Split intein mediated protein polymerization for microbial production of materials
The present disclosure is directed to systems and methods for synthesizing a spidroin. In some embodiments, the methods comprise synthesizing a monomer in vivo in a heterologous host, the monomer comprising an N-terminus IntC domain and a C-terminus IntN domain, and post-translationally polymerizing the synthesized monomer via in vitro split-intein mediated polymerization.
1. A method for synthesizing a spidroin, the method comprising:
synthesizing a spidroin fragment in vivo in a heterologous host, the spidroin fragment comprising an N-terminus Int C domain comprising the amino acid sequence of SEQ ID NO: 10 and a C-terminus Int N domain comprising the amino acid sequence of SEQ ID NO: 11;
producing the spidroin fragment from the heterologous host; and
post-translationally polymerizing the synthesized spidroin fragment via in vitro split-intein mediated polymerization;
wherein the spidroin fragment is a silk amino acid sequence from a spider species.
2. The method of claim 1 , wherein the heterologous host is a protein-expressing microbial host.
3. The method of claim 1 , wherein the synthesized spidroin has a molecular weight of at least 300 kDa.
4. The method of claim 1 , further comprising spinning the synthesized spidroin into fibers.
5. The method of claim 4 , wherein the fibers have a tensile strength of from about 0.9 GPa to about 1.3 GPa.
6. The method of claim 4 , wherein the fibers have an extensibility of from about 10% to about 25%.
7. The method of claim 4 , wherein the fibers have a toughness of from about 60 MJ/m 3 to about 170 MJ/m 3 .
8. The method of claim 4 , wherein the fibers have a β-sheet content of from about 35% to about 45%.
9. A method for synthesizing a spidroin, the method comprising:
synthesizing a seed protein in vivo in a heterologous host, the seed protein comprising a C-terminus Int N domain;
synthesizing a spidroin fragment in vivo in the heterologous host, the spidroin fragment comprising an N-terminus Int C domain comprising the amino acid sequence of SEQ ID NO: 10 and a C-terminus Int N domain comprising the amino acid sequence of SEQ ID NO: 11; and
co-translationally polymerizing the spidroin fragment via in vivo split-intein mediated polymerization;
wherein the spidroin fragment is a silk amino acid sequence from a spider species.
10. The method of claim 9 , wherein the heterologous host is a protein-expressing microbial host.
11. The method of claim 9 , wherein the synthesized spidroin is a linear polymer.
12. The method of claim 9 , wherein the synthesized spidroin has a molecular weight of at least 300 kDa.
13. The method of claim 9 , further comprising spinning the synthesized spidroin into fibers.
14. The method of claim 13 , wherein the fibers have a tensile strength of from about 170 MPa to about 310 MPa.
15. The method of claim 13 , wherein the fibers have a toughness of from about 55 MJ/m 3 to about 105 MJ/m 3 .