Rhodococcus opacus PD630 Genetic Tool Development to Enable the Conversion of Biomass

The discovery of fossil fuels facilitated a new era in human history and allowed many firsts, such as the mass production of goods, the ability to travel and communicate long distances, the formation of population dense cities, and unprecedented improvements in quality of life. Alternative sources of energy and chemicals are needed, however, as hydrocarbon reserves continue to deplete and the effects of burning fossils on the planet become better understood. Lignocellulosic biomass is the most abundant raw material in the world and a viable alternative to petroleum-derived products. The pre-treatment of lignocellulose (e.g., thermocatalytic depolymerization, enzymatic hydrolysis, pyrolysis, etc.) generates a range of products, including readily available sugars for microbial fermentation. One of the typically unused fractions of biomass is the structural component, referred to as lignin, that makes up 15 to 30% of the material and when depolymerized generates a heterogeneous mixture of toxic aromatic compounds. Generally, lignin is separated from the carbohydrate fraction and burned, but its utilization has been identified as a key factor in biorefinery profitability. One possible option for lignin valorization is to find a microbe that not only ferments lignocellulose-derived sugars into a valuable commodity, but also the lignin-derived aromatics.Rhodococcus opacus PD630 (hereafter R. opacus) is a non-model, gram-positive bacterium that possesses desirable traits for biomass conversion, including consumption capabilities for both lignocellulose-derived sugars and aromatic compounds, significant accumulation of the biodiesel precursor triacylglycerol, a relatively rapid growth rate, and genetic tractability. Few genetic elements and molecular biology techniques, however, have been directly characterized in R. opacus, limiting its application for lignocellulose bioconversion. The goal of this dissertation is to greatly expand the genetic toolbox available in R. opacus in order to provide insight into its aromatic catabolism and to promote its use as a microbial chassis for the conversion of biomass-derived products into biofuels or other value-added products. The contributions developed as part of this dissertation include 1) the development of strong constitutive promoters for the overexpression of heterologous genes, 2) the development of chemical and metabolite sensors for tunable gene expression, 3) the characterization of native and endogenous plasmid backbones and resistance markers, 4) a heterologous T7 RNA polymerase expression platform for gene expression, 5) the demonstration of genetic logic circuits for programable gene expression, 6) a recombinase-based recombineering platform for gene knockouts and insertions, 7) a CRISPR interference (CRISPRi) platform for targeted gene repression, 8) the identification of stable reference genes for RT-qPCR applications, 9) insight into aromatic degradation through the β-ketoadipate pathway via gene knockouts, and 10) insight into the role of aromatic transporters via gene knockouts. Taken together, this work greatly advances the ability to engineer R. opacus for any desired application, in addition to providing understanding into its catabolism of aromatic compounds

The logic layout of the TOL network of Pseudomonas putida pWW0 plasmid stems from a metabolic amplifier motif (MAM) that optimizes biodegradation of m-xylene

<p>Abstract</p> <p>Background</p> <p>The genetic network of the TOL plasmid pWW0 of the soil bacterium <it>Pseudomonas putida </it>mt-2 for catabolism of <it>m-</it>xylene is an archetypal model for environmental biodegradation of aromatic pollutants. Although nearly every metabolic and transcriptional component of this regulatory system is known to an extraordinary molecular detail, the complexity of its architecture is still perplexing. To gain an insight into the inner layout of this network a logic model of the TOL system was implemented, simulated and experimentally validated. This analysis made sense of the specific regulatory topology out on the basis of an unprecedented network motif around which the entire genetic circuit for <it>m-</it>xylene catabolism gravitates.</p> <p>Results</p> <p>The most salient feature of the whole TOL regulatory network is the control exerted by two distinct but still intertwined regulators (XylR and XylS) on expression of two separated catabolic operons (<it>upper </it>and <it>lower</it>) for catabolism of <it>m</it>-xylene. Following model reduction, a minimal modular circuit composed by five basic variables appeared to suffice for fully describing the operation of the entire system. <it>In silico </it>simulation of the effect of various perturbations were compared with experimental data in which specific portions of the network were activated with selected inducers: <it>m-</it>xylene, <it>o-</it>xylene, 3-methylbenzylalcohol and 3-methylbenzoate. The results accredited the ability of the model to faithfully describe network dynamics. This analysis revealed that the entire regulatory structure of the TOL system enables the action an unprecedented metabolic amplifier motif (MAM). This motif synchronizes expression of the <it>upper </it>and <it>lower </it>portions of a very long metabolic system when cells face the head pathway substrate, <it>m-</it>xylene.</p> <p>Conclusion</p> <p>Logic modeling of the TOL circuit accounted for the intricate regulatory topology of this otherwise simple metabolic device. The found MAM appears to ensure a simultaneous expression of the <it>upper </it>and <it>lower </it>segments of the <it>m-</it>xylene catabolic route that would be difficult to bring about with a standard substrate-responsive single promoter. Furthermore, it is plausible that the MAM helps to avoid biochemical conflicts between competing plasmid-encoded and chromosomally-encoded pathways in this bacterium.</p

