Studies of growth yield and substrate utilization in Escherichia coli

Orr, James Stephen

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Description

Title

Studies of growth yield and substrate utilization in Escherichia coli

Author(s)

Orr, James Stephen

Issue Date

2019-07-08

Director of Research (if dissertation) or Advisor (if thesis)

Rao, Christopher V

Doctoral Committee Chair(s)

Rao, Christopher V

Committee Member(s)

• Kraft, Mary L
• Schroeder, Charles M
• Jin, Yong-Su

Department of Study

Chemical & Biomolecular Engr

Discipline

Chemical Engineering

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• E. coli
• fermentation
• growth
• acetate
• bacteria

Abstract

Escherichia coli is an important micro-organism used for production of various chemicals and proteins. The bacterium has been studied extensively for over 100 years, and it has been instrumental to both the basic understanding of biology and modern developments in the medical and biotechnology fields. In order to further knowledge in E. coli, it is important to fully understand metabolic regulation. Topics of particular interest include: maximization of growth yield, which is important for maximizing production of proteins both in lab and industrial scale processes; regulation of substrate utilization, which can allow engineering of strains that can efficiently consume any desirable substrate/mixture of substrates; and finally, elimination of undesirable side products, such as acetate, which reduces productivity and can be toxic to the cells. In the following studies, we discuss three major projects which study one or more of the above topics. The first is a study of the impact of amino acids in complex mixtures of nutrients on sugar utilization. We specifically study growth and glucose consumption in the common laboratory growth medium, tryptone broth. We next demonstrate that without a buffer, the excess carbon with go to acetate which will dramatically reduce the culture pH and eventually inhibit acetate re-assimilation. Finally, we discuss a method of isolating high growth yield strains of E. coli by privatizing nutrients. Growth and substrate utilization in complex media Complex media are often used for cultivation of various bacteria due to the abundance and variety of nutrients available. A lot of metabolic regulation has been studied in defined media, often consisting of one or two carbon sources. There can be more complicated regulatory behavior to uncover by studying growth and substrate utilization in complex media. However, the complexity of the medium can obscure the reasons for culture behavior – including components that may regulate carbon and nitrogen metabolism or those that limit cell growth. In Chapter 2, we investigated glucose consumption and growth in a complex peptide-based medium. We observed that Escherichia coli consumed glucose slowly during exponential growth and only consumed 25% of the sugar before the culture entered stationary phase. We found that amino acids are consumed simultaneously with glucose during early and middle exponential growth. We determined that magnesium limitation is the reason growth is limited. Magnesium supplementation to TB7/glucose permits complete glucose utilization before stationary phase, which also increases cell growth three-fold by OD600. Finally, since acetylation is intimately linked to glucose consumption and acetate fermentation, we asked how acetylation compared between magnesium-limited and magnesium-replete cells. Cells with the magnesium-dependent growth increase had reduced acetylation compared to their magnesium-limited counterparts. We propose that peptide-based media have potential for more cell growth, which can be achieved through supplementation with excess carbon and magnesium. Furthermore, we can reduce acetylation without genetically manipulating E. coli. This has the potential to reduce formation of deleterious acetylated isoforms of recombinant proteins without negatively affecting cell growth. The acetate switch is inhibited in acidic growth media Escherichia coli produces acetate during aerobic growth on various carbon sources. Following consumption of the carbon substrate, E. coli can further grow on the acetate. This phenomenon is known as the acetate switch, where cells transition from producing acetate to consuming it. In Chapter 3, we investigated how pH governs the acetate switch. When E. coli was grown on a glucose-supplemented medium initially buffered to pH 7, the cells produced and then consumed the acetate. However, when the initial pH was dropped to 6, the cells still produced acetate but were only able to consume it when little (<10 mM) acetate was produced. When significant acetate was produced in acidic media, which occurs when the growth medium contains magnesium, amino acids, and sugar, the cells were unable to consume the acetate. To determine the mechanism, we characterized a set of metabolic mutants and found that those defective in the TCA cycle or glyoxylate shunt exhibited reduced rates of acetate consumption. We further found that expression of the genes in these pathways was reduced during growth in acidic media. Expression of the genes involved in the AckA-Pta pathway, which provides the principal route for both acetate production and consumption, were also inhibited in acidic media but only after glucose was depleted, which correlates with the acetate consumption phase. Based on these results, we conclude that growth in acidic environments inhibits the expression of the acetate catabolism genes, which in turn prevents acetate consumption. Enriching strains of E. coli with a high growth yield It is often desirable to obtain strains with a high growth yield for bioproduction purposes. These are strains that are capable of most efficiently converting some feed substrate into biomass, including protein. However, standard evolutionary procedures involving serial passage in liquid media in flasks select for strains with a high growth rate, but a low growth yield. In Chapter 4, we attempted to isolate high growth yield strains on xylose by privatizing resources to individual cells. We chose xylose because it is the second most abundant sugar in lignocellulosic feedstocks. We accomplished this using an emulsion-based media approach which isolates individual cells, or small numbers of cells, into single droplets of nutrients. The cells which utilize their allotment of nutrients to produce the most biomass will be present in the highest number once the growth is stopped. Using this approach, we enriched two different high growth yield strains with different mutations in carbon catabolite repression relevant genes. We were able to confirm the mutations we found allowed for improved growth yield by moving them over into a wild type background. The strains showed similar growth rate to wild type, though their sugar utilization what slightly slower and thus mixed acid fermentation was reduced. We are still working to characterize the impact these mutations have on gene expression.

Graduation Semester

2019-08

Type of Resource

text

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Copyright 2019, James Orr

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