Engineering Cellular Transport Systems to Enhance Lignocellulose Bioconversion

abstract: Lignocellulosic biomass represents a renewable domestic feedstock that can support large-scale biochemical production processes for fuels and specialty chemicals. However, cost-effective conversion of lignocellulosic sugars into valuable chemicals by microorganisms still remains a challenge. Biomass recalcitrance to saccharification, microbial substrate utilization, bioproduct titer toxicity, and toxic chemicals associated with chemical pretreatments are at the center of the bottlenecks limiting further commercialization of lignocellulose conversion. Genetic and metabolic engineering has allowed researchers to manipulate microorganisms to overcome some of these challenges, but new innovative approaches are needed to make the process more commercially viable. Transport proteins represent an underexplored target in genetic engineering that can potentially help to control the input of lignocellulosic substrate and output of products/toxins in microbial biocatalysts. In this work, I characterize and explore the use of transport systems to increase substrate utilization, conserve energy, increase tolerance, and enhance biocatalyst performance.Dissertation/ThesisDoctoral Dissertation Biological Design 201

Genome-scale architecture of small molecule regulatory networks and the fundamental trade-off between regulation and enzymatic activity

Metabolic flux is in part regulated by endogenous small molecules that modulate the catalytic activity of an enzyme, e.g., allosteric inhibition. In contrast to transcriptional regulation of enzymes, technical limitations have hindered the production of a genome-scale atlas of small molecule-enzyme regulatory interactions. Here, we develop a framework leveraging the vast, but fragmented, biochemical literature to reconstruct and analyze the small molecule regulatory network (SMRN) of the model organism Escherichia coli, including the primary metabolite regulators and enzyme targets. Using metabolic control analysis, we prove a fundamental trade-off between regulation and enzymatic activity, and we combine it with metabolomic measurements and the SMRN to make inferences on the sensitivity of enzymes to their regulators. Generalizing the analysis to other organisms, we identify highly conserved regulatory interactions across evolutionarily divergent species, further emphasizing a critical role for small molecule interactions in the maintenance of metabolic homeostasis.P30 CA008748 - NCI NIH HHS; R01 GM121950 - NIGMS NIH HH

Quantifying the benefit of a proteome reserve in fluctuating environments.

The overexpression of proteins is a major burden for fast-growing bacteria. Paradoxically, recent characterization of the proteome of Escherichia coli found many proteins expressed in excess of what appears to be optimal for exponential growth. Here, we quantitatively investigate the possibility that this overexpression constitutes a strategic reserve kept by starving cells to quickly meet demand upon sudden improvement in growth conditions. For cells exposed to repeated famine-and-feast cycles, we derive a simple relation between the duration of feast and the allocation of the ribosomal protein reserve to maximize the overall gain in biomass during the feast

A General Process-Based Model for Describing the Metabolic Shift in Microbial Cell Cultures

The metabolic shift between respiration and fermentation at high glucose concentration is a widespread phenomenon in microbial world, and it is relevant for the biotechnological exploitation of microbial cell factories, affecting the achievement of high-cell-densities in bioreactors. Starting from a model already developed for the yeast Saccharomyces cerevisiae, based on the System Dynamics approach, a general process-based model for two prokaryotic species of biotechnological interest, such as Escherichia coli and Bacillus subtilis, is proposed. The model is based on the main assumption that glycolytic intermediates act as central catabolic hub regulating the shift between respiratory and fermentative pathways. Furthermore, the description of a mixed fermentation with secondary by-products, characteristic of bacterial metabolism, is explicitly considered. The model also represents the inhibitory effect on growth and metabolism of self-produced toxic compounds relevant in assessing the late phases of high-cell density culture. Model simulations reproduced data from experiments reported in the literature with different strains of non-recombinant and recombinant E. coli and B. subtilis cultured in both batch and fed-batch reactors. The proposed model, based on simple biological assumptions, is able to describe the main dynamics of two microbial species of relevant biotechnological interest. It demonstrates that a reductionist System Dynamics approach to formulate simplified macro-kinetic models can provide a robust representation of cell growth and accumulation in the medium of fermentation by-products

Modelling overflow metabolism in Escherichia coli by acetate cycling

A new set of mathematical equations describing overflow metabolism and acetate accumulation in E. coli cultivation is presented. The model is a significant improvement of already existing models in the literature, with modifications based on the more recent concept of acetate cycling in E. coli, as revealed by proteomic studies of overflow routes. This concept opens up new questions regarding the speed of response of the acetate production and its consumption mechanisms in E. coli. The model is formulated as a set of continuous differentiable equations, which significantly improves model tractability and facilitates the computation of dynamic sensitivities in all relevant stages of fermentation (batch, fed-batch, starvation). The model is fitted to data from a simple 2 L fed-batch cultivation of E. coli W3110 M, where twelve (12) out of the sixteen (16) parameters were exclusively identified with relative standard deviation less than 10%. The framework presented gives valuable insight into the acetate dilemma in industrial fermentation processes, and serves as a tool for the development, optimization and control of E. coli fermentation processes.EC/H2020/643056/EU/Rapid Bioprocess Development/Biorapi

The inflection point hypothesis: The relationship between the temperature dependence of enzyme-catalyzed reaction rates and microbial growth rates.

