Engineering E. coli strain for conversion of short chain fatty acids to bioalcohols

Abstract

BackgroundRecent progress in production of various biofuel precursors and molecules, such as fatty acids, alcohols and alka(e)nes, is a significant step forward for replacing the fossil fuels with renewable fuels. A two-step process, where fatty acids from sugars are produced in the first step and then converted to corresponding biofuel molecules in the second step, seems more viable and attractive at this stage. We have engineered an Escherichia coli strain to take care of the second step for converting short chain fatty acids into corresponding alcohols by using butyrate kinase (Buk), phosphotransbutyrylase (Ptb) and aldehyde/alcohol dehydrogenase (AdhE2) from Clostridium acetobutylicum.ResultsThe engineered E. coli was able to convert butyric acid and other short chain fatty acids of chain length C3 to C7 into corresponding alcohols and the efficiency of conversion varied with different E. coli strain type. Glycerol proved to be a better donor of ATP and electron as compared to glucose for converting butyric acid to butanol. The engineered E. coli was able to tolerate up to 100 mM butyric acid and produced butanol with the conversion rate close to 100% under anaerobic condition. Deletion of native genes, such as fumarate reductase (frdA) and alcohol dehydrogenase (adhE), responsible for side products succinate and ethanol, which act as electron sink and could compete with butyric acid uptake, did not improve the butanol production efficiency. Indigenous acyl-CoA synthetase (fadD) was found to play no role in the conversion of butyric acid to butanol. Engineered E. coli was cultivated in a bioreactor under controlled condition where 60 mM butanol was produced within 24 h of cultivation. A continuous bioreactor with the provision of cell recycling allowed the continuous production of butanol at the average productivity of 7.6 mmol/l/h until 240 h.ConclusionsE. coli engineered with the pathway from C. acetobutylicum could efficiently convert butyric acid to butanol. Other short chain fatty acids with the chain length of C3 to C7 were also converted to the corresponding alcohols. The ability of engineered strain to convert butyric acid to butanol continuously demonstrates commercial significance of the system.

Potential production platform of n-butanol in Escherichia coli

“…Most recently, one study has reported an engineered E. coli that carries a synthetic pathway consisting of Clostridium buk, ptb, and adhE2 for n-butanol production from butyrate (Mattam and Yazdani, 2013). Under the controlled fermenter condition, their E. coli strain grown on TB rich medium produces 1.85 g/L (25 mM) n-butanol from glycerol (5 g/L) and butyrate (2.2 g/L) at 100 h. To improve the production, a high cell density of the strain is used and the n-butanol titer reaches 4.4 g/L (60 mM) with consumption of 5.3 g/L butyrate at 24 h. In addition, there is around 4 g/L acetate production.…”

Genetic engineering of non-native hosts for 1-butanol production and its challenges: a review

“…In another study, the butyrate-conversion strain (BUT-3EA) of E. coli was engineered by deleting ldhA, adhE, frdA, and pta genes and inserting native atoDA, acs and Clostridium adhE2 genes. The recombinant strain (BuT-3EA) was able to accumulate a titer of 6.2 g/L of 1-butanol using glucose and butyrate as a carbon sources [39], the titers were 1.5fold higher than the previously engineered strain using glycerol and butyrate as carbon sources [104].…”

Model-assisted metabolic engineering of Escherichia coli for long chain alkane and alcohol production

“…The zwf gene was amplified from E. coli DH5α genomic DNA and cloned in the pZS21mcs plasmid at KpnI and HindIII restriction sites to generate the pZF23 plasmid. Gene deletions in DH5α were achieved through the P1 transduction method (Fatma et al, 2016;Mattam and Yazdani, 2013;Munjal et al, 2012). The phage lysate was prepared and enriched by using the single gene knockout strain obtained from CGSC, and the targeted gene was replaced by an FRT flanked-kanamycin resistance cassette.…”

“…Validation of the in silico findings required selection of a suitable E. coli host strain as the genetic background of the host makes a significant impact on the metabolite profiles (Liu et al, 2014;Mattam and Yazdani, 2013;Song et al, 2016). Therefore we compared hydrocarbon profiles in eight different E. coli strains, i.e., E. coli B, DH5α, M15, MG1655, BW25113, Top10, BL21, and JM109, by transforming them with plasmid pZS22 and found that DH5α showed the highest alkane production ( Fig.…”

“…S6). Further, sequential knockouts of edd, pps, ldhA, aceA, poxB, ptaA and pflB were created in DH5α through phage transduction (Baba et al, 2006;Mattam and Yazdani, 2013). Gene knockouts were confirmed by PCR using external primers unique to the flanking regions of the genes (Table S1, Fig.…”