Étude structure-fonction des fructose-1,6-bisphosphate aldolases métallo-dépendantes : mécanisme catalytique et développement d’antimicrobiens

Les fructose-1,6-bisphosphate aldolases (FBPA) sont des enzymes glycolytiques (EC 4.1.2.13) qui catalysent la transformation réversible du fructose-1,6-bisphosphate (FBP) en deux trioses-phosphates, le glycéraldéhyde-3-phosphate (G3P) et le dihydroxyacétone phosphate (DHAP). Il existe deux classes de FBPA qui diffèrent au niveau de leur mécanisme catalytique. Les classes I passent par la formation d’un intermédiaire covalent de type iminium alors que les classes II, métallodépendantes, utilisent généralement un zinc catalytique. Contrairement au mécanisme des classes I qui a été très étudié, de nombreuses interrogations subsistent au sujet de celui des classes II. Nous avons donc entrepris une analyse détaillée de leur mécanisme réactionnel en nous basant principalement sur la résolution de structures cristallographiques. De nombreux complexes à haute résolution furent obtenus et ont permis de détailler le rôle de plusieurs résidus du site actif de l’enzyme. Nous avons ainsi corrigé l’identification du résidu responsable de l’abstraction du proton de l’O4 du FBP, une étape cruciale du mécanisme. Ce rôle, faussement attribué à l’Asp82 (chez Helicobacter pylori), est en fait rempli par l’His180, un des résidus coordonant le zinc. L’Asp82 n’en demeure pas moins essentiel car il oriente, active et stabilise les substrats. Enfin, notre étude met en évidence le caractère dynamique de notre enzyme dont la catalyse nécessite la relocalisation du zinc et de nombreux résidus. La dynamique de la protéine ne permet pas d’étudier tous les aspects du mécanisme uniquement par l’approche cristallographique. En particulier, le résidu effectuant le transfert stéréospécifique du proton pro(S) sur le carbone 3 (C3) du DHAP est situé sur une boucle qui n’est visible dans aucune de nos structures. Nous avons donc développé un protocole de dynamique moléculaire afin d’étudier sa dynamique. Validé par l’étude d’inhibiteurs de la classe I, l’application de notre protocole aux FBPA de classe II a confirmé l’identification du résidu responsable de cette abstraction chez Escherichia coli (Glu182) mais pointe vers un résidu diffèrent chez H. pylori (Glu149 au lieu de Glu142). Nos validations expérimentales confirment ces observations et seront consolidées dans le futur. Les FBPA de classe II sont absentes du protéome humain mais sont retrouvées chez de nombreux pathogènes, pouvant même s'y révéler essentielles. Elles apparaissent donc comme étant une cible idéale pour le développement de nouveaux agents anti-microbiens. L’obtention de nouveaux analogues des substrats pour ces enzymes a donc un double intérêt, obtenir de nouveaux outils d’étude du mécanisme mais aussi développer des molécules à visée pharmacologique. En collaboration avec un groupe de chimistes, nous avons optimisé le seul inhibiteur connu des FBPA de classe II. Les composés obtenus, à la fois plus spécifiques et plus puissants, permettent d’envisager une utilisation pharmacologique. En somme, c’est par l’utilisation de techniques complémentaires que de nouveaux détails moléculaires de la catalyse des FBPA de classe II ont pu être étudiés. Ces techniques permettront d’approfondir la compréhension fine du mécanisme catalytique de l’enzyme et offrent aussi de nouvelles perspectives thérapeutiques.Fructose-1,6-bisphosphate aldolases (FBPA) are glycolytic enzymes (EC 4.1.2.13) that catalyze the reversible cleavage of fructose-1,6-bisphosphate (FBP) into the triose phosphates, glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). There are two classes of FBPAs that differ at the level of their mechanism. Class I FBPAs form a covalent iminium intermediate whereas class II FBPAs, being metalloenzymes, generally use a catalytic zinc in their reaction mechanism. In contrast to the mechanism of the class I FBPAs, which has been thoroughly studied, there are several unresolved inquiries as to the mechanism of class II FBPAs. We have therefore pursued a detailed analysis of the reaction mechanism using as a primary tool the elucidation of crystallographic structures. Several high resolution complexes have been resolved and have provided critical evidence to help us suggest the implication and role of several key residues in the active site. Consequently, we have correctly identified the residue which is responsible for the abstraction of the O4 proton from FBP, a vital step in the reaction mechanism. The residue responsible for this abstraction, which had incorrectly been assigned to Asp82 (in Helicobacter pylori), has been appropriately consigned to His180, a residue which is involved in coordinating the zinc molecule. Nevertheless, Asp82 remains an important residue as it orients, activates and stabilizes substrates. Finally, our study brings to evidence the dynamic character of our enzyme in which catalysis entails the relocalization of the catalytic zinc and several residues. The complexity of this reaction, notably one of the proton exchanges in the mechanism, could not be resolved solely by crystallographic means. In fact, the residue responsible for the stereospecific transfer of the pro(S) proton on carbon 3 (C3) of DHAP is situated on a loop that was not resolved in any of our structures. We therefore developed a molecular dynamics approach to study this intricate movement. After preliminary validation by inhibitor studies with class I FBPAs, the protocol was applied to class II FBPAs and several remarkable observations emerged: the residue responsible for this abstraction in Escherichia coli is Glu182 whereas a different residue, Glu149 (instead of Glu142) appears to assume this role in H. pylori. Our preliminary validations have confirmed this observation and shall be further consolidated in the future. Class II FBP aldolases, although absent from the human proteome, are prevalently found in several pathogens, and have further been found to be essential to a number of these organisms. As such, they are ideal targets for the development of novel anti-microbial agents. Developing new analogues of the cognate substrates of these enzymes is therefore not only advantageous for mechanistic studies, but has endless pharmacological potential. In the context of a collaborative effort involving a group of chemists, a compound that initially had an inhibition constant in the millimolar range was optimized and produced a series of compounds that inhibit in the nanomolar range

