If small isoprenoids, the starting component of carotenoids, can be efficiently excreted from thermophilic cyanobacteria, they could help satisfy the demand for sustainably produced hydrocarbons. This is the driving force behind wanting to understand the response and regulation of isoprenoid pathways to environmental stimuli in the thermophilic cyanobacterium, Thermosynechococcus elongatus, BP-1. The portion of the isoprenoid pathway studied here is the carotenoid pathway since these products are critical to adaptation and they encompass the largest pool of isoprenoid compounds in cyanobacteria. Although synthetic biology in cyanboacteria has improved in recent years, there are many undiscovered metabolic complexities that make large-scale commercial production challenging. To address this need, I quantify and report for the first time metabolic shifts within the carotenoid pathway of BP-1 due to combined effects of temperature, pH and blue light. I show that metabolism shifts from the dicyclic into the monocyclic carotenoid pathway in response to pH, and that decreasing temperature drives flux into the end products of both pathways. Also, I report that the productivity of an uncommon carotenoid, 2-hydroxymyxol 2’-fucoside (HMF), approached 500 μg/L-day in cultures grown at 45 °C, high light intensity, and pH 8. In order to further elucidate these responses, I analyzed 42 RNAseq samples taken over time of BP-1 induced by cold and heat stress and compared these results to metabolomics data. I showed that crtR and crtG, two central carotenogenesis genes, are transcriptionally controlled and used weighted gene co-expression network analysis (WGCNA) to determine eight separate co-expressed modules of biological significance. Among the co-regulated heat response and cold response genes there were three and five non-coding RNA, respectively, providing targets for future investigation. Using subtractive genomics and transcriptional data I narrowed the potential missing steps of the myxol pathway in cyanobacteria to seven unknown BP-1 genes, two of which were confirmed not to be involved in the missing step(s). Finally, by generating a ΔcrtG mutant and testing it under different environmental parameters, I showed that HMF does not protect against high pH or low temperature (despite up-regulation at these conditions), and that CrtG has a higher affinity for monocyclic than dicyclic carotenoids. / text

The field of Synthetic Biology seeks to develop engineering principles for biological systems. Modular biological parts are repurposed and recombined to develop new synthetic biological devices with novel functions. The proper functioning of these devices is dependent on the cellular context provided by the host organism, and the interaction of these devices with host systems. The field of Systems Biology seeks to measure and model the properties of biological phenomena at the network scale. We present the application of systems biology approaches to synthetic biology, with particular emphasis on understanding and remodeling metabolic networks. Chapter 2 demonstrates the use of a Flux Balance Analysis model of the Saccharomyces cerevisiae metabolic network to identify and construct strains of S. cerevisiae that produced increased amounts of formic acid. Chapter 3 describes the development of synthetic metabolic pathways in Escherichia coli for the production of hydrogen, and a directed evolution strategy for hydrogenase enzyme improvement. Chapter 4 introduces the use of metabolomic profiling to investigate the role of circadian regulation in the metabolic network of the photoautotrophic cyanobacterium Synechococcus elongatus PCC 7942. Together, this work demonstrates the utility of network-scale approaches to understanding biological systems, and presents novel strategies for engineering metabolism.

Inexpensive petroleum is the cornerstone of the modern global economy despite its huge environmental costs and its nature as a non-renewable resource. While ninety percent of petroleum is ultimately used as fuel and can in principle be replaced by sources of renewable electricity, ten percent is used as a feedstock to produce societally important chemicals that cannot currently be made at a reasonable cost through alternative processes. In this dissertation, I will discuss my efforts, together with several colleagues, to apply synthetic biology approaches to the challenge of producing renewable petrochemical replacements. In Chapter 2, I discuss our efforts to engineer E. coli to produce fatty acids with a wide range of chain lengths at high yield, thereby providing an alternative platform for the production of diverse petrochemicals. In Chapter 3, I describe a novel method of DNA assembly that we developed to facilitate synthetic biology efforts such as those in Chapter 2. This method is capable of simultaneously assembling multiple DNA pieces with substantial sequence homology, a common challenge in synthetic biology. In Chapter 4, I discuss the development of a "bionic leaf": a hybrid microbial-inorganic catalyst that marries the advantages of photovoltaic-based light capture and microbial carbon fixation to achieve solar biomass yields greater than those observed in terrestrial plants. This technology offers a potentially low-cost alternative to photosynthesis as a source of biomass and derived chemicals and fuels. The work described in this dissertation demonstrates the capacity of synthetic biology to address the problem of renewable chemical production, and offers proof of principle demonstrations that both the scope and efficiency of biological chemical production may be improved.

