RESUMEN
Acyclic monoterpenes constitute a large and highly abundant class of secondary plant metabolites and are, therefore, attractive low-cost raw materials for the chemical industry. To date, numerous biocatalysts for their transformation are known, giving access to highly sought-after monoterpenoids. In view of the high selectivity associated with many of these reactions, the demand for enzymes generating commercially important target molecules is unabated. Here, linalool (de)hydratase-isomerase (Ldi, EC 4.2.1.127) from Castellaniella defragrans was examined for the regio- and stereoselective hydration of the acyclic monoterpene ß-myrcene to (S)-(+)-linalool. Expression of the native enzyme in Escherichia coli allowed for identification of bottlenecks limiting enzyme activity, which were investigated by mutating selected residues implied in enzyme assembly and function. Combining these analyses with the recently published 3D structures of Ldi highlighted the precisely coordinated reduction-oxidation state of two cysteine pairs in correct oligomeric assembly and the catalytic mechanism, respectively. Subcellular targeting studies upon fusion of Ldi to different signal sequences revealed the significance of periplasmic localization of the mature enzyme in the heterologous expression host. This study provides biochemical and mechanistic insight into the hydration of ß-myrcene, a nonfunctionalized terpene, and emphasizes its potential for access to scarcely available but commercially interesting tertiary alcohols.
Asunto(s)
Alquenos/metabolismo , Betaproteobacteria/metabolismo , Hidroliasas/metabolismo , Monoterpenos/metabolismo , Monoterpenos Acíclicos , Alcoholes/química , Alcoholes/metabolismo , Alquenos/química , Catálisis , Escherichia coli/metabolismo , Hidroliasas/química , Hidrólisis , Isomerasas , Monoterpenos/químicaRESUMEN
The addition of water to non-activated carbon-carbon double bonds catalyzed by fatty acid hydratases (FAHYs) allows for highly regio- and stereoselective oxyfunctionalization of renewable oil feedstock. So far, the applicability of FAHYs has been limited to free fatty acids, mainly owing to the requirement of a carboxylate function for substrate recognition and binding. Herein, we describe for the first time the hydration of oleic acid (OA) derivatives lacking this free carboxylate by the oleate hydratase from Elizabethkingia meningoseptica (OhyA). Molecular docking of OA to the OhyA 3D-structure and a sequence alignment uncovered conserved amino acid residues at the entrance of the substrate channel as target positions for enzyme engineering. Exchange of selected amino acids gave rise to OhyA variants which showed up to an 18-fold improved conversion of OA derivatives, while retaining the excellent regio- and stereoselectivity in the olefin hydration reaction.
Asunto(s)
Ácidos Grasos/metabolismo , Flavobacteriaceae/enzimología , Hidroliasas/química , Hidroliasas/metabolismo , Ácido Oléico/química , Ácido Oléico/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Catálisis , Modelos Moleculares , Simulación del Acoplamiento Molecular , Conformación Proteica , Estereoisomerismo , Especificidad por SustratoRESUMEN
Cytochrome P450 enzymes (CYPs) play an essential role in the biosynthesis of various natural compounds by catalyzing regio- and stereospecific hydroxylation reactions. Thus, CYP activities are of great interest in the production of fine chemicals, pharmaceutical compounds or flavors and fragrances. Industrial applicability of CYPs has driven extensive research efforts aimed at improving the performance of these enzymes to generate robust biocatalysts. Recently, our group has identified CYP-mediated hydroxylation of (+)-valencene as a major bottleneck in the biosynthesis of trans-nootkatol and (+)-nootkatone in Pichia pastoris. In the current study, we aimed at