Fructose-1,6-bisphosphate aldolase de classe II : aspects structural et dynamique dans le mécanisme réactionnel

La D-fructose-1,6-bisphosphate aldolase (FBPA) catalyse la réaction réversible d'aldolisation dans la voie métabolique du glucose, c'est-à-dire l'interconversion du dihydroxyacétone phosphate (DHAP) et du D-glyceraldéhyde 3-phosphate (G3P) en D-fructose 1,6-bisphosphate (FBP). Les aldolases sont regroupées en deux classes selon le mécanisme réactionnel : la classe I, dont fait partie l'enzyme humaine, catalyse la réaction en passant par la formation d'un intermédiaire covalent (base de Schiff), alors que les aldolases de classe II sont des métalloenzymes - un cation métallique divalent est requis pour son activité catalytique. L'aldolase de classe II, absente des mammifères, se retrouve notamment chez des agents pathogènes, par exemples Mycobacterium tuberculosis (tuberculose), Giardia lamblia (giardiase), Escherichia coli (infections diverses) et Helicobacter pylori (ulcère et cancer gastrique). Cette distribution en fait une cible potentielle dans la découverte de médicaments. La conception d'inhibiteurs spécifiques pour l'aldolase de classe II requiert une fine connaissance de sa catalyse enzymatique et de sa structure tridimensionnelle. Cette connaissance demeure incomplète, alors que l'ensemble des structures de complexes enzyme-inhibiteur ou enzyme-intermédiaire ne supporte pas une partie du mécanisme publié dans la littérature. Nous étudions le rôle catalytique de deux résidus situés chacun sur une boucle de surface mobile de l'aldolase de classe II de H. pylori et impliqués dans des étapes d'échange de proton. Les mutants simples H180Q et E142A ont été caractérisés cinétiquement et cristallisés pour la détermination de structure sur la base de la diffraction aux rayons X. Les structures cristallines des mutants complexés à des intermédiaires réactionnels ont été résolues. La déprotonation du groupe hydroxyle en C4 du FBP initie le clivage de la liaison en C3-C4 du cétohexose, première étape du mécanisme catalytique de rétro-aldolisation. Nos résultats identifient His180, sur la boucle beta6-alpha8, comme responsable de cet échange de proton. Ce résidu est un ligand de l'ion de zinc dans la structure native; le changement conformationnel observé suite à l'amarrage du phosphate en C1 de FBP libère His180 pour permettre le clivage. L'ion de zinc migre par la suite vers le site actif afin de faciliter la liaison du substrat et la stabilisation de l'intermédiaire énediolate. Nos résultats vont à l'encontre de l'hypothèse publiée précédemment sur le rôle catalytique de Asp82 dans cet échange de proton du groupe hydroxyle en C4, le rôle de ce dernier résidu se limitant plutôt au maintien de l'intégrité structurale du site actif. La libération du G3P nouvellement produit est suivie de la protonation stéréospécifique de l'intermédiaire énediolate générant le DHAP. La libération du DHAP complète ainsi le cycle catalytique. La protonation de l'intermédiaire énediolate est effectuée par l'intermédiaire du résidu Glu142, situé sur la boucle beta5-alpha7, ce qui concorde avec des études cinétiques publiées sur d'autres FBPA de classe II. Ces études ont attribué le même rôle à ce résidu conservé entre homologues. Nous avons par la suite établi un protocole de simulation de dynamique moléculaire pour évaluer le repliement de ladite boucle et ainsi comprendre le mode d'action du résidu Glu142. Des détails mécanistiques de l'étape de clivage s'ajoutent à nos connaissances actuelles; des questions subsistent quant à leur implication au reste de la catalyse. En attribuant un rôle crucial à la boucle beta6-alpha8 dans la catalyse et non