Current Trends in Methylotrophy -based Biotechnology
Yuri A Trotsenko* and Maria L Torgonskaya
GK Skryabin Institute of Biochemistry and Physiology of Microorganisms of Russian Academy of Sciences, Russia
Submission: April 28, 2018; Published: May 24, 2018
*Corresponding author: Yuri A Trotsenko, Laboratory of Methylotrophy, GK Skryabin Institute of Biochemistry and Physiology of Microorganisms of Russian Academy of Sciences, Russia.
How to cite this article: Yuri A T, Maria L T. Current Trends in Methylotrophy -based Biotechnology. Adv Biotech & Micro. 2018; 9(3): 555763. DOI: 10.19080/AIBM.2018.09.555763
Abstract
The review briefly summarizes the viewpoints on biotechnological potential of methylotrophic bacteria and yeasts, present state of art and new trends of application of their unique metabolism for development of novel technologies for biodegradation, bioanalytics and biosynthesis.
Keywords: Methylotrophy; Biotechnology; Bioremediation; Biosynthesis; Biocatalysis; Bioanalytics
Introduction
Methylotrophy is an amazing lifestyle, which allows prokaryotic and eukaryotic microorganisms to use single-carbon compounds as the sole sources of carbon and energy. Specialized group within methylotrophs is represented by methanotrophs capable of oxidizing a greenhouse gas methane, whereas the majority of methylotrophic bacteria and yeasts use methanol and other methylated compounds for their growth [1]. The presence of natural sources of C1-compounds such as plants, biomass burning, volcanic activity, etc. is evidently responsible for the almost ubiquitous distribution of microorganisms implementing methylotrophic metabolism [2-5]. Being biological sink for toxic C1-compounds methylotrophs prevent their release into the atmosphere and decrease the impact (as greenhouse gases) to global climate. At the same time C1-utilizers are able to synthesize a wide spectrum of valuable products from these abundant and potentially cheap substrates [6,7]. Together with a well-studied metabolism, availability of genomic data and actively developing methods of metabolic engineering, this peculiarity makes methylotrophs an attractive instrument for biotechnologists. In the present review current directions and modern tendencies of biotechnological application of methylotrophs and their specific metabolic abilities are listed.
Biodegradation and bioremediation
The most obvious application of methylotrophic metabolism lies in use of corresponding bacterial degraders and their enzymes in processes of biodegradation of industrially produced C1-compounds and bioremediation of polluted ecosystems. Methylotrophic bacteria are usually capable of oxidizing methanol, formaldehyde and formate utilizing these substances without formation of undesirable by-products [8-10]. High phenotypic plasticity allows to methylotrophs occupy different (soil, water, sediments), even extreme habitats, colonize plants and animals [11,12]. These microorganisms are phylogenetically diverse and adapt to environmental challenges by tuning of metabolic modes, therefore strains from different ecological niches can be used for selection of starting platforms for further development of desired technologies and products based on single-carbon substrates [7]. Some C1-utilizing bacteria can effectively remove methylated amines, methylated sulfur compounds (dimethylsulfide, dimethyldisulfide, methanesulfonic acid, etc.) [5,13] and halogenated methanes (chloro, bromo- and iodomethanes, DCM) [4,14]. However, the latter metabolic possibilities are rather rare and likely represent a result of evolution under conditions of long selective pressure of specific C1-compounds [15,16]. The ability to mineralize C1-compounds can be further improved by genetic engineering. For example, it was shown, that heterologous expression of methylotrophic genes encoding key enzymes of the ribulose monophosphate C1-assimilation pathway hexulose phosphate synthase (EC 4.1.2.43) and 6-phospho-3-hexuloisomerase (EC 5.3.1.27), increase the efficiency of removing formaldehyde by bacterial strains and transgenic plants [9,17,18].
