Natural production of fluorinated compounds and biotechnological prospects of the fluorinase enzyme

Fluorinated compounds are finding increasing uses in several applications. They are employed in almost all areas of modern society. These compounds are all produced by chemical synthesis and their abundance highly contrasts with fluorinated molecules of natural origin. To date, only some plants and a handful of actinomycetes species are known to produce a small number of fluorinated compounds that include fluoroacetate (FA), some ω-fluorinated fatty acids, nucleocidin, 4-fluorothreonine (4-FT), and the more recently identified (2R3S4S)-5-fluoro-2,3,4-trihydroxypentanoic acid. This largely differs from other naturally produced halogenated compounds, which totals more than 5000. The mechanisms underlying biological fluorination have been uncovered after discovering the first actinomycete species, Streptomyces cattleya, that is capable of producing FA and 4-FT, and a fluorinase has been identified as the enzyme responsible for the formation of the C–F bond. The discovery of this enzyme has opened new perspectives for the biotechnological production of fluorinated compounds and many advancements have been achieved in its application mainly as a biocatalyst for the synthesis of [¹⁸F]-labeled radiotracers for medical imaging. Natural fluorinated compounds may also be derived from abiogenic sources, such as volcanoes and rocks, though their concentrations and production mechanisms are not well known. This review provides an outlook of what is currently known about fluorinated compounds with natural origin. The paucity of these compounds and the biological mechanisms responsible for their production are addressed. Due to its relevance, special emphasis is given to the discovery, characterization and biotechnological potential of the unique fluorinase enzyme.     

真菌漆酶异源表达研究进展

由于漆酶能够氧化芳香类化合物和其它一些非芳香类有机物,具有广泛的底物特异性,因此在纸浆漂白、纺织品染料脱色、有毒废弃物的去除、生物修复和生物传感器等方面具有巨大的应用潜力。但是缺少大量廉价的酶源供应阻碍了漆酶商业化的应用,解决这个问题的一个主要方法就是通过漆酶的异源表达来获得大量的漆酶。综述了真菌漆酶在酵母表达系统和丝状真菌表达系统中表达的研究结果,着重总结了影响漆酶异源表达的因素和提高漆酶表达的策略。     

Production of rhamnolipids by a Pseudomonas alcaligenes strain

Brazil is one of the main producers of palm oil (Ellaus guineeusis). It is a low-cost product that has some interesting industrial qualities, such as its use as the raw material for the production of glycerin and soap as well as its use in the preparation of food. Some renewable sources and agroindustrial wastes have been used extensively in research on the production of biosurfactants of the Pseudomonas strains. However, to our knowledge, no studies have been published on the use of palm oil as a substrate for the synthesis of biosurfactants by Pseudomonas alcaligenes. This paper describes the production and characterization of biosurfactants synthesized by a strain of P. alcaligenes PCL previously isolated from soil that was contaminated with crude-oil. Furthermore, the paper presents the optimization of the production of biological surface-active compounds by applying experimental design tools and their capacity to emulsify hydrocarbons.     

Expression systems for heterologous production of antimicrobial peptides

Antimicrobial peptides (AMPs) consist of molecules that act on the defense systems of numerous organisms toward multiple pathogens such as bacteria, fungi, parasites and viruses. These compounds have become extremely significant due to the increasing resistance of microorganisms to common antibiotics. However, the low quantity of peptides obtained from direct purification is, to date, still a remarkable bottleneck for scientific and industrial research development. Therefore, this review describes the main heterologous systems currently used for AMP production, including bacteria, fungi and plants, and also the related strategies for reaching greater functional peptide production. The main difficulties of each system are also described in order to provide some directions for AMP production. In summary, data revised here indicate that large-scale production of AMPs can be obtained using biotechnological tools, and the products may be applied in the pharmaceutical industry as well as in agribusiness.     