limité à la liaison de substrats, cette boucle des aldolases de classe II peut devenir une cible dans le développement d'inhibiteurs. De plus, la migration de l'ion de zinc non dépendante de ligand suggère la possibilité de chélater et restreindre l'ion loin du site actif.Fructose-1,6-bisphosphate aldolase catalyzes the reversible aldol reaction in glucose metabolism interconverting dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (G3P) into D-fructose 1,6-bisphosphate (FBP). Aldolases are furthermore classified based on their reaction mechanism: class I aldolase (e. g. human aldolase) forms a covalent Schiff base intermediate with substrate, whereas class II aldolase utilizes a divalent metal cation in catalysis. Class II aldolase is commonly found in pathogenic organisms such as Mycobacterium tuberculosis (tuberculosis), Giardia lamblia (giardiasis), Escherichia coli (diverse infections) and Helicobacter pylori (ulcer and gastric cancer) but not in mammals. This distribution makes class II aldolase a potential target for drug discovery. Structure driven drug design depends on an explicit knowledge of the reaction mechanism of class II aldolase and its three-dimensional structure. Our current knowledge is lacking; existing aldolase crystal structures with reaction intermediates and with competitive inhibitors are not coherent with proposed mechanisms in literature. The present study focuses on the catalytic role of two residues, each located on a mobile loop of H. pylori class II aldolase and each implicated in a critical proton transfer step. Single mutants H180Q and E142A were characterized enzymatically and crystallized for X-ray structure determination. Crystal structures of reaction intermediates formed with substrate were determined. The catalytic mechanism requires proton abstraction at the FBP C4 hydroxyl group to initiate C3-C4 bond cleavage, first step of the retroaldol reaction. Our data supports His180 situated on the mobile loop beta6-alpha8, as the residue responsible for this proton transfer. Notably, His180 chelates the zinc ion in the native structure. The structural change induced due to C1 phosphate binding of FBP releases His180 to promote cleavage. Displacement of the catalytic zinc ion ensues, facilitating substrate binding and subsequent stabilization of the enediolate intermediate. Our results do not support the previous hypothesis of a catalytic role for Asp82 in C4 hydroxyl group proton abstraction; it rather plays an important role in maintaining structural integrity for active site binding. Displacement of the nascent aldehyde G3P and concomitant stereospecific protonation of the enediolate species generates the obligate triose phosphate, DHAP. Dissociation of DHAP from the active site completes the catalytic cycle. The residue responsible for initiating enediolate protonation was identified as residue Glu142, situated on mobile loop beta5-alpha7, and this is in agreement with previous kinetic studies of enediolate protonation in other class II aldolases, attributing the same role to this conserved residue. We devised a molecular dynamic simulation method to follow the catalytic loop folding event, further investigating details of the role of Glu142 in catalysis. We gained further knowledge of the cleavage event, although work remains to elucidate missing details of the catalysis and integrate our findings. By attributing a role in catalysis to loop beta6-alpha8 not limited to substrate binding, this loop of class II aldolases becomes a potential target in drug design. In addition, ligand independent zinc ion migration suggest it is possible to chelate the metal and restrain it far from the active site