The temperature dependence of biological rates at different scales (from individual enzymes to isolated organisms to ecosystem processes such as soil respiration and photosynthesis) is the subject of much historical and contemporary research. The precise relationship between the temperature dependence of enzyme rates and those at larger scales is not well understood. We have developed macromolecular rate theory (MMRT) to describe the temperature dependence of biological processes at all scales. Here we formalize the scaling relationship by investigating MMRT both at the molecular scale (constituent enzymes) and for growth of the parent organism. We demonstrate that the inflection point (ᵢₙ) for the temperature dependence of individual metabolic enzymes coincides with the optimal growth temperature for the parent organism, and we rationalize this concordance in terms of the necessity for linearly correlated rates for metabolic enzymes over fluctuating environmental temperatures to maintain homeostasis. Indeed, ᵢₙ is likely to be under strong selection pressure to maintain coordinated rates across environmental temperature ranges. At temperatures at which rates become uncorrelated, we postulate a regulatory catastrophe and organism growth rates precipitously decline at temperatures where this occurs. We show that the curvature in the plots of the natural log of the rate versus temperature for individual enzymes determines the curvature for the metabolic process overall and the curvature for the temperature dependence of the growth of the organism. We have called this "the inflection point hypothesis", and this hypothesis suggests many avenues for future investigation, including avenues for engineering the thermal tolerance of organisms

Redox Homeostasis in Cyanobacteria

Oxygenic photosynthetic organisms utilize high-energy electron transfer chains comprised of redox active intermediates and light harvesting complexes. While oxygen is a necessary byproduct of water oxidation and the source of photosynthetic electrons, its presence is also dangerous because leakage of electrons and excitation energy can interact with molecular oxygen to generate reactive oxygen species: ROS). Elaborate antioxidant networks and redox buffering systems have evolved to protect photosynthetic organisms from the threat of ROS. Glutathione: GSH) is a multifunctional molecule that is involved in core metabolism, detoxification of xenobiotics and in maintenance of cellular redox poise. The ubiquitous nature of glutathione and its importance to cellular metabolism has been observed in many organisms, however the specific roles of glutathione in photosynthetic organisms are not fully understood. To address these questions, we have generated several mutants in the glutathione biosynthesis and degradation pathways in the model organism Synechocystis sp. PCC 6803: Synechocystis 6803), an oxygenic photosynthetic cyanobacterium. We utilized targeted homologous recombination to generate deletion mutants of glutamate-cysteine ligase: GshA) and glutathione synthetase: GshB) in Synechocystis 6803. Our results indicate that GshA activity is essential for growth in cyanobacteria because we were unable to isolate a fully segregated ∆gshA deletion mutant. We did isolate a ∆gshB mutant strain that accumulates the biosynthetic intermediate γ-glutamylcysteine: γ-EC) instead of GSH. In this work, I have characterized the physiology of the ∆gshB mutant following environmental, genetic and redox perturbations. The results presented here also shed light on the dynamic nature of the low-molecular weight thiol pool in cyanobacteria. We quantified the levels of cellular thiols in Synechocystis 6803 during exposure to multiple environmental and redox perturbations and found that conditions promoting increased cellular metabolism and increased ROS production, including during high-light treatment and photomixotrophic growth, lead to higher cellular thiol levels. Furthermore, the intracellular pools of thiols decrease when the cell exhibits reduced metabolic capacity during conditions such as nutrient deprivation and dark incubation. Sulfate limitation results in dramatically decreased cellular thiol contents in a short period of time. We found that the ∆gshB strain is sensitive to sulfate limitation and exhibits delayed recovery upon sulfate repletion, indicating that GSH is important for acclimation to sulfate limiting conditions. To facilitate our understanding of GSH degradation in Synechocystis 6803 during sulfate limitation, we generated a mutant lacking γ-glutamyltranspeptidase: Ggt), an enzyme with GSH degradation activity. However, the ∆ggt mutant still exhibited GSH degradation during sulfate depletion, indicating the presence of an alternative system or mechanism. We did find increased levels of GSH in the growth media of the ∆ggt strain compared to the WT, which suggests a role in GSH uptake or prevention of leakage. Our results demonstrate that GSH is essential for protection from multiple environmental and redox perturbations in cyanobacteria. However, there are many pathways involved in maintenance of redox homeostasis in cyanobacteria. Therefore, we also aimed to determine whether these pathways function cooperatively to ameliorate damage from ROS. Several flavodiiron: Flv) proteins have been identified in Synechocystis 6803 that are involved in reduction of O2 to H2O without the formation of ROS intermediates. However, single ∆flv3 mutants do not exhibit severe growth defects under normal conditions. Therefore, we generated a ∆gshB/∆flv3 mutant to examine whether these systems cooperate to maintain redox homeostasis. Our results show that the ∆gshB/∆flv3 mutant exhibits reduced growth than either of the single mutants when grown on solid media, suggesting a degree of interaction between these pathways in cyanobacteria

