Bacterial microcompartment virus-like particles
The present invention relates to a method for producing a bacterial microcompartment virus-like particle (VLP) carrying a cargo molecule, the method comprising introducing and expressing in a host cell or organism one or more polynucleotides comprising (a) a first sequence encoding bacterial microcompartment shell protomers and a second sequence encoding a cargo molecule fused to an encapsulation peptide comprising the sequence SKITGSSGNDTQGSLITYSGGARG, and forming a microcompartment that encapsulates the cargo molecule, or (b) a first sequence encoding bacterial microcompartment shell protomers and a second sequence encoding at least one of said protomers fused with a cargo molecule or a biochemical tag, and forming a microcompartment that expresses the cargo molecule or biochemical tag on an exterior surface. In one embodiment, the bacterial microcompartment protomers are CsoSIA and CsoS4A from Halothiobacillus neapolitanus , or HO-H, HO-P and HO-T1 from Haliangium ochraceum.
1 . A method for producing a bacterial microcompartment virus-like particle (VLP) carrying a cargo molecule, said method comprising
A) introducing into a host cell or organism one or more heterologous polynucleotides comprising
(i) a first sequence encoding bacterial microcompartment shell protomers; and
(ii) a second sequence encoding a cargo molecule fused to an encapsulation peptide, wherein the encapsulation peptide comprises the amino acid sequence set forth in SEQ ID NO: 1 (SKITGSSGNDTQGSLITYSGGARG) or SEQ ID NO: 94 (KPEKPGSKITGSSGNDTQGSLITYSGGARG), or a functional variant thereof;
a) expressing the first and second sequences; and
b) forming a microcompartment that encapsulates the cargo molecule; or
B) introducing into a host cell or organism one or more polynucleotides comprising
(i) a first sequence encoding bacterial microcompartment shell protomers, wherein the bacterial microcompartment protomers consist of HO-H comprising the amino acid sequence set forth in SEQ ID NO: 4, HO-P comprising the amino acid sequence set forth in SEQ ID NO: 5 and HO-T1 comprising the amino acid sequence set forth in SEQ ID NO: 6 from Haliangium ochraceum, and variants thereof; and
(ii) a second sequence encoding at least one of said protomers fused with a cargo molecule or a biochemical tag;
a) expressing the first and second sequences; and
b) forming a microcompartment that expresses the cargo molecule on an exterior surface, or
c) forming a microcompartment that expresses the biochemical tag on an exterior surface to which a cargo molecule comprising a complementary tag can bind.
2 . The method of claim 1 , wherein the functional variant of the encapsulation peptide set forth in SEQ ID NO: 1 comprises, at its amino terminus, 1, 2, 3, 4, or 5 of the additional amino acids at the amino terminus of SEQ ID NO: 94.
3 . The method of claim 1 , wherein:
the bacterial microcompartment protomers in A)(i) are CsoS1A (SEQ ID NO: 2) and CsoS4A (SEQ ID NO: 3) from Halothiobacillus neapolitanus ; and variants thereof.
4 . The method of claim 1 , wherein:
i) the cargo molecule is at least one peptide, and/or
ii) the biochemical tag is selected from the group comprising Streptavidin-binding Strep-tag II peptide (Strep-Tag II), Streptococcus pyogenes -derived SpyCatcher protein/SpyTag peptide covalent binding pair (SpyCatcher/SpyTag pair) and Coiled-Coil Dimer A peptide/Coiled-Coil Dimer B peptide (CC-Di-A/B pair).
5 . The method of claim 3 , wherein the expression of CsoS1A is controlled by promoter P T7 ; CsoS4A is controlled by promoter P CON5 ; HO-H is controlled by yeast promoter P TDH3 ; HO-P is controlled by yeast promoter P PYK1 and HO-T1 is controlled by yeast promoter P YEF3 .
6 . The method of claim 1 , wherein the host organism is E. coli or S. cerevisiae.