Terpenes comprise a large diverse class of natural products and many of them attract interest because of their physiological function, therapeutic and industrial values. Triterpene oils including squalene (C30), botrycococcene (C30) and their methylated derivatives (C31-C37) generated by the green algae Botryococcus braunii race B, which have recently received significant attention because of their utility for advanced biofuels. However, the slow growth habit of B. braunii makes it impractical as a robust biofuel production system. In this thesis, we firstly evaluated the potential of generating high levels of triterpene (C30) production in tobacco plants by diverting carbon flux from cytosolic MVA pathway or plastidic MEP pathway by overexpressing avian farnesyl diphosphate synthase along with triterpene synthase targeted to the cytoplasm or the chloroplast of cells. Up to 1,000 µg/g fresh weight of squalene and 544 µg/g fresh weight of botryococcene was achieved in our transgenic plants with this metabolism direct to the chloroplasts, which is about approximately 100-times greater than that accumulating in the plants engineered for cytosolic production. To test if methylated triterpenes can be produced in tobacco, we also engineered triterpene methyltransferases (TMTs) into wild type plants and transgenic tobacco plants selected for high level triterpene accumulation. We observed that up to 91% of the total triterpene content was converted to methylated forms (C31, C32) by targeting the TMTs to the chloroplasts of transgenic plants, whereas only 4-14% of total triterpenes were methylated when TMTs were directed to the cytoplasm. Select transgenic lines were growing in field studies from 2011 to 2014 to evaluate their physiological performance under field conditions. Surprisingly, the field studies suggested that the growth and agronomic performance of the transgenic lines accumulating squalene were not compromised, while those accumulating high levels of botryococcene were only 72%-76% as tall, had about 59%-75% of the leaf area, and about 55%-75% of the biomass as wild type plants. Yet, these transgenic plants had photosynthetic capacity equal to the wild type plants.

The diminishing of fossil fuels and growing concerns towards climate change have intensified biofuel production from renewable resources. Recently, progresses are made in microbial production of biofuels. Among various biofuels, isobutanol is gaining an increasing attention due to its high energy content and suitable chemical and physical properties, enabling it to be a suitable substitution of fossil fuel. In this study, instead of using heterotrophic microorganisms, we performed metabolic engineering of Synechocystis PCC 6803 (Synechocystis) for isobutanol production under autotrophic condition. After introduced 2-keto acid pathway, Synechocystis is able to produce isobutanol when provided with water, carbon dioxide and solar energy. When cultivated in an optimal condition (50 μmol photons m-1s-2 and adjusted pH to 7-8 with HCl), the engineered strain pEEK2-ST was able to produce 425 mg L-1 in-flask isobutanol titer and 911 mg L-1 cumulative isobutanol titer, respectively, in 46 days. There should be bottlenecks existing in 2-keto acid pathway based on the similar isobutanol production of strain pEEK2-ST with and without pyruvate addition. However, the attempt to identify potential bottlenecks of upstream genes by overexpressing ST and one of the three upstream genes failed, instead what we conclude is that the isobutanol production is tightly correlated to Kivd (ST) expression level. Thus, more strategies will be employed for identifying bottlenecks successfully and further improvement of isobutanol production in the future. In conclusion, this study demonstrates the importance of cultivation condition on isobutanol production in Synechocystis.

abstract: Biomass synthesis is a competing factor in biological systems geared towards generation of commodity and specialty chemicals, ultimately limiting maximum titer and yield; in this thesis, a widely generalizable, modular approach focused on decoupling biomass synthesis from the production of the phenylalanine in a genetically modified strain of E. coli BW25113 was explored with the use of synthetic trans-encoded small RNA (sRNA) to achieve greater efficiency. The naturally occurring sRNA MicC was used as a scaffold, and combined on a plasmid with a promoter for anhydrous tetracycline (aTc) and a T1/TE terminator. The coding sequence corresponding to the target binding site for fourteen potentially growth-essential gene targets as well as non-essential lacZ was placed in the seed region of the of the sRNA scaffold and transformed into BW25113, effectively generating a unique strain for each gene target. The BW25113 strain corresponding to each gene target was screened in M9 minimal media; decreased optical density and elongated cell morphology changes were observed and quantified in all induced sRNA cases where growth-essential genes were targeted. Six of the strains targeting different aspects of cell division that effectively suppressed growth and resulted in increased cell size were then screened for viability and metabolic activity in a scaled-up shaker flask experiment; all six strains were shown to be viable during stationary phase, and a metabolite analysis showed increased specific glucose consumption rates in induced strains, with unaffected specific glucose consumption rates in uninduced strains. The growth suppression, morphology and metabolic activity of the induced strains in BW25113 was compared to the bacteriostatic additives chloramphenicol, tetracycline, and streptomycin. At this same scale, the sRNA plasmid targeting the gene murA was transformed into BW25113 pINT-GA, a phenylalanine overproducer with the feedback resistant genes aroG and pheA overexpressed. Two induction times were explored during exponential phase, and while the optimal induction time was found to increase titer and yield amongst the BW25113 pINT-GA murA sRNA variant, overall this did not have as great a titer or yield as the BW25113 pINT-GA strain without the sRNA plasmid; this may be a result of the cell filamentation. / Dissertation/Thesis / Masters Thesis Chemical Engineering 2016