The private life of environmental bacteria: pollutant biodegradation at the single cell level

Bacteria display considerable cell-to-cell heterogeneity in a number of genetic and physiological traits. Stochastic differences in regulatory patterns (e.g., at the transcriptional level) propagate into the metabolic and physiological status of otherwise isogenic cells, which ultimately results in appearance of sub-populations within the community. As new technologies emerge and because novel single cell strategies are constantly being refined, our knowledge on microbial individuality is in burgeoning and constant expansion. These approaches encompass not only molecular biology tools (e.g., fluorescent protein based reporters) but also a suite of sophisticated, non-invasive technologies to gain insight into the metabolic state of individual cells. Defining the role of individual heterogeneities is thus instrumental for the population-level understanding of macroscopic processes, in both environmental and industrial setups. The present article reviews the state-of-the-art methodologies for the investigation of single bacteria at both the genetic and metabolic level, and places the application of currently available tools in the context of microbial ecology and environmental microbiology. As a case example, we examine the stochastic and multi-stable behaviour of the TOL-encoded pathway of Pseudomonas putida mt-2 for the biodegradation of aromatic compounds. Bet-hedging strategies and division of labour are considered as factors pushing forward the evolution of environmental microorganisms.This study was supported by the BIO and FEDER CONSOLIDER INGENIO Program of the Spanish Ministry of Science and Innovation, the MICROME, ST-FLOW and ARYSIS Contracts of the EU, the ERANET-IB Program and the PROMT Project of the CAM.Peer reviewe

Genetic Circuits for Transcriptional Regulation in Synechocystis sp. PCC 6803

Microbial biosynthesis has produced a variety of complex compounds using processes that are more environmentally-friendly than many conventional methods. The most common hosts are heterotrophs, which require the addition of an organic carbon source; while cyanobacteria possess many traits that make them a more sustainable biotechnology platform. As phototrophs, cyanobacteria can employ sunlight and carbon dioxide to create many value-added compounds. A wealth of tools has been developed to engineer the commonly used heterotrophs for higher yields and titers; yet, few synthetic biology tools have been designed for cyanobacteria. Furthermore, many of the tools created for heterotrophs do not function as designed in the photosynthetic organisms. We developed a multi-input and several single-input transcriptional regulators for the model cyanobacterium Synechocystis sp. PCC 6803 to address this problem. These circuits were designed to respond to industrially-relevant signals, including oxygen, light and the cells\u27 nitrogen status, in addition to an inexpensive sugar. The two-input AND logic gate we built adds more sophisticated heterologous gene expression to the cyanobacterium\u27s synthetic biology toolbox. The addition of these regulators provides engineers more options when looking for a part that meets the needs of the situation. This was demonstrated by our use of the oxygen-responsive promoter to express, in a heterologous host, genes from a cluster that encodes nitrogenase. This new device can be used to probe the regulation of nitrogen fixation in a photosynthetic cell. Our development of genetic circuits for transcriptional regulation in Synechocystis sp. PCC 6803 improves the viability of this photosynthetic host in biotechnology

