Abstract: Recombinant Escherichia coli (E. coli) bacteria that have enhanced acetyl-CoA synthetase activity and the ability to produce glycerol and glycerol-derived products, such as 3-hydroxypropionic acid, methylglyoxal, 1,2-propanediol, and 1,3-propanediol, are described. The recombinant E. coli comprise a promoter operably linked to a nucleotide sequence encoding a polypeptide having acetyl-CoA synthetase enzyme activity, wherein the promoter and nucleotide sequence are each independently either native or non-native.

Abstract: Methods are provided to obtain recombinant microbial cells having at least one genetic modification that increase the buoyant density of a recombinant microbial cell or the buoyant density of inclusion bodies produced within a recombinant microbial cell. Exemplified are genetic modifications that increase the buoyant density of a recombinant microbial cell expressing heterologous peptides and polypeptides. Increasing expression of the genes ysaB, glyQ, glyS or a combination thereof within the recombinant microbial cell produces cells or inclusion bodies having higher buoyant density. A similar effect was achieved by decreasing or disrupting expression of the endogenous gltA gene. Increases in buoyant density render peptide production more efficient with respect to time and costs.

Abstract: Recombinant E. coli having enhanced acetyl-CoA synthetase activity and the ability to produce glycerol and glycerol-derived products, such as 3-hydroxypropionic acid, methylglyoxal, 1,2-propanediol, and 1,3-propanediol, are described. The recombinant E. coli comprise a promoter operably linked to a nucleotide sequence encoding a polypeptide having acetyl-CoA synthetase enzyme activity, wherein the promoter and nucleotide sequence are each independently either native or non-native.

Abstract: The invention relates to the recombinant expression of a peptide of interest in the form of a fusion protein comprising a solubility tag. The fusion protein comprises at least two portions separated by a cleavable peptide sequence wherein one portion is devoid of cysteine residues and the second portion comprises an effective number of cross-linkable cysteine residues. After cell lysis and isolation of the fusion protein, the fusion protein is subsequently cleaved into a mixture of first and second portions. Oxidative cross-linking is used to selectively precipitate one of the two portions to facilitate simple and effective separation of the peptide of interest.

Abstract: The invention relates to the recombinant expression of a peptide of interest in the form of a fusion protein comprising a solubility tag. The fusion protein comprises at least two portions separated by a cleavable peptide sequence wherein one portion is devoid of cysteine residues and the second portion comprises an effective number of cross-linkable cysteine residues. After cell lysis and isolation of the fusion protein, the fusion protein is subsequently cleaved into a mixture of first and second portions. Oxidative cross-linking is used to selectively precipitate one of the two portions to facilitate simple and effective separation of the peptide of interest.

Abstract: Disrupting the expression of endogenous Escherichia host cell genes gcvA and spr provides mutant host cells having increased heterologous peptide production. The addition of a genetic modification to the coding region of gene yejM further enhances peptide production and facilitates easier downstream processing. Recombinant Escherichia host cells are provided as well as methods of using such host cells for heterologous peptide production.

Abstract: A process of in vivo labeling and identifying recombinantly produced peptides or proteins within an unpermeabilized prokaryotic host cell. Recombinant prokaryotic cells expressing a fusion peptide comprising at least one tetracysteine tag were labeled in vivo using a biarsenical labeling reagent. A fluorescent activated cell sorter was used to identify and select subpopulations of fluorescent cells wherein the amount of fusion peptide in the cell was proportional to the amount of fluorescence detected.

Abstract: Peptide tags, referred to here as inclusion body tags, are disclosed useful for the generation of insoluble fusion peptides. The fusion peptides comprise at least one inclusion body tag operably linked to a peptide of interest. Expression of the fusion peptide in a host cell results in a product that is insoluble and contained within inclusion bodies in the cell and/or cell lysate. The inclusion bodies may then be purified and the protein of interest may be isolated after cleavage from the inclusion body tag.

Abstract: Disrupting the expression of endogenous Escherichia host cell genes gcvA and spr provides mutant host cells having increased heterologous peptide production. The addition of a genetic modification to the coding region of gene yejM further enhances peptide production and facilitates easier downstream processing. Recombinant Escherichia host cells are provided as well as methods of using such host cells for heterologous peptide production.

