Background Hydrocarbon-based biofuels—so-called drop-in fuels—have gained attention as sustainable alternatives to petroleum-derived fuels, yet their biological production remains limited by the availability of efficient enzymatic pathways for generating hydrocarbon precursors. Medium-chain alkanes produced by microorganisms represent a promising target, but the aldehyde-producing capabilities of acyl-CoA reductases (ACRs) from bacteria, plants, and animals have not been systematically compared. Because ACRs generate fatty aldehydes—key intermediates in hydrocarbon biosynthesis—understanding their diversity is essential for expanding biological fuel production strategies. In this study, we established a whole‑cell proxy screening framework in E. coli, a widely used microbial production host, to identify promising ACR candidates in this chassis and to provide new insights into ACR diversity and performance in a microbial production host that can help inform future development of microbial hydrocarbon bioproduction pathways. Results Sixteen acyl-CoA reductases (ACRs) from microorganisms, plants, and animals were cloned and expressed in Escherichia coli and screened using hydrocarbon formation as a practical proxy readout by coexpressing each enzyme with a cyanobacterial aldehyde decarbonylase. Several Arabidopsis thaliana ACRs produced higher alkane levels than microbial and animal enzymes. To further examine plant-derived enzymes, ACR homologs with high amino acid similarity to A. thaliana ACR1 and ACR2 were cloned from multiple plant species and tested. In the E. coli proxy‑screening context used here, some plant ACR homologs yield detectable medium‑chain hydrocarbon proxy readouts. Among these, introduction of ACR2 from Glycine max resulted in the highest alkane and alkene formation levels. Phylogenetic analysis of fourteen plant ACRs with considerable medium chain hydrocarbon proxy-readout showed that ACRs similar to GmACR2 generated higher levels of C13 alkanes, although no clear trend was observed for C15 alkane or C17 alkene. In an E. coli strain coexpressing GmACR2 and SpALDH, we detected a small C17 alkene (1‑heptadecene) signal above the empty‑vector background under the conditions tested, although enzyme‑specific attribution remains unresolved in the absence of single‑expression controls. This preliminary observation suggests that a C17 alkene signal can be detected in an ACR–ALDH coexpression background, suggesting a potentially broader space of ACR–ALDH combinations for future exploration in microbial hydrocarbon production. Conclusion This study identifies multiple microbial and plant-derived ACRs, particularly GmACR2, as a strong candidate in this E. coli whole‑cell proxy screening assay for medium-chain hydrocarbon biosynthesis. Coexpression of GmACR2 with S. pombe aldehyde dehydrogenase provides an initial proof-of-concept indication that hydrocarbon formation can be detected in an ACR–ALDH coexpression background in E. coli. Because ACR and ALDH homologs are widely distributed across taxa, our findings highlight broadly accessible enzymatic components that can be repurposed for engineered microbial hydrocarbon biosynthesis.

Acyl CoA reductases useful for bioproduction of hydrocarbons

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2026年6月2日

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Masakazu Ito,
Shigenobu Kishino,
Masayoshi Muramatsu,
Jun Ogawa

摘要

Background

Hydrocarbon-based biofuels—so-called drop-in fuels—have gained attention as sustainable alternatives to petroleum-derived fuels, yet their biological production remains limited by the availability of efficient enzymatic pathways for generating hydrocarbon precursors. Medium-chain alkanes produced by microorganisms represent a promising target, but the aldehyde-producing capabilities of acyl-CoA reductases (ACRs) from bacteria, plants, and animals have not been systematically compared. Because ACRs generate fatty aldehydes—key intermediates in hydrocarbon biosynthesis—understanding their diversity is essential for expanding biological fuel production strategies. In this study, we established a whole‑cell proxy screening framework in E. coli, a widely used microbial production host, to identify promising ACR candidates in this chassis and to provide new insights into ACR diversity and performance in a microbial production host that can help inform future development of microbial hydrocarbon bioproduction pathways.

Results

Sixteen acyl-CoA reductases (ACRs) from microorganisms, plants, and animals were cloned and expressed in Escherichia coli and screened using hydrocarbon formation as a practical proxy readout by coexpressing each enzyme with a cyanobacterial aldehyde decarbonylase. Several Arabidopsis thaliana ACRs produced higher alkane levels than microbial and animal enzymes. To further examine plant-derived enzymes, ACR homologs with high amino acid similarity to A. thaliana ACR1 and ACR2 were cloned from multiple plant species and tested. In the E. coli proxy‑screening context used here, some plant ACR homologs yield detectable medium‑chain hydrocarbon proxy readouts. Among these, introduction of ACR2 from Glycine max resulted in the highest alkane and alkene formation levels. Phylogenetic analysis of fourteen plant ACRs with considerable medium chain hydrocarbon proxy-readout showed that ACRs similar to GmACR2 generated higher levels of C13 alkanes, although no clear trend was observed for C15 alkane or C17 alkene. In an E. coli strain coexpressing GmACR2 and SpALDH, we detected a small C17 alkene (1‑heptadecene) signal above the empty‑vector background under the conditions tested, although enzyme‑specific attribution remains unresolved in the absence of single‑expression controls. This preliminary observation suggests that a C17 alkene signal can be detected in an ACR–ALDH coexpression background, suggesting a potentially broader space of ACR–ALDH combinations for future exploration in microbial hydrocarbon production.

Conclusion

This study identifies multiple microbial and plant-derived ACRs, particularly GmACR2, as a strong candidate in this E. coli whole‑cell proxy screening assay for medium-chain hydrocarbon biosynthesis. Coexpression of GmACR2 with S. pombe aldehyde dehydrogenase provides an initial proof-of-concept indication that hydrocarbon formation can be detected in an ACR–ALDH coexpression background in E. coli. Because ACR and ALDH homologs are widely distributed across taxa, our findings highlight broadly accessible enzymatic components that can be repurposed for engineered microbial hydrocarbon biosynthesis.