Accelerating innovation in biotechnology through knowledge in cyanobacterial photosynthesis

Microalgae are potential hosts for the sustainable production of valuable chemicals using CO2 as feedstock and light as an energy source. In whole-cell applications, biosynthetic reactions of interest are supplied with cellular reductants (e.g., ferredoxin or NADPH) which are recycled by the native photosynthetic apparatus. Using gene-editing techniques, photosynthesis can be engineered to increase the supply of reductants to the reactions of interest. However, such efforts require specialized knowledge about the photosynthetic machinery. Here, regulatory mechanisms of photosynthesis were investigated using the cyanobacterium Synechocystis sp. PCC 6803, a model organism of prokaryotic microalgae. In a primary regulatory process, excess photosynthetic electrons are transferred from ferredoxin to O2 via flavodiiron proteins. However, under controlled cultivation conditions, this process is dispensable and wastes reducing power. Here, it is shown that the heterodimeric flavodiiron proteins Flv1/Flv3 and Flv2/Flv4 have distinct electron sink capacities. Flv1/Flv3 disposes of electrons at a higher capacity and faster rate than Flv2/Flv4. The applicability of such knowledge is demonstrated by disrupting Flv1/Flv3 that consequently, enhanced the supply of reductants to a targeted chemical modification catalysed by a heterologous ene reductase. FLVB, a homolog of Flv3 in green algae has previously been shown to reduce not only O2 but nitric oxide (NO). This implies that flavodiiron proteins in cyanobacteria may be able to sink photosynthetic electrons into NO. However, it is shown here that NO inhibits photosynthesis in Synechocystis thus is unlikely to act as an efficient terminal electron acceptor in photosynthesis. Lastly, the promising cultivation conditions, photomixotrophy, were found to gradually decrease the photosynthetic capacity in Synechocystis. This decrease was reversed by deleting the cytochrome cM protein which appears to regulate the bioenergetic processes under photomixotrophic conditions. For developing an economically feasible and robust chassis to produce targeted compounds, scientific dilemmas are still to be solved at the laboratory scale. It is demonstrated that specialized knowledge created by fundamental research in photosynthesis provides a strong basis for innovative activity in the space of algae (cyanobacteria)-related biotechnologies.Mikrolevien avulla voidaan mahdollisesti tuottaa arvokkaita kemikaaleja kestävästi käyttäen raaka-aineena hiilidioksidia ja energianlähteenä valoa. Koko solun sovelluksissa fotosynteesikoneiston kierrättämät solunsisäiset pelkistäjät (esim. ferredoksiini tai NADPH) mahdollistavat kyseiset biosynteettiset reaktiot. Geenieditointitekniikoilla voidaan muokata fotosynteesiä tuottamaan enemmän pelkistäjiä kyseisissä reaktioissa. Tämä vaatii kuitenkin fotosynteesikoneiston erityistä tuntemusta. Tässä työssä tutkittiin fotosynteesin säätelymekanismeja prokaryoottisen mikrolevä malliorganismin, Synechocystis sp. PCC 6803 - syanobakteerin, avulla. Primaarisessa säätelyprosessissa ylimääräiset fotosynteettiset elektronit kuljetetaan ferredoksiinilta O2:lle flavoproteiinien välityksellä. Kontrolloiduissa kasvatusolosuhteissa prosessi on kuitenkin tarpeeton ja tuhlaa pelkistysvoimaa. Tässä työssä osoitetaan, että flavoproteiinien Flv1/Flv3 ja Flv2/Flv4 heterodimeerien elektroninvastaanottajan ominaisuudet ovat erilaiset. Flv1/Flv3 poistaa elektroneja tehokkaammin ja nopeammin kuin Flv2/Flv4. Näiden tietojen sovellettavuus voidaan osoittaa estämällä Flv1/Flv3 heterodimeeri, minkä seurauksena fotosynteettisiä pelkistäjiä saadaan lisää kemialliseen reaktioon, jota katalysoi heterologinen ene-reduktaasi. Aiempi tutkimus on osoittanut, että FLVB, Flv3:n homologi viherlevissä, pelkistää O2:n lisäksi myös typpioksidia (NO). Syanobakteerien flavoproteiinit pystyvätkin mahdollisesti poistamaan fotosynteettisiä elektroneja typpimonoksidiin. Tässä työssä kuitenkin osoitetaan, että NO estää fotosynteesin Synechocystis-lajissa, joten on epätodennäköistä, että NO toimisi tehokkaana fotosynteesin viimeisenä elektronin vastaanottajana. Fotomiksotrofian, joka on lupaava kasvatusolosuhde, huomattiin asteittain vähentävän Synechocystislajin fotosynteettistä kapasiteettiä. Fotosynteettisen kapasiteetin väheneminen kumoutui poistamalla arvoituksellinen sytokromi cM -proteiini, joka ilmeisesti sääntelee bioenergeettisiä prosesseja fotomiksotrofisissa olosuhteissa. Tieteellisiä dilemmoja on vielä ratkaistavana laboratoriotasolla, jotta voidaan kehittää taloudellisesti järkevä ja vankka alusta haluttujen yhdisteiden tuottamiseksi. Tässä työssä osoitetaan, että fotosynteesin perustutkimuksen luoma erityistietous tarjoaa vahvan pohjan innovaatioille leviin (ja syanobakteereihin) liittyvissä bioteknologioissa