Switching in bacterial gene expression networks

An ability of a bacterium to appropriately respond to its environmental cues ultimately decides its fate. Bacteria deal with the fluctuating environment as a population instead of individual cells. By allowing individual cells to stochastically switch between multiple phenotypes, the cell population can make sure some cells are always fit for the environmental change. The underlying genetic circuitry plays a key role in eliciting multiple phenotypes by an isogenic population of bacteria. Understanding the underlying mechanism requires careful and systematic approach. In this study, we investigated two very well-known systems: the motility in Salmonella enterica serovar Typhimurium and the sugar utilization in Escherichia coli. Many bacteria are motile only when nutrients are scarce. By contrast, Salmonella enterica is motile only when nutrients are plentiful, suggesting this bacterium uses motility for purposes other than foraging, most likely host colonization. We investigated how nutrients affect motility in S. enterica and found that nutrients tune the fraction of motile cells. In particular, we observed co-existing populations of motile and non-motile cells, where the distribution was determined by the concentration of nutrients in the growth medium. Interestingly, S. enterica does not respond to a single nutrient but apparently a complex mixture of them. We investigated the mechanism governing this behavior and found that it results from two antagonizing regulatory proteins, FliZ and YdiV. We further demonstrated that the response is bistable: namely, that genetically identical cells can exhibit different phenotypes under identical growth conditions. We further characterized the differences within class 2 and class 3 gene expression and showed that a secretion-dependent feedback loop involving flagellar specific sigma factor, σ28, is responsible for partitioning cells into two fractions. Together, these results uncover a new facet to the regulation of the flagellar genes in S. enterica and further demonstrate how bacteria employ phenotypic diversity as general mechanism for adapting to change in their environment. We then investigated the sugar utilization system in E. coli. Glucose is known to inhibit the transport and metabolism of many sugars in Escherichia coli. This mechanism leads to its preferential consumption. Far less, however, is known about the preferential utilization of non-glucose sugars in E. coli. One notable exception is arabinose and xylose. Previous studies have shown that E. coli will consume arabinose ahead of xylose. Selective utilization results from arabinose-bound AraC binding to the promoter of the xylose metabolic genes and inhibiting their expression. This mechanisms, however, has not been explored in single cells. Both the arabinose and xylose utilization systems are known to exhibit a bimodal induction response to their cognate sugar, where mixed populations of cells either expressing the metabolic genes or not are observed at intermediate sugar concentrations. This suggests that arabinose can only inhibit xylose metabolism in arabinose-induced cells. To understand how crosstalk between these systems affects their response, we investigated E. coli during growth on mixtures of arabinose and xylose at single-cell resolution. Our results show that mixed, multimodal populations of arabinose and xylose-induced cells occur at some intermediate sugar concentrations. We also found that xylose can inhibit the expression of the arabinose metabolic genes and that this repression is due to XylR. We further found that xylose-bound XylR binds to the divergent promoter region of the regulator araC and the arabinose metabolic genes and inhibit expression. These results demonstrate that a strict hierarchy does not exist between arabinose and xylose as previously thought and this may aid in the design of E. coli strains capable of simultaneous sugar consumption

Transcriptional regulation and responses in filamentous fungi exposed to lignocellulose

Biofuels derived from lignocellulose are attractive alternative fuels but their production suffers from a costly and inefficient saccharification step that uses fungal enzymes. One route to improve this efficiency is to understand better the transcriptional regulation and responses of filamentous fungi to lignocellulose. Sensing and initial contact of the fungus with lignocellulose is an important aspect. Differences and similarities in the responses of fungi to different lignocellulosic substrates can partly be explained with existing understanding of several key regulators and their mode of action, as will be demonstrated for Trichoderma reesei, Neurospora crassa and Aspergillus spp. The regulation of genes encoding Carbohydrate Active enZymes (CAZymes) is influenced by the presence of carbohydrate monomers and short oligosaccharides, as well as the external stimuli of pH and light. We explore several important aspects of the response to lignocellulose that are not related to genes encoding CAZymes, namely the regulation of transporters, accessory proteins and stress responses. The regulation of gene expression is examined from the perspective of mixed cultures and models are presented for the nature of the transcriptional basis for any beneficial effects of such mixed cultures. Various applications in biofuel technology are based on manipulating transcriptional regulation and learning from fungal responses to lignocelluloses. Here we critically access the application of fungal transcriptional responses to industrial saccharification reactions. As part of this chapter, selected regulatory mechanisms are also explored in more detail

