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.

1 . A method for making a chimeric polypeptide comprising an S-layer polypeptide, or a self-aggregating or self-assembling fragment thereof, and a heterologous polypeptide or peptide, the method comprising recombinantly engineering a methylotrophic or methanotrophic bacteria to recombinantly express a chimeric polypeptide comprising an S-layer polypeptide and a heterologous polypeptide or peptide,
 wherein optionally the S-layer polypeptide is on the carboxy terminal end of the heterologous polypeptide or peptide,   and optionally the recombinant or isolated chimeric polypeptide has assembled or is self-assembled to form a monomolecular layer.   
     
     
         2 . A method for displaying or immobilizing proteins on a methanotrophic S-layer protein, or a self-aggregating or self-assembling fragment thereof, comprising recombinantly engineering a methylotrophic or methanotrophic bacteria to recombinantly express a chimeric polypeptide comprising an S-layer polypeptide or a self-aggregating or self-assembling fragment thereof and a heterologous polypeptide or peptide,
 wherein optionally the S-layer polypeptide or self-aggregating or self-assembling fragment thereof is on the carboxy terminal end of the heterologous polypeptide or peptide,   wherein optionally the recombinant or isolated chimeric S-layer polypeptide or self-aggregating or self-assembling fragment thereof has assembled or is self-assembled to form a monomolecular layer.   
     
     
         3 . A recombinant or isolated chimeric S-layer polypeptide, wherein the recombinant or isolated chimeric S-layer polypeptide comprises an S-layer polypeptide or self-aggregating or self-assembling fragment thereof and a heterologous polypeptide or peptide,
 wherein optionally the S-layer polypeptide is on the carboxy terminal end of the heterologous polypeptide or peptide,   wherein optionally the recombinant or isolated chimeric S-layer polypeptide has assembled or is self-assembled to form a monomolecular layer.   
     
     
         4 . (canceled) 
     
     
         5 . A recombinant or genetically engineered methylotrophic or methanotrophic bacteria comprising the recombinant or isolated chimeric S-layer polypeptide or a self-aggregating or self-assembling fragment thereof of  claim 3 ,
 wherein optionally the recombinant or chimeric polypeptide has assembled or is self-assembled to form a monomolecular layer on the extracellular surface of the recombinant methylotrophic or methanotrophic bacteria, and optionally the heterologous polypeptide or peptide is at least partially exposed, or is fully exposed, to an extracellular environment or milieu.   
     
     
         6 . The recombinant or isolated chimeric S-layer polypeptide of  claim 3 , wherein the S-layer polypeptide or self-aggregating or self-assembling fragment thereof comprises or is a lipoprotein. 
     
     
         7 . The recombinant or genetically engineered methylotrophic or methanotrophic bacteria of  claim 5 , wherein:
 the methylotrophic or methanotrophic bacteria is selected 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; or,   the S-layer polypeptide is derived from a methylotrophic or methanotrophic bacteria, or a  Methylococcus , a  Methylomonas , a  Methylomicrobium , a  Methylobacter , a  Methylomarinum , a  Methylovulum , a  Methylocaldum , a  Methylothermus , a  Methylomarinovum , a  Methylosphaera , a  Methylocystis , or a  Methylosinus  bacteria.   
     
     
         8 . The recombinant or genetically engineered methylotrophic or methanotrophic bacteria of  claim 5 , wherein the methylotrophic or methanotrophic bacteria is a  Methylomicrobium alcaliphilum  ( M. alcaliphilum ), or a  M. alcaliphilum  sp. 20Z. 
     
     
         9 . The recombinant or genetically engineered methylotrophic or methanotrophic bacteria of  claim 5 , wherein the chimeric polypeptide, or the recombinant or isolated chimeric S-layer polypeptide or self-aggregating or self-assembling fragment thereof, is expressed on the surface of a methylotrophic or methanotrophic bacteria, and the heterologous polypeptide, or the recombinant or isolated chimeric S-layer polypeptide or self-aggregating or self-assembling fragment thereof, is at least in part (partially) exposed to an extracellular environment or milieu. 
     
     
         10 . The recombinant or isolated chimeric S-layer polypeptide or self-aggregating or self-assembling fragment thereofof  claim 3 , wherein the methanotrophic S-layer polypeptide or self-assembling fragment thereof is isolated from the methylotrophic or methanotrophic bacteria. 
     
     
         11 . The recombinant or genetically engineered methylotrophic or methanotrophic bacteria of  claim 5 , wherein the heterologous polypeptide or peptide, or recombinant or isolated chimeric S-layer polypeptide, comprises or is an enzyme, a structural protein, a fluorescent or a chemiluminescent protein, a ligand, a receptor, an antibody or antigen binding protein, or an antigen, a tolerogen or an immunogen. 
     
     
         12 . The recombinant or genetically engineered methylotrophic or methanotrophic bacteria of  claim 11 , wherein the enzyme is an industrial enzyme, or the enzyme is a lipase, a protease, a nitrogenase, a hydrogenase, a monooxygenase, an amylase, an isomerase, a cellulase or hemicellulase, a laccase, an epimerase, a decarboxylase, a glucanase or a fl-glucanase, a glucosidase, a phosphorylase, a phosphatase, a halogenase or a dehalogenase, a GlcNAc transferase, an N-acetylglucosamine, a GlcNAc transferase, a neuraminidase or sialidase, a nuclease, a peroxidase or an oxidase, or a metalloproteinase. 
     
