A 13C-Análise de Fluxos Metabólicos (13C-MFA) tornou-se uma técnica de alta precisão para estimar fluxos metabólicos e obter informações importantes sobre o metabolismo. Este método consiste em procedimentos experimentais, técnicas de medição e em cálculos para análise de dados. Neste contexto, os grupos de pesquisa de engenharia metabólica necessitam de ferramentas computacionais precisas e adequadas aos seus objetos de estudo. No presente trabalho, foi construída uma ferramenta computacional na plataforma MATLAB que executa cálculos de 13C-MFA, com balanços de metabólitos e cumômeros. Além disso, um módulo para estimar os fluxos metabólicos e um módulo para quantificar as incertezas das estimativas também foram implementados. O programa foi validado com dados presentes na literatura e aplicado a estudos de caso. Na estimação de fluxos de Pseudomonas sp. LFM046, identificou-se que esse micro-organismo possivelmente utiliza a Via das Pentoses em conjunto com a Via Entner-Doudoroff para a biossíntese de Polihidroxialcanoato (PHA). No design ótimo de experimentos para uma rede genérica de Pseudomonas, identificou-se a glicose marcada no átomo cinco como um substrato que permitirá determinar o fluxo na Via das Pentoses com menor incerteza. / 13C-Metabolic Flux Analysis (13C-MFA) has become a high-precision technique to estimate metabolic fluxes and get insights into metabolism. This method consists of experimental procedures, measurement techniques and data analysis calculations. In this context, metabolic engineering research groups demand accurate and suitable computational tools to perform the calculations. A computational tool was implemented in MATLAB platform that performs 13C-MFA calculation, using metabolite and cumomer balances, as well as a module to estimate the fluxes and a module to quantify their uncertainty. The program was validated with some classical cases from literature. From the flux estimates of Pseudomonas sp. LFM046, it was identified that the microorganism possibly uses the Pentose Phosphate Pathway along with the Entner-Doudoroff Pathway for Polyhydroxyalkanoate (PHA) biosynthesis. From the optimal experimental design for a generic Pseudomonas network, it was possible to conclude that glucose labeled at atom five is the best option to determine the flux in the Pentose Phosphate Pathway with smaller uncertainty.

The rapid postharvest deterioration of the roots, known as postharvest physiological deterioration (PPD), has been a major problem to the utilisation and development of cassava (Manihot esculenta Crantz) as a food and industrial crop. PPD usually occurs within two to three days after harvest and it is characterised by a blue-black discoloration of the roots, which renders the roots unpalatable and unmarketable. Scopoletin, which is synthesised de novo and released from its glucoside, scopolin, during PPD, plays a central role in this discoloration response. Interconversion of scopoletin and scopolin, which is catalysed by scopoletin-glucosyltransferase (scopoletin-GT) and scopolin-beta-glucosidase (scopolin-BG), regulates homeostasis of scopoletin in the cells. However, how this interconversion contributes to root discoloration development is poorly understood. In the present study, we identified and characterised cassava genes for the enzymes that are responsible for the interconversion of scopoletin and scopolin, subsequently manipulated their expression in transgenic cassava through scopoletin-GT RNAi gene silencing and scopolin-BG overexpression constructs. These approaches would potentially alter scopoletin and scopolin content in the root, thereby affecting PPD response. A BLAST search for homologous cassava genes revealed that scopoletin-GT and scopolin-BG are encoded by multiple genes, most of which belong to glucosyltransferase family-1 (GT1) and glycosyl hydrolase family-1 (GH1), respectively. Scopoletin-GT-down-regulated and scopolin-BG overexpressed transgenic cassava lines showed reduced not only scopolin but surprisingly also scopoletin, and delayed PPD. Additionally, other coumarins esculetin and esculin were also identified and both scopoletin-GT, MeSGT1, and scopolin-BG, BGLU23, were up-regulated during PPD development at day 4 and day 2, respectively. Our study reveals that disrupting the interconversion of scopoletin and scopolin by inhibiting scopoletin-GT and overexpressing scopolin-BG led to the decrease of both scopoletin and scopolin content and delayed PPD in cassava. These findings provide useful insights into the role of interconversion of scopoletin and scopolin in cassava PPD response and may suggest alternative ways to tackle PPD.