Novel approaches for the evolutionary engineering of pathways in saccharomyces cerevisiae

Modern biotechnological tools are making microbial production of chemicals, fuels, and pharmaceuticals increasingly practical and economically feasible. The field of metabolic engineering aims to enable this production by hijacking cellular systems to modify metabolism, converting each cell into an efficient chemical reactor. Traditionally, this has been accomplished through combining various knockouts and/or overexpressions of metabolic genes, but directed evolution strategies are often critical for improving metabolic pathways beyond native activity. Due to the complexity of cellular metabolism, simply evolving single genetic parts in a stepwise fashion can be limiting. In this work, we develop novel and powerful methods for applying directed evolution to the engineering of metabolic pathways in the yeast Saccharomyces cerevisiae. First, we develop a method for in vivo Continuous Evolution (ICE), which uses a synthetic retrotransposon element to allow generation of the largest mutant libraries of any in vivo mutational generation approach in yeast. This method is then validated by using it to rapidly evolve a variety of diverse genetic systems, including single enzymes, global transcriptional regulators, and multi-gene pathways. Next, we apply a modeling approach to create novel biosensors that can rapidly screen for production and secretion phenotypes in these large mutant libraries. These biosensors are then imported into microfluidic droplet systems to apply this screen in a high-throughput manner, creating a novel platform for screening libraries finally commensurate with the high level of diversity generation previously enabled. This method is validated by evolving an overproduction and secretion phenotype, resulting in strains that produce significantly elevated levels of aromatic amino acids. Finally, we develop and characterize new tools to enable the expression and evolution of multiple genes in a single genetic cassette. Taken together, these novel technologies significantly advance the state of the art for evolutionary engineering of metabolic pathways and will enable the evolution of pathways of enzymes for rapidly improving production of a number of desirable high-value biochemicals.Chemical Engineerin

