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Research
Jufang Wang
South China University of Technology
Corresponding Author
ORCiD: https://orcid.org/0000-0002-9908-1001
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This work is licensed under a CC BY 4.0 License. Read Full License
Background: Thermophilic microbes for biofuels and chemicals have attracted great attention due to their tolerance of high temperature and wide range of substrate utilization. Thermoanaerobacterium aotearoense SCUT27 has the ability of glucose and xylose co-utilization in lignocellulosic biomass. Polygene manipulation was a bottleneck since it was hindered by available markers for selection. In this study, the endogenous Type I-B CRISPR/Cas system was developed for multiplex genome editing in SCUT27.
Results: The protospacer-adjacent motif (PAM) was identified by in silico and orotidine-5’-phosphate decarboxylase (pyrF) and then lactate dehydrogenase (ldh) were chosen as the editing target to assess the toxicity of this immune system and gene editing efficiency. The mutants could be repeatedly obtained with an editing efficiency of 58.3-100%. Higher transformation efficiency was observed after optimization of some editing strategies. Furthermore, a new method was performed for screening mutants of plasmid curing (recycling of the editing plasmid) for multiplex genome editing based on the negative selection marker tdk, and then ldh and arginine repressor (argR) were knocked out successively. The mutant SCUT27/Δldh/ΔargR had the prominent advantages over SCUT27 for ethanol production with enhanced ability to metabolize xylose. When cultured under various lignocellulosic hydrolysates, the mutant showed a satisfactory performance with the ethanol titer and yield improved by 147.42–739.40% and 112.67–267.89%, respectively, compared with SCUT27, as well as the enhanced tolerance to inhibitors.
Conclusion: The multi-gene editing by native CRISPR/Cas system is a promising strategy to engineer SCUT27 for higher ethanol production with lignocellulosic hydrolysates.
Keywords
Thermoanaerobacterium aotearoense, Type I-B CRISPR, Genome editing, Bioethanol
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CURRENT STATUS: Under Review
Version 1
Posted 03 Nov, 2021
No community comments so far
Reviews received
Received 17 Nov, 2021
Reviewer #1 agreed
On 16 Nov, 2021
Reviewers invited
Invitations sent on 31 Oct, 2021
Editor invited
On 30 Oct, 2021
Editor assigned
On 30 Oct, 2021
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On 29 Oct, 2021
First submitted
On 27 Oct, 2021
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This preprint is under consideration at Biotechnology for Biofuels. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Jufang Wang
South China University of Technology
Corresponding Author
ORCiD: https://orcid.org/0000-0002-9908-1001
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 03 Nov, 2021
No community comments so far
Reviews received
Received 17 Nov, 2021
Reviewer #1 agreed
On 16 Nov, 2021
Reviewers invited
Invitations sent on 31 Oct, 2021
Editor invited
On 30 Oct, 2021
Editor assigned
On 30 Oct, 2021
Submission checks complete
On 29 Oct, 2021
First submitted
On 27 Oct, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Biotechnology and Bioengineering
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Thermophilic microbes for biofuels and chemicals have attracted great attention due to their tolerance of high temperature and wide range of substrate utilization. Thermoanaerobacterium aotearoense SCUT27 has the ability of glucose and xylose co-utilization in lignocellulosic biomass. Polygene manipulation was a bottleneck since it was hindered by available markers for selection. In this study, the endogenous Type I-B CRISPR/Cas system was developed for multiplex genome editing in SCUT27.
Results: The protospacer-adjacent motif (PAM) was identified by in silico and orotidine-5’-phosphate decarboxylase (pyrF) and then lactate dehydrogenase (ldh) were chosen as the editing target to assess the toxicity of this immune system and gene editing efficiency. The mutants could be repeatedly obtained with an editing efficiency of 58.3-100%. Higher transformation efficiency was observed after optimization of some editing strategies. Furthermore, a new method was performed for screening mutants of plasmid curing (recycling of the editing plasmid) for multiplex genome editing based on the negative selection marker tdk, and then ldh and arginine repressor (argR) were knocked out successively. The mutant SCUT27/Δldh/ΔargR had the prominent advantages over SCUT27 for ethanol production with enhanced ability to metabolize xylose. When cultured under various lignocellulosic hydrolysates, the mutant showed a satisfactory performance with the ethanol titer and yield improved by 147.42–739.40% and 112.67–267.89%, respectively, compared with SCUT27, as well as the enhanced tolerance to inhibitors.
Conclusion: The multi-gene editing by native CRISPR/Cas system is a promising strategy to engineer SCUT27 for higher ethanol production with lignocellulosic hydrolysates.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
This is a list of supplementary files associated with this preprint. Click to download.
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