Expression and secretion of a lytic polysaccharide monooxygenase by a fast-growing cyanobacterium

D. A. Russo, J. A.Z. Zedler, D. N. Wittmann, B. Möllers, R. K. Singh, T. S. Batth, B. Van Oort, J. V. Olsen, M. J. Bjerrum, P. E. Jensen

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

Background: Cyanobacteria have the potential to become next-generation cell factories due to their ability to use CO 2 , light and inorganic nutrients to produce a range of biomolecules of commercial interest. Synechococcus elongatus UTEX 2973, in particular, is a fast-growing, genetically tractable, cyanobacterium that has garnered attention as a potential biotechnological chassis. To establish this unique strain as a host for heterologous protein production, we aimed to demonstrate expression and secretion of the industrially relevant TfAA10A, a lytic polysaccharide monooxygenase from the Gram-positive bacterium Thermobifida fusca. Results: Two variations of TfAA10A were successfully expressed in S. elongatus UTEX 2973: One containing the native N-terminal, Sec-targeted, signal peptide and a second with a Tat-targeted signal peptide from the Escherichia coli trimethylamine-N-oxide reductase (TorA). Although the TorA signal peptide correctly targeted the protein to the plasma membrane, the majority of the TorA-TfAA10A was found unprocessed in the plasma membrane with a small fraction of the mature protein ultimately translocated to the periplasm. The native Sec signal peptide allowed for efficient secretion of TfAA10A into the medium with virtually no protein being found in the cytosol, plasma membrane or periplasm. TfAA10A was demonstrated to be correctly cleaved and active on the model substrate phosphoric acid swollen cellulose. Additionally, expression and secretion only had a minor impact on cell growth. The secretion yield was estimated at 779 ± 40 μg L -1 based on densitometric analysis. To our knowledge, this is the highest secretion yield ever registered in cyanobacteria. Conclusions: We have shown for the first time high-titer expression and secretion of an industrially relevant and catalytically active enzyme in S. elongatus UTEX 2973. This proof-of-concept study will be valuable for the development of novel and sustainable applications in the fields of bioremediation and biocatalysis.

Original languageEnglish
Article number74
Pages (from-to)1-13
Number of pages13
JournalBiotechnology for Biofuels
Volume12
DOIs
Publication statusPublished - 1 Apr 2019

Fingerprint

Cyanobacteria
Polysaccharides
Protein Sorting Signals
Mixed Function Oxygenases
polysaccharide
secretion
cyanobacterium
Cell membranes
Proteins
peptide
Periplasm
Cell Membrane
Oxides
protein
Biocatalysis
oxide
membrane
Synechococcus
plasma
Environmental Biodegradation

Keywords

  • Bacterial secretion
  • Cyanobacteria
  • General secretory pathway
  • Lytic polysaccharide monooxygenase
  • Synechococcus elongatus UTEX 2973
  • TfAA10A
  • Twin-arginine-translocation

Cite this

Russo, D. A., Zedler, J. A. Z., Wittmann, D. N., Möllers, B., Singh, R. K., Batth, T. S., ... Jensen, P. E. (2019). Expression and secretion of a lytic polysaccharide monooxygenase by a fast-growing cyanobacterium. Biotechnology for Biofuels, 12, 1-13. [74]. https://doi.org/10.1186/s13068-019-1416-9
Russo, D. A. ; Zedler, J. A.Z. ; Wittmann, D. N. ; Möllers, B. ; Singh, R. K. ; Batth, T. S. ; Van Oort, B. ; Olsen, J. V. ; Bjerrum, M. J. ; Jensen, P. E. / Expression and secretion of a lytic polysaccharide monooxygenase by a fast-growing cyanobacterium. In: Biotechnology for Biofuels. 2019 ; Vol. 12. pp. 1-13.
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abstract = "Background: Cyanobacteria have the potential to become next-generation cell factories due to their ability to use CO 2 , light and inorganic nutrients to produce a range of biomolecules of commercial interest. Synechococcus elongatus UTEX 2973, in particular, is a fast-growing, genetically tractable, cyanobacterium that has garnered attention as a potential biotechnological chassis. To establish this unique strain as a host for heterologous protein production, we aimed to demonstrate expression and secretion of the industrially relevant TfAA10A, a lytic polysaccharide monooxygenase from the Gram-positive bacterium Thermobifida fusca. Results: Two variations of TfAA10A were successfully expressed in S. elongatus UTEX 2973: One containing the native N-terminal, Sec-targeted, signal peptide and a second with a Tat-targeted signal peptide from the Escherichia coli trimethylamine-N-oxide reductase (TorA). Although the TorA signal peptide correctly targeted the protein to the plasma membrane, the majority of the TorA-TfAA10A was found unprocessed in the plasma membrane with a small fraction of the mature protein ultimately translocated to the periplasm. The native Sec signal peptide allowed for efficient secretion of TfAA10A into the medium with virtually no protein being found in the cytosol, plasma membrane or periplasm. TfAA10A was demonstrated to be correctly cleaved and active on the model substrate phosphoric acid swollen cellulose. Additionally, expression and secretion only had a minor impact on cell growth. The secretion yield was estimated at 779 ± 40 μg L -1 based on densitometric analysis. To our knowledge, this is the highest secretion yield ever registered in cyanobacteria. Conclusions: We have shown for the first time high-titer expression and secretion of an industrially relevant and catalytically active enzyme in S. elongatus UTEX 2973. This proof-of-concept study will be valuable for the development of novel and sustainable applications in the fields of bioremediation and biocatalysis.",
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Russo, DA, Zedler, JAZ, Wittmann, DN, Möllers, B, Singh, RK, Batth, TS, Van Oort, B, Olsen, JV, Bjerrum, MJ & Jensen, PE 2019, 'Expression and secretion of a lytic polysaccharide monooxygenase by a fast-growing cyanobacterium' Biotechnology for Biofuels, vol. 12, 74, pp. 1-13. https://doi.org/10.1186/s13068-019-1416-9

