Modulation of acetate utilization in Komagataella phaffii by metabolic engineering of tolerance and metabolism
Abstract Background Acetate, an economical industrial chemical, which is also the precursor of acetyl-CoA, could serve as an alternative substrate for biomanufacturing. This nontraditional substrate can be widely produced from syngas via hydrolysis or pyrolysis of the cellulosic biomass, chemical or...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2019-03-01
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| Series: | Biotechnology for Biofuels |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13068-019-1404-0 |
| _version_ | 1817977992425504768 |
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| author | Qin Xu Chenxiao Bai Yiqi Liu Lili Song Lin Tian Yunfeng Yan Jinfeng Zhou Xiangshan Zhou Yuanxing Zhang Menghao Cai |
| author_facet | Qin Xu Chenxiao Bai Yiqi Liu Lili Song Lin Tian Yunfeng Yan Jinfeng Zhou Xiangshan Zhou Yuanxing Zhang Menghao Cai |
| author_sort | Qin Xu |
| collection | DOAJ |
| description | Abstract Background Acetate, an economical industrial chemical, which is also the precursor of acetyl-CoA, could serve as an alternative substrate for biomanufacturing. This nontraditional substrate can be widely produced from syngas via hydrolysis or pyrolysis of the cellulosic biomass, chemical or microbial catalysis, anaerobic fermentation in treated wastewater, etc. However, the toxicity of acetate to microorganisms has held back its utilization, especially for the eukaryotes that are good hosts for production of complicated pharmaceuticals or chemicals. This study seeks to improve acetate utilization in a widely used yeast host, Komagataella phaffii (previously Pichia pastoris), by metabolic engineering of acetate tolerance, transport, and metabolism. Results A kinase-deficient library of K. phaffii was firstly used to screen acetate-resistant kinases. The HRK1 knockout strain was sensitive to acetate and overexpression of this gene improved acetate tolerance and cell growth of the strain. Also, overexpression of HRK1 caused a 55% productivity improvement of acetyl-CoA-dependent 6-methylsalicylic acid (6-MSA). However, activation of Hrk1 on membrane H(+)-ATPase Pma1 seemed not to work in the engineered strain. Acetate transporter gene ScFPS1* was further overexpressed, despite of not improving 6-MSA biosynthesis. To enhance acetate metabolism, acetyl-CoA synthesizing related genes, yeast PpACS1, ScACS1*, and E. coli ackA/pta were overexpressed separately. Introduction of PpACS1 and ScACS1* each increased biosynthesis of 6-MSA by approximately 20% on 20 mM acetate. Finally, co-overexpression of HRK1 and ScACS1* improved 6-MSA productivity by 51% on 20 mM acetate, despite that a low expression level of HRK1 happened when genes were expressed under the same promoter. Conclusions HRK1 screened by K. phaffii kinase-deficient library played an important role in acetate tolerance and was proved to profit the biosynthesis of acetyl-CoA-derived chemicals. It could be a potential target for metabolic engineering of acetate utilization in other eukaryotic hosts as well. A combined strategy of introducing genes for acetate tolerance and metabolism further improved biosynthesis of acetyl-CoA derived reporter compound in K. phaffii. This makes it a good choice for acetyl-CoA-derived chemicals with acetate as substrate or precursor in K. phaffii, which would also extend the use of this chassis host. |
| first_indexed | 2024-04-13T22:23:37Z |
| format | Article |
| id | doaj.art-372069ca1b764c54a669afc25629001c |
| institution | Directory Open Access Journal |
| issn | 1754-6834 |
| language | English |
| last_indexed | 2024-04-13T22:23:37Z |
| publishDate | 2019-03-01 |
| publisher | BMC |
| record_format | Article |
| series | Biotechnology for Biofuels |
| spelling | doaj.art-372069ca1b764c54a669afc25629001c2022-12-22T02:27:07ZengBMCBiotechnology for Biofuels1754-68342019-03-0112111410.1186/s13068-019-1404-0Modulation of acetate utilization in Komagataella phaffii by metabolic engineering of tolerance and metabolismQin Xu0Chenxiao Bai1Yiqi Liu2Lili Song3Lin Tian4Yunfeng Yan5Jinfeng Zhou6Xiangshan Zhou7Yuanxing Zhang8Menghao Cai9State Key Laboratory of Bioreactor Engineering, East China University of Science and TechnologyState Key Laboratory of Bioreactor Engineering, East China University of Science and TechnologyState Key Laboratory of Bioreactor Engineering, East China University of Science and TechnologyState Key Laboratory of Bioreactor Engineering, East