This document discusses engineering E. coli to produce 1-butanol as a biofuel. 1-butanol is typically produced through Clostridium fermentation which produces undesirable byproducts. E. coli was engineered as it is well-characterized, does not produce spores, and is easier to genetically modify. The researchers deleted genes competing for acetyl-CoA and NADH, cloned genes from Clostridium, and were able to produce 1-butanol in E. coli without undesirable byproducts. Future work could optimize production levels to make E. coli a viable platform for scalable 1-butanol biofuel production.
Genetically modified organisms for the production of biofuels
1. GENETICALLY MODIFIED
ORGANISMS FOR THE
PRODUCTION OF BIOFUELS
(E. coli)
AMBICA BORA
BBT1503
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Metabolic engineering of Escherichia coli
for 1-butanol production by Liao et. al
3. • Typically, 1-butanol is produced by Clostridium in a
mixed-product fermentation.
– Mixed as butyrate, acetone and ethanol are produced with
it as byproducts
– Difficult to separate out
– Yield difficult to control
– Clostridium has slow growth rate and spore forming life
cycle
– Hard to genetically engineer the genome for C.
acetobutylicum, as it is relatively unknown.
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5. Steps for engineering the pathway
1. Growing the bacteria in specific conditions and media
(semi aerobically in M9 media). Strain specific uses –
plasmid propagation and knockout strains
2. Deletion and inactivation of host genes
3. Sequencing of all plasmids to be inserted carried out
to verify accuracy
4. Cloning of C. acetobutylicum using PCR templates.
5. Digestion of PCR products and their ligation in specific
plasmids at already sequenced sites (primers
synthesized for the same).
6. Inoculation and growth of the organisms and
preliminary assays for metabolite detection
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6. 7. Detection of metabolites
– Alcohol compounds were detected and separated using GC
(with helium as the carrier gas). Supernatant of culture
broth was injected and iso-butanol was used as an internal
standard.
– For secondary metabolites filtered supernatant was
applied to a HPLC column, and organic acids and glucose
were detected.
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8. The Pathway Taken
• In Clostridium, 1-Butanol pathway branches to butyrate and
acetone. This branching step was eliminated in E. coli by removing
the genes which caused it to happen. Hence other by products
were eliminated.
• To further improve 1-butanol production, the host pathways that
compete with the 1-butanol pathway for acetyl-CoA and NADH
were deleted.
• All expression in E. coli is under the control of the IPTG-inducible
(an allolactose mimic) PLlacO1 promoter.
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9. Other Factors Taken Into Consideration
• The transfer of a biosynthetic pathway from a native producer
to a non-native producer may face several difficulties.
Overexpression of non-native pathways may disturb the
native metabolism in the hosts by competing for precursors
necessary for growth or maintenance.
• The re-engineering of pathways often leads to imbalanced
gene expression, creating a bottleneck in the biosynthetic
pathway that diminishes production of the target compound.
• Dependence on NADH and Acetyl coA established
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10. FUTURE PROSPECTS
• E. coli can tolerate 1-butanol up to a concentration of
1.5% , which is similar to published results found for
the native producer C. acetobutylicum.
• This can be scaled up and optimized to produce
Biobutanol more easily and economically.
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