METABOLIC ENGINEERING OF BACILLUS FOR ENHANCED PRODUCT AND CELLULAR YIELDS

Microbial cultures usually produce a significant amount of acidic byproducts which can represses cell growth and product synthesis. In addition, the production of acids is a waste of carbon source thereby reduces the product yield and productivity. Metabolic engineering provides a powerful approach to optimize the cellular activities and improve product yields by genetically manipulating specific metabolic pathways. Previous work has identified mutation of Pyruvate Kinase (PYK) as an efficient way to reduce acids production; however, complete abolishment of PYK in Bacillus subtilis resulted in dramatically reduced cell growth rate. In this study, an inducible PYK (iPYK) mutant of B. subtilis was constructed and extensively characterized. The results demonstrated that good cell growth rate and low acetate formation can be attained at an appropriate PYK expression level. In addition, mutation at phosphofructokinase (PFK) on the glycolysis pathway also provides an alternative approach to reduce acetate formation.Two outcomes of the pyk mutant of B. subtilis, high phosphoenolpyruvate (PEP) pool and low acetate concentration, prompted us to investigate the deployment of pyk mutation as an efficient way to improve folic acid and recombinant protein production. The high intracellular PEP and glucose-6-phosphate (G6P) concentration in the pyk mutant led to higher folic acid production by providing abundant synthetic precursors. Additional mutations in the folic acid synthesis pathway, along with the pyk mutation, resulted in 8-fold increase in folic acid production. Recombinant protein was improved two-fold by the pyk mutation due to low acetate formation and longer production time in the pyk mutant. In addition, using glycerol instead of glucose as the carbon source reduced acetate production and improved protein production by 60%.The effect of citrate on acetate production in Bacillus thuringiensis (Bt) was investigated and the continuous culture results showed the effectiveness of citrate on reducing acetate formation. These results indicated pyk may be a potential mutation target to reduce acetate formation in Bt

Understanding Control of Metabolite Dynamics and Heterogeneity

Microbes live in complex and continually changing environments. Rapid shifts in nutrient availability are a common challenge for microbes, and cause changes in intracellular metabolite levels. Microbial response to dynamic environments requires coordination of multiple levels of cellular machinery including gene expression and metabolite concentrations. This coordination is achieved through metabolic control systems, which sense metabolite concentrations and direct cellular activity in response. Several reoccurring control architectures are found throughout diverse metabolic systems, which suggests underlying evolutionary advantages for using these control systems to coordinate metabolism. One common, yet understudied, control architecture is the positive feedback metabolite uptake loop, which features a metabolite responsive-transcription factor (MRTF) that activates genes necessary to uptake its cognate metabolite. Understanding the design principles behind these complex metabolic control systems is a fundamental issue across many biological sub-disciplines since metabolism is a central feature of cellular behavior.The goal of this dissertation is to elucidate how the architecture and parameters of a MRTF-based control system shape metabolite dynamics and heterogenous metabolic response to changing nutrient environments. This dissertation focuses on the Escherichia coli fatty acid degradation system, which employs the positive feedback uptake loop architecture. The function and performance of these control systems to three common metabolic tasks was evaluated. First, after a nutrient depletion, microbes must rapidly turn off metabolic pathways to conserve resources. Second, microbes must maintain sensing ability in the face of metabolic conditions which impact cellular growth rate. Finally, upon abrupt shifts between nutrients, microbes must shift metabolic resources to uptake the new nutrient or otherwise cease growth. This shifting process can be heterogenous, with a sub-population which maintains a non-growing state that confers tolerance to antimicrobial compounds. Taken together, this work provides deeper understanding of the design principles for the control of metabolite dynamics and heterogeneity for applications in metabolic engineering and synthetic biology