The present invention provides regulatable cell lines with an engineered genome encoding an auxotrophic response system or element, and methods of use thereof. Growth and protein production of the cell lines is controlled by exposure of the cell line to an auxotrophic factor to activate the auxotrophic response system or element. Examples of auxotrophic factors include a nutrient, an enzyme, a moiety that alters pH, a moiety that alters temperature, a non-organic molecule, a non-essential amino acid, an altered concentration of a moiety, and a cellular niche environment. These regulatable cell lines may be used in regenerative medicine and enzyme replacement therapy.

Provided is a recombinant Hansenula polymorpha-based high dosage hepatitis B vaccine, an HBsAg pure stock solution yield of a recombinant Hansenula polymorpha fermentation broth used for producing the hepatitis B vaccine being 300 mg/L-400 mg/L.

The present invention relates to methods of producing a polypeptide, comprising: (a) cultivating a mutant of a parent Fusarium venenatum strain in a medium for the production of the polypeptide, wherein the mutant strain comprises a polynucleotide encoding the polypeptide and one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA, wherein the one or more (several) genes are modified rendering the mutant strain deficient in the production of one or more (several) enzymes selected from the group consisting of orotidine-5-monophosphate decarboxylase, alpha-amylase, and alkaline protease, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions; and (b) recovering the polypeptide from the cultivation medium. The present invention also relates to enzyme-deficient mutants of Fusarium venenatum strains and methods for producing such mutants.

The present invention relates to methods of producing a polypeptide, comprising: (a) cultivating a mutant of a parent Fusarium venenatum strain in a medium for the production of the polypeptide, wherein the mutant strain comprises a polynucleotide encoding the polypeptide and one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA, wherein the one or more (several) genes are modified rendering the mutant strain deficient in the production of one or more (several) enzymes selected from the group consisting of orotidine-5-monophosphate decarboxylase, alpha-amylase, and alkaline protease, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions; and (b) recovering the polypeptide from the cultivation medium. The present invention also relates to enzyme-deficient mutants of Fusarium venenatum strains and methods for producing such mutants.

The present invention relates to methods of producing a polypeptide, comprising: (a) cultivating a mutant of a parent Fusarium venenatum strain in a medium for the production of the polypeptide, wherein the mutant strain comprises a polynucleotide encoding the polypeptide and one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA, wherein the one or more (several) genes are modified rendering the mutant strain deficient in the production of one or more (several) enzymes selected from the group consisting of orotidine-5-monophosphate decarboxylase, alpha-amylase, and alkaline protease, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions; and (b) recovering the polypeptide from the cultivation medium. The present invention also relates to enzyme-deficient mutants of Fusarium venenatum strains and methods for producing such mutants.

The instant disclosure is generally related to compositions and methods for obtaining and constructing Bacillus licheniformis host cells (e.g., protein production host cells, cell factories) having increased protein production capabilities. Certain embodiments of the disclosure are directed to efficient genetic modifications of B. licheniformis cells and the subsequent selection of such B. licheniformis cells having increased protein production capabilities. Certain other embodiments of the disclosure are generally related to methods and compositions for producing/obtaining auxotrophic B. licheniformis cells, wherein certain other embodiments of the disclosure are directed to methods and compositions for restoring prototrophy in auxotrophic B. licheniformis cells, and expressing genes of interest (GOIs) in such restored prototrophy B. licheniformis cells.

The present invention relates to methods for making compounds of formula (I) including violacein, violacein analogues and derivatives thereof, and to compositions, cells, and fermentation liquids comprising the compounds resulting from these methods.