Dynamics of gene expression in the genotype-phenotype map

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Física Teórica de la Materia Condensada. Fecha de lectura: 08 de febrero de 2016Genetic and environmental components can combine in quite complex ways to determine the phenotype of living organisms. Broadly, the goal of this thesis is to understand some of the design principles and constraints driving this assembly. We first study how genetic interaction networks – composed of phenotypically relevant interactions between genes – change in response to perturbation in their elements. Such networks at the genome-scale are progressively contributing to map the molecular circuitry that determines cellular behaviour. To what extent this mapping changes in response to different environmental or genetic conditions is however largely unknown. In Chapter 1 we assembled a genetic network using an in silico model of yeast metabolism to explicitly ask how separate genetic backgrounds alter the overall structure of the network. Backgrounds defined by single deletions induce particularly strong rewiring when the deletion corresponds to a catabolic or central metabolic gene, evidencing compensatory versatility. We found as well that weak interactions and those linking functionally separate genes tend to be more unstable. Overall, these patterns reflect the distributed robustness of core metabolic pathways. We examined as well a second class of evolutionary-motivated background, defined as a neutral mutation accumulation. The observed genetic network instability (predominantly in negative interactions) together with an increase in essential genes reflects a global reduction in buffering. Notably, rewiring of the genetic network is associated as well to a diminished environmental plasticity, what emphasizes a mechanistic integration of genetic and environmental buffering. More generally, this work demonstrates how the specific mechanistic causes of robustness influence the architecture of multiconditional genetic interaction maps. In Chapters 2, 3 and 4, we shift to systems that regulate the expression of genes. The plastic expression of different phenotypes enables organisms to respond to a wide variety of environmental changes, adapting their homeostasis. The dynamics of this plasticity can gen particularly interesting when operating mechanisms involve feedback, for instance when a gene encodes its own activator or repressor. The integration of positive and negative feedbacks can establish intricate patterns such as multistability, pulsing or oscillations. This depends on the specific characteristics of each interlinked feedback. In Chapter 2, we investigate a circuit associated with a dual, positive and negative transcriptional autoregulatory motif derived from the multiple antibiotic resistance system (mar) of Escherichia coli. Our results show that this motif enhances response speedup when it incorporates a linear positive feedback. Linearity also anticipates a homogeneous population phenotype anda higher input sensitivity, which we corroborate experimentally. As the motif is embedded in a broader regulatory network, we also studied how the system integrates additional cross-talks. Notably, the presence of an accessory positive regulation scales the response so that the circuit becomes unresponsive to other (metabolic) stress signals. Overall, we found that an antagonistic autoregulatory motif genetically encoded as a bicistron represents a versatile stimulus-response mode of control through the action of the positive-feedback regulation. Beyond precise and specific regulatory systems such as mar, in Chapter 3 we explore the possibility that more broad and “stereotypic” expression programs also exist. We firstly analyzed a genome-scale expression dataset comprising single gene deletions in 25% of Saccharomyces cerevisiae genes. Our analyses suggest that tens of broad expression programs exist that explain more variation in this dataset than expected at random. We further find that these programs seem to be activated also in conditions different to gene deletion, such as environmental perturbation or upon experimental compensatory evolution. These results suggest the possibility that broad, unspecific, “educated guess” gene expression responses have evolved as an adaptation to uncertain environments. Finally, in Chapter 4, we focus on a phenomenon by which the ability of expression change (plasticity) appears coupled to uncontrolled, stochastic expression variation (noise). This coupling can constrain gene function and limit adaptation. We examine the factors that contribute at the molecular level to modulate this coupling. Both transcription re-initiation and strong chromatin regulation are generally associated to coupling. Alternatively we show that strong regulation can lead to plasticity without noise. The nature of this regulation is also relevant, with plastic but noiseless genes subjected to broad expression activation whereas plastic and noisy genes experience targeted repression. This differential action is particularly illustrated in how histones influence these genes. The cost of coupling plasticity to noise seems to be then compensated by a wider regulatory versatility. Contrarily, in genes with low plasticity, translational efficiency is the main determinant of noise, a pattern we found linked to gene length. Genome architecture (particularly, neighboring genes) appear then as a modifier only effective in highly plastic genes. In this class, we confirm bipromoters as a architecture capable to reduce coupling (by reducing noise) but also highlight its limitation (as they could also decrease plasticity). This presents ultimately a paradox between intergenic distances and modulation, with short intergenic distances both associated and disassociated to noise at different plasticity levels. In summary, balancing the coupling among different types of expression variability appears as a potential shaping force of genome architecture and regulation.El fenotipo de los organismos vivos es el resultado de una compleja combinación de componentes genéticos y ambientales. Desde un punto de vista general, esta tesis tiene como objetivo tratar de