enhancing CYP-mediated (+)-valencene hydroxylation by over-expressing target genes identified through transcriptome analysis in P. pastoris. Strikingly, over-expression of the DNA repair and recombination gene RAD52 had a distinctly positive effect on trans-nootkatol formation. Combining RAD52 over-expression with optimization of whole-cell biotransformation conditions, i.e. optimized media composition and cultivation at higher pH value, enhanced trans-nootkatol production 5-fold compared to the initial strain and condition. These engineering approaches appear to be generally applicable for enhanced hydroxylation of hydrophobic compounds in P. pastoris as confirmed here for two additional membrane-attached CYPs, namely the limonene-3-hydroxylase from Mentha piperita and the human CYP2D6.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Pichia/genética , Proteína Recombinante y Reparadora de ADN Rad52/genética , Biotransformación , Medios de Cultivo , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Perfilación de la Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Mentha piperita/enzimología , Oxidación-Reducción , Pichia/enzimología , Pichia/crecimiento & desarrollo , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Sesquiterpenos/metabolismo , Terpenos/metabolismo , Regulación hacia ArribaRESUMEN
ω-Hydroxy oleic acid is an important intermediate for the synthesis of certain polyesters and polyamides. In this study, a functional CYP153A/putidaredoxin (Pdx)/putidaredoxin reductase (Pdr) hybrid system was engineered for improved ω-hydroxylation activity towards oleic acid. By the combination of site-directed saturation mutagenesis (SDSM) and iterative saturation mutagenesis (ISM), a best mutant (Variant II) was obtained with mutations at two sites (S120 and P165) at the Pdx interaction interface with CYP153A, and one site (S453) in the substrate binding pocket. The in vitro-reconstituted activity of Variant II with purified Pdx and Pdr was 2.7-fold that of the template, while the whole cell transformation activity was 2.0-fold that of the template. A 96-well format-based screening scheme for CYP153A was also developed, which should be useful for engineering of other P450s with low activity. Kinetic analyses indicated that the activity improvement for CYP153A variants largely resulted from enhanced electron transfer. This further demonstrates the importance of the electron transfer between P450s and the non-native redox partners for the overall performance of hybrid P450 systems.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Marinobacter/enzimología , Ácido Oléico/metabolismo , Ingeniería de Proteínas , Transporte de Electrón , Ferredoxinas/metabolismo , Hidroxilación , Marinobacter/genética , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , NADH NADPH Oxidorreductasas/metabolismoRESUMEN
Hydratases provide access to secondary and tertiary alcohols by regio- and/or stereospecifically adding water to carbon-carbon double bonds. Thereby, hydroxy groups are introduced without the need for costly cofactor recycling, and that makes this approach highly interesting on an industrial scale. Here we present the first crystal structure of a recombinant oleate hydratase originating from Elizabethkingia meningoseptica in the presence of flavin adenine dinucleotide (FAD). A structure-based mutagenesis study targeting active site residues identified E122 and Y241 as crucial for the activation of a water molecule and for protonation of the double bond, respectively. Moreover, we also observed that two-electron reduction of FAD results in a sevenfold increase in the substrate hydration rate. We propose the first reaction mechanism for this enzyme class that explains the requirement for the flavin cofactor and the involvement of conserved amino acid residues in this regio- and stereoselective hydration.