Expression and secretion of a lytic polysaccharide monooxygenase by a fast-growing cyanobacterium. / Russo, D. A.; Zedler, J. A.Z.; Wittmann, D. N.; Möllers, B.; Singh, R. K.; Batth, T. S.; Van Oort, B.; Olsen, J. V.; Bjerrum, M. J.; Jensen, P. E.

In: Biotechnology for Biofuels, Vol. 12, 74, 01.04.2019, p. 1-13.

Research output: Contribution to JournalArticleAcademicpeer-review

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T1 - Expression and secretion of a lytic polysaccharide monooxygenase by a fast-growing cyanobacterium

AU - Russo, D. A.

AU - Zedler, J. A.Z.

AU - Wittmann, D. N.

AU - Möllers, B.

AU - Singh, R. K.

AU - Batth, T. S.

AU - Van Oort, B.

AU - Olsen, J. V.

AU - Bjerrum, M. J.

AU - Jensen, P. E.

PY - 2019/4/1

Y1 - 2019/4/1

N2 - Background: Cyanobacteria have the potential to become next-generation cell factories due to their ability to use CO 2 , light and inorganic nutrients to produce a range of biomolecules of commercial interest. Synechococcus elongatus UTEX 2973, in particular, is a fast-growing, genetically tractable, cyanobacterium that has garnered attention as a potential biotechnological chassis. To establish this unique strain as a host for heterologous protein production, we aimed to demonstrate expression and secretion of the industrially relevant TfAA10A, a lytic polysaccharide monooxygenase from the Gram-positive bacterium Thermobifida fusca. Results: Two variations of TfAA10A were successfully expressed in S. elongatus UTEX 2973: One containing the native N-terminal, Sec-targeted, signal peptide and a second with a Tat-targeted signal peptide from the Escherichia coli trimethylamine-N-oxide reductase (TorA). Although the TorA signal peptide correctly targeted the protein to the plasma membrane, the majority of the TorA-TfAA10A was found unprocessed in the plasma membrane with a small fraction of the mature protein ultimately translocated to the periplasm. The native Sec signal peptide allowed for efficient secretion of TfAA10A into the medium with virtually no protein being found in the cytosol, plasma membrane or periplasm. TfAA10A was demonstrated to be correctly cleaved and active on the model substrate phosphoric acid swollen cellulose. Additionally, expression and secretion only had a minor impact on cell growth. The secretion yield was estimated at 779 ± 40 μg L -1 based on densitometric analysis. To our knowledge, this is the highest secretion yield ever registered in cyanobacteria. Conclusions: We have shown for the first time high-titer expression and secretion of an industrially relevant and catalytically active enzyme in S. elongatus UTEX 2973. This proof-of-concept study will be valuable for the development of novel and sustainable applications in the fields of bioremediation and biocatalysis.

AB - Background: Cyanobacteria have the potential to become next-generation cell factories due to their ability to use CO 2 , light and inorganic nutrients to produce a range of biomolecules of commercial interest. Synechococcus elongatus UTEX 2973, in particular, is a fast-growing, genetically tractable, cyanobacterium that has garnered attention as a potential biotechnological chassis. To establish this unique strain as a host for heterologous protein production, we aimed to demonstrate expression and secretion of the industrially relevant TfAA10A, a lytic polysaccharide monooxygenase from the Gram-positive bacterium Thermobifida fusca. Results: Two variations of TfAA10A were successfully expressed in S. elongatus UTEX 2973: One containing the native N-terminal, Sec-targeted, signal peptide and a second with a Tat-targeted signal peptide from the Escherichia coli trimethylamine-N-oxide reductase (TorA). Although the TorA signal peptide correctly targeted the protein to the plasma membrane, the majority of the TorA-TfAA10A was found unprocessed in the plasma membrane with a small fraction of the mature protein ultimately translocated to the periplasm. The native Sec signal peptide allowed for efficient secretion of TfAA10A into the medium with virtually no protein being found in the cytosol, plasma membrane or periplasm. TfAA10A was demonstrated to be correctly cleaved and active on the model substrate phosphoric acid swollen cellulose. Additionally, expression and secretion only had a minor impact on cell growth. The secretion yield was estimated at 779 ± 40 μg L -1 based on densitometric analysis. To our knowledge, this is the highest secretion yield ever registered in cyanobacteria. Conclusions: We have shown for the first time high-titer expression and secretion of an industrially relevant and catalytically active enzyme in S. elongatus UTEX 2973. This proof-of-concept study will be valuable for the development of novel and sustainable applications in the fields of bioremediation and biocatalysis.

KW - Bacterial secretion

KW - Cyanobacteria

KW - General secretory pathway

KW - Lytic polysaccharide monooxygenase

KW - Synechococcus elongatus UTEX 2973

KW - TfAA10A

KW - Twin-arginine-translocation

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