Binding determinants for substrates and inhibitors of trehalose-6-phosphate phosphatase

Trehalose is a sugar commonly found in archaeon, bacteria, fungi, plants, and invertebrates. It is utilized as an energy source and upregulated during stress conditions such as thermal fluctuations and oxidative stress. As mammals do not synthesize trehalose, trehalose biosynthetic pathways have become therapeutic targets for infectious diseases. The enzyme trehalose-6-phosphate phosphatase (T6PP) catalyzes the dephosphorylation of trehalose 6-phosphate to form trehalose. In its absence, the viability and virulence of bacteria, fungi, plants and nematodes are decreased. Hence T6PP is the focus of this study as a target for therapeutics of the diseases tuberculosis and lymphatic filariasis. T6PP is a phosphohydrolase in the haloalkanoic acid dehalogenase superfamily. To identify the determinants for substrate specificity needed to guide structure-aided inhibitor design for therapeutics, atomic-resolution crystallographic information on the Michaelis complex is of great importance. Toward this goal, the structure of T6PP from Mycobacterium marinum was determined via X-ray crystallography in an unliganded form and the structure of T6PP from Salmonella typhimurium (St) was determined in the apo form, bound to the substrate analog, trehalose 6-phosphate, the product, trehalose, and the inhibitor, 4-n-octylphenyl α-D-glucopyranoside 6-sulfate. The enzyme confers specificity via hydrogen bonding to the phosphate and glucosyl group proximal to the phosphate. Specifically, the conserved residues Glu123, Lys125 and Glu167 form hydrogen bonds to the hydroxyl groups of the proximal glucose. However, the distal glucose binding sub-site can tolerate new chemotypes. To further aid inhibitor design, the two inhibitors of Brugia malayi T6PP discovered via screening the Johns Hopkins library of FDA-approved drugs, Cephalosporin C and Closantel, were computationally docked into StT6PP. The Cephalosporin C scaffold was optimized to provide an inhibitor with a KI of 20 M that comprises a 5,6-indole scaffold to afford hydrogen bonds to the Glu/Lys/Glu motif and a computationally discovered phosphate mimic tetrazole. Closantel acts as a slow-binding inhibitor and a series of analogs were synthesized to increase potency. Two analogs show enhanced efficacy relative to Closantel with IC50 values near 60 M. Future efforts will aim to optimize these scaffolds for inhibition of T6PP to develop therapeutics for tuberculosis and lymphatic filariasis

