US 11,629,173 B2 · Compositions and methods using methanotrophic S-layer proteins for expression of heterologous proteins

Abstract

In alternative embodiments, provided are compositions and methods for making a chimeric polypeptide comprising an S-layer polypeptide and a heterologous polypeptide or peptide. In alternative embodiments, the compositions and methods comprise recombinantly engineering a methylotrophic or methanotrophic bacteria to recombinantly express a chimeric polypeptide comprising an S-layer polypeptide and a heterologous polypeptide or peptide. Also provided are compositions and methods for displaying or immobilizing proteins on a methanotrophic S-layer. In alternative embodiments, provided are compositions and methods comprising recombinant methylotrophic or methanotrophic bacteria comprising assembled or self-assembled recombinant or isolated chimeric S-layer polypeptides. In alternative embodiments, provided are compositions and methods using recombinant methylotrophic or methanotrophic bacteria, optionally a Methylomicrobium alcaliphilum, optionally a M. alcaliphilum sp. 20Z, for ectoine ((4S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid), for the production or synthesis of a protein, e.g., an ectoine, or an enzyme, e.g., a lipase.

Claims20

Dependent claims

A method for making a chimeric S-layer fusion protein, or a self-aggregating or self-assembling fragment thereof, and a heterologous polypeptide or peptide, the method comprising:

(a) recombinantly engineering a methylotrophic or methanotrophic bacteria to recombinantly express a chimeric fusion polypeptide comprising an S-layer polypeptide and a heterologous polypeptide or peptide,
wherein the methylotrophic or methanotrophic bacteria comprises a type I secretion system,
and the methylotrophic or methanotrophic bacteria is selected from the group consisting of a Methylococcus, a Methylomonas, a Methylomicrobium, a Methylobacter, a Methylomarinum, a Methylovulum, a Methylocaldum, a Methylothermus, a Methylomarinovum, a Methylosphaera, a Methylocystis, and a Methylosinus bacteria;
and the S-layer polypeptide is derived from a methylotrophic or methanotrophic bacteria selected from the group consisting of a Methylococcus, a Methylomonas, a Methylomicrobium, a Methylobacter, a Methylomarinum, a Methylovulum, a Methylocaldum, a Methylothermus, a Methylomarinovum, a Methylosphaera, a Methylocystis, and a Methylosinus bacteria, and
(b) culturing the recombinantly engineered methylotrophic or methanotrophic bacteria under culture conditions wherein the chimeric S-lay fusion protein is secreted extracellularly or is at least in part partially exposed to an extracellular environment or milieu by the recombinantly engineered methylotrophic or methanotrophic bacteria.

2 more elements

A recombinant methylotrophic or methanotrophic bacteria, optionally a Methylomicrobium alcaliphilum (M. alcaliphilum), optionally a M. alcaliphilum sp. 20Z, for ectoine ((4S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) production or synthesis, wherein:

(a) the recombinant or engineered methylotrophic or methanotrophic bacteria comprises an ectoine biosynthetic gene cluster organized as one operon (ectABC-ask), wherein the operon comprises genes encoding:
a diaminobutyric acid (DABA) aminotransferase (EctB);
a DABA acetyltransferase (EctA), and an ectoine synthase (EctC);
and
(b) the recombinant or engineered methylotrophic or methanotrophic bacteria:
(i) is engineered to lack or not express a functional EctR1 repressor;
(ii) comprises an isocitrate lyase/malate synthase fusion (transcriptionally controlled by a hps promoter (Phps);
and/or,
(iii) comprises one or more of the genetic modifications selected from the group consisting of:
(1) deletion or inactivation of a MarR transcriptional regulator gene;
(2) deletion or inactivation of a sucrose-phosphate synthase gene;
(3) deletion or inactivation of a glycogen synthase 1 gene;
(4) deletion or inactivation of a glycogen synthase cluster 2 gene;
(5) deletion or inactivation of an amylosucrose gene;
(6) deletion or inactivation of a gene for exopolysaccharide (EPS) biosynthesis;
(7) deletion or inactivation of an ectoine hydrolase gene;
(8) overexpression of a gene encoding for a lipase;
(9) deletion or inactivation of an ectoine-R (EctR) gene;
(10) deletion or inactivation of an ectoine-R (EctR) gene and overexpression of a lipase gene;
(11) deletion or inactivation of an ectoine-R (EctR) gene and deletion or inactivation of a DoeA gene;
(12) overexpression of an Icl/ms gene;
(13) deletion or inactivation of:
20ZR SLCter-LipL1 ΔectR Δsps Δglg1 Δglg2 Δeps ΔdoeA::Popt-ectABC-icl-ms;
(14) deletion or inactivation of:
20ZRΔectR Δsps Δglg1 Δglg2 Δeps ΔdoeA::Popt-ectABC-icl-ms;
(15) expression of LipL from N-ter;
(16) expression of LipL from N-ter with intein;
and
(17) expression of GFP with SLP C-term fusion.

26 more elements

Dependencies are inferred from the claim text.

Compositions and methods using methanotrophic S-layer proteins for expression of heterologous proteins

Life of this patent

Compositions and methods using methanotrophic S-layer proteins for expression of heterologous proteins

Life of this patent

Abstract

Claims20