     
         13 . The recombinant or isolated monomolecular layer, or the recombinant or genetically engineered methylotrophic or methanotrophic bacteria of  claim 5 , wherein the chimeric protein, the recombinant or isolated chimeric S-layer polypeptide or self-assembling fragment thereof, the recombinant or isolated monomolecular layer, or the recombinant methylotrophic or methanotrophic bacteria, act as or are used as or used for: an ultrafiltration membrane; an affinity structure; nitrogen fixation; converting carbon dioxide into methane; methane uptake or methane oxidation; converting nitrogen gas to ammonia; a membrane of an enzyme membrane; a micro-carrier; a biosensor; a diagnostic device; a biocompatible surface; a vaccine; a device or composition for targeting, delivery and/or encapsulation; an anchor for extracellular production of a small molecule or a protein (optionally an enzyme or a structural protein), an enzymatic system for a bioremediation or a bio-mitigation, or a pharmaceutical or a protein-based biopharmaceutical. 
     
     
         14 . A membrane or an enzyme membrane; an ultrafiltration membrane; an affinity structure; a composition or device for nitrogen fixation; a composition or device for converting carbon dioxide into methane; a composition or device for methane uptake or methane oxidation; a composition or device for converting nitrogen gas to ammonia; a membrane of an enzyme membrane; a micro-carrier; a biosensor; a diagnostic device; a biocompatible surface; a vaccine; a device or composition for targeting, delivery and/or encapsulation; an implant; an anchor for extracellular production of a small molecule or a protein (optionally an enzyme or a structural protein), an enzymatic system for a bioremediation or a bio-mitigation, or a pharmaceutical or a protein-based biopharmaceutical, comprising:
 a chimeric polypeptide of  claim 3 .   
     
     
         15 . 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 under (transcriptionally controlled by) a hps promoter (P hps ); and/or,   (iii) comprises one or more of the genetic modifications set forth in Table 1 (see Example 2, below).   
     
     
         16 . The recombinant methylotrophic or methanotrophic bacteria of  claim 15 , wherein a doeA-gene encoding ectoine hydrolase is deleted or mutated such that a functional ectoine hydrolase is not expressed. 
     
     
         17 . The recombinant methylotrophic or methanotrophic bacteria of  claim 15 , wherein:
 (a) the recombinant methylotrophic or methanotrophic bacteria further comprises an exogenous nucleic acid capable of expressing a methanotrophic lipase, or a functional lipase fragment thereof (optionally a LipL1 expression plasmid), in the recombinant methylotrophic or methanotrophic bacteria;   (b) the recombinant methylotrophic or methanotrophic bacteria is engineered such that the ectoine and/or the lipase, or the functional lipase fragment thereof, is expressed as an S layer protein chimeric polypeptide, optionally as a lipase-S protein fusion protein (an S layer-lipase or an S layer-ectoine fusion protein), wherein optionally the S-layer protein is positioned at the amino terminus;   (c) the methylotrophic or methanotrophic bacteria is selected 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/or   (d) the methylotrophic or methanotrophic bacteria further comprises the ability to express: a heterologous or exogenous protein or enzyme, optionally an industrial enzyme; or a chimeric protein comprising an S-layer protein and the heterologous or exogenous protein or enzyme,   wherein optionally the protein or enzyme is a lipase, a protease, a nitrogenase, a hydrogenase, a monooxygenase, an amylase, an isomerase, a cellulase or hemicellulase, a laccase, an epimerase, a decarboxylase, a glucanase or a fl-glucanase, a glucosidase, a phosphorylase, a phosphatase, a halogenase or a dehalogenase, a GlcNAc transferase, an N-acetylglucosamine, a GlcNAc transferase, a neuraminidase or sialidase, a nuclease, a peroxidase or an oxidase, or a metalloproteinase.   
     
     
         18 - 20 . (canceled) 
     
     
         21 . The recombinant methylotrophic or methanotrophic bacteria of  claim 15 , wherein the recombinant methylotrophic or methanotrophic bacteria, act as or are used as or used for: an ultrafiltration membrane; an affinity structure; nitrogen fixation; converting carbon dioxide into methane; methane uptake or methane oxidation; converting nitrogen gas to ammonia; a membrane of an enzyme membrane; a micro-carrier; a biosensor; a diagnostic device; a biocompatible surface; a vaccine; a device or composition for targeting, delivery and/or encapsulation; an anchor for extracellular production of a small molecule or a protein (optionally an enzyme or a structural protein), an enzymatic system for a bioremediation or a bio-mitigation, or a pharmaceutical or a protein-based biopharmaceutical. 
     
     
         22 . A membrane or an enzyme membrane; an ultrafiltration membrane; an affinity structure; a composition or device for nitrogen fixation; a composition or device for converting carbon dioxide into methane; a composition or device for methane uptake or methane oxidation; a composition or device for converting nitrogen gas to ammonia; a membrane of an enzyme membrane; a micro-carrier; a biosensor; a diagnostic device; a biocompatible surface; a vaccine; a device or composition for targeting, delivery and/or encapsulation; an implant; an anchor for extracellular production of a small molecule or a protein (optionally an enzyme or a structural protein), an enzymatic system for a bioremediation or a bio-mitigation, or a pharmaceutical or a protein-based biopharmaceutical, comprising:
 a recombinant methylotrophic or methanotrophic bacteria of  claim 15 .