Isobutanol production by combined in vivo and in vitro metabolic engineering
The production of the biofuel, isobutanol, in E. coli faces limitations due to alcohol toxicity, product inhibition, product recovery, and long-term industrial feasibility. Here we demonstrate an approach of combining both in vivo with in vitro metabolic engineering to produce isobutanol. The in viv...
| Main Authors: | , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2022-12-01
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| Series: | Metabolic Engineering Communications |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2214030122000190 |
| _version_ | 1811178877939089408 |
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| author | Mamta Gupta Matthew Wong Kamran Jawed Kamil Gedeon Hannah Barrett Marcelo Bassalo Clifford Morrison Danish Eqbal Syed Shams Yazdani Ryan T. Gill Jiaqi Huang Marc Douaisi Jonathan Dordick Georges Belfort Mattheos A.G. Koffas |
| author_facet | Mamta Gupta Matthew Wong Kamran Jawed Kamil Gedeon Hannah Barrett Marcelo Bassalo Clifford Morrison Danish Eqbal Syed Shams Yazdani Ryan T. Gill Jiaqi Huang Marc Douaisi Jonathan Dordick Georges Belfort Mattheos A.G. Koffas |
| author_sort | Mamta Gupta |
| collection | DOAJ |
| description | The production of the biofuel, isobutanol, in E. coli faces limitations due to alcohol toxicity, product inhibition, product recovery, and long-term industrial feasibility. Here we demonstrate an approach of combining both in vivo with in vitro metabolic engineering to produce isobutanol. The in vivo production of α-ketoisovalerate (KIV) was conducted through CRISPR mediated integration of the KIV pathway in bicistronic design (BCD) in E. coli and inhibition of competitive valine pathway using CRISPRi technology. The subsequent in vitro conversion to isobutanol was carried out with engineered enzymes for 2-ketoacid decarboxylase (KIVD) and alcohol dehydrogenase (ADH). For the in vivo production of KIV and subsequent in vitro production of isobutanol, this two-step serial approach resulted in yields of 56% and 93%, productivities of 0.62 and 0.074 g L−1 h−1, and titers of 5.6 and 1.78 g L−1, respectively. Thus, this combined biosynthetic system can be used as a modular approach for producing important metabolites, like isobutanol, without the limitations associated with in vivo production using a consolidated bioprocess. |
| first_indexed | 2024-04-11T06:26:29Z |
| format | Article |
| id | doaj.art-ac03648bb6c645638fbd2ddc4000accd |
| institution | Directory Open Access Journal |
| issn | 2214-0301 |
| language | English |
| last_indexed | 2024-04-11T06:26:29Z |
| publishDate | 2022-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Metabolic Engineering Communications |
| spelling | doaj.art-ac03648bb6c645638fbd2ddc4000accd2022-12-22T04:40:21ZengElsevierMetabolic Engineering Communications2214-03012022-12-0115e00210Isobutanol production by combined in vivo and in vitro metabolic engineeringMamta Gupta0Matthew Wong1Kamran Jawed2Kamil Gedeon3Hannah Barrett4Marcelo Bassalo5Clifford Morrison6Danish Eqbal7Syed Shams Yazdani8Ryan T. Gill9Jiaqi Huang10Marc Douaisi11Jonathan Dordick12Georges Belfort13Mattheos A.G. Koffas14Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA; Department of Botany and Environmental Studies, DAV University, Jalandhar, 144 001, Punjab, IndiaHoward P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA; Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USAHoward P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA; DBT-ICGEB Advanced Bioenergy Research, International Centre for Genetic Engineering and Biotechnology, New Delhi, 110067, IndiaHoward P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180, USAHoward P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180, USADepartment of Chemical and Biological Engineering, University of Colorado, Boulder, CO, 80309, USAHoward P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180, USADBT-ICGEB Advanced Bioenergy Research, International Centre for Genetic Engineering and Biotechnology, New Delhi, 110067, IndiaDBT-ICGEB Advanced Bioenergy Research, International Centre for Genetic Engineering and Biotechnology, New Delhi, 110067, IndiaDepartment of Chemical and Biological Engineering, University of Colorado, Boulder, CO, 80309, USAHoward P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180, USAHoward P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180, USAHoward P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA; Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USAHoward P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA; Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USAHoward P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA; Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA; Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA; Corresponding author. Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.The production of the biofuel, isobutanol, in E. coli faces limitations due to alcohol toxicity, product inhibition, product recovery, and long-term industrial feasibility. Here we demonstrate an approach of combining both in vivo with in vitro metabolic engineering to produce isobutanol. The in vivo production of α-ketoisovalerate (KIV) was conducted through CRISPR mediated integration of the KIV pathway in bicistronic design (BCD) in E. coli and inhibition of competitive valine pathway using CRISPRi technology. The subsequent in vitro conversion to isobutanol was carried out with engineered enzymes for 2-ketoacid decarboxylase (KIVD) and alcohol dehydrogenase (ADH). For the in vivo production of KIV and subsequent in vitro production of isobutanol, this two-step serial approach resulted in yields of 56% and 93%, productivities of 0.62 and 0.074 g L−1 h−1, and titers of 5.6 and 1.78 g L−1, respectively. Thus, this combined biosynthetic system can be used as a modular approach for producing important metabolites, like isobutanol, without the limitations associated with in vivo production using a consolidated bioprocess.http://www.sciencedirect.com/science/article/pii/S2214030122000190α-KetoisovalerateIsobutanolCRISPRCRISPRiBicistronicFed-batch |
| spellingShingle | Mamta Gupta Matthew Wong Kamran Jawed Kamil Gedeon Hannah Barrett Marcelo Bassalo Clifford Morrison Danish Eqbal Syed Shams Yazdani Ryan T. Gill Jiaqi Huang Marc Douaisi Jonathan Dordick Georges Belfort Mattheos A.G. Koffas Isobutanol production by combined in vivo and in vitro metabolic engineering Metabolic Engineering Communications α-Ketoisovalerate Isobutanol CRISPR CRISPRi Bicistronic Fed-batch |
| title | Isobutanol production by combined in vivo and in vitro metabolic engineering |
| title_full | Isobutanol production by combined in vivo and in vitro metabolic engineering |
| title_fullStr | Isobutanol production by combined in vivo and in vitro metabolic engineering |
| title_full_unstemmed | Isobutanol production by combined in vivo and in vitro metabolic engineering |
| title_short | Isobutanol production by combined in vivo and in vitro metabolic engineering |
| title_sort | isobutanol production by combined in vivo and in vitro metabolic engineering |
| topic | α-Ketoisovalerate Isobutanol CRISPR CRISPRi Bicistronic Fed-batch |
| url | http://www.sciencedirect.com/science/article/pii/S2214030122000190 |
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