Besides C1-compounds some methylotrophic bacteria can degrade a variety of other organic toxicants. Pseudomonas esterophilus 27RD, Pseudomonas esterovorus 24RA utilize methyl- and ethyl-acetates [19]. Two strains of Methylobacterium populi (V2 and BJ001) were shown to destruct polyaromatic hydrocarbons [20] and even toxic explosives, such as 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5- triazene (hexogen) and octahydro-1,3,5,7-tetranitro-1,3,5- tetrazocine (octagen) [21]. Methylobacterium sp. GPE1 degrades a heterocyclic constituent of tobacco smoke carbazole [22]. Bacterial consortia, containing methylotrophic bacteria, were demonstrated to efficiently remove phenol [23] and also common soil pollutants methyl-tert-butyl ether [24,25] and trichloroethylene [26]. Genomic prediction of methylotrophic lifestyle, basing on the concept of minimal metabolic modules [1], constantly expands our understanding of distribution and environmental roles of methylotrophic bacteria. In particular, available genomic data of newly discovered poly (ethylene terephtalate)-degrading bacteria Ideonella sakaiensis 201- F6 [27], suggest that this strain can also employ autotrophic methylotrophy.
Bioanalytics
Whole cells and enzymes of methylotrophs are also used in bioanalytics for detection and monitoring of the content of toxic C1-compounds in the environment (e.g., industrial gas emissions, wastewaters, and indoor air), drinking water, various products, foods, therapeutic drugs, and biological fluids. Highly sensitive and selective biosensor technology is an accessible alternative to the expensive and complex physicochemical methods of detection. Biosensors are hybrid devices containing a bioelement (immobilized bioactive substance), which defines a degree of selectivity, and a physical transformer (transducer). To date a number of biosensors and bioreporter systems on the basis of cells, enzymes and even specific cofactors of methylotrophs were designed for methanol, ethanol, dichloromethane, formaldehyde, mono-, di-, and trimethylamines, glucose, lactate, aspartam, n-butanol, benzyl alcohol [6, 28,29].
Another type of bioreporter systems is represented by genetically modified cells demonstrating visible signals in response to chemical stimuli. Such a system constructed by introducing into intact cells of the chloromethane destructor Methylobacterium chloromethanicum CM4 of a plasmid with the gene of the yellow fluorescent protein under control of the promoter of the chloromethane dehalogenase (EC 2.1.1.-) cmuA gene allowed marking of the natural sources of methyl halides by biofluorescence [30].
Biosynthesis and biocatalysis
Historically, methylotrophic microorganisms represent a widely used platform for bioproduction of chemicals for agricultural and chemical industry from non-sugar substrates. Methylotroph-based single cell protein (SCP) production has been active already in the 1980s, but has not yet exhausted its potential and can progress with use of new perspective strains [7,31]. Remarkable outcomes were reached for methane- and methanolbased production of biopolymers - polyhydroxyalkanoates, which have a wide range of applications in packaging, medicine, or as textile and household materials, and represent a fully biodegradable environmentally friendly alternative to traditional petroleum-based plastics [6,32]. Stiffness and brittleness of natural product poly-3-hydroxybutyrate (PHB) can be overcomed by thorough selection of strains (≥3000 kDa for Methyloligella halotolerans C2 polymer vs. 50-200 kDa for PHB from Ideonella sakaiensis) [6,33] and carbon sources ensuring the synthesis of high-molecular polymer or by functionalizing of PHB by addition of copolymers with other hydroxyalkanoate co-monomer(s), like 3-hydroxyvalerate or 3-hydroxyhexanoate [34]. Second way needs an addition of precursors increasing production cost, therefore as a less expensive scenario an artificial limitation of flux through ethyl-malonyl-CoA pathway (EMCP) was suggested, thus providing propionyl- and butyryl- CoA precursors for synthesis of functionalized PHB [34]. Methylotrophic bacteria and yeasts are also commonly used as natural producers in methanol-based synthesis of a series of chemicals for pharmaceutic, cosmetic, perfume and chemical industry like aldehydes, formate, glycerol, hydrogen peroxide, pyruvate, citrate, amino acids (L-serine, L-glutamate, L-lysine), glyoxylate, gamma-aminobutyric acid, cadaverine, dicarboxylic acids from EMCP ((2S)-methylsuccinic and mesaconic acid), cofactors (FAD, GSH, ATP), homological methylotrophic and antioxidant enzymes, exopolysaccharides, bioprotectant ectoine, antioxidant carotenoid compounds [6,12,35-37]. The possibility of additional increase of the naturally high lipid content in cells of proteobacterial methanotrophs by genetic engineering makes these bacteria promising platform for biodiesel production from methane [7].