China University of Science and TechnologyState Key Laboratory of Bioreactor Engineering, East China University of Science and TechnologyState Key Laboratory of Bioreactor Engineering, East China University of Science and TechnologyState Key Laboratory of Bioreactor Engineering, East China University of Science and TechnologyState Key Laboratory of Bioreactor Engineering, East China University of Science and TechnologyState Key Laboratory of Bioreactor Engineering, East China University of Science and TechnologyState Key Laboratory of Bioreactor Engineering, East China University of Science and TechnologyAbstract Background Acetate, an economical industrial chemical, which is also the precursor of acetyl-CoA, could serve as an alternative substrate for biomanufacturing. This nontraditional substrate can be widely produced from syngas via hydrolysis or pyrolysis of the cellulosic biomass, chemical or microbial catalysis, anaerobic fermentation in treated wastewater, etc. However, the toxicity of acetate to microorganisms has held back its utilization, especially for the eukaryotes that are good hosts for production of complicated pharmaceuticals or chemicals. This study seeks to improve acetate utilization in a widely used yeast host, Komagataella phaffii (previously Pichia pastoris), by metabolic engineering of acetate tolerance, transport, and metabolism. Results A kinase-deficient library of K. phaffii was firstly used to screen acetate-resistant kinases. The HRK1 knockout strain was sensitive to acetate and overexpression of this gene improved acetate tolerance and cell growth of the strain. Also, overexpression of HRK1 caused a 55% productivity improvement of acetyl-CoA-dependent 6-methylsalicylic acid (6-MSA). However, activation of Hrk1 on membrane H(+)-ATPase Pma1 seemed not to work in the engineered strain. Acetate transporter gene ScFPS1* was further overexpressed, despite of not improving 6-MSA biosynthesis. To enhance acetate metabolism, acetyl-CoA synthesizing related genes, yeast PpACS1, ScACS1*, and E. coli ackA/pta were overexpressed separately. Introduction of PpACS1 and ScACS1* each increased biosynthesis of 6-MSA by approximately 20% on 20 mM acetate. Finally, co-overexpression of HRK1 and ScACS1* improved 6-MSA productivity by 51% on 20 mM acetate, despite that a low expression level of HRK1 happened when genes were expressed under the same promoter. Conclusions HRK1 screened by K. phaffii kinase-deficient library played an important role in acetate tolerance and was proved to profit the biosynthesis of acetyl-CoA-derived chemicals. It could be a potential target for metabolic engineering of acetate utilization in other eukaryotic hosts as well. A combined strategy of introducing genes for acetate tolerance and metabolism further improved biosynthesis of acetyl-CoA derived reporter compound in K. phaffii. This makes it a good choice for acetyl-CoA-derived chemicals with acetate as substrate or precursor in K. phaffii, which would also extend the use of this chassis host.http://link.springer.com/article/10.1186/s13068-019-1404-0Komagataella phaffiiAcetate utilizationAcetyl-CoAKinase screeningMetabolic engineering |
| spellingShingle | Qin Xu Chenxiao Bai Yiqi Liu Lili Song Lin Tian Yunfeng Yan Jinfeng Zhou Xiangshan Zhou Yuanxing Zhang Menghao Cai Modulation of acetate utilization in Komagataella phaffii by metabolic engineering of tolerance and metabolism Biotechnology for Biofuels Komagataella phaffii Acetate utilization Acetyl-CoA Kinase screening Metabolic engineering |
| title | Modulation of acetate utilization in Komagataella phaffii by metabolic engineering of tolerance and metabolism |
| title_full | Modulation of acetate utilization in Komagataella phaffii by metabolic engineering of tolerance and metabolism |
| title_fullStr | Modulation of acetate utilization in Komagataella phaffii by metabolic engineering of tolerance and metabolism |
| title_full_unstemmed | Modulation of acetate utilization in Komagataella phaffii by metabolic engineering of tolerance and metabolism |
| title_short | Modulation of acetate utilization in Komagataella phaffii by metabolic engineering of tolerance and metabolism |
| title_sort | modulation of acetate utilization in komagataella phaffii by metabolic engineering of tolerance and metabolism |
| topic | Komagataella phaffii Acetate utilization Acetyl-CoA Kinase screening Metabolic engineering |
| url | http://link.springer.com/article/10.1186/s13068-019-1404-0 |
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