Lichens: a promising source of antibiotic and anticancer drugs

Lichens are symbiotic associations between fungi and a photosynthetic alga and/or cyanobacteria. Lichenized fungi have been found to produce a wide array of secondary metabolites, most of which are unique to the lichenized condition. These secondary metabolites have shown an impressive range of biological activities including antibiotics, antifungal, anti-HIV, anticancer, anti-protozoan, etc. This review focuses primarily on the antibiotic and anticancer properties of lichen secondary chemicals. We have reviewed various publications related to antibiotic and anticancer drug therapies emphasizing results about specific lichens and/or lichen compounds, which microbes or cancer cells were involved and the main findings of each study. We found that crude lichen extracts and various isolated lichen compounds often demonstrate significant inhibitory activity against various pathogenic bacteria and cancer cell lines at very low concentrations. There were no studies examining the specific mechanism of action against pathogenic bacteria; however, we did find a limited number of studies where the mechanism of action against cancer cell lines had been explored. The molecular mechanism of cell death by lichen compounds includes cell cycle arrest, apoptosis, necrosis, and inhibition of angiogenesis. Although lichens are a reservoir for various biologically active compounds, only a limited number have been tested for their biological significance. There is clearly an urgent need for expanding research in this area of study, including in depth studies of those compounds which have shown promising results as well as a strong focus on identifying specific mechanisms of action and extensive clinical trials using the most promising lichen based drug therapies followed by large scale production of the best of those compounds.     

Opportunities and Challenges of Plant Bioactive Compounds for Food and Agricultural-Related Areas

The growing development of biological products highlights the social and environmental responsibility that several industrial companies are facing in recent years. In this context, the advancement of bioprocessing as an alternative for exploring the potential of ecologically based products, especially in biofuels, food, and agro-industrial business, exposes the rational efficiency of the application of renewable sources in different industrial segments. Industries strongly associated with food production concentrate large amounts of wastes rich in bioactive compounds. A range of highly effective technologies has been highly explored to recover large concentrations of prominent compounds present in these materials. The advances in this scenario assurance value addition to these by-products, in addition to highlighting their various technological applications, considering the biorefinery and ecologically based production concepts. Accordingly, this review article described a detailed and systematic approach to the importance of using bioactive compounds and exploring the main sources of these elements. Also, some recent and innovative research that has achieved encouraging results was highlighted. Furthermore, the study included the main extraction technologies that have been investigated as a strategy of prospecting the application of bioactive compounds and optimizing the processes for obtaining natural compounds from plant sources. Finally, future outlooks were presented to contribute to the innovative opportunities and applicability of highly promising technologies and manipulations of bioactive compounds from a range of perspectives.     

Major secondary metabolites produced by two commercial Trichoderma strains active against different phytopathogens

AIMS: Trichoderma harzianum strains T22 and T39 are two micro-organisms used as active agents in a variety of commercial biopesticides and biofertilizers and widely applied amongst field and greenhouse crops. The production, isolation, biological and chemical characterization of the main secondary metabolites produced by these strains are investigated. METHODS AND RESULTS: Of the three major compounds produced by strain T22, one is a new azaphilone that shows marked in vitro inhibition of Rhizoctonia solani, Pythium ultimum and Gaeumannomyces graminis var. tritici. In turn, filtrates from strain T39 were demonstrated to contain two compounds previously isolated from other T. harzianum strains and a new butenolide. The production of the isolated metabolites was also monitored by liquid chromatography/mass spectrometry during in vitro interaction with R. solani. CONCLUSIONS: This paper reports the isolation and characterization of the main secondary metabolites obtained from culture filtrates of two T. harzianum strains and their production during antagonistic interaction with the pathogen R. solani. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first work on secondary metabolites produced by the commercially applied strains T22 and T39. Our results provide a better understanding of the metabolism of these fungi, which are both widely used as biopesticides and/or biofertilizers in biocontrol.     

