Biocatalytic application of rare PLP-dependent aminotransferases for the synthesis of high value amino acids and amines

Optically active amines and amino acids are ubiquitously distributed in nature where they play many crucial roles. Moreover, it was recently estimated that around 40% of blockbuster drugs and 20% of agrochemicals contain chiral amines in their structure, thus there has been considerable effort in developing efficient, low cost and widely-applicable methods for their production. Compared to classical chemical synthesis, aminotransferase (AT) enzymes have been widely explored as a more efficient and sustainable method for the preparation of optically pure amines from the corresponding ketones. In several cases these biocatalysts have replaced the existing chemical catalysts, which are limited by their high cost and poor regio- and stereoselectivity. Two very interesting, distantly-related class III bacterial ATs have been investigated in this study: the D-phenylglycine (D-Phg) aminotransferase (D-PhgAT) and the amino-pentol AT (FumI). The D-PhgAT from Pseudomonas stutzeri ST-201 catalyses the reversible transamination from L-glutamic acid to benzoylformate, yielding α-ketoglutarate and D-Phg with high (>99%) enantiomeric excess (% ee). The D-PhgAT possesses the unique feature that the amino acid donor and amino acid product display an inverted absolute configuration. Thus, D-PhgAT is a very promising biocatalyst as it yields high value D-amino acids from inexpensive L-amino acid donors. By carrying out a detailed kinetic analysis of recombinant D-PhgAT the best substrates were identified and an optimized method, delivering a range of enantiopure aromatic D-amino acids at 1 g scale, has been developed. Moreover, the x-ray crystal structure of D-PhgAT at 2.25 Å resolution has been determined with its pyridoxal 5’- phosphate (PLP) cofactor bound as an internal aldimine. The active site architecture highlights various residues potentially involved in catalysis and illuminates the basis of the exquisite enantioselectivity of this unique member of the AT superfamily. These studies promote D-PhgAT as a useful tool for the sustainable production of high value, aromatic D-amino acids. Fatty amines (C8-C20) are high value products with a wide variety of applications in fabric softeners, detergents and cosmetic formulations. The unusual Sphingopyxis FumI detoxifies the C20 mycotoxin natural product fumonisin B1 (FB1). FumI catalyses transamination at the C2 position of a hydrolyzed fumonisin B1 (HFB1) derivative with pyruvate as the amino acceptor. A screen of prochiral C2 ketones of varying chain length (C3-C17 2K) with L-alanine as amino donor has revealed that the recombinant FumI is capable of producing alkyl-amines of various chain lengths (C3-C17) by working in the reverse direction. Chiral HPLC analysis has revealed that the enzyme generates (S)-amines with high ee (>99%). The regioselectivity of FumI was also determined using prochiral keto-substrates with substitutions along the chain (such as 3-decanone (C10-3K) and 5-decanone (C10-5K)). Interestingly, C10-3K, but not C10-5K, was converted to the corresponding amine. A range of long chain aldehydes (C10-C20) were also converted by FumI to the corresponding amines with L-Ala as the amino donor. The x-ray crystal structure of the PLP-bound, internal aldimine form of FumI was solved to a resolution of 1.6 Å by molecular replacement using a Bacillus subtilis AT as a model. The structure suggested residues potentially involved in catalysis and revealed a potential hydrophobic binding site for the long alkyl chain substrates. The combination of aldehyde and ketone alkyl chain substrate promiscuity, the high enantioselectivity and the 3D structures of this rare AT suggests that FumI is a very useful addition to the biocatalytic toolbox for the synthesis of high value fatty amines

Biorefinery Gets Hot: Thermophilic Enzymes and Microorganisms for Second-Generation Bioethanol Production

To mitigate the current global energy and the environmental crisis, biofuels such as bioethanol have progressively gained attention from both scientific and industrial perspectives. However, at present, commercialized bioethanol is mainly derived from edible crops, thus raising serious concerns given its competition with feed production. For this reason, lignocellulosic biomasses (LCBs) have been recognized as important alternatives for bioethanol production. Because LCBs supply is sustainable, abundant, widespread, and cheap, LCBs-derived bioethanol currently represents one of the most viable solutions to meet the global demand for liquid fuel. However, the cost-effective conversion of LCBs into ethanol remains a challenge and its implementation has been hampered by several bottlenecks that must still be tackled. Among other factors related to the challenging and variable nature of LCBs, we highlight: (i) energy-demanding pretreatments, (ii) expensive hydrolytic enzyme blends, and (iii) the need for microorganisms that can ferment mixed sugars. In this regard, thermophiles represent valuable tools to overcome some of these limitations. Thus, the aim of this review is to provide an overview of the state-of-the-art technologies involved, such as the use of thermophilic enzymes and microorganisms in industrial-relevant conditions, and to propose possible means to implement thermophiles into second-generation ethanol biorefineries that are already in operation

Systematic screening for the biocatalytic hydration of fatty acids from different oily substrates by Elizabethkingia meningoseptica oleate hydratase through a Design-of-experiments approach

