Production of the Biocommodities Butanol and Acetone from Methanol with Fluorescent FAST-tagged Proteins using Metabolically Engineered Strains of Eubacterium Limosum
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.
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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.
Posted 04 Feb, 2021
Received 18 Feb, 2021
Received 17 Feb, 2021
On 03 Feb, 2021
Received 03 Feb, 2021
On 01 Feb, 2021
Invitations sent on 28 Jan, 2021
On 28 Jan, 2021
On 27 Jan, 2021
On 27 Jan, 2021
On 27 Jan, 2021
On 27 Jan, 2021
Production of the Biocommodities Butanol and Acetone from Methanol with Fluorescent FAST-tagged Proteins using Metabolically Engineered Strains of Eubacterium Limosum
Posted 04 Feb, 2021
Received 18 Feb, 2021
Received 17 Feb, 2021
On 03 Feb, 2021
Received 03 Feb, 2021
On 01 Feb, 2021
Invitations sent on 28 Jan, 2021
On 28 Jan, 2021
On 27 Jan, 2021
On 27 Jan, 2021
On 27 Jan, 2021
On 27 Jan, 2021
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