Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursor
Abstract Background Butadiene is a platform chemical used as an industrial feedstock for the manufacture of automobile tires, synthetic resins, latex and engineering plastics. Currently, butadiene is predominantly synthesized as a byproduct of ethylene production from non-renewable petroleum resourc...
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2018-12-01
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Series: | Microbial Cell Factories |
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Online Access: | http://link.springer.com/article/10.1186/s12934-018-1042-4 |
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Article |
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DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Jing Yang Chang-Tai Zhang Xiao-Jie Yuan Min Zhang Xu-Hua Mo Ling-Ling Tan Li-Ping Zhu Wen-Jing Chen Ming-Dong Yao Bo Hu Song Yang |
spellingShingle |
Jing Yang Chang-Tai Zhang Xiao-Jie Yuan Min Zhang Xu-Hua Mo Ling-Ling Tan Li-Ping Zhu Wen-Jing Chen Ming-Dong Yao Bo Hu Song Yang Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursor Microbial Cell Factories Methylobacterium extorquens Butadiene Crotyl diphosphate High throughput screening In vitro reaction Pathway engineering |
author_facet |
Jing Yang Chang-Tai Zhang Xiao-Jie Yuan Min Zhang Xu-Hua Mo Ling-Ling Tan Li-Ping Zhu Wen-Jing Chen Ming-Dong Yao Bo Hu Song Yang |
author_sort |
Jing Yang |
title |
Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursor |
title_short |
Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursor |
title_full |
Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursor |
title_fullStr |
Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursor |
title_full_unstemmed |
Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursor |
title_sort |
metabolic engineering of methylobacterium extorquens am1 for the production of butadiene precursor |
publisher |
BMC |
series |
Microbial Cell Factories |
issn |
1475-2859 |
publishDate |
2018-12-01 |
description |
Abstract Background Butadiene is a platform chemical used as an industrial feedstock for the manufacture of automobile tires, synthetic resins, latex and engineering plastics. Currently, butadiene is predominantly synthesized as a byproduct of ethylene production from non-renewable petroleum resources. Although the idea of biological synthesis of butadiene from sugars has been discussed in the literature, success for that goal has so far not been reported. As a model system for methanol assimilation, Methylobacterium extorquens AM1 can produce several unique metabolic intermediates for the production of value-added chemicals, including crotonyl-CoA as a potential precursor for butadiene synthesis. Results In this work, we focused on constructing a metabolic pathway to convert crotonyl-CoA into crotyl diphosphate, a direct precursor of butadiene. The engineered pathway consists of three identified enzymes, a hydroxyethylthiazole kinase (THK) from Escherichia coli, an isopentenyl phosphate kinase (IPK) from Methanothermobacter thermautotrophicus and an aldehyde/alcohol dehydrogenase (ADHE2) from Clostridium acetobutylicum. The K m and k cat of THK, IPK and ADHE2 were determined as 8.35 mM and 1.24 s−1, 1.28 mM and 153.14 s−1, and 2.34 mM and 1.15 s−1 towards crotonol, crotyl monophosphate and crotonyl-CoA, respectively. Then, the activity of one of rate-limiting enzymes, THK, was optimized by random mutagenesis coupled with a developed high-throughput screening colorimetric assay. The resulting variant (THKM82V) isolated from over 3000 colonies showed 8.6-fold higher activity than wild-type, which helped increase the titer of crotyl diphosphate to 0.76 mM, corresponding to a 7.6% conversion from crotonol in the one-pot in vitro reaction. Overexpression of native ADHE2, IPK with THKM82V under a strong promoter mxaF in M. extorquens AM1 did not produce crotyl diphosphate from crotonyl-CoA, but the engineered strain did generate 0.60 μg/mL of intracellular crotyl diphosphate from exogenously supplied crotonol at mid-exponential phase. Conclusions These results represent the first step in producing a butadiene precursor in recombinant M. extorquens AM1. It not only demonstrates the feasibility of converting crotonol to key intermediates for butadiene biosynthesis, it also suggests future directions for improving catalytic efficiency of aldehyde/alcohol dehydrogenase to produce butadiene precursor from methanol. |
topic |
Methylobacterium extorquens Butadiene Crotyl diphosphate High throughput screening In vitro reaction Pathway engineering |
url |
http://link.springer.com/article/10.1186/s12934-018-1042-4 |
