Production of the biocommodities butanol and acetone from methanol with fluorescent FAST-tagged proteins using metabolically engineered strains of Eubacterium limosum.
Acetogens
Anaerobes
C1-substrates
Fluorescence reporter system
Fluorescence-activating and absorption shifting tag
Fusion protein
Journal
Biotechnology for biofuels
ISSN: 1754-6834
Titre abrégé: Biotechnol Biofuels
Pays: England
ID NLM: 101316935
Informations de publication
Date de publication:
10 May 2021
10 May 2021
Historique:
received:
27
01
2021
accepted:
29
04
2021
entrez:
11
5
2021
pubmed:
12
5
2021
medline:
12
5
2021
Statut:
epublish
Résumé
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. 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 concentrations during growth. 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.
Sections du résumé
BACKGROUND
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
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 concentrations during growth.
CONCLUSIONS
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.
Identifiants
pubmed: 33971948
doi: 10.1186/s13068-021-01966-2
pii: 10.1186/s13068-021-01966-2
pmc: PMC8111989
doi:
Types de publication
Journal Article
Langues
eng
Pagination
117Subventions
Organisme : ERACoBioTech
ID : 161B0609A
Références
Sci Rep. 2018 Jul 4;8(1):10137
pubmed: 29973667
ACS Synth Biol. 2019 Sep 20;8(9):2059-2068
pubmed: 31373788
Chem Soc Rev. 2009 Oct;38(10):2887-921
pubmed: 19771335
J Biotechnol. 2018 Jan 10;265:119-126
pubmed: 29158189
Chem Sci. 2017 Aug 1;8(8):5598-5605
pubmed: 28970939
Biochemistry. 2018 Oct 2;57(39):5648-5653
pubmed: 30204425
Metab Eng. 2018 Nov;50:173-191
pubmed: 30055325
Annu Rev Physiol. 2017 Feb 10;79:93-117
pubmed: 27860833
Int J Mol Sci. 2020 Oct 15;21(20):
pubmed: 33076477
Curr Protoc Cell Biol. 2005 Jul;Chapter 21:21.4.1-21.4.13
pubmed: 18228466
Mol Microbiol. 2002 Sep;45(5):1191-6
pubmed: 12207688
Nat Biotechnol. 2007 Apr;25(4):443-5
pubmed: 17351616
Bioresour Technol. 2017 Dec;245(Pt A):560-566
pubmed: 28898856
J Bacteriol. 1997 Sep;179(17):5442-7
pubmed: 9286999
Mol Cell. 2020 Oct 15;80(2):193-209
pubmed: 33010203
J Microbiol Methods. 2015 Dec;119:37-43
pubmed: 26427827
Appl Environ Microbiol. 2020 Oct 1;86(20):
pubmed: 32769192
Biomacromolecules. 2019 Sep 9;20(9):3271-3282
pubmed: 31066546
J Bacteriol. 1951 Sep;62(3):293-300
pubmed: 14888646
Yeast. 2004 Jun;21(8):661-70
pubmed: 15197731
Water Sci Technol. 2001;44(8):7-14
pubmed: 11730139
Sci Rep. 2016 Mar 21;6:23463
pubmed: 26996606
Biotechnol Biofuels. 2016 Oct 18;9:219
pubmed: 27777621
Antimicrob Agents Chemother. 2003 Dec;47(12):3682-7
pubmed: 14638465
World J Microbiol Biotechnol. 2016 Jul;32(7):119
pubmed: 27263014
Front Microbiol. 2016 Jul 07;7:1036
pubmed: 27458439
Appl Environ Microbiol. 1987 Mar;53(3):471-6
pubmed: 3579266
Proc Natl Acad Sci U S A. 2016 Jan 19;113(3):497-502
pubmed: 26711992
Appl Environ Microbiol. 2012 Nov;78(22):8112-21
pubmed: 22983967
J Bacteriol. 2002 Feb;184(3):821-30
pubmed: 11790753
Appl Environ Microbiol. 2013 Feb;79(4):1102-9
pubmed: 23204413
J Microbiol Methods. 2009 Jul;78(1):79-85
pubmed: 19445976
ACS Synth Biol. 2020 Jun 19;9(6):1426-1440
pubmed: 32379961
Microb Biotechnol. 2021 Feb 25;:
pubmed: 33629808
Appl Microbiol Biotechnol. 2020 Jan;104(2):687-699
pubmed: 31807888
Trends Biotechnol. 2009 Feb;27(2):107-15
pubmed: 19111927
Biotechnol Biofuels. 2016 Apr 26;9:92
pubmed: 27118994
Phys Biol. 2015 Dec 10;12(6):066016
pubmed: 26656747
Appl Environ Microbiol. 1988 Nov;54(11):2717-22
pubmed: 16347774
J Biotechnol. 2019 Nov 10;305:18-22
pubmed: 31472166
Metab Eng. 2018 Jul;48:243-253
pubmed: 29906505
PLoS One. 2016 Aug 15;11(8):e0160711
pubmed: 27525986
Appl Environ Microbiol. 2014 Apr;80(8):2410-6
pubmed: 24509933
Appl Environ Microbiol. 1981 Jul;42(1):12-9
pubmed: 6791591
Metab Eng. 2017 May;41:202-211
pubmed: 28442386
Adv Drug Deliv Rev. 2013 Oct;65(10):1357-69
pubmed: 23026637
Nat Commun. 2016 Sep 30;7:12800
pubmed: 27687501
Appl Environ Microbiol. 2019 Jul 1;85(14):
pubmed: 31076434
Cell. 2014 Oct 23;159(3):635-46
pubmed: 25307933
Nature. 2009 May 21;459(7245):393-7
pubmed: 19458715
Appl Environ Microbiol. 1997 Nov;63(11):4543-51
pubmed: 9361441
Proc Natl Acad Sci U S A. 1997 Jul 22;94(15):8168-72
pubmed: 9223333
Appl Environ Microbiol. 2011 Jan;77(2):471-8
pubmed: 21097603
Appl Environ Microbiol. 2015 Mar;81(5):1652-60
pubmed: 25527559
Sci Rep. 2016 Aug 16;6:31518
pubmed: 27527841
Science. 1994 Feb 11;263(5148):802-5
pubmed: 8303295
Chem Rev. 2002 Mar;102(3):759-81
pubmed: 11890756
mBio. 2020 Sep 1;11(5):
pubmed: 32873766
Curr Opin Biotechnol. 2020 Apr;62:168-180
pubmed: 31733545
Proc Natl Acad Sci U S A. 2010 Jul 20;107(29):13087-92
pubmed: 20616070
Nat Chem Biol. 2021 Jan;17(1):30-38
pubmed: 32778846
Sci Rep. 2018 Aug 14;8(1):12088
pubmed: 30108248
Nat Biotechnol. 2004 Dec;22(12):1567-72
pubmed: 15558047
FEMS Microbiol Rev. 2021 Mar 16;45(2):
pubmed: 32901799
Environ Microbiol. 2018 Dec;20(12):4369-4384
pubmed: 30003650
Adv Appl Microbiol. 2018;103:143-221
pubmed: 29914657
Front Microbiol. 2020 Mar 11;11:402
pubmed: 32218779
Metab Eng. 2016 Jul;36:37-47
pubmed: 26971669
ACS Synth Biol. 2016 Dec 16;5(12):1355-1361
pubmed: 27276212
Angew Chem Int Ed Engl. 2020 Oct 5;59(41):17917-17923
pubmed: 32568417