Natural Saccharomyces cerevisiae Strain Reveals Peculiar Genomic Traits for Starch-to-Bioethanol Production: the Design of an Amylolytic Consolidated Bioprocessing Yeast
Natural yeast with superior fermentative traits can serve as a platform for the development of recombinant strains that can be used to improve the sustainability of bioethanol production from starch. This process will benefit from a consolidated bioprocessing (CBP) approach where an engineered strai...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-01-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2021.768562/full |
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| author | Nicoletta Gronchi Nicola De Bernardini Rosemary A. Cripwell Laura Treu Stefano Campanaro Marina Basaglia Maria R. Foulquié-Moreno Johan M. Thevelein Johan M. Thevelein Willem H. Van Zyl Lorenzo Favaro Sergio Casella |
| author_facet | Nicoletta Gronchi Nicola De Bernardini Rosemary A. Cripwell Laura Treu Stefano Campanaro Marina Basaglia Maria R. Foulquié-Moreno Johan M. Thevelein Johan M. Thevelein Willem H. Van Zyl Lorenzo Favaro Sergio Casella |
| author_sort | Nicoletta Gronchi |
| collection | DOAJ |
| description | Natural yeast with superior fermentative traits can serve as a platform for the development of recombinant strains that can be used to improve the sustainability of bioethanol production from starch. This process will benefit from a consolidated bioprocessing (CBP) approach where an engineered strain producing amylases directly converts starch into ethanol. The yeast Saccharomyces cerevisiae L20, previously selected as outperforming the benchmark yeast Ethanol Red, was here subjected to a comparative genomic investigation using a dataset of industrial S. cerevisiae strains. Along with Ethanol Red, strain L20 was then engineered for the expression of α-amylase amyA and glucoamylase glaA genes from Aspergillus tubingensis by employing two different approaches (delta integration and CRISPR/Cas9). A correlation between the number of integrated copies and the hydrolytic abilities of the recombinants was investigated. L20 demonstrated important traits for the construction of a proficient CBP yeast. Despite showing a close relatedness to commercial wine yeast and the benchmark Ethanol Red, a unique profile of gene copy number variations (CNVs) was found in L20, mainly encoding membrane transporters and secretion pathway proteins but also the fermentative metabolism. Moreover, the genome annotation disclosed seven open reading frames (ORFs) in L20 that are absent in the reference S288C genome. Genome engineering was successfully implemented for amylase production. However, with equal amylase gene copies, L20 proved its proficiency as a good enzyme secretor by exhibiting a markedly higher amylolytic activity than Ethanol Red, in compliance to the findings of the genomic exploration. The recombinant L20 dT8 exhibited the highest amylolytic activity and produced more than 4 g/L of ethanol from 2% starch in a CBP setting without the addition of supplementary enzymes. Based on the performance of this strain, an amylase/glucoamylase ratio of 1:2.5 was suggested as baseline for further improvement of the CBP ability. Overall, L20 showed important traits for the future construction of a proficient CBP yeast. As such, this work shows that natural S. cerevisiae strains can be used for the expression of foreign secreted enzymes, paving the way to strain improvement for the starch-to-bioethanol route. |
| first_indexed | 2024-12-20T16:07:44Z |
| format | Article |
| id | doaj.art-3192abb9dc5a40fb8bc0ffa3c56bf5a7 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-20T16:07:44Z |
| publishDate | 2022-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-3192abb9dc5a40fb8bc0ffa3c56bf5a72022-12-21T19:34:04ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2022-01-011210.3389/fmicb.2021.768562768562Natural Saccharomyces cerevisiae Strain Reveals Peculiar Genomic Traits for Starch-to-Bioethanol Production: the Design of an Amylolytic Consolidated Bioprocessing YeastNicoletta Gronchi0Nicola De Bernardini1Rosemary A. Cripwell2Laura Treu3Stefano Campanaro4Marina Basaglia5Maria R. Foulquié-Moreno6Johan M. Thevelein7Johan M. Thevelein8Willem H. Van Zyl9Lorenzo Favaro10Sergio Casella11Department of Agronomy, Food, Natural Resources, Animals and Environment (DAFNAE), University of Padua, Legnaro, ItalyDepartment of Biology, University of Padua, Padua, ItalyDepartment of Microbiology, Stellenbosch University, Stellenbosch, South AfricaDepartment of Biology, University of Padua, Padua, ItalyDepartment of Biology, University of Padua, Padua, ItalyDepartment of Agronomy, Food, Natural Resources, Animals and Environment (DAFNAE), University of Padua, Legnaro, ItalyDepartment of Molecular Microbiology, VIB, KU