Asunto(s)
Flavobacteriaceae/enzimología , Hidroliasas/química , Hidroliasas/metabolismo , Ácido Oléico/química , Dominio Catalítico , Estructura MolecularRESUMEN
The sesquiterpenoid (+)-nootkatone is a highly demanded and highly valued aroma compound naturally found in grapefruit, pummelo or Nootka cypress tree. Extraction of (+)-nootkatone from plant material or its production by chemical synthesis suffers from low yields and the use of environmentally harmful methods, respectively. Lately, major attention has been paid to biotechnological approaches, using cell extracts or whole-cell systems for the production of (+)-nootkatone. In our study, the yeast Pichia pastoris initially was applied as whole-cell biocatalyst for the production of (+)-nootkatone from (+)-valencene, the abundant aroma compound of oranges. Therefore, we generated a strain co-expressing the premnaspirodiene oxygenase of Hyoscyamus muticus (HPO) and the Arabidopsis thaliana cytochrome P450 reductase (CPR) that hydroxylated extracellularly added (+)-valencene. Intracellular production of (+)-valencene by co-expression of valencene synthase from Callitropsis nootkatensis resolved the phase-transfer issues of (+)-valencene. Bi-phasic cultivations of P. pastoris resulted in the production of trans-nootkatol, which was oxidized to (+)-nootkatone by an intrinsic P. pastoris activity. Additional overexpression of a P. pastoris alcohol dehydrogenase and truncated hydroxy-methylglutaryl-CoA reductase (tHmg1p) significantly enhanced the (+)-nootkatone yield to 208mg L(-1) cell culture in bioreactor cultivations. Thus, metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of (+)-nootkatone from simple carbon sources.
Asunto(s)
Proteínas de Arabidopsis , Ingeniería Metabólica , Pichia , Sesquiterpenos/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/biosíntesis , Proteínas de Arabidopsis/genética , Cupressus/enzimología , Cupressus/genética , Hyoscyamus/enzimología , Hyoscyamus/genética , Pichia/enzimología , Pichia/genética , Sesquiterpenos PolicíclicosRESUMEN
Kievitone hydratase catalyzes the addition of water to the double bond of the prenyl moiety of plant isoflavonoid kievitone and, thereby, forms the tertiary alcohol hydroxy-kievitone. In nature, this conversion is associated with a defense mechanism of fungal pathogens against phytoalexins generated by host plants after infection. As of today, a gene sequence coding for kievitone hydratase activity has only been identified and characterized in Fusarium solani f. sp. phaseoli. Here, we report on the identification of a putative kievitone hydratase sequence in Nectria haematococca (NhKHS), the teleomorph state of F. solani, based on in silico sequence analyses. After heterologous expression of the enzyme in the methylotrophic yeast Pichia pastoris, we have confirmed its kievitone hydration activity and have assessed its biochemical properties and substrate specificity. Purified recombinant NhKHS is obviously a homodimeric glycoprotein. Due to its good activity for the readily available chalcone derivative xanthohumol (XN), this compound was selected as a model substrate for biochemical studies. The optimal pH and temperature for hydratase activity were 6.0 and 35°C, respectively, and apparent Vmax and Km values for hydration of XN were 7.16 µmol min-1 mg-1 and 0.98 ± 0.13 mM, respectively. Due to its catalytic properties and apparent substrate promiscuity, NhKHS is a promising enzyme for the biocatalytic production of tertiary alcohols.
Asunto(s)
Hidroliasas/metabolismo , Nectria/enzimología , Secuencia de Aminoácidos , Reactores Biológicos , Glicosilación , Hidroliasas/química , Hidroliasas/genética , Hidroliasas/aislamiento & purificación , Cinética , Resonancia Magnética Nuclear Biomolecular , Pichia/genética , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Espectrofotometría UltravioletaRESUMEN
Cytochrome P450 monooxygenases are a unique family of enzymes that are able to catalyze regio- and stereospecific oxidations for a broad substrate range. However, due to limited enzyme activities and stabilities, hydrophobicity of substrates, as well as the necessity of a continuous electron and oxygen supply the implementation of P450s for industrial processes remains challenging. Aim of this study was to point out key aspects for the development of an efficient synthesis concept for cytochrome P450 catalyzed oxidations. In order to regenerate the natural cofactor NADPH, a glucose dehydrogenase was applied. The low water soluble terpene α-ionone was used as substrate for the model reaction system. The studies reveal that an addition of surfactants in combination with low volumetric amounts of co-solvent can significantly increase substrate availability and reaction rates. Furthermore, these additives facilitated a reliable sampling procedure during the process. Another key factor for the process design was the oxygen supply. Based on various investigations, a bubble-aerated stirred tank reactor in batch mode represents a promising reactor concept for P450 oxidations. Main restriction of the investigated reaction system was the low process stability of the P450 monooxygenase, characterized by maximum total turnover numbers of â¼4100molα-ionone/molP450.