Laccases: A Useful Group of Oxidoreductive Enzymes

Using enzymes as decontaminating agents has received great attention. One of the most promising groups of enzymes, laccases, are used to decontaminate phenol-polluted systems and for bio technological applications. Higher plants and fungi, mostly wood-rotting fungi, are the main producers of laccases, but bacterial laccases also have been found. Belonging to the class of phenoloxidases, laccases catalyze the polymerization of several phenolic substances to polymeric products. In addition, they have transformed lignin and lignin-related compounds, showing a very broad substrate specificity. Specific compounds acting as protein-synthesis inducers historically have been used to improve the production of the enzyme. Recent success in fungal molecular and cellular engineering technology has contributed to significantly increase the industrial production of recombinant laccase. Kinetic (Michaelis-Menten parameters, optimum pH, kcat) and stability properties of laccases may vary according to the source of the enzymes. Laccases are used in a variety of applications, such as to remove toxic compounds from aquatic and terrestrial systems, to produce and treat beverages, as analytical tools, and as biosensors to estimate the quantity of phenols in natural juices or the presence of other enzymes. Laccases have been used successfully in immobilized form as well as dissolved in organic solvents.     

利用海洋硅藻生产生物活性物质研究进展*

生物活性物质在食品、饵料、化妆品、保健品和医药等行业具有广阔的应用前景,其研究早已受到广泛关注。鉴于海洋硅藻具有生长速度快、生物活性物质含量高、易于规模培养、便于提取等诸多优势,为理想的生物活性物质生产者。尽管国内外已进行了大量利用海洋硅藻生产生物活性物质的研究,但是受限于培养工艺老旧、生产成本过高等缺陷,商业化利用海洋硅藻开发生物活性物质依然停滞不前。阐述海洋硅藻五种常见生物活性物质的应用价值,进一步探讨海洋硅藻高产生物活性物质的策略,就如何低成本、高效开发利用硅藻源生物活性物质提出建议,为海洋硅藻商业化开发利用提供参考。     

Production of halogenated compounds byBjerkandera adusta

The white-rot fungusBjerkandera adusta produces volatile chlorinated phenyl compounds. The main compounds identified were 3-chloro-4-methoxybenzaldehyde (3-chloro-p-anisaldehyde), 3-chloro-4-methoxybenzyl alcohol (3-chloro-p-anisyl alcohol), 3,5-dichloro-4-methoxybenzaldehyde (3,5-dichloro-p-anisaldehyde), and 3,5-dichloro, 4-methoxybenzyl alcohol (3,5-dichloro-p-anisyl alcohol).p-Anisaldehyde, veratraldehyde and the corresponding alcohols,p-anisyl alcohol and veratryl alcohol were produced simultaneously. Even with a very low concentration of chloride in the medium (< 10^–5 m), chlorinated aromatic compounds were still observed. Addition of bromide to the culture medium led to the production of brominated compounds: 3-bromo-4-methoxybenzaldehyde, 3-bromo-4-methoxybenzyl alcohol, 3,5-dibromo-4-methoxybenzaldehyde and 3-bromo-5-chloro-4-methoxybenzaldehyde. These brominated compounds have not previously been reported as natural products. Although iodo-aromatic compounds were not produced by supplementation of the medium with iodide, isovanillin was found in the culture broth under these conditions. This compound may be formed by substitution of the iodine intermediate by a hydroxyl group on the third carbon of the ring. Diiodomethane or chloroiodomethane were also found. It is the first time that the production of halomethane has been related to the production of halogenated aromatic compounds. All the strains tested have these capabilities.     

Nonribosomal peptides in fungal cell factories: from genome mining to optimized heterologous production

Fungi are notoriously prolific producers of secondary metabolites including nonribosomal peptides (NRPs). The structural complexity of NRPs grants them interesting activities such as antibiotic, anti-cancer, and anti-inflammatory properties. The discovery of these compounds with attractive activities can be achieved by using two approaches: either by screening samples originating from various environments for their biological activities, or by identifying the related clusters in genomic sequences thanks to bioinformatics tools. This genome mining approach has grown tremendously due to recent advances in genome sequencing, which have provided an incredible amount of genomic data from hundreds of microbial species. Regarding fungal organisms, the genomic data have revealed the presence of an unexpected number of putative NRP-related gene clusters. This highlights fungi as a goldmine for the discovery of putative novel bioactive compounds. Recent development of NRP dedicated bioinformatics tools have increased the capacity to identify these gene clusters and to deduce NRPs structures, speeding-up the screening process for novel metabolites discovery. Unfortunately, the newly identified compound is frequently not or poorly produced by native producers due to a lack of expression of the related genes cluster. A frequently employed strategy to increase production rates consists in transferring the related biosynthetic pathway in heterologous hosts. This review aims to provide a comprehensive overview about the topic of NRPs discovery, from gene cluster identification by genome mining to the heterologous production in fungal hosts. The main computational tools and methods for genome mining are herein presented with an emphasis on the particularities of the fungal systems. The different steps of the reconstitution of NRP biosynthetic pathway in heterologous fungal cell factories will be discussed, as well as the key factors to consider for maximizing productivity. Several examples will be developed to illustrate the potential of heterologous production to both discover uncharacterized novel compounds predicted in silico by genome mining, and to enhance the productivity of interesting bio-active natural products.     