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doaj-c6643bed8dff41beb1b5382c3383329d2020-11-25T00:51:53ZengBMCMicrobial Cell Factories1475-28592018-12-0117111510.1186/s12934-018-1042-4Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursorJing Yang0Chang-Tai Zhang1Xiao-Jie Yuan2Min Zhang3Xu-Hua Mo4Ling-Ling Tan5Li-Ping Zhu6Wen-Jing Chen7Ming-Dong Yao8Bo Hu9Song Yang10School of Life Sciences, Shandong Province Key Laboratory of Applied Mycology, and Qingdao International Center on Microbes Utilizing Biogas, Qingdao Agricultural UniversitySchool of Life Sciences, Shandong Province Key Laboratory of Applied Mycology, and Qingdao International Center on Microbes Utilizing Biogas, Qingdao Agricultural UniversitySchool of Life Sciences, Shandong Province Key Laboratory of Applied Mycology, and Qingdao International Center on Microbes Utilizing Biogas, Qingdao Agricultural UniversitySchool of Life Sciences, Shandong Province Key Laboratory of Applied Mycology, and Qingdao International Center on Microbes Utilizing Biogas, Qingdao Agricultural UniversitySchool of Life Sciences, Shandong Province Key Laboratory of Applied Mycology, and Qingdao International Center on Microbes Utilizing Biogas, Qingdao Agricultural UniversitySchool of Life Sciences, Shandong Province Key Laboratory of Applied Mycology, and Qingdao International Center on Microbes Utilizing Biogas, Qingdao Agricultural UniversitySchool of Life Sciences, Shandong Province Key Laboratory of Applied Mycology, and Qingdao International Center on Microbes Utilizing Biogas, Qingdao Agricultural UniversitySchool of Life Sciences, Shandong Province Key Laboratory of Applied Mycology, and Qingdao International Center on Microbes Utilizing Biogas, Qingdao Agricultural UniversityKey Laboratory of Systems Bioengineering, Ministry of Education, Tianjin UniversityIndustrial Product Division, Intrexon CorporationSchool of Life Sciences, Shandong Province Key Laboratory of Applied Mycology, and Qingdao International Center on Microbes Utilizing Biogas, Qingdao Agricultural UniversityAbstract Background Butadiene is a platform chemical used as an industrial feedstock for the manufacture of automobile tires, synthetic resins, latex and engineering plastics. Currently, butadiene is predominantly synthesized as a byproduct of ethylene production from non-renewable petroleum resources. Although the idea of biological synthesis of butadiene from sugars has been discussed in the literature, success for that goal has so far not been reported. As a model system for methanol assimilation, Methylobacterium extorquens AM1 can produce several unique metabolic intermediates for the production of value-added chemicals, including crotonyl-CoA as a potential precursor for butadiene synthesis. Results In this work, we focused on constructing a metabolic pathway to convert crotonyl-CoA into crotyl diphosphate, a direct precursor of butadiene. The engineered pathway consists of three identified enzymes, a hydroxyethylthiazole kinase (THK) from Escherichia coli, an isopentenyl phosphate kinase (IPK) from Methanothermobacter thermautotrophicus and an aldehyde/alcohol dehydrogenase (ADHE2) from Clostridium acetobutylicum. The K m and k cat of THK, IPK and ADHE2 were determined as 8.35 mM and 1.24 s−1, 1.28 mM and 153.14 s−1, and 2.34 mM and 1.15 s−1 towards crotonol, crotyl monophosphate and crotonyl-CoA, respectively. Then, the activity of one of rate-limiting enzymes, THK, was optimized by random mutagenesis coupled with a developed high-throughput screening colorimetric assay. The resulting variant (THKM82V) isolated from over 3000 colonies showed 8.6-fold higher activity than wild-type, which helped increase the titer of crotyl diphosphate to 0.76 mM, corresponding to a 7.6% conversion from crotonol in the one-pot in vitro reaction. Overexpression of native ADHE2, IPK with THKM82V under a strong promoter mxaF in M. extorquens AM1 did not produce crotyl diphosphate from crotonyl-CoA, but the engineered strain did generate 0.60 μg/mL of intracellular crotyl diphosphate from exogenously supplied crotonol at mid-exponential phase. Conclusions These results represent the first step in producing a butadiene precursor in recombinant M. extorquens AM1. It not only demonstrates the feasibility of converting crotonol to key intermediates for butadiene biosynthesis, it also suggests future directions for improving catalytic efficiency of aldehyde/alcohol dehydrogenase to produce butadiene precursor from methanol.http://link.springer.com/article/10.1186/s12934-018-1042-4Methylobacterium extorquensButadieneCrotyl diphosphateHigh throughput screeningIn vitro reactionPathway engineering |