abstract: The basic scheme for photosynthesis suggests the two photosystems existing in parity with one another. However, cyanobacteria typically maintain significantly more photosystem I (PSI) than photosystem II (PSII) complexes. I set out to evaluate this disparity through development and analysis of multiple mutants of the genetically tractable cyanobacterium Synechocystis sp. PCC 6803 that exhibit a range of expression levels of the main proteins present in PSI (Chapter 2). One hypothesis was that the higher abundance of PSI in this organism is used to enable more cyclic electron flow (CEF) around PSI to contribute to greater ATP synthesis. Results of this study show that indeed CEF is enhanced by the high amount of PSI present in WT. On the other hand, mutants with less PSI and less cyclic electron flow appeared able to maintain healthy levels of ATP synthesis through other compensatory mechanisms. Reduction in PSI abundance is naturally associated with reduced chlorophyll content, and mutants with less PSI showed greater primary productivity as light intensity increased due to increased light penetration in the cultures. Another question addressed in this research project involved the effect of deletion of flavoprotein 3 (an electron sink for PSI-generated electrons) from mutant strains that produce and secrete a fatty acid (Chapter 3). Removing Flv3 increased fatty acid production, most likely due to increased abundance of reducing equivalents that are key to fatty acid biosynthesis. Additional components of my dissertation research included examination of alkane biosynthesis in Synechocystis (Chapter 4), and effects of attempting to overexpress fibrillin genes for enhancement of stored compounds (Chapter 5). Synechocystis is an excellent platform for metabolic engineering studies with its photosynthetic capability and ease of genetic alteration, and the presented research sheds light on multiple aspects of its fundamental biology. / Dissertation/Thesis / Doctoral Dissertation Biology 2017

abstract: The production of monomer compounds for synthesizing plastics has to date been largely restricted to the petroleum-based chemical industry and sugar-based microbial fermentation, limiting its sustainability and economic feasibility. Cyanobacteria have, however, become attractive microbial factories to produce renewable fuels and chemicals directly from sunlight and CO2. To explore the feasibility of photosynthetic production of (S)- and (R)-3-hydroxybutyrate (3HB), building-block monomers for synthesizing the biodegradable plastics polyhydroxyalkanoates and precursors to fine chemicals, synthetic metabolic pathways have been constructed, characterized and optimized in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803). Both types of 3HB molecules were produced and readily secreted from Synechocystis cells without over-expression of transporters. Additional inactivation of the competing PHB biosynthesis pathway further promoted the 3HB production. Analysis of the intracellular acetyl-CoA and anion concentrations in the culture media indicated that the phosphate consumption during the photoautotrophic growth and the concomitant elevated acetyl-CoA pool acted as a key driving force for 3HB biosynthesis in Synechocystis. Fine-tuning of the gene expression levels via strategies, including tuning gene copy numbers, promoter engineering and ribosome binding site optimization, proved critical to mitigating metabolic bottlenecks and thus improving the 3HB production. One of the engineered Synechocystis strains, namely R168, was able to produce (R)-3HB to a cumulative titer of ~1600 mg/L, with a peak daily productivity of ~200 mg/L, using light and CO2 as the sole energy and carbon sources, respectively. Additionally, in order to establish a high-efficiency transformation protocol in cyanobacterium Synechocystis 6803, methyltransferase-encoding genes were cloned and expressed to pre-methylate the exogenous DNA before Synechocystis transformation. Eventually, the transformation efficiency was increased by two orders of magnitude in Synechocystis. This research has demonstrated the use of cyanobacteria as cell factories to produce 3HB directly from light and CO2, and developed new synthetic biology tools for cyanobacteria. / Dissertation/Thesis / Ph.D. Biological Design 2014

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.

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