The potential value of the chitin biomass (e.g. food waste) is recently considered being ignored by landfill. Chitin can be a potential cheap carbon source for converting into value-added chemicals by microorganisms. Serratia marcescens is a chitinolytic bacterium that harbors endogenous chitinase systems. With goals of characterzing S. marcescens chitinolytic capabilities and applying S. marcescens to chemical production from chitin, my dissertation main content includes five chapters: 1) Chapter 1 highlights background information of chitin source, S. marcescens and potential metabolic engineering targets using chitin as a substrate; 2) Chapter 2 demonstrates that ChiR is a key regulator in regulating 9 chitinase-related genes in S. marcescens Db11 and manipulation of chiR can be a useful and efficient genetic target to enhance chitin utilization; 3) Chapter 3 reports the production of N-acetylneuraminic acid (Neu5Ac) from chitin by a bottom-up approach of engineering the nonconventional chitinolytic bacterium, Serratia marcescens, including native constitutive promoter characterization and transcriptional and translational pathway balancing; 4) Chapter 4 describes improvement of S. marcescens chitinolytic capability by an adaptive evolution approach; 5) Chapter 5 elucidates S. marcescens intracellular metabolite profile using a constraint-based genome-scale metabolic model (iSR929) based on genomic annotation of S. marcescens Db11. Overall, the dissertation work is the first report of demonstrating the concept of chitin-based CBP using S. marcescens and the computational model and genetic molecular tools developed in this dissertation are valuable but not limited to design-build-test of S. marcescens for contributing to the field of biological science and metabolic engineering applications.

This research focuses on the understanding and engineering of nonribosomal peptide biosynthetic pathways in Streptomyces coelicolor CH999, Escherichia coli BAP1 and Saccharomyces cerevisiae BJ5464-NpgA. The biosynthetic systems of indigoidine from bacteria and beauvericin/bassianolide from fungi were studied in this research. The production of these valuble products was significantly increased by enhancing their synthetic pathway with metabolic engineering approaches. Indigoidine is a bacterial natural product with antioxidant and antimicrobial activities. Its bright blue color resembles the industrial dye indigo, thus representing a new natural blue dye that may find uses in industry. Indigo is a dark blue crystalline powder and has been known for more than 4,000 years. It is commonly used to dye cotton yarn for the production of denim cloth to make blue jeans but the chemical synthesis of indigo requires harsh conditions and use of a strong base. Indigoidine is a new natural blue dye that is vi assembled from two molecules of L-glutamine under the catalysis of indigoidine synthetase. We identified a novel indigoidine synthetic gene from the genome of Streptomyces chromofuscus ATCC 49982. The successful heterologous expression of Sc-indC in E. coli BAP1 give us a pretty good yield of indigoidine under the optimized conditions. The production of this blue dye was then further improved by introducing two additional genes, sc-indB and glnA, into the biosynthetic pathway. Beauvericins and bassianolide are anticancer natural products from fungi and are assembled by corresponding iterative nonribosomal peptide synthetases. The beauvericin (BbBEAS) and bassianolide (BbBSLS) synthetases were successfully reconstituted in S. cerevisiae BJ5464-NpgA, leading to the production of beauvericins and bassianolide, respectively. The production of beauvericins was significantly improved by co-expression of BbBEAS and ketoisovalerate reductase (KIVR). To better understand the synthetic strategy of fungal iterative NRPs, the module/domain of BbBSLS and BbBEAS were dissected and reconstituted in S. cerevisiae. The result shows the intermodular linker is essential for the reconstitution of the separate modules and the domain swapping results indicated the fungal iterative NRPSs use a liner biosynthetic route which is different than bacterial iterative NRPs. The in vitro reactions of C2 and C3 with monomer/dimer/trimerN-acetylcysteamines demonstrated that C2 forms the amide bond and C3 catalyses the synthesis of the ester bond. Beauvericin could be reconstituted in vitro through co-reaction of C2(BbBEAS) and C3(BbBEAS) with D-Hiv-SNAC and N-Me-L-Phe- SNAC. This work also provides an unprecedented tool for engineering fungal iterative NRPSs to yield ‘unnatural’ cyclooligomer depsipeptides with varied chain lengths.

