Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species
Abstract Background Fuels and chemicals derived from non-fossil sources are needed to lessen human impacts on the environment while providing a healthy and growing economy. 3-hydroxypropionic acid (3-HP) is an important chemical building block that can be used for many products. Biosynthesis of 3-HP...
| Main Authors: | , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-03-01
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| Series: | Biotechnology for Biofuels and Bioproducts |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13068-023-02288-1 |
| _version_ | 1797853997280788480 |
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| author | Ziyu Dai Kyle R. Pomraning Shuang Deng Joonhoon Kim Kristen B. Campbell Ana L. Robles Beth A. Hofstad Nathalie Munoz Yuqian Gao Teresa Lemmon Marie S. Swita Jeremy D. Zucker Young-Mo Kim Kristin E. Burnum-Johnson Jon K. Magnuson |
| author_facet | Ziyu Dai Kyle R. Pomraning Shuang Deng Joonhoon Kim Kristen B. Campbell Ana L. Robles Beth A. Hofstad Nathalie Munoz Yuqian Gao Teresa Lemmon Marie S. Swita Jeremy D. Zucker Young-Mo Kim Kristin E. Burnum-Johnson Jon K. Magnuson |
| author_sort | Ziyu Dai |
| collection | DOAJ |
| description | Abstract Background Fuels and chemicals derived from non-fossil sources are needed to lessen human impacts on the environment while providing a healthy and growing economy. 3-hydroxypropionic acid (3-HP) is an important chemical building block that can be used for many products. Biosynthesis of 3-HP is possible; however, low production is typically observed in those natural systems. Biosynthetic pathways have been designed to produce 3-HP from a variety of feedstocks in different microorganisms. Results In this study, the 3-HP β-alanine pathway consisting of aspartate decarboxylase, β-alanine-pyruvate aminotransferase, and 3-hydroxypropionate dehydrogenase from selected microorganisms were codon optimized for Aspergillus species and placed under the control of constitutive promoters. The pathway was introduced into Aspergillus pseudoterreus and subsequently into Aspergillus niger, and 3-HP production was assessed in both hosts. A. niger produced higher initial 3-HP yields and fewer co-product contaminants and was selected as a suitable host for further engineering. Proteomic and metabolomic analysis of both Aspergillus species during 3-HP production identified genetic targets for improvement of flux toward 3-HP including pyruvate carboxylase, aspartate aminotransferase, malonate semialdehyde dehydrogenase, succinate semialdehyde dehydrogenase, oxaloacetate hydrolase, and a 3-HP transporter. Overexpression of pyruvate carboxylase improved yield in shake-flasks from 0.09 to 0.12 C-mol 3-HP C-mol−1 glucose in the base strain expressing 12 copies of the β-alanine pathway. Deletion or overexpression of individual target genes in the pyruvate carboxylase overexpression strain improved yield to 0.22 C-mol 3-HP C-mol−1 glucose after deletion of the major malonate semialdehyde dehydrogenase. Further incorporation of additional β-alanine pathway genes and optimization of culture conditions (sugars, temperature, nitrogen, phosphate, trace elements) for 3-HP production from deacetylated and mechanically refined corn stover hydrolysate improved yield to 0.48 C-mol 3-HP C-mol−1 sugars and resulted in a final titer of 36.0 g/L 3-HP. Conclusions The results of this study establish A. niger as a host for 3-HP production from a lignocellulosic feedstock in acidic conditions and demonstrates that 3-HP titer and yield can be improved by a broad metabolic engineering strategy involving identification and modification of genes participated in the synthesis of 3-HP and its precursors, degradation of intermediates, and transport of 3-HP across the plasma membrane. |
| first_indexed | 2024-04-09T19:58:43Z |
| format | Article |
| id | doaj.art-8683f00131474c27bb32b907d4c7cade |
| institution | Directory Open Access Journal |
| issn | 2731-3654 |
| language | English |
| last_indexed | 2024-04-09T19:58:43Z |
| publishDate | 2023-03-01 |
| publisher | BMC |
| record_format | Article |
| series | Biotechnology for Biofuels and Bioproducts |
