EP4627098A1 - Compositions and methods for using previously cultured cells - Google Patents

Compositions and methods for using previously cultured cells

Info

Publication number
EP4627098A1
EP4627098A1 EP23822111.3A EP23822111A EP4627098A1 EP 4627098 A1 EP4627098 A1 EP 4627098A1 EP 23822111 A EP23822111 A EP 23822111A EP 4627098 A1 EP4627098 A1 EP 4627098A1
Authority
EP
European Patent Office
Prior art keywords
protein
cells
proteins
concentration
hours
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23822111.3A
Other languages
German (de)
French (fr)
Inventor
Carla Cristina MARQUES DE OLIVEIRA
Ana Catarina BARROS LOPES
Erdem ÇARSANBA
Ana Paula TABOADA DA COSTA SANTOS CARVALHO
Ana Sofia DA SILVA OLIVEIRA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BIORBIS, LDA
UNIVERSIDADE CATOLICA PORTUGUESA
Original Assignee
Universidade Catolica Portuguesa
Amyris Bio Products Portugal Unipessoal Ltda
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidade Catolica Portuguesa, Amyris Bio Products Portugal Unipessoal Ltda filed Critical Universidade Catolica Portuguesa
Publication of EP4627098A1 publication Critical patent/EP4627098A1/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • a biological product may be obtained by culturing a population of host cells capable of producing the biological product in a culture medium.
  • the culture medium may include a peptide extract that has been obtained from a population of previously cultured cells (e.g., previously cultured yeast cells).
  • the previously cultured cells may, for example, no longer produce a significant quantity of a biological product as a result of being previously cultured.
  • the previously cultured cells may have been fermented for a desired period of time, such as a period of time sufficient to produce a peptide composition described herein.
  • the present disclosure is based, in part, on the surprising discovery that peptide extracts isolated from such previously cultured cells may confer antioxidant activity to a population of host cells that are presently being fermented with the aim of biosynthesizing a biological product of interest. This antioxidant activity may, in turn, have the beneficial effect of improving the amount of the desired biological product that is ultimately biosynthesized by the host cells.
  • the disclosure provides a method of producing a biological product including: providing a population of host cells capable of producing the biological product, and culturing the population of host cells in a culture medium that includes a peptide extract obtained from a population of previously cultured cells.
  • the disclosure provides a method of culturing a population of host cells including: providing a population of host cells capable of producing a biological product, and culturing 1 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT the population of host cells in a culture medium that includes a peptide extract obtained from a population of previously cultured cells.
  • the peptide extract includes proteins.
  • the peptide extract has a protein concentration of between 20% (w/w) and 95% (w/w) (e.g., between 20% (w/w) and 75% (w/w), 20% (w/w) and 50% (w/w), 20% (w/w) and 30% (w/w), 30% (w/w) and 95% (w/w), 50% (w/w) and 95% (w/w), 70% (w/w) and 95% (w/w), or 80% (w/w) and 95% (w/w)).
  • w/w w/w
  • 95% w/w
  • the peptide extract has a protein concentration of between 45% (w/w) and 90% (w/w) (e.g., between 50% (w/w) and 90% (w/w), 60% (w/w) and 90% (w/w), 70% (w/w) and 90% (w/w), 80% (w/w) and 90% (w/w), 45% (w/w) and 80% (w/w), 45% (w/w) and 70% (w/w), 45% (w/w) and 60% (w/w), or 45% (w/w) and 50% (w/w)).
  • w/w protein concentration of between 45% (w/w) and 90% (w/w) (e.g., between 50% (w/w) and 90% (w/w), 60% (w/w) and 90% (w/w), 70% (w/w) and 90% (w/w), 80% (w/w) and 90% (w/w), 45% (w/w) and 70% (w/w), 45% (w/w) and 60% (w
  • the proteins comprise one or more amino acid amino acid residues selected from histidine (His), threonine (Thr), arginine (Arg), valine (Val), phenylalanine (Phe), tyrosine (Tyr), Isoleucine (Ile), leucine (Leu), and lysine (Lys).
  • the proteins have a molecular weight of greater than 1 kDa.
  • the proteins comprise a His residue, optionally wherein proteins comprising His are at a concentration of between about 20 mg/g protein and 30 mg/g protein (e.g., between about 20 mg/g protein and about 26 mg/g protein, about 20 mg/g protein and about 24 mg/g protein, about 24 mg/g protein and about 30 mg/g protein, or about 28 mg/g protein and about 30 mg/g protein).
