Directed evolution of acyltransferases for the improvement of triglyceride production in Escherichia coli

Póster presentado al XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Granada del 9 al 12 de septiembre de 2014.Wax ester synthase/diacylglicerol acyltransferase (WS/DGAT) is a family of enzymes able to perform the esterifi cation of diacylglycerol or fatty alcohols and acyl-CoA to produce triglycerides (TAGs) or wax esters, respectively. Both products can be easily transformed onto biodiesel. Although the specifi city determinants of substrate recognition are still unknown and there is no crystal structure solved from any bacterial WS/DGATs, we recently published a study where we used limited proteolysis and directed mutagenesis approaches to identify key folding domains and motifs critical for the catalysis. We have also recently patented a diacylglycerol acyltransferase (tDGAT) (ES201200967) from the thermophilic organism Thermomonospora curvata that is able to produce TAGs and waxes in E. coli. We have used this system for TAG production in E.coli, but we believe it can be signifi cantly improved through directed evolution of tDGAT. Thus, the main goal of our work is to enhance TAG production in E. coli by directed evolution. Moreover, this work will provide us some information about the amino acids residues involved in substrate recognition. For this purpose we have developed a direct evolution protocol where we constructed mutant libraries by mutagenic PCR in order to obtain variants of the protein. Using Nile Red, a fluorescent dye that binds to neutral lipids we can select different variants of tDGAT through a high throughput selection system based on fluorimetry and flow cytometry. Mutants carrying interesting phenotypes are further selected and sequenced. This way we were able to detect mutations that lead to a 2-fold increase in the TAG production.This work was supported by Ministerio de Ciencia e Innovación (BIO2010-240512).Peer Reviewe

Loading of malonyl-CoA Onto Tandem Acyl Carrier Protein Domains of Polyunsaturated Fatty Acid Synthases

Omega-3 polyunsaturated fatty acids (PUFA) are produced in some unicellular organisms, such as marine gammaproteobacteria, myxobacteria, and thraustochytrids, by large enzyme complexes called PUFA synthases. These enzymatic complexes resemble bacterial antibiotic-producing proteins known as polyketide synthases (PKS). One of the PUFA synthase subunits is a conserved large protein (PfaA in marine proteobacteria) that contains three to nine tandem acyl carrier protein (ACP) domains as well as condensation and modification domains. In this work, a study of the PfaA architecture and its ability to initiate the synthesis by selecting malonyl units has been carried out. As a result, we have observed a self-acylation ability in tandem ACPs whose biochemical mechanism differ from the previously described for type II PKS. The acyltransferase domain of PfaA showed a high selectivity for malonyl-CoA that efficiently loads onto the ACPs domains. These results, together with the structural organization predicted for PfaA, suggest that this protein plays a key role at early stages of the anaerobic pathway of PUFA synthesis

Zoonotic "Enterocytozoon bieneusi" genotypes in free-ranging and farmed wild ungulates in Spain

Microsporidia comprises a diverse group of obligate, intracellular, and spore-forming parasites that infect a wide range of animals. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and other mammals and birds. Data on the epidemiology of E. bieneusi in wildlife are limited. Hence, E. bieneusi was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from five Spanish bioregions. The parasite was detected only in red deer (10.4%, 68/653) and wild boar (0.8%, 3/359). Enterocytozoon bieneusi infections were more common in farmed (19.4%, 63/324) than in wild (1.5%, 5/329) red deer. A total of 11 genotypes were identified in red deer, eight known (BEB6, BEB17, EbCar2, HLJD-V, MWC_d1, S5, Type IV, and Wildboar3) and three novel (DeerSpEb1, DeerSpEb2, and DeerSpEb3) genotypes. Mixed genotype infections were detected in 15.9% of farmed red deer. Two genotypes were identified in wild boar, a known (Wildboar3) and a novel (WildboarSpEb1) genotypes. All genotypes identified belonged to E. bieneusi zoonotic Groups 1 and 2. This study provides the most comprehensive epidemiological study of E. bieneusi in Spanish ungulates to date, representing the first evidence of the parasite in wild red deer populations worldwide. Spanish wild boars and red deer are reservoir of zoonotic genotypes of E. bieneusi and might play an underestimated role in the transmission of this microsporidian species to humans and other animal