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Ingeniería Metabólica/métodos , Proteínas Recombinantes/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Estabilidad de Enzimas , Escherichia coli/genética , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Biológicos , NADP/metabolismo , Norisoprenoides/metabolismo , Oxidación-Reducción , Oxígeno/metabolismo , Proteínas Recombinantes/genéticaRESUMEN
Membrane-anchored cytochrome P450 enzymes (CYPs) are a versatile and interesting class of enzymes for industrial applications, as they are capable of regio- and stereoselectively hydroxylating hydrophobic molecules. However, CYP activity requires sufficient levels of suitable cytochrome P450 reductases (CPRs) for regeneration of catalytic capacity, which is a bottleneck in many industrial applications. Searching for positive effectors of membrane-anchored CYP/CPR function, we transformed and screened selected strains from a Saccharomyces cerevisiae knockout collection for Hyoscyamus muticus premnaspirodiene oxygenase (HPO; CYP) and Arabidopsis thaliana CPR (AtCPR) expression levels, as well as for activity towards (+)-valencene. We found that in cells lacking the type III membrane protein Ice2p, AtCPR was destabilized. Remarkably, over-expression of ICE2 improved (+)-valencene hydroxylation to trans-nootkatol by 40-50%, both in resting cells and in vivo. Time-resolved immunoblot analysis and cytochrome c reductase activity assays revealed that Ice2 up-regulation stabilized AtCPR levels and activity over extended periods of bioconversion. To underscore that we had identified a novel positive effector of recombinant CYP/CPR function, we confirmed the beneficial effect of ICE2 over-expression for two further CYP/CPR combinations and the alternative host Pichia pastoris. Thus, we propose Ice2 up-regulation as a general tool for improving the applications of recombinant CYPs in yeasts.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas Fúngicas/metabolismo , Pichia/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Arabidopsis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Proteínas Fúngicas/genética , Pichia/genética , Estabilidad Proteica , Saccharomyces cerevisiae/genética , Sesquiterpenos/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Threonine aldolases catalyze the pyridoxal phosphate (PLP) dependent cleavage of threonine into glycine and acetaldehyde and play a major role in the degradation of this amino acid. In nature, L- as well as D-specific enzymes have been identified, but the exact physiological function of D-threonine aldolases (DTAs) is still largely unknown. Both types of enantio-complementary enzymes have a considerable potential in biocatalysis for the stereospecific synthesis of various ß-hydroxy amino acids, which are valuable building blocks for the production of pharmaceuticals. While several structures of L-threonine aldolases (LTAs) have already been determined, no structure of a DTA is available to date. Here, we report on the determination of the crystal structure of the DTA from Alcaligenes xylosoxidans (AxDTA) at 1.5 Å resolution. Our results underline the close relationship of DTAs and alanine racemases and allow the identification of a metal binding site close to the PLP-cofactor in the active site of the enzyme which is consistent with the previous observation that divalent cations are essential for DTA activity. Modeling of AxDTA substrate complexes provides a rationale for this metal dependence and indicates that binding of the ß-hydroxy group of the substrate to the metal ion very likely activates this group and facilitates its deprotonation by His193. An equivalent involvement of a metal ion has been implicated in the mechanism of a serine dehydratase, which harbors a metal ion binding site in the vicinity of the PLP cofactor at the same position as in DTA. The structure of AxDTA is completely different to available structures of LTAs. The enantio-complementarity of DTAs and LTAs can be explained by an approximate mirror symmetry of crucial active site residues relative to the PLP-cofactor.