The edible plant oils production is associated with the release of different types of by-products. The latter represent cheap and available substrates to produce valuable compounds, such as flavours and fragrances, biologically active compounds and bio-based polymers. Elizabethkingia meningoseptica Oleate hydratases (Em_OhyA) can selectively catalyze the conversion of unsaturated fatty acids, specifically oleic acid, into hydroxy fatty acids, which find different industrial applications. In this study, Design-of-experiment (DoE) strategy was used to screen and identify conditions for reaching high yields in the reaction carried out by Escherichia coli whole-cell carrying the recombinant enzyme Em_OhyA using Waste Cooking Oils (WCO)-derived free fatty acids (FFA) as substrate. The identified reaction conditions for high oleic acid conversion were also tested on untreated triglycerides-containing substrates, such as pomace oil, sunflower oil, olive oil and oil mill wastewater (OMW), combining the triglyceride hydrolysis by the lipase from Candida rugosa and the E. coli whole-cell containing Em_OhyA for the production of hydroxy fatty acids. When WCO, sunflower oil and OMW were used as substrate, the one-pot bioconversion led to an increase of oleic acid conversion compared to the standard reaction. This work highlights the efficiency of the DoE approach to screen and identify conditions for an enzymatic reaction for the production of industrially-relevant products

Pseudomonas germanica sp. nov., isolated from Iris germanica rhizomes

Through bacterial plant-endophyte extraction from rhizomes of Iris germanica plant, a Gram-stain-negative, aerobic, catalase- and oxidase-positive gammaproteobacterial strain, referred to as FIT28T, was isolated. FIT28T shows vigorous growth on nutrient rich media within the temperature range of 4-35 °C, with optimal growth at 28 °C, a wide pH tolerance from pH 5 to 11, and salt tolerance up to 6 % (w/v) NaCl. Colonies are white-yellow and quickly become mucoid. The results of analysis of the 16S rRNA gene sequence placed the strain within the genus Pseudomonas, and multilocus sequence analysis (MLSA) using 16S rRNA, rpoB, gyrB and rpoD concatenated sequences revealed that the closest relatives of FIT28T are Pseudomonas zeae OE48.2T, 'Pseudomonas crudilactis' UCMA 17988, Pseudomonas tensinigenes ZA5.3T, Pseudomonas helmanticensis OHA11T, Pseudomonas baetica a390T, Pseudomonas iridis P42T, Pseudomonas atagonensis PS14T and Pseudomonas koreensis Ps 9-14T, within the Pseudomonas koreensis subgroup of the Pseudomonas fluorescens lineage. The genome size of FIT28T is about 6.7 Mb with 59.09 mol% DNA G+C content. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values calculated from the genomic sequences of FIT28T, and the closely related P. zeae OE48.2T are 95.23 and 63.4 %, respectively. Biochemical, metabolic and chemotaxonomic studies further support our proposal that Pseudomonas germanica sp. nov., should be considered a novel species of the genus Pseudomonas. Hence, the type strain FIT28T (=LMG 32353T=DSM 112698T) has been deposited in public cell-type culture centres

Pseudomonas fitomaticsae sp. nov., isolated at Marimurtra Botanical Garden in Blanes, Catalonia, Spain

In the framework of the research project called fitomatics, we have isolated and characterized a bacterial plant-endophyte from the rhizomes of Iris germanica, hereafter referred to as strain FIT81T. The bacterium is Gram negative, rod-shaped with lophotrichous flagella, and catalase- and oxidase-positive. The optimal growth temperature of strain FIT81T is 28 °C, although it can grow within a temperature range of 4-32 °C. The pH growth tolerance ranges between pH 5 and 10, and it tolerates 4% (w/v) NaCl. A 16S rRNA phylogenetic analysis positioned strain FIT81T within the genus Pseudomonas, and multilocus sequence analysis revealed that Pseudomonas gozinkensis IzPS32dT, Pseudomonas glycinae MS586T, Pseudomonas allokribbensis IzPS23T, 'Pseudomonas kribbensis' 46-2 and Pseudomonas koreensis PS9-14T are the top five most closely related species, which were selected for further genome-to-genome comparisons, as well as for physiological and chemotaxonomic characterization. The genome size of strain FIT81T is 6 492 796 base-pairs long, with 60.6 mol% of G+C content. Average nucleotide identity and digital DNA-DNA hybridization analyses yielded values of 93.6 and 56.1%, respectively, when the FIT81T genome was compared to that of the closest type strain P. gozinkensis IzPS32dT. Taken together, the obtained genomic, physiologic and chemotaxonomic data indicate that strain FIT81T is different from its closest relative species, which lead us to suggest that it is a novel species to be included in the list of type strains with the name Pseudomonas fitomaticsae sp. nov. (FIT81T=CECT 30374T=DSM 112699T)

Marine-Derived Surface Active Agents: Health-Promoting Properties and Blue Biotechnology-Based Applications

Surface active agents are characterized for their capacity to adsorb to fluid and solid-water interfaces. They can be classified as surfactants and emulsifiers based on their molecular weight (MW) and properties. Over the years, the chemical surfactant industry has been rapidly increasing to meet consumer demands. Consequently, such a boost has led to the search for more sustainable and biodegradable alternatives, as chemical surfactants are non-biodegradable, thus causing an adverse effect on the environment. To these ends, many microbial and/or marine-derived molecules have been shown to possess various biological properties that could allow manufacturers to make additional health-promoting claims for their products. Our aim, in this review article, is to provide up to date information of critical health-promoting properties of these molecules and their use in blue-based biotechnology (i.e., biotechnology using aquatic organisms) with a focus on food, cosmetic and pharmaceutical/biomedical applications

Single Crystal X-ray diffraction data for D-phgAT

Single Crystal X-ray diffraction data for Dpg-AT from Pseudomonas stutzeri. Data collected at Diamond Light Source, on beamline I03 on 18/02/2017

The art of exploiting plant endophytes as a source of novel bioactive compounds production

Trabajo presentado al XV Meeting of Plant Molecular Biology, celebrado online del 26 al 27 de noviembre de 2020.Peer reviewe