Heterologous expression of a thermophilic diacylglycerol acyltransferase triggers triglyceride accumulation in Escherichia coli

Triglycerides (TAGs), the major storage molecules of metabolic energy and source of fatty acids, are produced as single cell oil by some oleogenic microorganisms. However, these microorganisms require strict culture conditions, show low carbon source flexibilities, lack efficient genetic modification tools and in some cases pose safety concerns. TAGs have essential applications such as behaving as a source for added-value fatty acids or giving rise to the production of biodiesel. Hence, new alternative methods are urgently required for obtaining these oils. In this work we describe TAG accumulation in the industrially appropriate microorganism Escherichia coli expressing the heterologous enzyme tDGAT, a wax ester synthase/triacylglycerol:acylCoA acyltranferase (WS/DGAT). With this purpose, we introduce a codon-optimized gene from the thermophilic actinomycete Thermomonospora curvata coding for a WS/DGAT into different E. coli strains, describe the metabolic effects associated to the expression of this protein and evaluate neutral lipid accumulation. We observe a direct relation between the expression of this WS/DGAT and TAG production within a wide range of culture conditions. More than 30% TAGs were detected within the bacterial neutral lipids in 90 minutes after induction. TAGs were observed to be associated with the hydrophobic enzyme while forming round intracytoplasmic bodies, which could represent a bottleneck for lipid accumulation in E. coli. We detected an increase of almost 3- fold in the monounsaturated fatty acids (MUFA) occurring in the recombinant strains. These MUFA were predominant in the accumulated TAGs achieving 46% of the TAG fatty acids. These results set the basis for further research on the achievement of a suitable method towards the sustainable production of these neutral lipids

Caracterización estructural y bioquímica de proteínas implicadas en la síntesis de triglicéridos y ácidos grasos poliinsaturados

[EN]: Fatty acids are carboxylic acids with an aliphatic chain and an even number of carbon atoms. When they have two or more unsaturations are called polyunsaturated fatty acids and have many beneficial properties for health. Fatty acids can be accumulated in the form of triglycerides, which are the main form of energy storage in living cells and can be easily converted into biofuels and other valuable products. In this doctoral thesis an in vitro evolution protocol has been developed for a bacterial protein that is able to synthesize triglycerides, achieving in this way a significant improvement in its catalytic capacity. In addition, the synthesis of polyunsaturated fatty acids in marine bacteria has been studied from a biochemical and structural biology point of view, improving in this way the general understanding of the complex of proteins responsible for this biological process.[ES]: Los ácidos grasos son ácidos carboxílicos con una cadena alifática y con un número par de átomos de carbono. Cuando éstos poseen dos o más insaturaciones se denominan ácidos grasos poliinsaturados y poseen muchas propiedades beneficiosas para la salud. Los ácidos grasos se pueden acumular en forma de triglicéridos, que constituyen la forma principal de almacenamiento de energía en los seres vivos y además pueden ser fácilmente convertidos en biocombustibles y otros productos valiosos. En esta tesis doctoral se ha desarrollado un método de evolución in vitro para una proteína bacteriana productora de triglicéridos, consiguiéndose una mejora significativa en su capacidad catalítica. Además es esto se ha estudiado la síntesis de ácidos grasos poliinsaturados en bacterias marinas desde un punto de vista bioquímico y de la biología estructural, mejorando de esta forma el entendimiento del complejo de proteínas responsable de este proceso biológico.Peer Reviewe

Caracterización estructural y bioquímica de proteínas implicadas en la síntesis de triglicéridos y ácidos grasos poliinsaturados