Asunto(s)
Alcaligenes/enzimología , Aldehído-Liasas/química , Proteínas Bacterianas/química , Acetaldehído/metabolismo , Alanina Racemasa/química , Alanina Racemasa/genética , Alcaligenes/genética , Aldehído-Liasas/genética , Aldehído-Liasas/aislamiento & purificación , Aldehído-Liasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Sitios de Unión , Catálisis , Dominio Catalítico , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Escherichia coli , Glicina/biosíntesis , Manganeso/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Familia de Multigenes , Conformación Proteica , Estructura Terciaria de Proteína , Protones , Fosfato de Piridoxal/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Serina/análogos & derivados , Serina/química , Serina/metabolismo , Relación Estructura-Actividad , Treonina/metabolismoRESUMEN
GH29 α-l-fucosidases catalyze the hydrolysis of α-l-fucosidic linkages. Deficiency in human lysosomal α-l-fucosidase (FUCA1) leads to the recessively inherited disorder, fucosidosis. Herein we describe the development of fucopyranose-configured cyclophellitol aziridines as activity-based probes (ABPs) for selective in vitro and in vivo labeling of GH29 α-l-fucosidases from bacteria, mice and man. Crystallographic analysis on bacterial α-l-fucosidase confirms that the ABPs act by covalent modification of the active site nucleophile. Competitive activity-based protein profiling identified l-fuconojirimycin as the single GH29 α-l-fucosidase inhibitor from eight configurational isomers.
RESUMEN
Soluble water-forming NAD(P)H oxidases constitute a promising NAD(P)(+) regeneration method as they only need oxygen as cosubstrate and produce water as sole byproduct. Moreover, the thermodynamic equilibrium of O2 reduction is a valuable driving force for mostly energetically unfavorable biocatalytic oxidations. Here, we present the generation of an NAD(P)H oxidase with high activity for both cofactors, NADH and NADPH. Starting from the strictly NADH specific water-forming Streptococcus mutans NADH oxidase 2 several rationally designed cofactor binding site mutants were created and kinetic values for NADH and NADPH conversion were determined. Double mutant 193R194H showed comparable high rates and low K m values for NADPH (k cat 20 s(-1), K m 6 µM) and NADH (k cat 25 s(-1), K m 9 µM) with retention of 70% of wild type activity towards NADH. Moreover, by screening of a SeSaM library S. mutans NADH oxidase 2 variants showing predominantly NADPH activity were found, giving further insight into cofactor binding site architecture. Applicability for cofactor regeneration is shown for coupling with alcohol dehydrogenase from Sphyngobium yanoikuyae for 2-heptanone production.
RESUMEN
Polymersomes (Ps) based on a biodegradable and biocompatible block copolymer of methoxy poly(ethylene glycol) (mPEG) and poly(D,L-lactide) (PDLLA) in which apeptide sequence, Gly-Phe-Leu-Gly-Phe (GFLGF), was introduced in between the two blocks(mPEG-pep-PDLLA) were developed. The peptide linker is cleavable by the lysosomal enzymecathepsin B (Cath B). Ps containing the peptide linker (Ps(pep)) with an average diameter of about 124 nm were prepared by injecting a THF solution of the block copolymer into DI water. The Ps had a membrane thickness of about 15 nm as determined by transmission electron microscopy (TEM). In order to investigate the enzymatic degradation of the Ps (pep), dynamic light scattering (DLS) measurements of Ps(pep) dispersions with different concentrations of Cath B at pH 5.5 and 7.4 were performed as a function of time. A gradual decrease in kilo counts per second (Kcps) of the Ps (pep) over 7 d was observed after incubation of the Ps (pep) dispersions with 5 units/ml of Cath B at pH 5.5 at 37 °C. The size distribution became also bimodal, indicating that aggregation and precipitation of Ps (pep) occurred by disintegration of the Ps (pep) as a result of cleavage of the peptide. The rate of disintegration of the Ps (pep) was depending on the concentration of Cath Band the pH. No changes by DLS were seen when the dispersions