Date of Award

8-2019

Degree Type

Report

Degree Name

Master of Science (MS)

Department

Biological Engineering

Committee Chair(s)

Jixun Zhan

Committee

Jixun Zhan

Committee

Charles Miller

Committee

David Britt

Abstract

Indigoidine is a natural blue dye with antioxidant and antimicrobial activities. It has also been used as an indicator for gene expression based on its distinctive blue color. Similar to the industry blue dye indigo, indigoidine has a promising potential to be applied in industry as a blue dye. However, the indigoidine production level in the original microorganisms was very low. Heterologous expression of the responsible synthetase gene in Escherichia coli can facilitate the fast and large-scale production of indigoidine. Also, a good understating of the working mechanism of the synthetase is favorable for the industrial application.

In our previous study, a putative indigoidine synthetase gene (Sc-indC) has been heterologously expressed in E. coli, and 20 g/L of indigoidine was produced under optimized culture conditions. To further improve the production, we intended to explore the indigoidine biosynthetic process by studying the working mechanism of two indigoidine synthetases BpsA and Sc-IndC from two different bacteria.

Both BpsA and Sc-IndC were predicted to have similar domain architecture that consists of an adenylation domain (A) with an embedded oxidation (Ox) domain, thiolation (T) domain, and thioesterase (TE) domain, except that Sc-IndC has an additional C-terminal. To explore the enzymatic mechanism, I dissected the gene between domains to get A-Ox-T/TE and A-Ox/T-TE, and co-expressed the dissected fragments to find out whether they can work in the combination of pieces as the intact enzymes do. On the other hand, I separately expressed the domains in E. coli BAP1 and purified the resulting proteins, and tested the in vitro activities on precursors individually or in different combinations. My results showed that the intact enzymes can convert glutamine, glutamine-SNAC and pyor-Gln to indigoidine in the presence of ATP. However, none of the dissected enzymes showed the ability to form the blue dye in vitro or in vivo.

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