Recent Developments in Flavin-Based Catalysis:Enzymatic Sulfoxidation

The synthesis of optically active sulfoxides, compounds due to their unique properties, has been a main target for synthetic organic chemistry. Recent efforts in the field of biocatalysis have allowed the preparation of enantiopure sulfoxides starting from the corresponding sulfides while using relatively mild conditions. In fact, several different types of redox biocatalysts have been found that can catalyze enantio- and/or regioselective sulfoxidations. The most promising group of enzymes able to perform selective sulfoxidations is the flavin-containing monooxygenases (FMOs). Enzymes containing a flavin cofactor have already been widely studied and used in organic synthesis, especially in reduction and/or oxidation processes. This chapter highlights the recent efforts in the preparation of chiral sulfoxides catalyzed by different types of flavoenzymes, with special attention to the parameters that can improve their catalytic properties. Novel approaches that allow performing selective sulfoxidations in which modified flavin systems are used are also discussed.</p

An investigation into the chloroplast transformation of wheat, and the use of a cyanobacterial CCM gene for improving photosynthesis in a C3 plant

Wheat is a major component of the UK diet, and provides approximately 20% of global caloric intake. Wheat is grown on more land area than any other crop, and the continued supply of wheat is essential for global food security. Biotechnology is likely to play an important role in the sustainable increase of wheat yields, and the genetic manipulation of chloroplasts for photosynthetic improvement has many potential advantages over transformation of the nuclear genome. The genetic modification of the chloroplast genome via transformation was first demonstrated in the late 1980’s, and since then, chloroplast transformation of many Dicotyledonous (dicot) plant species such as Nicotiana tabaccum has been routinely performed. In comparison, the transformation of chloroplasts in Monocotyledons (monocot) plant species, which includes all cereal crops, has made far less progress. To date, there has been no reproducible homoplasmic plastid transformation event in the monocots. This study identifies a number of bottlenecks responsible for the prevention of chloroplast transformation in wheat. One such bottleneck is the lack of a suitable explant for plastid transformation, as traditional nuclear transformation targets are absent of metabolically active plastids. This study has developed a robust regeneration protocol for a previously undescribed tissue, termed the primary inflorescence leaf sheath (piLS), which is rich in active chloroplasts. Functional wheat specific chloroplast transformation vectors have been generated, and bombardment studies have been conducted with these on piLS and a second tissue, the immature embryosderived callus. Immature embryo callus (IEC) does not contain active plastids, however contains pro-plastids and is highly embryogenic. To uncover novel ways of increasing photosynthesis in C3 plants, a number of transplastomic tobacco lines expressing the Synechococcus elongatus PCC 7942 ictB gene were generated. Previous studies suggest that ictB may be an inorganic carbon transporter. In a number of transplastomic lines produced in this study, the intercellular carbon concentration (Ci) is significantly increased. This increased Ci did not result in an increased photosynthetic rate, however did cause a number of phenotypic differences, such as smaller plants, wider leaves, and earlier seed pod formation. The results, with regards to chloroplast transformation, and its implications in improving photosynthesis within C3 plants, are discussed in this thesis