Abstract: Short single chain peptides having affinity for a target surface often lack the binding durability required for certain commercial applications. One way to improve durability is to promote multivalent binding by linking together binding sequences using peptide linkers. However, the resulting single chain binding peptides often suffer from linker entropy. It has been discovered that the use of rigid peptide linkers when linking together multiple binding sequences enhances the binding affinity of the resulting single chain peptide.

Abstract: Several endogenous genes have been identified in Escherichia coli, the overexpression of which increases recombinant peptide production. Increasing the copy number of aroB, aroK, proB, or crl increases the amount of a recombinant peptide produced by a host cell.

Abstract: Peptide tags, referred to here as inclusion body tags, are disclosed useful for the generation of insoluble fusion peptides. The fusion peptides comprise at least one inclusion body tag operably linked to a peptide of interest. Expression of the fusion peptide in a host cell results in a product that is insoluble and contained within inclusion bodies in the cell and/or cell lysate. The inclusion bodies may then be purified and the protein of interest may be isolated after cleavage from the inclusion body tag.

Abstract: Peptide tags, referred to here as inclusion body tags, are disclosed useful for the generation of insoluble fusion peptides. The fusion peptides comprise at least one inclusion body tag operably linked to a peptide of interest. Expression of the fusion peptide in a host cell results in a product that is insoluble and contained within inclusion bodies in the cell and/or cell lysate. The inclusion bodies may then be purified and the protein of interest may be isolated after cleavage from the inclusion body tag.

Abstract: Disrupting the expression of endogenous Escherichia host cell genes gcvA and spr provides mutant host cells having increased heterologous peptide production relative to control Escherichia host cells. Recombinant Escherichia host cells are provided as well as methods of using such host cells for heterologous peptide production.

Abstract: A process of in vivo labeling and identifying recombinantly produced peptides or proteins within an unpermeabilized prokaryotic host cell. Recombinant prokaryotic cells expressing a fusion peptide comprising at least one tetracysteine tag were labeled in vivo using a biarsenical labeling reagent. A fluorescent activated cell sorter was used to identify and select subpopulations of fluorescent cells wherein the amount of fusion peptide in the cell was proportional to the amount of fluorescence detected.

Abstract: The invention relates to the recombinant expression of a peptide of interest in the form of a fusion protein comprising a solubility tag. The fusion protein comprises at least two portions separated by a cleavable peptide sequence wherein one portion is devoid of cysteine residues and the second portion comprises an effective number of cross-linkable cysteine residues. After cell lysis and isolation of the fusion protein, the fusion protein is subsequently cleaved into a mixture of first and second portions. Oxidative cross-linking is used to selectively precipitate one of the two portions to facilitate simple and effective separation of the peptide of interest.

Abstract: Peptide tags, referred to here as inclusion body tags, are disclosed useful for the generation of insoluble fusion peptides. The fusion peptides comprise at least one inclusion body tag operably linked to a peptide of interest. Expression of the fusion peptide in a host cell results in a product that is insoluble and contained within inclusion bodies in the cell and/or cell lysate. The inclusion bodies may then be purified and the protein of interest may be isolated after cleavage from the inclusion body tag.

Abstract: Protein engineered CrtO ketolases isolated from Rhodococcus erythropolis AN12 are provided having increased carotenoid ketolase activity. Methods using the present CrtO ketolases are also provided for increasing ketocarotenoid production in suitable production hosts.

Abstract: A plasmid has been isolated from Rhodococcus erythropolis strain AN12 comprising a unique replication protein. The replication protein may be used in a variety of cloning and expression vectors and particularly in shuttle vectors for the expression of heterologous genes in Rhodococcus sp.

Abstract: CrtW carotenoid ketolases are provided that are useful for the production of astaxanthin and other cyclic ketocarotenoids. The mutant ketolase genes of the present invention encode polypeptides characterized by an improvement in astaxanthin synthesis activity when converting cyclic hydroxylated carotenoid intermediates into astaxanthin. Expression of the mutant carotenoid ketolases in heterologous hosts enabled increased production of astaxanthin relative to the Sphingomonas melonis DC18 CrtW.