ABSTRACT: Fatty acids are carboxylic acids with an aliphatic chain and an even number of carbon atoms. When they have two or more unsaturations are called polyunsaturated fatty acids and have many beneficial properties for health. Fatty acids can be accumulated in the form of triglycerides, which are the main form of energy storage in living cells and can be easily converted into biofuels and other valuable products. In this doctoral thesis an in vitro evolution protocol has been developed for a bacterial protein that is able to synthesize triglycerides, achieving in this way a significant improvement in its catalytic capacity. In addition, the synthesis of polyunsaturated fatty acids in marine bacteria has been studied from a biochemical and structural biology point of view, improving in this way the general understanding of the complex of proteins responsible for this biological process.RESUMEN: Los ácidos grasos son ácidos carboxílicos con una cadena alifática y con un número par de átomos de carbono. Cuando éstos poseen dos o más insaturaciones se denominan ácidos grasos poliinsaturados y poseen muchas propiedades beneficiosas para la salud. Los ácidos grasos se pueden acumular en forma de triglicéridos, que constituyen la forma principal de almacenamiento de energía en los seres vivos y además pueden ser fácilmente convertidos en biocombustibles y otros productos valiosos. En esta tesis doctoral se ha desarrollado un método de evolución in vitro para una proteína bacteriana productora de triglicéridos, consiguiéndose una mejora significativa en su capacidad catalítica. Además es esto se ha estudiado la síntesis de ácidos grasos poliinsaturados en bacterias marinas desde un punto de vista bioquímico y de la biología estructural, mejorando de esta forma el entendimiento del complejo de proteínas responsable de este proceso biológico.El presente trabajo ha sido realizado entre el Departamento de Biología Molecular de la Universidad de Cantabria y el Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), bajo la dirección del Dr. Gabriel Moncalián Montes, gracias a un contrato de la Universidad de Cantabria asociado a los proyectos "Aceites a la carta: bioingeniería de la síntesis de monoésteres y triglicéridos en microorganismos” (BIO2010-14809, del Ministerio de Ciencia e Innovación) y PLASMID OFFENSIVE: PROTEIN ENGINEERING OPERATIONS (BFU2014-55534-C2, Ministerio de Economía y Competitividad) Durante este periodo se ha realizado una estancia de tres meses en el laboratorio del doctor Abel Baerga Ortiz (Universidad de Puerto Rico, Recinto de Ciencias Médicas

Structure of a ketosynthase-chain length factor of a polyunsaturated fatty acids synthase

Resumen del póster presentado a la 16th International Conference on the Crystallization of Biological Macromolecules, celebrada en Praga (República Checa) del 2 al 7 de julio de 2016.The use of omega-3 polyunsaturated fatty acids (O3PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), has increased in recent years because of its benefits for human health. . For the production of these O3PUFAs, marine bacteria such as moritella marina or colwellia psychrerythraea (DHA producers) or Shewanella baltica (EPA producer) possess large enzyme complexes called PUFA synthases (Pfa), which resemble bacterial antibiotic production proteins known as polyketide synthases (PKS). For growing PUFA carbon chain, some essential enzyme modules are needed to produce the first decarboxylation and successive condensations rounds. These modules consist of heterodimers of keto synthases (KS) and acyltransferase (AT) that, in coordination with acyl carrier proteins (ACPs), incorporate acyl groups to the growing chain, performing the so-called claisen condensations. In cases where long-chain PUFAs are synthesized, the chain elongation is guided by a special heterodimeric Ketosynthase-Chain length factor (KS-CLF) domain. In contrast to homodimeric KS-KS domains found in regular fatty acid synthases, the active site cysteine is absent from the C-terminal subunit of this KS-CLF heterodimer. A hydrophobic tunnel that holds the growing chain is a critical determinant of the final polyketide chain length. Besides determining the final length of PUFAs, KS-CLF heterodimer can decarboxylate malonyl units, thus >seeding> the acyl chain in the first cyclization of the polyketide and placing the new molecule within the KS-CLF tunnel. We have recently obtained crystals of a 100 kDa Ketosynthase-Chain length factor heterodimer from a PUFA synthase. These crystals were diffracted at ALBA synchrotron in Barcelona (Spain) and the protein structure was solved by molecular replacement at 1.9 A resolution. This first solved structure of a Pfa KS-CLF heterodimer resulted to be very informative to understand the role of this domain in O3PUFA synthesis. Moreover, biochemical studies in combination with modeling of the other Pfa modules allowed us to propose a model of the overall Pfa architecture. These structural studies could be used for the modification and optimization of O3PUFA synthesis in different microorganisms.Peer Reviewe

