OptSSeq explores enzyme expression and function landscapes to maximize isobutanol production rate
Efficient microbial production of the next-generation biofuel isobutanol (IBA) is limited by metabolic bottlenecks. Overcoming these bottlenecks will be aided by knowing the optimal ratio of enzymes for efficient flux through the IBA biosynthetic pathway. OptSSeq (Optimization by Selection and Sequencing) accomplishes this goal by tracking growth rate-linked selection of optimal expression elements from a combinatorial library. The 5-step pathway to IBA consists of Acetolactate synthase (AlsS), Keto-acid reductoisomerase (KARI), Di-hydroxy acid dehydratase (DHAD), Ketoisovalerate decarboxylase (Kivd) and Alcohol dehydrogenase (Adh). Using OptSSeq, we identified gene expression elements leading to optimal enzyme levels that enabled theoretically maximal productivities per cell biomass in Escherichia coli. We identified KARI as the rate-limiting step, requiring the highest levels of enzymes expression, followed by AlsS and AdhA. DHAD and Kivd required relatively lower levels of expression for optimal IBA production. OptSSeq also enabled the identification of an Adh enzyme variant capable of an improved rate of IBA production. Using models that predict impacts of enzyme synthesis costs on cellular growth rates, we found that optimum levels of pathway enzymes led to maximal IBA production, and that additional limitations lie in the E. coli metabolic network. Our optimized constructs enabled the production of ~3 g IBA per hour per gram dry cell weight and was achieved with 20 % of the total cell protein devoted to IBA-pathway enzymes in the molar ratio 2.5:6.7:2:1:5.2 (AlsS:IlvC:IlvD:Kivd:AdhA). These enzyme levels and ratios optimal for IBA production in E. coli provide a useful starting point for optimizing production of IBA in diverse microbes and fermentation conditions.
- Research Organization:
- Great Lakes Bioenergy Research Center (GLBRC), Madison, WI (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- Grant/Contract Number:
- SC0018409; FC02-07ER64494
- OSTI ID:
- 1547915
- Alternate ID(s):
- OSTI ID: 1506451
- Journal Information:
- Metabolic Engineering, Journal Name: Metabolic Engineering Vol. 52 Journal Issue: C; ISSN 1096-7176
- Publisher:
- ElsevierCopyright Statement
- Country of Publication:
- Belgium
- Language:
- English
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