific ratio could be predicted. This information was then implemented in the co-cultivation process, following the concept of a DBTL-cycle. In total, the streamlining of the process resulted in an increased maximal PHA titer of 393 mg/L and a PHA production rate of 42.1 mg/(L•day).     

Biocatalytic degradation of pollutants

Microbial reactions play key roles in biocatalysis and biodegradation. The recent genome sequencing of environmentally relevant bacteria has revealed previously unsuspected metabolic potential that could be exploited for useful purposes. For example, oxygenases and other biodegradative enzymes are benign catalysts that can be used for the production of industrially useful compounds. In conjunction with their biodegradative capacities, bacterial chemotaxis towards pollutants might contribute to the ability of bacteria to compete with other organisms in the environment and to be efficient agents for bioremediation. In addition to the bacterial biomineralization of organic pollutants, certain bacteria are also capable of immobilizing toxic heavy metals in contaminated aquifers, further illustrating the potential of microorganisms for the removal of pollutants.     

Systematic and biotechnological aspects of halophilic and halotolerant actinomycetes

More than 70 species of halotolerant and halophilic actinomycetes belonging to at least 24 genera have been validly described. Halophilic actinomycetes are a less explored source of actinomycetes for discovery of novel bioactive secondary metabolites. Degradation of aliphatic and aromatic organic compounds, detoxification of pollutants, production of new enzymes and other metabolites such as antibiotics, compatible solutes and polymers are other potential industrial applications of halophilic and halotolerant actinomycetes. Especially new bioactive secondary metabolites that are derived from only a small fraction of the investigated halophilic actinomycetes, mainly from marine habitats, have revealed the huge capacity of this physiological group in production of new bioactive chemical entities. Combined high metabolic capacities of actinomycetes and unique features related to extremophilic nature of the halophilic actinomycetes have conferred on them an influential role for future biotechnological applications.     

Omega-3 biotechnology: Thraustochytrids as a novel source of omega-3 oils

Thraustochytrids are large-celled marine heterokonts and classified as oleaginous microorganisms due to their production of docosahexaenoic (DHA) and eicosapentaenoic (EPA) ω-3-fatty acids. The applications of microbial DHA and EPA for human health are rapidly expanding, and a large number of clinical trials have been carried out to verify their efficacy. The development of refined isolation and identification techniques is important for the cultivation of thraustochytrids. With a high proportion of lipid biomass, thraustochytrids are also amenable to various production strategies which increase omega-3 oil output. Modifications to the existing lipid extraction methods and utilisation of sophisticated analytical instruments have increased extraction yields of DHA and EPA. Other metabolites such as enzymes, carotenoids and extracellular polysaccharides can also be obtained from these marine protists. Approaches such as the exploration for more diverse isolates having fast growth rates, metabolic engineering including gene cloning, and growing thraustochytrids on alternate low cost carbon source, will further enhance the biotechnological potential of thraustochytrids.     

Bacterial communities in full-scale wastewater treatment systems

Bacterial metabolism determines the effectiveness of biological treatment of wastewater. Therefore, it is important to define the relations between the species structure and the performance of full-scale installations. Although there is much laboratory data on microbial consortia, our understanding of dependencies between the microbial structure and operational parameters of full-scale wastewater treatment plants (WWTP) is limited. This mini-review presents the types of microbial consortia in WWTP. Information is given on extracellular polymeric substances production as factor that is key for formation of spatial structures of microorganisms. Additionally, we discuss data on microbial groups including nitrifiers, denitrifiers, Anammox bacteria, and phosphate- and glycogen-accumulating bacteria in full-scale aerobic systems that was obtained with the use of molecular techniques, including high-throughput sequencing, to shed light on dependencies between the microbial ecology of biomass and the overall efficiency and functional stability of wastewater treatment systems. Sludge bulking in WWTPs is addressed, as well as the microbial composition of consortia involved in antibiotic and micropollutant removal.