(444c) Genome Scale Model Analysis of Clostridium Tyrobutyricum for Butyl Butyrate Production

1.1pt"> " times new roman major-bidi>Genome Scale Model Analysis of Clostridium
tyrobutyricum for Butyl Butyrate Production

Kiumars
Badr¹, Jie Zhang², Yi Wang², and Jin Wang¹*

¹Department
of Chemical Engineering, Auburn University, Auburn, AL, 36849, USA. ²Department of
Biosystems Engineering, Auburn University, Auburn, AL 36849, USA.    *Email: wang@auburn.edu

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12.0pt;font-family:" times new roman mso-hansi-theme-font:major-bidi>Butyl butyrate
(BB) which has a unique fragrant smell like pineapple and banana, widely has been
applied as a food ingredient to enhance the flavor. BB is also employed in
plastic and fiber industries as a solvent, as well as important extractant in
the processing of petroleum products [1]. Moreover, BB has a high-octane rating
of 97.3 with excellent compatibility and properties as gasoline, aviation
kerosene, and diesel components and can be used as a valuable fuel source [2,3].

12.0pt;font-family:" times new roman mso-hansi-theme-font:major-bidi>The
conventional industrial production of BB, as for most other esters, needs high
temperature (200–250 °C) and concentrated sulfuric acid as catalyst (the
Fischer esterification process). The reaction consumes a lot of energy and generates
significant amount of hazardous wastes, further increasing downstream
processing and treatment cost [4]. An alternative approach for ester production
would be to employ lipase, one of the most commonly existing enzymes in nature,
to catalyze the highly specific and efficient reaction with renewable
substrates under mild reaction conditions [5]. It is energy saving and
environment friendly. Therefore, less byproduct or waste will be generated,
reducing the downstream processing cost.

Our
previous work has showed that if butanol (BOH) and lipase were supplemented to
the fermentation with C. tyrobutyricum, high
level of BB production can be achieved, through in situ esterification
and under the optimized conditions [3]. However, it is desirable to engineer C.
tyrobutyricum for enhanced butyryl-CoA/butyrate
and butanol co-production simultaneously.

It
is well-known that genome-scale metabolic model allows the analysis of the cellular
metabolism from a systems perspective to predict whole-cell effects of genetic
changes, and to simulate known and hypothesized phenotypes, which is not
possible with standard experiments. Considering the redox and flux balancing
requirements, a genome-scale metabolic model is imperative to guide our further
metabolic optimization for enhanced BB production. To this end, we developed the
first draft reconstruction of genome-scale metabolic model for C. tyrobutrycum, manually curated based on a published
model iCM925 for C. beijerinckii strain [6]. iCM925
is the largest genome-scale model for  Clostridial
species and contains a very close overall pathway to C. tyrobutyricum.
Using published data in literature, the developed model can accurately predict
cell growth and product formation given specific substrate uptakes for both batch
and continuous culture experiments, and correctly captures the relationships
between the formation of products such as butyrate and/or butanol. Applying the
system-identification based framework that we developed previously for GEM
analysis [7,8], we identified reactions that have significant impacts on
butyrate production, which include hydrogen production, as well as electron
transfer reactions. Since the wild type can product small amount of ethanol, we
also used the model to test a hypothesis that could enable small amount of
ethanol production, and discovered that the model could use threonine to
produce acetaldehyde and glycine, and enable ethanol production. Finally, our
system identification based analysis reveals several key reactions that play
important role in understanding the cellular metabolism of the C. tyrobutyrium,
in particular the interplay between ATP production and redox balance. These
findings are being validated using designed wet-lab experiment under both batch
and continuous culture conditions.

Reference:

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