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Title: Harnessing a P450 fatty acid decarboxylase from Macrococcus caseolyticus for microbial biosynthesis of odd chain terminal alkenes

Abstract

Alkenes are industrially important platform chemicals with broad applications. In this study, we report a direct microbial biosynthesis of terminal alkenes from fermentable sugars by harnessing a P450 fatty acid (FA) decarboxylase from Macrococcus caseolyticus (OleTMC). We first characterized OleTMC and demonstrated its in vitro H2O2-independent activities towards linear C10:0-C18:0 FAs, with higher activity for C16:0-C18:0 FAs. Next, we engineered a de novo alkene biosynthesis pathway, consisting of OleTMC and an engineered E. coli thioesterase (TesA) with compatible substrate specificities, and introduced this pathway into E. coli for terminal alkene biosynthesis from glucose. The recombinant E. coli EcNN101 produced a total of 17.78 ± 0.63 mg/L odd-chain terminal alkenes, comprising of 0.9% ± 0.5% C11 alkene, 12.7% ± 2.2% C13 alkene, 82.7% ± 1.7% C15 alkene, and 3.7% ± 0.8% C17 alkene, and a yield of 0.87 ± 0.03 (mg/g) on glucose. To improve alkene production, we identified and overcame the electron transfer limitation in OleTMC, by introducing a twocomponent redox system, consisting of a putidaredoxin reductase (CamA) and a putidaredoxin (CamB) from Pseudomonas putida, into EcNN101, and demonstrated the alkene production increased ~2.8 fold. Finally, to better understand the substrate specificities of OleTMC observed, we employed in silico proteinmore » modeling to illuminate the functional role of FA binding pocket.« less

Authors:
; ;
Publication Date:
Research Org.:
Oak Ridge National Lab (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1566205
Alternate Identifier(s):
OSTI ID: 1629029
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Published Article
Journal Name:
Metabolic Engineering Communications
Additional Journal Information:
Journal Name: Metabolic Engineering Communications Journal Volume: 7 Journal Issue: C; Journal ID: ISSN 2214-0301
Publisher:
Elsevier
Country of Publication:
Netherlands
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; P450 decarboxylase; Terminal alkene; Protein homology modeling; Alanine scan; Escherichia coli; Macrococcus caseolyticus

Citation Formats

Lee, Jong-Won, Niraula, Narayan P., and Trinh, Cong T. Harnessing a P450 fatty acid decarboxylase from Macrococcus caseolyticus for microbial biosynthesis of odd chain terminal alkenes. Netherlands: N. p., 2018. Web. doi:10.1016/j.mec.2018.e00076.
Lee, Jong-Won, Niraula, Narayan P., & Trinh, Cong T. Harnessing a P450 fatty acid decarboxylase from Macrococcus caseolyticus for microbial biosynthesis of odd chain terminal alkenes. Netherlands. https://doi.org/10.1016/j.mec.2018.e00076
Lee, Jong-Won, Niraula, Narayan P., and Trinh, Cong T. Sat . "Harnessing a P450 fatty acid decarboxylase from Macrococcus caseolyticus for microbial biosynthesis of odd chain terminal alkenes". Netherlands. https://doi.org/10.1016/j.mec.2018.e00076.
@article{osti_1566205,
title = {Harnessing a P450 fatty acid decarboxylase from Macrococcus caseolyticus for microbial biosynthesis of odd chain terminal alkenes},
author = {Lee, Jong-Won and Niraula, Narayan P. and Trinh, Cong T.},
abstractNote = {Alkenes are industrially important platform chemicals with broad applications. In this study, we report a direct microbial biosynthesis of terminal alkenes from fermentable sugars by harnessing a P450 fatty acid (FA) decarboxylase from Macrococcus caseolyticus (OleTMC). We first characterized OleTMC and demonstrated its in vitro H2O2-independent activities towards linear C10:0-C18:0 FAs, with higher activity for C16:0-C18:0 FAs. Next, we engineered a de novo alkene biosynthesis pathway, consisting of OleTMC and an engineered E. coli thioesterase (TesA) with compatible substrate specificities, and introduced this pathway into E. coli for terminal alkene biosynthesis from glucose. The recombinant E. coli EcNN101 produced a total of 17.78 ± 0.63 mg/L odd-chain terminal alkenes, comprising of 0.9% ± 0.5% C11 alkene, 12.7% ± 2.2% C13 alkene, 82.7% ± 1.7% C15 alkene, and 3.7% ± 0.8% C17 alkene, and a yield of 0.87 ± 0.03 (mg/g) on glucose. To improve alkene production, we identified and overcame the electron transfer limitation in OleTMC, by introducing a twocomponent redox system, consisting of a putidaredoxin reductase (CamA) and a putidaredoxin (CamB) from Pseudomonas putida, into EcNN101, and demonstrated the alkene production increased ~2.8 fold. Finally, to better understand the substrate specificities of OleTMC observed, we employed in silico protein modeling to illuminate the functional role of FA binding pocket.},
doi = {10.1016/j.mec.2018.e00076},
journal = {Metabolic Engineering Communications},
number = C,
volume = 7,
place = {Netherlands},
year = {2018},
month = {12}
}