Directed evolution of a bacterial WS/DGAT acyltransferase: improving tDGAT from Thermomonospora curvata

Some bacteria belonging to the actinobacteria and proteobacteria groups can accumulate neutral lipids expressing enzymes of the wax ester synthase/acyl coenzyme A: diacylglycerol acyltransferase (WS/DGAT) family. tDGAT is a WS/DGAT-like enzyme from Thermomonospora curvata able to produce TAGs and WEs when heterologously expressed in Escherichia coli. In this study, a protocol for the directed evolution of bacterial lipid-producing enzymes based on fluorimetry is developed and tested. tDGAT has been successfully evolved towards the improvement of TAG production with an up to 2.5 times increase in TAG accumulation. Mutants with no ability to produce TAGs but able to accumulate waxes were also selected during the screening. The localization of the mutations that enhance TAG production in the outer surface of tDGAT points out possible new mechanisms that contribute to the activity of this family of enzymes. This Nile red-based high throughput screening provides an evolution platform for other WS/DGAT-like enzymes.This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness [BFU2014-55534-C2-2-P]

Structure and Mechanism of the Ketosynthase-Chain Length Factor Didomain from a Prototypical Polyunsaturated Fatty Acid Synthase

Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential ingredients of the human diet. They are synthesized by LC-PUFA synthases (PFASs) expressed in marine bacteria and other organisms. PFASs are large enzyme complexes that are homologous to mammalian fatty acid synthases and microbial polyketide synthases. One subunit of each PFAS harbors consecutive ketosynthase (KSc) and chain length factor (CLF) domains that collectively catalyze the elongation of a nascent fatty acyl chain via iterative carbon–carbon bond formation. We report the X-ray crystal structure of the KS–CLF didomain from a well-studied PFAS in Moritella marina. Our structure, in combination with biochemical analysis, provides a foundation for understanding the mechanism of substrate recognition and chain length control by the KS–CLF didomain as well as its interaction with a cognate acyl carrier protein partner.This work was supported by Spanish Ministry of Science, Innovation and Universities Grants BFU2014-55534-C2-2-P and PGC2018-093885-B-I00 (MCIU/AEI/FEDER, UE) to G.M. and by a grant from the National Institutes of Health (R01 GM087934) to C.K

Ultracentrifugation experiments reveal tDGAT remains associated to the neutral lipids accumulated by the recombinant bacteria.

<p><b>A and B, cell lysate of <i>E</i>. <i>coli</i> C41 (DE3) cells expressing tDGAT were sonicated and separated into phases by centrifugation. Samples from 1 to 3 correspond to consecutive centrifugations of the same sample at 2,000 g, 10,000 g and 55,000 g respectively</b>. (A) Electrophoretic 8% SDS-PAGE gel analysis of the supernatants (S1-S3) and pellets (P1-P3) of the different centrifugation fractions. tDGAT forming inclusion bodies falls at 2,000g. Lane M: Low Range SDS-PAGE Molecular Weight Standards (BioRad). (B) Thin layer chromatography (TLC) of lipid fractions extracted from 50 ml cultures expressing tDGAT. Lane +: Full pellet centrifuged at 55,000g. Lanes 1–4: Centrifugation pellets from P1 to P3. C and D, lipid bodies purification by sucrose gradient. (C) TLC of lipid fractions extracted from a cell lysate of <i>E</i>. <i>coli</i> C41 (DE3) cells expressing tDGAT: Lane M, triolein; lane 1, pellet obtained after centrifugation at 3,000 g and lane 2, the supernantant of the sucrose gradient centrifugation at 180,000 g. (D) Electrophoretic 12% SDS-PAGE gel analysis of the total <i>E</i>. <i>coli</i> C41 (DE3) cell lysate (1), proteins in the pellet obtained at 3,000 g (2) and proteins in the supernantant of the sucrose gradient centrifugation (3). Lane M shows the PageRuler Plus Prestained Protein Ladder (ThermoFisher). Position of TAGs are marked with black arrows in the TLC images. White arrowheads correspond to the protein tDGAT.</p