| spelling | doaj.art-8683f00131474c27bb32b907d4c7cade2023-04-03T05:19:54ZengBMCBiotechnology for Biofuels and Bioproducts2731-36542023-03-0116111610.1186/s13068-023-02288-1Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus speciesZiyu Dai0Kyle R. Pomraning1Shuang Deng2Joonhoon Kim3Kristen B. Campbell4Ana L. Robles5Beth A. Hofstad6Nathalie Munoz7Yuqian Gao8Teresa Lemmon9Marie S. Swita10Jeremy D. Zucker11Young-Mo Kim12Kristin E. Burnum-Johnson13Jon K. Magnuson14DOE Agile BiofoundryDOE Agile BiofoundryDOE Agile BiofoundryDOE Agile BiofoundryEnergy and Environment Directorate, Pacific Northwest National LaboratoryDOE Agile BiofoundryDOE Agile BiofoundryDOE Agile BiofoundryDOE Agile BiofoundryEnergy and Environment Directorate, Pacific Northwest National LaboratoryEnergy and Environment Directorate, Pacific Northwest National LaboratoryDOE Agile BiofoundryDOE Agile BiofoundryDOE Agile BiofoundryDOE Agile BiofoundryAbstract Background Fuels and chemicals derived from non-fossil sources are needed to lessen human impacts on the environment while providing a healthy and growing economy. 3-hydroxypropionic acid (3-HP) is an important chemical building block that can be used for many products. Biosynthesis of 3-HP is possible; however, low production is typically observed in those natural systems. Biosynthetic pathways have been designed to produce 3-HP from a variety of feedstocks in different microorganisms. Results In this study, the 3-HP β-alanine pathway consisting of aspartate decarboxylase, β-alanine-pyruvate aminotransferase, and 3-hydroxypropionate dehydrogenase from selected microorganisms were codon optimized for Aspergillus species and placed under the control of constitutive promoters. The pathway was introduced into Aspergillus pseudoterreus and subsequently into Aspergillus niger, and 3-HP production was assessed in both hosts. A. niger produced higher initial 3-HP yields and fewer co-product contaminants and was selected as a suitable host for further engineering. Proteomic and metabolomic analysis of both Aspergillus species during 3-HP production identified genetic targets for improvement of flux toward 3-HP including pyruvate carboxylase, aspartate aminotransferase, malonate semialdehyde dehydrogenase, succinate semialdehyde dehydrogenase, oxaloacetate hydrolase, and a 3-HP transporter. Overexpression of pyruvate carboxylase improved yield in shake-flasks from 0.09 to 0.12 C-mol 3-HP C-mol−1 glucose in the base strain expressing 12 copies of the β-alanine pathway. Deletion or overexpression of individual target genes in the pyruvate carboxylase overexpression strain improved yield to 0.22 C-mol 3-HP C-mol−1 glucose after deletion of the major malonate semialdehyde dehydrogenase. Further incorporation of additional β-alanine pathway genes and optimization of culture conditions (sugars, temperature, nitrogen, phosphate, trace elements) for 3-HP production from deacetylated and mechanically refined corn stover hydrolysate improved yield to 0.48 C-mol 3-HP C-mol−1 sugars and resulted in a final titer of 36.0 g/L 3-HP. Conclusions The results of this study establish A. niger as a host for 3-HP production from a lignocellulosic feedstock in acidic conditions and demonstrates that 3-HP titer and yield can be improved by a broad metabolic engineering strategy involving identification and modification of genes participated in the synthesis of 3-HP and its precursors, degradation of intermediates, and transport of 3-HP across the plasma membrane.https://doi.org/10.1186/s13068-023-02288-1Beta-alanine pathway3-hydroxypropionic acidAspergillus nigerAspergillus pseudoterreusMetabolic engineering |
| spellingShingle | Ziyu Dai Kyle R. Pomraning Shuang Deng Joonhoon Kim Kristen B. Campbell Ana L. Robles Beth A. Hofstad Nathalie Munoz Yuqian Gao Teresa Lemmon Marie S. Swita Jeremy D. Zucker Young-Mo Kim Kristin E. Burnum-Johnson Jon K. Magnuson Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species Biotechnology for Biofuels and Bioproducts Beta-alanine pathway 3-hydroxypropionic acid Aspergillus niger Aspergillus pseudoterreus Metabolic engineering |
| title | Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species |
| title_full | Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species |
| title_fullStr | Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species |
| title_full_unstemmed | Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species |
| title_short | Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species |
| title_sort | metabolic engineering to improve production of 3 hydroxypropionic acid from corn stover hydrolysate in aspergillus species |
| topic | Beta-alanine pathway 3-hydroxypropionic acid Aspergillus niger Aspergillus pseudoterreus Metabolic engineering |
| url | https://doi.org/10.1186/s13068-023-02288-1 |
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