Plant in vitro culture for the production of antioxidants--a review

Plant in vitro cultures are able to produce and accumulate many medicinally valuable secondary metabolites. Antioxidants are an important group of medicinal preventive compounds as well as being food additives inhibiting detrimental changes of easily oxidizable nutrients. Many different in vitro approaches have been used for increased biosynthesis and the accumulation of antioxidant compounds in plant cells. In the present review some of the most active antioxidants derived from plant tissue cultures are described; they have been divided into the main chemical groups of polyphenols and isoprenoids, and several examples also from other chemical classes are presented. The strategies used for improving the antioxidants in vitro production efficiency are also highlighted, including media optimization, biotransformation, elicitation, Agrobacterium transformation and scale-up.     

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.     

Visible light-dependent degradation of lipidic phytoplanktonic components during senescence: a review

Though most of the organic components of phytoplankton are susceptible to being photodegraded during senescence, until recent years there has been very little research in this area. Recently, however, there have been a renewal of interest, and the heterogeneous visible light-induced degradative reactions of lipidic compounds associated with phytodetritus have been studied. The present paper reviews the results obtained in the course of these studies. In the first part, the production and quenching of excited states of chlorophyll and toxic oxygen species (singlet oxygen, superoxide ion, hydroxyl radical and hydrogen peroxide) in healthy and senescent phytoplanktonic cells are discussed. Then, the photo-oxidation of the main lipidic cell components (chlorophyll, chlorophyll phytyl chain, carotenoids, sterols, unsaturated fatty acids, alkenones and unsaturated alkenes) in senescent phytoplanktonic cells is examined. Emphasis is given to the degradation rates and the mechanisms of visible light-induced degradation of the main lipidic components of phytoplankton and to the structure of the photoproducts formed. In each case, the selectivity of these compounds is discussed in order to select a "pool" of photoproducts able to act as tracers of photo-oxidative alterations in the marine medium. This "pool" of photoproducts could provide very useful information concerning the physiological state of phytoplanktonic communities and current environmental problems related to ozone depletion.     

Microalgae as platforms for production of recombinant proteins and valuable compounds: progress and prospects

Over the last few years microalgae have gained increasing interest as a natural source of valuable compounds and as bioreactors for recombinant protein production. Natural high-value compounds including pigments, long-chain polyunsaturated fatty acids, and polysaccharides, which have a wide range of applications in the food, feed, cosmetics, and pharmaceutical industries, are currently produced with nontransgenic microalgae. However, transgenic microalgae can be used as bioreactors for the production of therapeutic and industrially relevant recombinant proteins. This technology shows great promise to simplify the production process and significantly decrease the production costs. To date, a variety of recombinant proteins have been produced experimentally from the nuclear or chloroplast genome of transgenic Chlamydomonas reinhardtii. These include monoclonal antibodies, vaccines, hormones, pharmaceutical proteins, and others. In this review, we outline recent progress in the production of recombinant proteins with transgenic microalgae as bioreactors, methods for genetic transformation of microalgae, and strategies for highly efficient expression of heterologous genes. In particular, we highlight the importance of maximizing the value of transgenic microalgae through producing recombinant proteins together with recovery of natural high-value compounds. Finally, we outline some important issues that need to be addressed before commercial-scale production of high-value recombinant proteins and compounds from transgenic microalgae can be realized.     

