Part:BBa_J45900:Experience
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Applications of BBa_J45900
Production of isoamyl acetate (banana odor) from cellular metabolites.
User Reviews
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The banana odor biosynthetic system BBa_J45900 may not be functional since no isoamyl acetate is detected. |
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Characterization
Failure analysis
In engineering a system for producing a wintergreen odor during exponential growth and banana odor in stationary phase, we were unable to construct a system for banana odor production from cellular metabolites. We attempted to engineer a heterologous pathway for conversion of leucine to isoamyl alcohol, the precursor for the banana odor generator. The enzyme BAT2 catalyzes the conversion of leucine to α-ketoisocaproate and the enzyme THI3 catalyzes the conversion of α-isocaproate to 3-methylbutanal which is subsequently reduced to isoamyl alcohol by the chassis. Cultures of the banana odor generator BBa_J45200 supplemented with either 3-methylbutanal or isoamyl alcohol produced a strong banana odor. Thus, the banana odor biosynthetic system (BBa_J45900) produced insufficient quantities of 3-methylbutanal from leucine. The precursor to 3-methylbutanal in the engineered biosynthetic system, α-ketoisocaproate, is naturally used in the E. coli leucine biosynthesis pathway. Thus, α-ketoisocaproate is likely present at least at low levels in E. coli even if the heterologous BAT2 enzyme is poorly expressed or not functional. Given that α-ketoisocaproate is present naturally in E. coli and that a strong banana odor was obtained if exogenous 3-methylbutanal is supplied, the problem with the banana biosynthetic system likely lies in the conversion of α-ketoisocaproate to 3-methylbutanal catalyzed by the enzyme THI3. Specifically, likely issues are (1) there is insufficient α-ketoisocaproate available for conversion to 3-methylbutanal possibly because α-ketoisocaproate is preferentially converted to leucine, (2) THI3 is poorly expressed perhaps due in part to its seven rare codons in a 610 amino acid sequence, and/or (3) THI3 is not functional in E. coli for some reason. Interestingly, although much work has been done on isoamyl acetate production from isoamyl alcohol in E. coli, production of isoamyl alcohol in E. coli has not been reported to our knowledge. Our results lead us to speculate whether past failures to synthesize isoamyl alcohol in E. coli have gone unreported. Classical metabolic engineering techniques like flux balance analysis are likely needed to understand the operation of the banana odor biosynthetic system.
Gas chromatography analysis of banana odor biosynthetic system
We designed and constructed a system for banana odor production from intracellular metabolites (BBa_J45900). We analyzed cultures with the banana odor biosynthetic system for isoamyl acetate production but did not measure detectable levels of isoamyl acetate by gas chromatography (A). The banana odor generator (BBa_J45200) in the absence of exogenous isoamyl acetate does not produce measurable quantities of isoamyl acetate (B). For reference, we analyzed a sample with 100 ppm pure isoamyl acetate as well (E). Most E. coli strains produce indole. Octyl acetate was used as an internal standard for all samples containing isoamyl acetate.