(586g) Understanding the Redox Shift in a Clostridium Tyrobutyricum mutant Strain for Butanol Production through Genome-Scale Metabolic Modeling

Clostridium tyrobutyricum is well known for its hyper-butyrate production, with both very high production and selectivity (the fraction of butyrate out of the total endproducts) [1]. In addition, high-level butanol production has been achieved by overexpressing aldehyde/alcohol dehydrogenase (adhE2) to convert the butyryl-CoA to butanol [2]. Therefore, C. tyrobutyricum can be considered as an excellent platform for butyl butyrate production through rational metabolic engineering. Butyl butyrate is a valuable fuel source and industrially important biochemical. It is estimated that the current US market demand for BB is $0.63 billion per year [3].

The bioproduction of butyl butyrate will require more reducing power, which might impair the redox balance of the metabolic pathways within C. tyrobutyricum. Therefore, it is desirable to design mutant strategies that will consider the energy and redox balance, as well as precursor supply for optimal butyl butyrate production. 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 energy and redox balance requirements, a genome-scale metabolic model is imperative to guide the development of metabolic strategies for enhanced butyl butyrate production. To this end, previously, we developed the first draft reconstruction of genome-scale metabolic model for Wide Type (WT) C. tyrobutyricum, manually curated based on a published model iCM925 for C. beijerinckii strain [4]. 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 could accurately predict cell growth and product formation given specific substrate uptakes for batch culture experiments, and correctly captures the relationships between the formation of products such as butyrate.

In his work, by adding/removing the corresponding reactions associated with inserted/deleted genes, we obtained a modified GEM for a C. tyrobutyricum mutant strain (adh2::Δcat1). The modified GEM could capture the shifted product excretion pattern but underestimate the biomass growth, suggesting the mutant strain contains more efficient route for energy and reducing power supply than the GEM does. Applying system identification based framework for GEM analysis that we previously developed [5,6], we designed various in silico experiments to elucidate the redox shift in the mutant model. With the additional information from transcriptomic data collected for the mutant strain, we were able to further improve the modified GEM to better understand the redox and energy balance in the mutant strain.

Reference:

[1] Zigová, J., Šturdík, E., Advances in biotechnological production of butyric acid. J Ind Microbiol Biotech 2000, 24, 153-160.

[2] Yu, M. R., Zhang, Y. L., Tang, I. C., Yang, S. T., Metabolic engineering of Clostridium tyrobutyricum for n-butanol production. Metabolic Engineering 2011, 13, 373-382.

[3] https://www.grandviewresearch.com/press-release/us-esters-market-analysis 2016.

[4] Milne CB, Eddy JA, Raju R, Ardekani S, Kim PJ, Senger RS, et al. “Metabolic network reconstruction and genome-scale model of butanol-producing strain Clostridium beijerinckii NCIMB 8052.” BMC Systems Biology. 2011,5(130):1–15.

[5] Damiani AL, He QP, Jeffries TW, Wang J. “Comprehensive evaluation of two genome-scale metabolic network models for Scheffersomyces stipitis.” Biotechnol Bioeng. 2015,112:1250–1262.

[6] Hilliard, M.; Damiani, A.; He, Q. P.; Jeffries, T.; Wang, J. “Elucidating Redox Balance Shift in Scheffersomyces Stipitis’ Fermentative Metabolism Using a Modified Genome-Scale Metabolic Model.” Microb. Cell Fact. 2018, 17 (1), 140.