Pathway optimization is, in general, a very demanding task due to the complex, nonlinear and largely unknown interactions of enzymes, regulators and metabolites. While in vitro reconstruction and pathway analysis is a viable alternative, a major limitation of this approach is the availability of the pathway enzymes for reliable pathway reconstruction. Here, we report the application of RNA display methods for the construction of fusion (chimeric) molecules, comprising mRNA and the protein they express, that can be used for the above purpose. The chimeric molecule is immobilized via hybridization of its mRNA end with homologous capture DNA spotted on a substrate surface. We show that the protein (enzyme) end of the fusion molecule retains its function under immobilized conditions and that the enzymatic activity is proportional to the amount of capture DNA spotted on the surface of a microarray or 96-well microplate. The relative amounts of all pathway enzymes can thus be changed at will by changing the amount of the corresponding capture DNA. Hence, entire pathways can be reconstructed and optimized in vitro from genomic information alone by generating chimeric molecules for all pathway enzymes in a single in vitro translation step and hybridizing on 96-well microplates where each well contains a different combination of capture DNA. We provide validation of this concept with the sequential reactions catalyzed by luciferase and nucleoside diphosphate kinase and further illustrate this method with the optimization of the five-step pathway for trehalose synthesis. Multi-enzyme pathways leading to the synthesis of specialty molecules can thus be optimized from genomic information about the pathway enzymes, provided the latter retain their activity under the in vitro immobilized conditions. / Singapore-MIT Alliance (SMA)

Synechocystis PCC 6803 is a photosynthetic bacterium that has the potential to make bioproducts from carbon dioxide and light. Biochemical production from photosynthetic organisms is attractive because it replaces the typical bioprocessing steps of crop growth, milling, and fermentation, with a one-step photosynthetic process. However, low yields and slow growth rates limit the economic potential of such endeavors. Rational metabolic engineering methods are hindered by limited cellular knowledge and inadequate models of Synechocystis. Instead, inverse metabolic engineering, a scheme based on combinatorial gene searches which does not require detailed cellular models, but can exploit sequence data and existing molecular biological techniques, was used to find genes that (1) improve the production of the biopolymer poly-3-hydroxybutyrate (PHB) and (2) increase the growth rate. A fluorescence activated cell sorting assay was developed to screen for high PHB producing clones. Separately, serial sub-culturing was used to select clones that improve growth rate. Novel gene knock-outs were identified that increase PHB production and others that increase the specific growth rate. These improvements make this system more attractive for industrial use and demonstrate the power of inverse metabolic engineering to identify novel phenotype-associated genes in poorly understood systems. / Singapore-MIT Alliance (SMA)

A critical concern in metabolic engineering is the need to balance the demand and supply of redox intermediates. Bioelectrochemical techniques offer a promising method to alleviate redox imbalances during the synthesis of biochemicals. Broadly, these techniques reduce intracellular NAD+ to NADH and therefore manipulate the cell’s redox balance. The cellular response to such redox changes and the additional reducing can be harnessed to produce desired metabolites. In the context of microbial fermentation, these bioelectrochemical techniques can improve product yields and/or productivity. We have developed a method to characterize the role of bioelectrosynthesis in chemical production using the genome-scale metabolic model of E. coli. The results elucidate the role of bioelectrosynthesis and its impact on biomass growth, cellular ATP yields and biochemical production. The results also suggest that strain design strategies can change for fermentation processes that employ microbial electrosynthesis and suggest that dynamic operating strategies lead to maximizing productivity.

A critical concern in metabolic engineering is the need to balance the demand and supply of redox intermediates. Bioelectrochemical techniques offer a promising method to alleviate redox imbalances during the synthesis of biochemicals. Broadly, these techniques reduce intracellular NAD+ to NADH and therefore manipulate the cell’s redox balance. The cellular response to such redox changes and the additional reducing can be harnessed to produce desired metabolites. In the context of microbial fermentation, these bioelectrochemical techniques can improve product yields and/or productivity. We have developed a method to characterize the role of bioelectrosynthesis in chemical production using the genome-scale metabolic model of E. coli. The results elucidate the role of bioelectrosynthesis and its impact on biomass growth, cellular ATP yields and biochemical production. The results also suggest that strain design strategies can change for fermentation processes that employ microbial electrosynthesis and suggest that dynamic operating strategies lead to maximizing productivity.