7 . An engineered bacterial microcompartment VLP carrying a cargo molecule, comprising:
i) bacterial microcompartment shell protomers, and a cargo molecule fused to an encapsulation peptide, wherein the encapsulation peptide comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 94, or a functional variant thereof; or
ii) bacterial microcompartment shell protomers and a cargo molecule, wherein the cargo molecule is fused to an end of at least one of said protomers, or wherein at least one of said protomers is fused to a tag and a cargo molecule comprising a complementary tag is bound to it on the exterior surface of the VLP, wherein the bacterial microcompartment protomers consist of HO-H comprising the amino acid sequence set forth in SEQ ID NO: 4, HO-P comprising the amino acid sequence set forth in SEQ ID NO: 5 and HO-T1 comprising the amino acid sequence set forth in SEQ ID NO: 6 from Haliangium ochraceum , and variants thereof.
8 . The engineered VLP of claim 7 , wherein the functional variant of the encapsulation peptide set forth in SEQ ID NO: 1 comprises, at its amino terminus, 1, 2, 3, 4, or 5 of the additional amino acids at the amino terminus of SEQ ID NO: 94.
9 . The engineered VLP of claim 7 , wherein
the bacterial microcompartment protomers of i) are CsoS1A comprising the amino acid sequence set forth in SEQ ID NO: 2 and CsoS4A comprising the amino acid sequence set forth in SEQ ID NO: 3 from Halothiobacillus neapolitanus ; and variants thereof.
10 . The engineered VLP of claim 7 , wherein the cargo molecule is at least one peptide.
11 . The engineered VLP of claim 7 , wherein the biochemical tag is selected from the group comprising Strep-Tag II peptide (Strep-Tag II), Streptococcus pyogenes -derived SpyCatcher protein/SpyTag peptide covalent binding pair (SpyCatcher/SpyTag pair) and Coiled-Coil Dimer A peptide/Coiled-Coil Dimer B peptide (CC-Di-A/B pair).
12 . An isolated plasmid or vector comprising:
a) a first DNA sequence that encodes bacterial microcompartment shell protomers, each of which is operably linked to a promoter, and
b) a second DNA sequence that encodes a cargo molecule fused to an encapsulation peptide, operably linked to a promoter, wherein the encapsulation peptide comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 94, or a functional variant thereof; or c) a first DNA sequence that encodes bacterial microcompartment shell protomers, each of which is operably linked to a promoter, wherein the bacterial microcompartment protomers consist of HO-H comprising the amino acid sequence set forth in SEQ ID NO: 4, HO-P comprising the amino acid sequence set forth in SEQ ID NO: 5 and HO-T1 comprising the amino acid sequence set forth in SEQ ID NO: 6 from Haliangium ochraceum , and variants thereof, and d) a second DNA sequence encoding at least one of said protomers fused with a cargo molecule or a biochemical tag.
13 . The isolated plasmid or vector of claim 12 , wherein the functional variant of the encapsulation peptide set forth in SEQ ID NO: 1 comprises, at its amino terminus, 1, 2, 3, 4, or 5 of the additional amino acids at the amino terminus of SEQ ID NO: 94.
14 . The isolated plasmid or vector of claim 12 , wherein the DNA sequences encoding said bacterial microcompartment shell protomers, cargo molecules and tags have at least 70%, at least 80%, at least 90%, or 100% identity with SEQ ID Nos 7-12 and 95 due to redundancy of the genetic code.
15 . A composition comprising at least one engineered VLP of claim 7 .
16 . The composition of claim 15 , wherein the at least one engineered VLP comprises;
i) an enzyme for conversion of a prodrug; and/or
ii) one or more additional therapeutic agents.
17 . The isolated plasmid or vector nucleic acid of claim 12 , wherein the bacterial microcompartment protomers of a) are CsoS1A comprising the amino acid sequence set forth in SEQ ID NO: 2 and CsoS4A comprising the amino acid sequence set forth in SEQ ID NO: 3 from Halothiobacillus neapolitanus and variants thereof.