Hydrocarbon-Degrading Microbes as Sources of New Biocatalysts

Coscolín, Cristina; Bargiela, Rafael; Martínez-Martínez, Mónica; Alonso, Sandra; Bollinger, Alexander; Thies, Stephan; Chernikova, Tatyana; Tran, Hai; Golyshina, Olga V.; Jaeger, Karl‐Erich; Yakimov, Michail M.; Golyshin, Peter N.; Ferrer, Manuel

doi:10.1007/978-3-030-14796-9_13

Cited by 3 publications

(2 citation statements)

References 75 publications

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“…H. aestusnigri VGXO14 was isolated from a crude oil‐contaminated sand sample of the intertidal zone (Sánchez et al., 2014). It harbours a remarkable number of lipolytic enzymes, some with astonishing substrate promiscuity and resistance to organic solvents (Bollinger, Molitor, Thies, et al., 2020; Coscolín et al., 2019). The soil bacterium H. bauzanensis BZ93 was isolated from an industrial site (Zhang et al., 2011).…”

Section: Resultsmentioning

confidence: 99%

Halopseudomonas species: Cultivation and molecular genetic tools

Kruse,

Loeschcke,

de Witt

et al. 2023

Microbial Biotechnology

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The Halopseudomonas species, formerly classified as Pseudomonas pertucinogena lineage, form a unique phylogenetic branch within the Pseudomonads. Most strains have recently been isolated from challenging habitats including oil‐ or metal‐polluted sites, deep sea, and intertidal zones, suggesting innate resilience to physical and chemical stresses. Despite their comparably small genomes, these bacteria synthesise several biomolecules with biotechnological potential and a role in the degradation of anthropogenic pollutants has been suggested for some Halopseudomonads. Until now, these bacteria are not readily amenable to existing cultivation and cloning methods. We addressed these limitations by selecting four Halopseudomonas strains of particular interest, namely H. aestusnigri, H. bauzanensis, H. litoralis, and H. oceani to establish microbiological and molecular genetic methods. We found that C4‐C10 dicarboxylic acids serve as viable carbon sources in both complex and mineral salt cultivation media. We also developed plasmid DNA transfer protocols and assessed vectors with different origins of replication and promoters inducible with isopropyl‐β‐d‐thiogalactopyranoside, l‐arabinose, and salicylate. Furthermore, we have demonstrated the simultaneous genomic integration of expression cassettes into one and two attTn7 integration sites. Our results provide a valuable toolbox for constructing robust chassis strains and highlight the biotechnological potential of Halopseudomonas strains.

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Section: Resultsmentioning

confidence: 99%

Halopseudomonas species: Cultivation and molecular genetic tools

Kruse,

Loeschcke,

de Witt

et al. 2023

Microbial Biotechnology

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“…The metabolic diversity of bacteria is among their most useful properties for synthetic biology; this feature is the product of millions of years of evolutionary adaptation to diverse niches, nutrients, and stresses. Hydrocarbon-metabolising bacteria are especially interesting for synthetic biology applications, including bioremediation ( Das and Chandran, 2011 ), biocatalysis ( Coscolín et al, 2019 ), and biosensors ( Sticher et al, 1997 ).…”

Section: Introductionmentioning

confidence: 99%

Synthetic Biology Approaches to Hydrocarbon Biosensors: A Review

Moratti

Scott

Coleman

2022

Front. Bioeng. Biotechnol.

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Monooxygenases are a class of enzymes that facilitate the bacterial degradation of alkanes and alkenes. The regulatory components associated with monooxygenases are nature’s own hydrocarbon sensors, and once functionally characterised, these components can be used to create rapid, inexpensive and sensitive biosensors for use in applications such as bioremediation and metabolic engineering. Many bacterial monooxygenases have been identified, yet the regulation of only a few of these have been investigated in detail. A wealth of genetic and functional diversity of regulatory enzymes and promoter elements still remains unexplored and unexploited, both in published genome sequences and in yet-to-be-cultured bacteria. In this review we examine in detail the current state of research on monooxygenase gene regulation, and on the development of transcription-factor-based microbial biosensors for detection of alkanes and alkenes. A new framework for the systematic characterisation of the underlying genetic components and for further development of biosensors is presented, and we identify focus areas that should be targeted to enable progression of more biosensor candidates to commercialisation and deployment in industry and in the environment.

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Organic-Solvent-Tolerant Carboxylic Ester Hydrolases for Organic Synthesis

Bollinger

Molitor

Thies

et al. 2020

Appl Environ Microbiol

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Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by carboxylic ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of carboxylic ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant esterase from Pseudomonas aestusnigri. In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard ester hydrolase CalB from Candida antarctica. IMPORTANCE Major challenges hampering biotechnological applications of esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant carboxylic ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.

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Hydrocarbon-Degrading Microbes as Sources of New Biocatalysts

Cited by 3 publications

References 75 publications

Halopseudomonas species: Cultivation and molecular genetic tools

Halopseudomonas species: Cultivation and molecular genetic tools

Synthetic Biology Approaches to Hydrocarbon Biosensors: A Review

Organic-Solvent-Tolerant Carboxylic Ester Hydrolases for Organic Synthesis

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