Recientemente, el uso de técnicas de manipulación genética ha abierto la puerta a la obtención de microorganismos con fenotipos mejorados, lo que a su vez ha llevado a unas mejoras significativas en la síntesis de algunos productos bioquímicos. Sin embargo, la mutación y selección de estos nuevos organismos se ha llevado a cabo, en la mayoría de casos, por ensayo y error. Es de esperar que estos procesos puedan ser mejorados si se usan principios de diseño cuantitativos para guiar la búsqueda hacia el perfil enzimático ideal. Esta tesis está dedicada al desarrollo de un conjunto de herramientas de optimización avanzadas para asesorar en problemas de ingeniería metabólica y otras cuestiones emergentes en biología de sistemas. Concretamente, nos centramos en problemas en qué se modelan las redes metabólicas usando expresiones cinéticas. La utilidad de los algoritmos desarrollados para resolver tales problemas es demostrada por medio de varios casos de estudio. / In recent years, the use of genetic manipulation techniques has opened the door for obtaining microorganisms with enhanced phenotypes, which has in turn led to significant improvements in the synthesis of certain biochemical products. However, mutation and selection of these new organisms has been performed, in most cases, in a trial-and-error basis. It is expected that these processes could be further improved if quantitative design principles were used to guide the search towards the ideal enzymatic profiles. This thesis is devoted to developing a set of advanced global optimization tools to assess metabolic engineering problems and other questions arising in systems biology. In particular, we focus on problems where metabolic networks are modeled making use of kinetic expressions. The usefulness of the algorithms developed to solve such problems is demonstrated by means of several case studies.

Oligosaccharides are important biomolecules that are targets and also components of many medical treatments, including treatments for cancer, HIV, and inflammation. While the demand for medically-relevant oligosaccharides is increasing, these compounds have proven difficult to synthesize. Whole-cell oligosaccharide synthesis is a promising method that requires relatively inexpensive substrates and can complete the synthesis in just one step. However, whole-cell oligosaccharide synthesis employing common microorganisms like E. coli have been plagued by low yields. This dissertation investigates an alternative microorganism for oligosaccharide production: Agrobacterium sp. ATCC 31749. This Agrobacterium strain produces high levels of curdlan polysaccharide, demonstrating its natural ability to produce the sugar nucleotide precursor for oligosaccharide production. The two main objectives of this dissertation are 1) to develop biocatalysts for oligosaccharide synthesis by engineering ATCC 31749 and 2) to determine what factors affect poly- and oligosaccharide production in this Agrobacterium strain. ATCC 31749 was engineered to produce two oligosaccharides of medical importance: N-acetyllactosamine and galactose-α 1,3-lactose. Oligosaccharide production in the biocatalyst was further improved with additional metabolic engineering. Substrate uptake was increased through expression of a lactose permease, and availability of the sugar nucleotide substrate improved with gene knockout of the curdlan synthase gene. Both of these engineering efforts led to increased oligosaccharide synthesis in the Agrobacterium biocatalyst. Overall, the engineered Agrobacterium strains synthesized gram-scale quantities of the oligosaccharide products in just one step and requiring only a few inexpensive substrates and cofactors. Additional improvement of the oligosaccharide-producing biocatalysts required further investigation of the factors influencing poly- and oligosaccharide production in ATCC 31749. In this dissertation, several environmental and intracellular factors are identified that affect both oligosaccharide and curdlan production. Sucrose was the preferred carbon source for oligosaccharide synthesis, and the addition of citrate to the synthesis reaction led to significant improvement in oligosaccharide production. To identify the genetic factors and possible mechanisms regulating curdlan production, the genome of ATCC 31749 was sequenced. The genome sequence was utilized for transcriptome analysis of ATCC 31749. In the transcriptome analysis, genes significantly up- and down-regulated during curdlan production were identified. Subsequent gene knockout experiments showed several factors to be important for curdlan synthesis, namely the nitrogen signaling cascade, polyphosphate, and the GTP-derived second messengers (p)ppGpp and c-di-GMP. In addition to the development of biocatalysts for oligosaccharide production, this investigation provides insight into the complex mechanisms regulating exopolysaccharide synthesis.