Antifouling enzymes and the biochemistry of marine settlement

Antifouling coatings are used extensively on marine vessels and constructions, but unfortunately they are found to pose a threat to the marine environment, notably due to content of metal-based biocides. Enzymes have repeatedly been proposed as an alternative to traditional antifouling compounds. In this review, the enzymes claimed to hold antifouling activity are classified according to catalytic functions. The enzyme functions are juxtaposed with the current knowledge about the chemistry of settlement and adhesion of fouling organisms. Specific focus will be on bacteria, microalgae, invertebrate larvae and macroalgae zoospores. Two main concepts in enzyme-based antifouling are identified: breakdown of adhesive components and catalytic production of repellent compounds in-situ. The validity of the various modes of action is evaluated and the groups of enzymes with the highest potential are highlighted.     

Biotransformation of terpenes

The main application of terpenes as fragrances and flavors depends on the absolute configuration of the compounds because enantiomers present different organoleptic properties. Biotransformations allow the production of regio- and stereoselective compounds under mild conditions. These products may be labeled as "natural". Commercially useful chemical building-blocks and pharmaceutical stereo isomers can also be produced by bioconversion of terpenes. Enzymes and extracts from bacteria, cyanobacteria, yeasts, microalgae, fungi, plants, and animal cells have been used for the production and/or bioconversion of terpenes. In addition, whole cell catalysis has also been used. A variety of media and reactors have been assessed for these biotransformations and have produced encouraging results, as discussed in this review.     

Modulation of volatile sulfur compounds by wine yeast

Sulfur compounds in wine can be a ‘double-edged sword’. On the one hand, certain sulfur-containing volatile compounds such as hydrogen sulfide, imparting a rotten egg-like aroma, can have a negative impact on the perceived quality of the wine, and on the other hand, some sulfur compounds such as 3-mercaptohexanol, imparting fruitiness, can have a positive impact on wine flavor and aroma. Furthermore, these compounds can become less or more attractive or repulsive depending on their absolute and relative concentrations. This presents an interesting challenge to the winemaker to modulate the concentrations of these quality-determining compounds in wine in accordance with consumer preferences. The wine yeast Saccharomyces cerevisiae plays a central role in the production of volatile sulfur compounds. Through the sulfate reduction sequence pathway, the HS^- is formed, which can lead to the formation of hydrogen sulfide and various mercaptan compounds. Therefore, limiting the formation of the HS^- ion is an important target in metabolic engineering of wine yeast. The wine yeast is also responsible for the transformation of non-volatile sulfur precursors, present in the grape, to volatile, flavor-active thiol compounds. In particular, 4-mercapto-4-methylpentan-2-one, 3-mercaptohexanol, and 3-mercaptohexyl acetate are the most important volatile thiols adding fruitiness to wine. This paper briefly reviews the metabolic processes involved in the production of important volatile sulfur compounds and the latest strategies in the pursuit of developing wine yeast strains as tools to adjust wine aroma to market specifications.     

施加外源物质对盐胁迫下水稻生长发育的影响

水稻是重要的粮食作物之一,属于不耐盐的甜土植物,土壤盐渍化是影响其产量和生长发育的主要因素。消除或缓解盐胁迫是当务之急,目前主要应用传统育种、转基因技术及外源物质缓解盐胁迫。拟从外源物质对盐胁迫下水稻的影响作一综述,旨在为水稻生产及抗性育种提供理论依据和实践方法。     

Perspectives and limits of engineering the isoprenoid metabolism in heterologous hosts

Terpenoids belong to the largest class of natural compounds and are produced in all living organisms. The isoprenoid skeleton is based on assembling of C5 building blocks, but the biosynthesis of a great variety of terpenoids ranging from monoterpenoids to polyterpenoids is not fully understood today. Terpenoids play a fundamental role in human nutrition, cosmetics, and medicine. In the past 10 years, many metabolic engineering efforts have been undertaken in plants but also in microorganisms to improve the production of various terpenoids like artemisinin and paclitaxel. Recently, inverse metabolic engineering and combinatorial biosynthesis as main strategies in synthetic biology have been applied to produce high-cost natural products like artemisinin and paclitaxel in heterologous microorganisms. This review describes the recent progresses made in metabolic engineering of the terpenoid pathway with particular focus on fundamental aspects of host selection, vector design, and system biotechnology.