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

Abstract

Background: Recent 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.

Results: The 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.

Conclusions: E. 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.

Figure 1

Metabolic pathway of Clostridium acetobutylicum engineered in E. coli. Abbreviations: Buk – butyrate kinase, Ptb – phosphotransbutyrylase, AdhE2 – aldehyde-alcohol dehydrogenase.

Figure 2

Expression of clostridial pathway enzymes in E. coli for conversion of butyric acid to butanol. (A) The DH5α strain containing test and control plasmids were grown in LB medium in presence or absence of IPTG and analyzed for the expression of aldehyde/alcohol dehydrogenase (AdhE2) and butyrate kinase (Buk) containing 6-histidine tag on Western blot. Lane M – Molecular weight marker; lane 1 – pQE30 – IPTG; lane 2 - pQE30 + IPTG; lane 3 – pQE-adhE2/ptb/buk – IPTG; lane 4 – pQE-adhE2/ptb/buk + IPTG. (B) The grown cells in the LB medium were permeabilized with chloroform and analyzed for the activity of phosphotransbutyrylase (Ptb), Buk and AdhE2. (C) Cells containing control and test plasmids were grown in LB medium containing 10 mM butyric acid and samples were withdrawn after 48 h and 120 h to test for butanol production.

Figure 3

Butyrate tolerance level of engineered E. coli. The MG1655 strain containing pQE-adhE2/ptb/buk plasmid was grown under anaerobic condition and resuspended in TB medium containing various concentration of butyric acid and 100 mM glycerol to achieve OD₆₀₀ of either 1 (A) or 5 (B). The butanol production and cell density were monitored after 120 h of growth in the sealed bottle under anaerobic condition.

Figure 4

Substrate specificity and substrate ratio for butanol production. (A) Impact of electron donor on butanol yield. Engineered E. coli MG1655 (pQE-adhE2/ptb/buk) strain was grown under anaerobic condition and resuspended in Terrific Broth with 40 mM butyric acid and 40 mM of either glucose or glycerol as electron donor. Various substrates consumed and butanol produced were analyzed through HPLC after 120 h of incubation. The butanol yield was calculated with respect to (wrt) each carbon source. (B) Different ratios of glycerol and butyric acid were tested for production of butanol using cells at the OD₆₀₀ of 1.0. (C) Different butyric acid concentrations were tested for production of butanol using cells at the OD₆₀₀ of 10 by keeping the glycerol to butyric acid ratio fixed at 1.5:1.

Figure 5

Substrate specificity of engineered cells towards various short chain fatty acids. Various short chain fatty acids were added in the growth medium (i.e. Terrific broth + 45 mM glycerol) of E. coli MG1655 carrying control or the test plasmid and their conversion to the corresponding alcohol were monitored through HPLC or GC.

Figure 6

Strain specificity of engineered cells on the uptake of butyric acid and production of butanol. Effect of strain type (A) and fadD gene deletion (B) on butyric acid uptake and butanol production was monitored by growing the strains anaerobically in Terrific broth medium containing 45 mM glycerol and 30 mM butyric acid and determining the metabolites through HPLC or GC.

Figure 7

Production of butanol in the bioreactor in batch and continuous mode. (A) Fermentation profile and (B) enzyme kinetics of the E. coli MG1655 (pQE-adhE2/ptb/buk) strain in a bioreactor cultivated in the batch mode with an initial OD₆₀₀ of 1.0. (C) Fermentation profile of the engineered strain with an initial OD₆₀₀ of 10. (D) Butyric acid consumption and butanol production kinetics in the bioreactor operated under continuous mode with cell recycling using hollow fiber module. Vertical bar at 24 hr indicates the position where fermentation was shifted from batch mode to continuous mode at the dilution rate of 0.2 h^-1.