Publication Date

2024

Document Type

Dissertation/Thesis

First Advisor

Zhou, Shengde

Degree Name

Ph.D. (Doctor of Philosophy)

Legacy Department

Department of Biological Sciences

Abstract

Riboflavin, a water-soluble vitamin B2, is essential for the growth and reproduction of living organisms. However, humans and animals lack a riboflavin synthesis pathway and must rely on diets derived from plants and microbes for this essential vitamin. To meet our needs for vitamin B2, industrial riboflavin production is currently being pursued using strains of Bacillus subtilis. The fermentation process is successful, but improvements are needed for better substrate-to-product conversion yield. To this end, it is imperative to develop alternative microbial strains for cost-effective riboflavin production.

Due to its simple growth requirement, well-understood genetic background, and widely available genetic tools, the bacterium Escherichia coli remains a host of choice for metabolic pathway engineering to produce a product of industrial and economic importance. This study engineered an E. coli strain for riboflavin production by cloning and functional expression of the riboflavin pathway genes (ribGBAH) from B. subtilis. The cloned operon was confirmed by DNA sequencing, qPCR analysis, and riboflavin production. The functionally expressed operon enabled the resulting strain E. coli W (pCR-XL-2-TOPO-RIB) to produce 4.80 ± 0.34 mg/L of riboflavin while the control strain produced no detectable riboflavin.

With a further stepwise metabolic pathway engineering by blocking the fermentation pathways, the glycolytic Enter-Doudoroff Shunt, and the glycolysis pathway, the resulting strain E. coli JAT819 metabolized glucose through only the pentose phosphate pathway, leading to increased production of riboflavin.

The engineered strain E. coli JAT819 (pCR-XL-2-TOPO-RIB) produced approximately 17.05 ± 0.25 mg/L of riboflavin, representing a 3.5-fold improvement of product titer and a 6.7-fold improvement of specific productivity compared to those achieved by the starting strain E. coli W (pCR-XL-2-TOPO-RIB). These results demonstrated that E. coli has excellent potential to produce riboflavin through genetic engineering. Nevertheless, further strain improvements are needed for a product titer comparable to current industrial strains.

Extent

59 pages

Language

en

Publisher

Northern Illinois University

Rights Statement

In Copyright

Rights Statement 2

NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.

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