  • the proteins comprise a Thr residue, optionally wherein proteins comprising Thr are at a concentration of between about 25 mg/g protein and about 55 mg/g protein (e.g., between about 35 mg/g protein and about 55 mg/g protein, about 45 mg/g protein and about 55 mg/g protein, about 25 mg/g protein and about 45 mg/g protein, or about 25 mg/g protein and about 35 mg/g protein).
  • the proteins comprise an Arg residue, optionally wherein proteins comprising Arg are at a concentration of between about 25 mg/g protein and about 40 mg/g protein (e.g., between about 25 mg/g protein and about 35 mg/g protein, about 25 mg/g protein and about 30 mg/g protein, about 30 mg/g protein and about 40 mg/g protein, or about 35 mg/g protein and about 40 mg/g protein).
  • proteins comprising Arg are at a concentration of between about 25 mg/g protein and about 40 mg/g protein (e.g., between about 25 mg/g protein and about 35 mg/g protein, about 25 mg/g protein and about 30 mg/g protein, about 30 mg/g protein and about 40 mg/g protein, or about 35 mg/g protein and about 40 mg/g protein).
  • the proteins comprise an Ile residue, optionally wherein proteins comprising Ile are at a concentration of between about 20 mg/g protein and about 50 mg/g protein (e.g., between about 30 mg/g protein and about 50 mg/g protein, about 40 mg/g protein and about 50 mg/g protein, about 20 mg/g protein and about 40 mg/g protein, or about 20 mg/g protein and about 30 mg/g protein).
  • proteins comprising Ile are at a concentration of between about 20 mg/g protein and about 50 mg/g protein (e.g., between about 30 mg/g protein and about 50 mg/g protein, about 40 mg/g protein and about 50 mg/g protein, about 20 mg/g protein and about 40 mg/g protein, or about 20 mg/g protein and about 30 mg/g protein).
  • the proteins comprise a Leu residue, optionally wherein proteins comprising Leu are at a concentration of between about 25 mg/g protein and about 60 mg/g protein (e.g., between about 35 mg/g protein and about 60 mg/g protein, about 45 mg/g protein and about 60 mg/g protein, about 55 mg/g protein and about 60 mg/g protein, about 25 mg/g protein and about 55 mg/g protein, about 25 mg/g protein and about 45 mg/g protein, or about 25 mg/g protein and about 35 mg/g protein).
  • proteins comprising Leu are at a concentration of between about 25 mg/g protein and about 60 mg/g protein (e.g., between about 35 mg/g protein and about 60 mg/g protein, about 45 mg/g protein and about 60 mg/g protein, about 55 mg/g protein and about 60 mg/g protein, about 25 mg/g protein and about 55 mg/g protein, about 25 mg/g protein and about 45 mg/g protein, or about 25 mg/g protein and about 35 mg/g protein).
  • the proteins comprise a Thr residue, optionally wherein proteins comprising Thr are at a concentration of between about 10 mg/g protein and about 30 mg/g protein (e.g., between about 10 mg/g protein and about 25 mg/g protein, about 10 mg/g protein and about 20 mg/g protein, about 10 mg/g protein and about 15 mg/g protein, about 15 mg/g protein and about 30 mg/g protein, about 20 mg/g protein and about 30 mg/g protein, or about 25 mg/g protein and about 30 mg/g protein).
  • proteins comprising Thr are at a concentration of between about 10 mg/g protein and about 30 mg/g protein (e.g., between about 10 mg/g protein and about 25 mg/g protein, about 10 mg/g protein and about 20 mg/g protein, about 10 mg/g protein and about 15 mg/g protein, about 15 mg/g protein and about 30 mg/g protein, about 20 mg/g protein and about 30 mg/g protein, or about 25 mg/g protein and about 30 mg/g protein).
  • the proteins comprise an Arg residue, optionally wherein proteins comprising Arg are at a concentration of about between 10 mg/g protein and about 20 mg/g (e.g., between about 10 mg/g protein and about 17 mg/g protein, about 10 mg/g protein and about 12 mg/g protein, about 12 mg/g protein and about 20 mg/g protein, about 15 mg/g protein and about 20 mg/g protein, or about 17 mg/g protein and about 20 mg/g protein).
