Title: Metabolic engineering of Escherichia coli for the utilization of ethanol
Journal: Journal of Biological Research-Thessaloniki
Published: 2020
Background
Ethanol, with the chemical formula C2H6O, has various applications in the chemical, food, medical, and health industries. It can be produced through fermentation using biomass or synthesized from petroleum-derived sources, often at a lower cost. While significant progress has been made in ethanol production, its utilization by microorganisms remains underexplored. Some microbial strains, such as acetic acid bacteria, Acinetobacter baylyi, and certain yeasts, can metabolize ethanol. However, these organisms are not easily genetically engineered to produce high-value chemicals. Escherichia coli, a commonly used industrial microorganism, has shown limited ethanol utilization capacity under anaerobic conditions. This study investigates the introduction of ethanol metabolic pathways from Aspergillus nidulans into E. coli to enable ethanol utilization for growth and production of value-added chemicals like mevalonic acid.
Materials & Methods
Reagents and Culture Media
- Enzymes (FastDigest, T4 DNA ligase) from Thermo Scientific, PrimerSTAR Max Taq DNA polymerase from Takara.
- DNA extraction and purification kits from Omega Bio-Tek.
- Mevalonic acid lactone from Acros Organics, ethanol-D6 from Aladdin.
- LB medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) for DNA manipulation.
- M9 medium (6 g/L Na₂HPO₄, 3 g/L KH₂PO₄, 1 g/L NH₄Cl, 0.5 g/L NaCl) for fermentation.
- Antibiotics: Ampicillin (100 mg/mL) and chloramphenicol (34 mg/mL) when required.
Plasmid Construction
- adhE, adhP, and aldA genes from E. coli were amplified and cloned into pTrcHis2B to generate pTrc-EcadhE, pTrc-EcadhP, and pTrc-EcaldA.
- pTrc-EcadhEaldA and pTrc-EcadhPaldA were constructed by inserting the aldA gene into pTrc-EcadhE and pTrc-EcadhP.
- Codon-optimized alcA and aldA from A. nidulans were cloned into pTrcHis2B to obtain pTrc-AnalcA, pTrc-AnaldA, and pTrc-AnalcdA.
- The acs gene from E. coli was cloned into pACYCDuet-1 (pA-Ecacs).
- Recombinant plasmid pA-EfmvaES containing mvaE and mvaS was used from previous work.
- All plasmids were sequence-verified.
Shake-Flask Fermentation
- E. coli strains with recombinant plasmids were grown in LB medium overnight, then inoculated (1:100) into M9 medium with glucose or ethanol at 37°C.
- Cells were induced with 0.2 mM IPTG at the early exponential phase, and the temperature was shifted to 30°C.
- Ethanol or ethanol-D6 was added upon glucose depletion.
- Samples were collected periodically for analysis.
Analytical Methods
- Cell Density: Measured at OD600 using a Varian Cary-50 UV–Vis spectrophotometer.
- Glucose Concentration: Determined using a YSI 2950D Biochemistry Analyzer.
- Ethanol, Acetaldehyde, and Acetic Acid: Analyzed using Agilent 7890B GC with FID and DB-FFAP column. Nitrogen was used as the carrier gas (1 mL/min).
- Mevalonic Acid: After acidification and conversion to lactone, extracted with ethyl acetate, dried, and analyzed by Agilent 7890A/5975C GC–MS with an HP-5ms column.
- Mevalonic acid lactone was identified by retention time comparison and mass spectrum analysis using the NIST library.
Results
E. coli Cannot Utilize Ethanol as the Sole Carbon Source:
- Several E. coli strains (BL21(DE3), DH5α, Top10, JM109) were tested for ethanol tolerance. Ethanol concentrations up to 50 g/L did not significantly affect growth.
- However, when ethanol (5 g/L) was added as the sole carbon source after glucose depletion, none of the strains could grow, indicating their inability to utilize ethanol.
Construction of an Ethanol Utilization Pathway:
- Three native E. coli dehydrogenases (AdhE, AdhP, and AldA) were overexpressed, but no ethanol utilization was observed.
- Genes from Aspergillus nidulans, alcA (encoding alcohol dehydrogenase I) and aldA (encoding aldehyde dehydrogenase), were introduced into E. coli.
- Expression of alcA alone enabled E. coli to utilize ethanol, while co-expression of both genes (alcA and aldA) further enhanced ethanol consumption.
Shake-Flask Fermentation:
- Engineered strains with alcA and aldA showed notable ethanol consumption (up to 2.24 g/L).
- Acetaldehyde was not detected due to rapid conversion to acetic acid, with a maximum acetic acid titer of 142 mg/L.
Effect of Acetyl-CoA Synthetase Overexpression:
- Overexpression of the native E. coli acetyl-CoA synthetase (Acs) did not significantly improve ethanol utilization, suggesting sufficient endogenous enzyme levels.
- ATP limitations during ethanol metabolism may restrict further enhancement through Acs overexpression.
Mevalonic Acid Production from Ethanol:
- A mevalonic acid biosynthesis pathway was introduced into the engineered E. coli strain.
- Mevalonic acid was successfully produced, though at lower yields compared to glucose-based fermentation.
- Use of deuterium-labeled ethanol confirmed the ethanol-derived carbon incorporation into mevalonic acid.
Identification of mevalonic acid lactones by GC-MS (Cao, Yujin et al., 2020).
Reference
- Cao, Yujin, et al. "Metabolic engineering of Escherichia coli for the utilization of ethanol." Journal of Biological Research-Thessaloniki 27 (2020): 1-10. https://doi.org/10.1186/s40709-020-0111-0
