Harnessing a P450 fatty acid decarboxylase from Macrococcus caseolyticus for microbial biosynthesis of odd chain terminal alkenes
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...
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| Format: | Article |
| Language: | English |
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Elsevier
2018-12-01
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| Series: | Metabolic Engineering Communications |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2214030118300270 |
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| author | Jong-Won Lee Narayan P. Niraula Cong T. Trinh |
| author_facet | Jong-Won Lee Narayan P. Niraula Cong T. Trinh |
| author_sort | Jong-Won Lee |
| collection | DOAJ |
| description | 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 two-component 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. Keywords: P450 decarboxylase, Terminal alkene, Protein homology modeling, Alanine scan, Escherichia coli, Macrococcus caseolyticus |
| first_indexed | 2024-04-12T17:37:03Z |
| format | Article |
| id | doaj.art-c55cb1c0367d4ca8a06129f13b5fd022 |
| institution | Directory Open Access Journal |
| issn | 2214-0301 |
| language | English |
| last_indexed | 2024-04-12T17:37:03Z |
| publishDate | 2018-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Metabolic Engineering Communications |
| spelling | doaj.art-c55cb1c0367d4ca8a06129f13b5fd0222022-12-22T03:22:56ZengElsevierMetabolic Engineering Communications2214-03012018-12-017Harnessing a P450 fatty acid decarboxylase from Macrococcus caseolyticus for microbial biosynthesis of odd chain terminal alkenesJong-Won Lee0Narayan P. Niraula1Cong T. Trinh2Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee, Knoxville, TN, USA; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, USADepartment of Chemical and Biomolecular Engineering, University of Tennessee, Knoxville, TN, USABredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee, Knoxville, TN, USA; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, USA; Department of Chemical and Biomolecular Engineering, University of Tennessee, Knoxville, TN, USA; Corresponding author at: Department of Chemical and Biomolecular Engineering, University of Tennessee, Knoxville, TN, USA.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 two-component 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. Keywords: P450 decarboxylase, Terminal alkene, Protein homology modeling, Alanine scan, Escherichia coli, Macrococcus caseolyticushttp://www.sciencedirect.com/science/article/pii/S2214030118300270 |
| spellingShingle | Jong-Won Lee Narayan P. Niraula Cong T. Trinh Harnessing a P450 fatty acid decarboxylase from Macrococcus caseolyticus for microbial biosynthesis of odd chain terminal alkenes Metabolic Engineering Communications |
| title | Harnessing a P450 fatty acid decarboxylase from Macrococcus caseolyticus for microbial biosynthesis of odd chain terminal alkenes |
| title_full | Harnessing a P450 fatty acid decarboxylase from Macrococcus caseolyticus for microbial biosynthesis of odd chain terminal alkenes |
| title_fullStr | Harnessing a P450 fatty acid decarboxylase from Macrococcus caseolyticus for microbial biosynthesis of odd chain terminal alkenes |
| title_full_unstemmed | Harnessing a P450 fatty acid decarboxylase from Macrococcus caseolyticus for microbial biosynthesis of odd chain terminal alkenes |
| title_short | Harnessing a P450 fatty acid decarboxylase from Macrococcus caseolyticus for microbial biosynthesis of odd chain terminal alkenes |
| title_sort | harnessing a p450 fatty acid decarboxylase from macrococcus caseolyticus for microbial biosynthesis of odd chain terminal alkenes |
| url | http://www.sciencedirect.com/science/article/pii/S2214030118300270 |
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