were incubated with the enzyme at pH 7.4. Acridine orange (AO) was encapsulated in Ps (pep)as a model drug and rapid release of AO triggered by Cath B degradation of Ps (pep) was observed at pH 5.5. Anti-epidermal growth factor receptor (anti-EGFR) antibody (abEGFR) was immobilized on the surface of Ps(pep)in order to enhance the cellular uptake of Ps (pep). Fluorescein isothiocyanate labeled dextran (40,000 g/mol) (FD40) was incorporated in the Ps (pep) for the cell study and Ps either without peptide or antibody or without both peptide and antibody were used as negative controls. After 3 d exposure to SKBR3 cells, abEGFR-conjugated Ps (pep) (abEGFR-Ps (pep)) were directly bound to the membrane of the cells and were endocytosed more rapidly as compared to Ps (pep)without abEGFR. Intracellular release of FD40 from Ps (pep) was observed, suggesting that the peptide linker in Ps (pep) was cleaved in the lysosomal compartments of the cells leading to Ps (pep) membrane disruption. An Alexa Fluor(®) 488 labeled fragment of anti-mouse IgG (F(ab')(2)A) was also coupled to Ps (pep). Specific binding of the Ps (pep) coupled IgG (F(ab')(2)A) onto SKBR3 cells treated with primary mouse antibody was observed, whereas no binding was found with SKBR3 cells treated with goat antibody.
Asunto(s)
Anticuerpos Inmovilizados/inmunología , Sistemas de Liberación de Medicamentos , Receptores ErbB/inmunología , Lisosomas/metabolismo , Péptidos/química , Poliésteres/química , Polietilenglicoles/química , Naranja de Acridina/administración & dosificación , Catepsina B/metabolismo , Línea Celular Tumoral , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Humanos , Neoplasias/tratamiento farmacológico , Péptidos/metabolismo , Poliésteres/metabolismo , Polietilenglicoles/metabolismoRESUMEN
Aldolases are emerging as powerful and cost efficient tools for the industrial synthesis of chiral molecules. They catalyze enantioselective carbon-carbon bond formations, generating up to two chiral centers under mild reaction conditions. Despite their versatility, narrow substrate ranges and enzyme inactivation under synthesis conditions represented major obstacles for large-scale applications of aldolases. In this study we applied directed evolution to optimize Escherichia coli 2-deoxy-D-ribose 5-phosphate aldolase (DERA) as biocatalyst for the industrial synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside. This versatile chiral precursor for vastatin drugs like Lipitor (atorvastatin) is synthesized by DERA in a tandem-aldol reaction from chloroacetaldehyde and two acetaldehyde equivalents. However, E. coli DERA shows low affinity to chloroacetaldehyde and is rapidly inactivated at aldehyde concentrations useful for biocatalysis. Using high-throughput screenings for chloroacetaldehyde resistance and for higher productivity, several improved variants have been identified. By combination of the most beneficial mutations we obtained a tenfold improved variant compared to wild-type DERA with regard to (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside synthesis, under industrially relevant conditions.
Asunto(s)
Aldehído-Liasas/química , Aldehído-Liasas/genética , Industria Química/métodos , Evolución Molecular Dirigida/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/químicaRESUMEN
Biocatalysis has emerged as an important tool in the industrial synthesis of bulk chemicals, pharmaceutical and agrochemical intermediates, active pharmaceuticals, and food ingredients. However, the number and diversity of the applications are modest, perhaps in part because of perceived or real limitations of biocatalysts, such as limited enzyme availability, substrate scope, and operational stability. Recent scientific breakthroughs in genomics, directed enzyme evolution, and the exploitation of biodiversity should help to overcome these limitations. As a result, we expect many new industrial applications of biocatalysis to be realized, from single-step enzymatic conversions to customized multistep microbial synthesis by means of metabolic pathway engineering.