Browsing by Subject "E. coli"

Malonyl-CoA (malonyl-CoA) is a platform chemical that serves as a precursor for a wide range of commercial products and pharmaceutical intermediates. In E. coli, malonyl-CoA levels are tightly regulated to remain at low levels. Two Stage Dynamic Metabolic Control (DMC) is a tool previously demonstrated to improve desired metabolite flux for several products. This work leverages DMC to improve malonyl-CoA fluxes. Specifically, we demonstrate coordinated dynamic reductions in the activities of fabI (enoyl-ACP reductase), gltA (citrate synthase), zwf (glucose-6-phosphate dehydrogenase) and glnB (nitrogen regulatory protein PII-1), during stationary phase lead to synergistic improvements in malonyl-CoA flux and the production of malonyl-CoA dependent products, 1,3,6,8-tetrahydroxynaphthalene (THN), Triacetic Lactone (TAL), and Phloroglucinol (PG). We also discuss the unique set of limitations that were observed for both TAL and PG biosynthesis as well as the strategies that were tested to overcome them. Additionally, we provide a historical review of the challenges associated with the production of Phloroglucinol. Finally, we end with a critical review focused on the bioproduction of an Acetyl-CoA and Succinyl-CoA derived product, Adipic Acid, to give perspective to common challenges associated with biobased product development.

Metabolic engineering is an effective strategy to optimize the biosynthesis of chemical molecules in genetically modified microbes. However, many current metabolic engineering strategies are limited by the requirements for cellular growth. To further optimize cell factories and overcome this limitation, we have applied 2-stage dynamic metabolic control strategies to optimize the biosynthesis of several compounds in E. coli. Using this strategy, cells grow without being impacted by product biosynthesis. We use phosphate depletion as a trigger to both force cells into a stationary production stage and initiate product synthesis. Phosphate depletion also dynamically removes enzymes involved in competitive or inhibitory metabolic pathways. This is accomplished by both inhibiting new transcription with CRISPR interference and degrading existing proteins via DAS+4 degron mediated proteolysis. Through my work, we demonstrate that the implementation of 2-stage dynamic metabolic control can indeed improve the biosynthesis of several small molecule chemicals in engineered E. coli, including: xylitol, citramalate and ethylene glycol. Rates, titers and yields were improved significantly. In addition, my work explored the mechanisms underlying improvements in performance. Specifically, we conclude that dynamic dysregulation of feedback control over central metabolism can lead to greatly improved stationary phase sugar uptake rates and pathway fluxes.

Since being developed as a tool in 2012, CRISPR-Cas9 and other CRISPR systems have revolutionized the way we manipulate biology. CRISPR systems broadly rely on a programmable guide RNA (gRNA) to enable targeted nuclease activity. Additionally, the ability to target a protein to a specific sequence of DNA has enabled a myriad of applications, including transcriptional silencing and/or activation, single base editing and targeted transposase activity. However, there remains a gap in knowledge as to what factors influence the activity of these systems. The gRNA sequence has been shown to at least partially predict activity but the mechanism behind this is not well understood. In addition, other contextual factors such as DNA repair, target accessibility, and others may play a role. Here, we present factors that influence Cas9 activity in E. coli and other organisms. Additionally, we find that gRNA sequence influences activity in large part by determining how fast Cas9 finds the target site. Together, the work presented improves our understanding of Cas9 and could lead to better gRNA prediction algorithms and new routes to improve Cas9 on-target activity.

Since the inception of recombinant protein expression, this technique has revolutionized our everyday lives with the number of protein products we have at our disposal ranging from household products to pharmaceuticals. E. coli was the first host used for recombinant protein expression and it has remained a mainstay expression host in biotechnology. However, the utility of E. coli as an expression host is limited by challenges that, if addressed, would make it a more suitable host for a wider variety of proteins, and thereby catalyze the translation of more protein products. Here, we address these challenges with dynamic control of the E. coli metabolism. This approach involves decoupling cell growth and protein expression and altering metabolism exclusively in the production phase. With this dynamic tool we have been able to: (1) engineer E. coli to improve the cytoplasmic solubility of redox sensitive proteins that are prone to aggregation, (2) improve the growth robustness of these engineered strains over the current state the art, (3) simply the bioprocess to purify these redox sensitive proteins, (4) identify uncharacterized redox regulation that impacts protein expression. Additionally, we provide a historical overview of biotechnological advances associated with a class of biopharmaceuticals, enzyme-based therapies, which have underutilized potential. Cumulatively, this work analyzes development trends of biologics, recognizes gaps in therapeutic and production capabilities, and provides solutions to challenges associated with redox sensitive protein expression and purification in E. coli.

Introduction: Municipalities often struggle to build and maintain basic infrastructure for informal slums in urban cities for its most vulnerable populations. One impact of inadequate water and sanitation access is the creation of an environment that breeds water borne pathogens that are the agents of infectious disease. Escherichia coli is a common bacteria found in water, often as an indicator of fecal contamination in the water supply. This study looks at one of the most common diseases found in women that results from E. coli growth, urinary tract infection. Specifically, this study aims to examine and describe factors of water, sanitation, and hygiene that are associated with positive E. coli urine results among women. The study took place in Ahmedabad, Gujarat, one of India's wealthier cities, in which heavy investments have been made in improving slum settlements throughout the rapidly expanding city.

Method: This was a cross-sectional study of 250 women recruited from households in urban Ahmedabad from October to December 2017. To determine positive cases of E. coli urinary tract infection, urine samples were collected from each participant. A commercial laboratory performed the urine analyses using a culture method. The threshold for positive cases was 10,000 CFU/mL or greater for E. coli. To obtain information on the water, sanitation, and hygiene practices, each participant completed a structured survey that included questions on demographics, working environment, reproductive health, sanitation access, family relationships, public toilets and social customs.

Results: Of the 250 participants, 23 (9.2%) were above the 100,000 CFU/mL threshold for E. coli, and therefore defined as a positive case. There were 124 (49.6%) participants who attempted a treat method, such as over the counter medicine or home self-treatments, for feminine health in the last three month. There were three factors that significantly correlated with positive cases. The first was the location of the handwashing facility, which could be either inside or outside of the dwelling. The second factor was antibiotic use in the last three weeks. The third factor was a participant living in a home with a child under the age of 5 years old, who experienced diarrhea.

Conclusion: This study identified a higher point prevalence of positive E. coli urine cultures than what we would want or would have expected for a sample population that all had access to piped water and a toilet inside of the dwelling. There is evidence to suggest that hygiene management around water use has an impact on a woman's susceptibility for E. coli causing infections in the urinary tract. Because half of the participants sought a form of treatment over the last three months for feminine health, a longitudinal study that tracks these women over a three month period, could provide relevant information on the incidence of new infections as well as prolonged urinary tract infections, particularly since multi-drug resistant E. coli infections are on the rise.