entender algunos de los principios de diseño y limitaciones de tiene este ensamblaje. En el primero de los trabajos presentados se estudia cómo las redes de interacción genética (compuestas de interacciones fenotípicamente relevantes entre genes) cambian en respuesta a perturbaciones en algunos de sus elementos. Este tipo de redes a escala genómica están contribuyendo de manera creciente a mapear los circuitos moleculares que determinan el comportamiento celular. Hasta qué punto este “mapa” cambia en respuesta a diferentes perturbaciones genéticas o ambientales? Tratando de responder a esta pregunta, en el Capítulo 1 hemos ensamblado este tipo de redes en de manera sistemática diferentes fondos genéticos usando un modelo in silico del metabolismo de la levadura. Los fondos genéticos correspondientes a enzimas del catabolismo o metabolismo central indujeron una reorganización de la red particularmente fuerte, indicando una versatilidad en los mecanismos de compensación. Asímismo, las interacciones más débiles y aquellas entre genes funcionalmente distantes aparecen como las más inestables. Estos patrones reflejan la robustez distribuída de las rutas catabólicas y del metabolismo central. Por otro lado, también hemos examinado un tipo de fondo genético evolutivamente motivado, definido por la acumulación sucesiva de deleciones neutrales. La inestabilidad observada (predominantemente en interacciones negativas), junto con un incremento en el número de genes esenciales, refleja una reducción global en los mecanismos de compensación. De manera particularmente interesante, hemos observado que la reorganización de la red genética está asociada a una reducción en la plasticidad ambiental. Esto pone de manifiesto que los mecanismos que subyacen a la robustez genética y a la ambiental son esencialmente los mismos. De manera más general, este trabajo muestra cómo los mecanismos específicos de robustez afectan la arquitectura multi-condicional de los mapas de interacción genética. En los capítulos 2, 3, y 4, estudiamos diferentes aspectos de los sistemas que regulan la expresión de los genes. La expresión plástica de diferentes fenotipos hace posible que los organismos puedan responder a un amplio rango de cambios ambientales, adaptando su homeostasis a éstos. Las dinámicas específicas de esta plasticidad son particularmente interesantes cuando el mecanismo implica retroalimentación; por ejemplo, cuando un gen codifica su propio activador o represor. La integración de auto-regulaciones positivas y negativas puede establecer complejos patrones fenotípicos, como multiestabilidad, pulsos de actividad o oscilaciones. Esto depende de las características específicas de cada uno de los sistemas de retroalimentación implicados. En el Capítulo 2, estudiamos un motivo que contiene tanto una autoregulación positiva como una negativa, usando como modelo el operón de resistencia múltiple a antibióticos (mar) de Escherichia coli. Nuestros resultados demuestran que eeste sistema acelera la respuesta al incorporar una retroalimentación positiva lineal. Se demuestra experimentalmente que esta linealidad también produce una respuesta homogénea en la población y una alta sensibilidad. Por otro lado, también estudiamos cómo se integra este “motivo” en la red de regulación mayor. En este sentido, observamos que la presencia de una autoregulación positiva adicional es capaz de desacoplar el sistema de señales metabólicas. Finalmente, examinamos la influencia de posibles arquitecturas alternativas, mostrando cómo codificar la autoregulación dual antagonística en forma de bi-cistrón representa un versátil sistema estímulo-respuesta. Además de sistemas regulatorios específicos y precisos como mar, en el Capítulo 3 exploramos la posible existencia adicional de sistemas regulatorios “estereotípicos”, más generales e inespecíficos. Para ello, analizamos en primer lugar un conjunto de datos experimentales en los que la expresión génica a escala genómica fue medida para deleciones en un único gen, que engloba un 25% de los genes de Saccharomyces cerevisiae. Nuestros análisis sugieren que existen decenas de programas globales e inespecíficos. Además, encontramos evidencia de que estos mismos programas también pueden encontrarse en otros tipos de perturbaciones, como las ambientales y tras evolución experimental compensatoria. Estos resultados indican la posibilidad de una respuesta global e inespecifica como potencial estrategia adaptativa en un ambiente incierto. Finalmente, en el Capítulo 4, transladamos nuestra atención al fenómeno por el que la capacidad de un gen de cambiar su expresión génica en respuesta a cambios ambientales (plasticidad) se correlaciona con una variabilidad incontrolada y estocástica (ruido). Este acoplamiento puede limitar la función génica y la adaptación. Examinamos por tanto los factores a nivel molecular que pueden contribuír a su modulación. Tanto la re-iniciación transcripcional como la regulación a nivel de cromatina se presentan asociados a este acoplamiento. Alternativamente, demostramos cómo una regulación fuerte también puede ser ejercida sin incrementar el ruido. La naturaleza de esta regulación también es relevante; la plasticidad desacoplada del ruido se obtiene mediante mecanismos de activación generales. Mientras tanto, la regulación por represión específica está asociada a ruido, como pone también de manifiesto la influencia de las histonas. Nuestros resultados indican que el coste del ruido se ve compensado por una mayor versatilidad regulatoria. Por el contrario, en genes poco plásticos el ruido viene determinado fundamentalmente por la eficiencia traduccional, un patrón que encontramos asociado a la longitud de los genes. En consecuencia, la arquitectura genómica (particularmente la influencia de genes vecinos) constituye un modificadorsólo en genes plásticos. En estos últimos, confirmamos que los promotores bi-direccionales pueden reducir el ruido, pero también reducen la plasticidad. Constituyen por tanto un mecanismo limitado para desacoplar plasticidad y ruido. En resumen, nuestros resultados sugieren que equilibrar diferentes tipos de variabilidad constituye potencialmente una fuerza modeladora de la arquitectura y regulación de los genomas