Heterologous expression of proteins represents another way of implementation of biotechnological potential of methylotrophs. At present, the most used methylotrophic expression systems suitable for investigations and production of industrial recombinant enzymes include well described yeast platforms of Pichia pastoris, Hansenula polymorpha, Pichia methanolica and Candida boidinii [35,38], which were subjected to adaptive laboratory evolution, resulting in populations with improved growth and production characteristics [39]. Examples of use of methylotrophic bacteria Ideonella sakaiensis for methanolbased expression of heterological proteins (green fluorescent protein, insecticidal protein Cry1Aa, a haloalkane dehalogenase, and Enterocin P) were also reported, but the achieved yields were not sufficient for commercial use. Facilitation of product recovery procedures by using of minimal media and relatively “clean” supernatant of methylotrophic cultures can be noted as advantage of methanol-based expression systems. However, the necessity to synthesize products for food and pharmaceutical industry often makes consider the use of toxic substrate as a shortage of these platforms and avoid it. Furthermore, bacterial hosts lack necessary posttranlational modification machinery and secretion machinery for these strains is not sufficiently studied [36].
Heterologous expression also opens new possibilities for synthesis of chemicals. Recently, this approach was used for engineering of methylotrophic bacteria for production of 2-hydroxybutyric acid, 3-hydroxypropionic acid, mevalonate, lactate and sesquiterpenoid a-humulene [7,36], and also for reversion of the native methanogenic pathway and addition of supplementary enzymes in archaeon Methanosarcina acetivorans for synthesis of acetate and L-lactate from methane [40]. An alternative way to gain benefit from using of simple C1-substrates is represented by so-called “synthetic methylotrophy” and implies an introduction of a minimal set of well studied methylotrophic modules/enzymes enabling methylotrophy into usual heterotrophic industrial platforms like E. coli and Corynebacterium glutamicum. Nowadays, the available instruments of this direction of synthetic biology include design of improved and even artificial variants of methylotrophic enzymes and pathways and also biosensors for monitoring of some C1-fluxes in heterologous hosts [7].
The ability of methylotrophic bacteria to form stable associations with non-methylotrophic microorganisms, supplying them with carbon sources [7], makes economically attractive the use of microbial communities for the synthesis of valuable products, which are not specific for C1-utilizers. It was shown, that in consortium with exoelectrogen Geobacter sulfurreducens and in the presence of humic acids the engineered Ms. acetivorans with reversed methanogenic pathway can convert methane into electrical current. Biotechnological potential of several platforms involving synthetic consortia of methanotrophs and non-methanotrophic methylotrophs or cyanobacteria for production of chemicals and biofuels is under analysis [7].
The last extensive area of application of methylotrophic bacteria lies in agricultural use for increasing of plant growth due to production of phytohormones (auxins, cytokinines, gibberelins) and siderophores, nitrogen fixation, ethylene production decreasing, solubilization of phosphorus, inhibition of plant pathogens and induction of systemic resistance in plants [11,12]. Interestingly, for some methylotrophic phytosymbionts the ability to increase production of furanoid compounds, responsible for fruit flavor, was also demonstrated [41]. Furthermore, the C1-utilizers decrease environmental stress by degrading toxic organic compounds and immobilizing heavy metals [12].
Recent unexpected discovery in methylotrophs of enzymes requiring rare Earth elements (REEs) - lanthanides for their functioning [42] suggest that they possess yet unknown specific mechanisms for sensing, transport and binding of these elements [7]. Identification of such systems can be potentially useful for engineering of new bioprocesses of REEs’ mining for industrial purposes basing on methylotrophic strains.
Conclusion
Broad functional diversity, unpretentiousness in terms of substrates and culture media, profoundly studied metabolism, cultivation technologies, and well-developed systems of genetic modification make methylotrophs increasingly popular agents of modern green biotechnologies. Conversion to valuable products of universally available renewable C1-substrates such as methane, methanol and carbon dioxide, is able not only reduce the cost of synthesis, but also decrease the harmful effects of these sustainable compounds on the ecosystem and the global climate by reducing their anthropogenic emissions. The use of C1-compounds, synthesized as by-products, as a raw material for other industrial processes, obviously represents an element potentially important for the development of technologies of non-waste production.
Acknowledgement
This work was supported by the Russian Foundation of Basic Research (no. 18-04-01148).
Conflict of Interest
The authors declare no conflicts of interest.
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