Leuven, Leuven, BelgiumDepartment of Molecular Microbiology, VIB, KU Leuven, Leuven, BelgiumNovelYeast Bv, Open Bio-Incubator, Erasmus High School, Jette, BelgiumDepartment of Microbiology, Stellenbosch University, Stellenbosch, South AfricaDepartment of Agronomy, Food, Natural Resources, Animals and Environment (DAFNAE), University of Padua, Legnaro, ItalyDepartment of Agronomy, Food, Natural Resources, Animals and Environment (DAFNAE), University of Padua, Legnaro, ItalyNatural yeast with superior fermentative traits can serve as a platform for the development of recombinant strains that can be used to improve the sustainability of bioethanol production from starch. This process will benefit from a consolidated bioprocessing (CBP) approach where an engineered strain producing amylases directly converts starch into ethanol. The yeast Saccharomyces cerevisiae L20, previously selected as outperforming the benchmark yeast Ethanol Red, was here subjected to a comparative genomic investigation using a dataset of industrial S. cerevisiae strains. Along with Ethanol Red, strain L20 was then engineered for the expression of α-amylase amyA and glucoamylase glaA genes from Aspergillus tubingensis by employing two different approaches (delta integration and CRISPR/Cas9). A correlation between the number of integrated copies and the hydrolytic abilities of the recombinants was investigated. L20 demonstrated important traits for the construction of a proficient CBP yeast. Despite showing a close relatedness to commercial wine yeast and the benchmark Ethanol Red, a unique profile of gene copy number variations (CNVs) was found in L20, mainly encoding membrane transporters and secretion pathway proteins but also the fermentative metabolism. Moreover, the genome annotation disclosed seven open reading frames (ORFs) in L20 that are absent in the reference S288C genome. Genome engineering was successfully implemented for amylase production. However, with equal amylase gene copies, L20 proved its proficiency as a good enzyme secretor by exhibiting a markedly higher amylolytic activity than Ethanol Red, in compliance to the findings of the genomic exploration. The recombinant L20 dT8 exhibited the highest amylolytic activity and produced more than 4 g/L of ethanol from 2% starch in a CBP setting without the addition of supplementary enzymes. Based on the performance of this strain, an amylase/glucoamylase ratio of 1:2.5 was suggested as baseline for further improvement of the CBP ability. Overall, L20 showed important traits for the future construction of a proficient CBP yeast. As such, this work shows that natural S. cerevisiae strains can be used for the expression of foreign secreted enzymes, paving the way to strain improvement for the starch-to-bioethanol route.https://www.frontiersin.org/articles/10.3389/fmicb.2021.768562/fullSaccharomyces cerevisiaedelta integrationCRISPR/Cas9starchEthanol Redconsolidated bioprocessing |
| spellingShingle | Nicoletta Gronchi Nicola De Bernardini Rosemary A. Cripwell Laura Treu Stefano Campanaro Marina Basaglia Maria R. Foulquié-Moreno Johan M. Thevelein Johan M. Thevelein Willem H. Van Zyl Lorenzo Favaro Sergio Casella Natural Saccharomyces cerevisiae Strain Reveals Peculiar Genomic Traits for Starch-to-Bioethanol Production: the Design of an Amylolytic Consolidated Bioprocessing Yeast Frontiers in Microbiology Saccharomyces cerevisiae delta integration CRISPR/Cas9 starch Ethanol Red consolidated bioprocessing |
| title | Natural Saccharomyces cerevisiae Strain Reveals Peculiar Genomic Traits for Starch-to-Bioethanol Production: the Design of an Amylolytic Consolidated Bioprocessing Yeast |
| title_full | Natural Saccharomyces cerevisiae Strain Reveals Peculiar Genomic Traits for Starch-to-Bioethanol Production: the Design of an Amylolytic Consolidated Bioprocessing Yeast |
| title_fullStr | Natural Saccharomyces cerevisiae Strain Reveals Peculiar Genomic Traits for Starch-to-Bioethanol Production: the Design of an Amylolytic Consolidated Bioprocessing Yeast |
| title_full_unstemmed | Natural Saccharomyces cerevisiae Strain Reveals Peculiar Genomic Traits for Starch-to-Bioethanol Production: the Design of an Amylolytic Consolidated Bioprocessing Yeast |
| title_short | Natural Saccharomyces cerevisiae Strain Reveals Peculiar Genomic Traits for Starch-to-Bioethanol Production: the Design of an Amylolytic Consolidated Bioprocessing Yeast |
| title_sort | natural saccharomyces cerevisiae strain reveals peculiar genomic traits for starch to bioethanol production the design of an amylolytic consolidated bioprocessing yeast |
| topic | Saccharomyces cerevisiae delta integration CRISPR/Cas9 starch Ethanol Red consolidated bioprocessing |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2021.768562/full |
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