Development of Synechococcus sp. PCC 11901 as a biotechnology platform

Cyanobacteria are key organisms in the global ecosystem and potential renewable platforms for production of chemicals. Many aspects of cyanobacterial biology are unique to this subset of prokaryotes. Characterising cyanobacterial metabolism and physiology is key to understanding their environmental role and unlocking their potential for biotechnology. This thesis provides a comprehensive summary of our knowledge on cyanobacterial physiology and the pathways in the model organism, Synechocystis sp. PCC 6803 (PCC 6803). One of the main issues within cyanobacterial bioindustry has been the lack of sustained fast-growing strains. The newly discovered Synechococcus sp. PCC 11901 (PCC 11901) reportedly demonstrates the highest, most sustained growth of any known cyanobacterium. Knowledge of PCC 11901 biology, including the factors underlying the sustained fast growth, is limited, which hinders its potential for biotechnology. Genetic tools for generating unmarked mutants in PCC 11901 are not established. This thesis confirms that PCC 11901 displays faster growth than other model cyanobacteria. Comparative genomics between PCC 11901 and PCC 6803 reveal conservation of most metabolic pathways but PCC 11901 has a simplified electron transport chain and reduced light-harvesting complex. This may underlie its efficient light use, reduced photoinhibition, and higher photosynthetic and respiratory rates. Attempts to generate unmarked knockouts using two negative selectable markers were unsuccessful, suggesting that recombinase or CRISPR-based approaches may be necessary for the industry requirement of repeated genetic manipulation. To further cement PCC 11901 as a future industrial strain, biomass and optical density measurements were carried out over a range of light intensities to aid photo-mechanistic modelling of the strain. This thesis establishes PCC 11901 as one of the most promising species currently available for cyanobacterial biotechnology and provides a useful set of bioinformatic tools and strains for advancing this field, in addition to insights into the factors underlying its fast growth phenotype

Multidimensional engineering for the production of fatty acid derivatives in Saccharomyces cerevisiae

Saccharomyces cerevisiae, also known as budding yeast, has been important for human society since ancient time due to its use during bread making and beer brewing, but it has also made important contribution to scientific studies as model eukaryote. The ease of genetic modification and the robustness and tolerance towards harsh conditions have established yeast as one of the most popular chassis in industrial-scale production of various compounds. The synthesis of oleochemicals derived from fatty acids (FAs), such as fatty alcohols and alka(e)nes, has been extensively studied in S. cerevisiae, which is due to their key roles as substitutes for fossil fuels as well as their wide applications in other manufacturing processes. Aiming to meet the commercial requirements, efforts in different engineering approaches were made to optimize the TRY (titer, rate and yield) metrics in yeast. The major aim of this thesis was to enable a versatile yeast platform for the production of FA derivatives through diverse engineering strategies. We tested several membrane transporters for the potential to mediate fatty alcohol export in S. cerevisiae. A novel function of the mammalian transporter FATP1 was identified as it was able to benefit fatty alcohol efflux in a high fatty alcohol production strain. According to the results, human FATP1 led to an improvement of extracellular fatty alcohols (2.6-fold increase) and cell fitness compared with the control strain. FATP1 was then introduced into an engineered S. cerevisiae strain carrying a heterologous 1-alkene biosynthetic pathway for improved 1-alkene secretion and production. Combined with an optimization of fatty acid metabolism and the electron transport system, a final titer of 35.3 mg/L of 1-alkenes was achieved with more than 80% being secreted. Medium-chain fatty acids (MCFAs) are non-inherent fatty acids in yeast whose microbial synthesis is considered to be challenging. Through expressing either an engineered native fatty acid synthase (FAS) or an engineered bacterial type I FAS, the synthesis of MCFAs has been successfully implemented in yeast. In our work, directed evolution of the native transporter Tpo1 and adaptive laboratory evolution were performed to increase the tolerance against MCFAs. Together with further augmentation of the metabolic flux towards MCFAs and optimization of the cultivation process this resulted in &gt;1 g/L MCFA production. Based on the MCFA production platform, we attempted to synthesize medium-chain fatty alcohols (MCFOHs, C6-C12) in yeast. Different protein engineering strategies were designed to engineer the carboxylic acid reductase from Mycobacterium marinum (MmCAR), a key enzyme involved in fatty acid conversion. We successfully changed the substrate specificity towards MCFAs and improved the enzyme catalytic activity via directed evolution, using both rational and semi-rational approaches. With further deleting the TPO1 transporter gene and combining different MmCAR mutations, a final production of 250 mg/L MCFOHs was achieved, a 3-fold increase compared with the control strain.\ua0 In conclusion, we provided new insight into the establishment of yeast platforms for the production of FA derivatives through multidimensional engineering strategies