Abstract
Short-chain fatty acids (SCFAs), such as butyric acid, have a broad range of applications in chemical and fuel industries. Worldwide demand of sustainable fuels and chemicals has encouraged researchers for microbial synthesis of SCFAs. In this study we compared three thioesterases, i.e., TesAT from Anaerococcus tetradius, TesBF from Bryantella formatexigens and TesBT from Bacteroides thetaiotaomicron, for production of SCFAs in Escherichia coli utilizing native fatty acid synthesis (FASII) pathway and modulated the genetic and bioprocess parameters to improve its yield and productivity. E. coli strain expressing tesBT gene yielded maximum butyric acid titer at 1.46 g L-1, followed by tesBF at 0.85 g L-1 and tesAT at 0.12 g L-1. The titer of butyric acid varied significantly depending upon the plasmid copy number and strain genotype. The modulation of genetic factors that are known to influence long chain fatty acid production, such as deletion of the fadD and fadE that initiates the fatty acid degradation cycle and overexpression of fadR that is a global transcriptional activator of fatty acid biosynthesis and repressor of degradation cycle, did not improve the butyric acid titer significantly. Use of chemical inhibitor cerulenin, which restricts the fatty acid elongation cycle, increased the butyric acid titer by 1.7-fold in case of TesBF, while it had adverse impact in case of TesBT. In vitro enzyme assay indicated that cerulenin also inhibited short chain specific thioesterase, though inhibitory concentration varied according to the type of thioesterase used. Further process optimization followed by fed-batch cultivation under phosphorous limited condition led to production of 14.3 g L-1 butyric acid and 17.5 g L-1 total free fatty acid at 28% of theoretical yield. This study expands our understanding of SCFAs production in E. coli through FASII pathway and highlights role of genetic and process optimization to enhance the desired product.
Highlights
Short chain fatty acids (SCFAs), such as butyric acid (C4), are promising intermediates of many chemical and biofuel molecules
Butyric acid is produced on industrial scale by chemical synthesis from crude oils that involves the oxidation of butyraldehyde
We found marked differences amongst the thioesterases and plasmid copy number used in terms of butyric acid titer obtained
Summary
Short chain fatty acids (SCFAs), such as butyric acid (C4), are promising intermediates of many chemical and biofuel molecules. Butyric acid is a precursor for short chain fatty alcohols like butanol, which is superior to ethanol in terms of energy density, vapor pressure and hygroscopicity, and is a direct replacement of gasoline [1, 2]. Butyric acid is produced on industrial scale by chemical synthesis from crude oils that involves the oxidation of butyraldehyde. It can be extracted from butter as its concentration in butter ranges from 2% to 4%. These chemical means to produce butyric acid is costly and non-ecofriendly [5, 9, 10]. There is a need to produce butyric acid from renewable carbon sources using a microbial platform to replace its chemical synthesis
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