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.
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Maximilian Flaiz
Ulm University: Universitat Ulm
Corresponding Author
ORCiD: https://orcid.org/0000-0003-0238-4995
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Background: The interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Eubacterium limosum is one of the few acetogens that can utilize methanol, is genetically accessible and, therefore, a promising candidate for the recombinant production of biocommodities from this C1 carbon source. Although several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited.
Results: In this study, we expanded the genetic toolbox of E. limosum by implementing the fluorescence-activating and absorption shifting tag (FAST) as a fluorescent reporter protein. Recombinant E. limosum strains that expressed the gene encoding FAST in an inducible and constitutive manner were constructed. Cultivation of these recombinant strains resulted in brightly fluorescent cells even under anaerobic conditions. Moreover, we produced the biocommodities butanol and acetone from methanol with recombinant E. limosum strains. Therefore, we used E. limosum cultures that produced FAST-tagged fusion proteins of the bifunctional acetaldehyde/alcohol dehydrogenase or the acetoacetate decarboxylase, respectively, and determined the fluorescence intensity and product yields during growth.
Conclusions: The addition of FAST as an oxygen-independent fluorescent reporter protein expands the genetic toolbox of E. limosum. Moreover, our results show that FAST-tagged fusion proteins can be constructed without negatively impacting the stability, functionality, and productivity of the resulting enzyme. Finally, butanol and acetone can be produced from methanol using recombinant E. limosum strains expressing genes encoding fluorescent FAST-tagged fusion proteins.
Keywords
acetogens, anaerobes, C1-substrates, fluorescence-activating and absorption shifting tag, fluorescence reporter system, fusion protein
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Additional file 1 Fig. S1 Growth experiment with E. limosum [pMTL83251_PbgaL_AdhE2] and E. limosum [pMTL83251]. Strains were cultivated using 30 mM glucose (A) or 100 mM methanol (B) as carbon source. Gene expression of cells was either induced by lactose or non-induced. Induction with lactose is indicated with the vertical dotted line. Monitored are OD600, methanol consumption, as well as acetate, butyrate, ethanol, and butanol production. Error bars indicate standard deviations. n=3. Fig. S2 Growth experiment with E. limosum [pMTL83251_PthlA_act] and E. limosum [pMTL83251]. Strains were cultivated using 30 mM glucose (A) or 100 mM methanol (B) as carbon source. Monitored are OD600, glucose and methanol consumption, as well as acetate, butyrate, and acetone production. Error bars indicate standard deviations. n=3. Table S1 Growth characteristics and product formation of recombinant E. limosum strains characterized in growth experiments using glucose as carbon source. Table S2 Growth characteristics and product formation of recombinant E. limosum strains characterized in growth experiments using methanol as carbon source.
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On 26 Mar, 2021
Editor assigned
On 26 Mar, 2021
Reviewers invited
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On 04 Mar, 2021
Review #1 received
Received 27 Feb, 2021
Review #2 received
Received 18 Feb, 2021
Review #3 received
Received 17 Feb, 2021
Reviewer #3 agreed
On 03 Feb, 2021
Reviews received
Received 03 Feb, 2021
Reviewer #2 agreed
On 01 Feb, 2021
Reviewers invited
Invitations sent on 28 Jan, 2021
Reviewer #1 agreed
On 28 Jan, 2021
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On 27 Jan, 2021
Submission checks complete
On 27 Jan, 2021
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On 27 Jan, 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
Maximilian Flaiz
Ulm University: Universitat Ulm
Corresponding Author
ORCiD: https://orcid.org/0000-0003-0238-4995
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 (no preprint)
Posted 27 Apr, 2021
Reviewer #1 agreed
On 27 Apr, 2021
Review #1 received
Received 27 Apr, 2021
Reviewers invited
Invitations sent on 25 Apr, 2021
Submission checks complete
On 20 Apr, 2021
Editor invited
On 20 Apr, 2021
Editor assigned
On 26 Mar, 2021
First submitted
On 23 Mar, 2021
This version was not preprinted
Version (no preprint)
Posted 08 Apr, 2021
Review #3 received
Received 08 Apr, 2021
Review #1 received
Received 04 Apr, 2021
Review #2 received
Received 04 Apr, 2021
Reviewer #3 agreed
On 29 Mar, 2021
Reviewer #2 agreed
On 26 Mar, 2021
Editor assigned
On 26 Mar, 2021
Reviewers invited
Invitations sent on 26 Mar, 2021
Reviewer #1 agreed
On 26 Mar, 2021
Submission checks complete
On 26 Mar, 2021
Editor invited
On 26 Mar, 2021
This version was not preprinted
Version 1
Posted 04 Feb, 2021
No community comments so far
Editorial decision: Minor revision
On 04 Mar, 2021
Review #1 received
Received 27 Feb, 2021
Review #2 received
Received 18 Feb, 2021
Review #3 received
Received 17 Feb, 2021
Reviewer #3 agreed
On 03 Feb, 2021
Reviews received
Received 03 Feb, 2021
Reviewer #2 agreed
On 01 Feb, 2021
Reviewers invited
Invitations sent on 28 Jan, 2021
Reviewer #1 agreed
On 28 Jan, 2021
Editor assigned
On 27 Jan, 2021
Submission checks complete
On 27 Jan, 2021
Editor invited
On 27 Jan, 2021
First submitted
On 27 Jan, 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: The interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Eubacterium limosum is one of the few acetogens that can utilize methanol, is genetically accessible and, therefore, a promising candidate for the recombinant production of biocommodities from this C1 carbon source. Although several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited.
Results: In this study, we expanded the genetic toolbox of E. limosum by implementing the fluorescence-activating and absorption shifting tag (FAST) as a fluorescent reporter protein. Recombinant E. limosum strains that expressed the gene encoding FAST in an inducible and constitutive manner were constructed. Cultivation of these recombinant strains resulted in brightly fluorescent cells even under anaerobic conditions. Moreover, we produced the biocommodities butanol and acetone from methanol with recombinant E. limosum strains. Therefore, we used E. limosum cultures that produced FAST-tagged fusion proteins of the bifunctional acetaldehyde/alcohol dehydrogenase or the acetoacetate decarboxylase, respectively, and determined the fluorescence intensity and product yields during growth.
Conclusions: The addition of FAST as an oxygen-independent fluorescent reporter protein expands the genetic toolbox of E. limosum. Moreover, our results show that FAST-tagged fusion proteins can be constructed without negatively impacting the stability, functionality, and productivity of the resulting enzyme. Finally, butanol and acetone can be produced from methanol using recombinant E. limosum strains expressing genes encoding fluorescent FAST-tagged fusion proteins.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
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