  • the proteins comprise a Phe residue, optionally wherein proteins comprising Phe are at a concentration of between about 5 mg/g protein and about 25 mg/g protein (e.g., between about 10 mg/g protein and about 25 mg/g protein, about 15 mg/g protein and about 25 mg/g protein, about 20 mg/g protein and 3 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT about 25 mg/g protein, about 5 mg/g protein and about 20 mg/g protein, about 5 mg/g protein and about 15 mg/g protein, or about 5 mg/g protein and about 10 mg/g ⁇ protein).
  • proteins comprising Phe are at a concentration of between about 5 mg/g protein and about 25 mg/g protein (e.g., between about 10 mg/g protein and about 25 mg/g protein, about 15 mg/g protein and about 25 mg/g protein, about 20 mg/g protein and 3 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS
  • the proteins comprise a Tyr residue, optionally wherein proteins comprising Tyr are at a concentration of between about 10 mg/g protein and about 30 mg/g protein (e.g., between about 10 mg/g protein and about 25 mg/g protein, about 10 mg/g protein and about 20 mg/g protein, about 10 mg/g protein and about 15 mg/g protein, about 15 mg/g protein and about 25 mg/g protein, or about 20 mg/g protein and about 25 mg/g protein).
  • proteins comprising Tyr are at a concentration of between about 10 mg/g protein and about 30 mg/g protein (e.g., between about 10 mg/g protein and about 25 mg/g protein, about 10 mg/g protein and about 20 mg/g protein, about 10 mg/g protein and about 15 mg/g protein, about 15 mg/g protein and about 25 mg/g protein, or about 20 mg/g protein and about 25 mg/g protein).
  • the proteins comprise an Ile residue, optionally wherein proteins comprising Ile are at a concentration of between about 10 mg/g protein and 35 mg/g (e.g., between about 15 mg/g protein and 35 mg/g protein, about 20 mg/g protein and 35 mg/g protein, about 25 mg/g protein and 35 mg/g protein, about 30 mg/g and 35 mg/g protein, about 10 mg/g protein and 30 mg/g protein, about 10 mg/g protein and 25 mg/g protein, about 10 mg/g protein and 20 mg/g protein, or about 10 mg/g protein and 15 mg/g protein).
  • proteins comprising Ile are at a concentration of between about 10 mg/g protein and 35 mg/g (e.g., between about 15 mg/g protein and 35 mg/g protein, about 20 mg/g protein and 35 mg/g protein, about 25 mg/g protein and 35 mg/g protein, about 30 mg/g and 35 mg/g protein, about 10 mg/g protein and 30 mg/g protein, about 10 mg/g protein and 25 mg/g protein
  • the proteins comprise a Leu residue, optionally wherein proteins comprising Leu are at a concentration of between about 15 mg/g protein and about 45 mg/g protein (e.g., between about 25 mg/g protein and about 45 mg/ ⁇ protein g, about 35 mg/g protein and about 45 mg/g ⁇ protein, about 15 mg/g protein and about 35 mg/g protein, or about 15 mg/g protein and about 25 mg/g protein).
  • proteins comprising Leu are at a concentration of between about 15 mg/g protein and about 45 mg/g protein (e.g., between about 25 mg/g protein and about 45 mg/ ⁇ protein g, about 35 mg/g protein and about 45 mg/g ⁇ protein, about 15 mg/g protein and about 35 mg/g protein, or about 15 mg/g protein and about 25 mg/g protein).
  • the proteins comprise a Lys residue, optionally wherein proteins comprising Lys are at a concentration of between about 15 mg/g protein and 45 mg/g protein (e.g., between about 25 mg/g protein and about 45 mg/g protein, about 35 mg/g protein and about 45 mg/g protein, about 15 mg/g protein and about 35 mg/g protein, or about 15 mg/g protein and about 25 mg/g ⁇ protein).
  • the peptide extract includes polysaccharides.
  • the peptide extract has a polysaccharide concentration of between 1% (w/w) and 50% (w/w) (e.g., between 1% (w/w) and 40% (w/w), 1% (w/w) and 30% (w/w), 1% (w/w) and 20% (w/w), 1% (w/w) and 10% (w/w), 10% (w/w) and 50% (w/w), 20% (w/w) and 50% (w/w), 30% (w/w) and 50% (w/w), or 40% (w/w) and 50% (w/w)).
  • a polysaccharide concentration of between 1% (w/w) and 50% (w/w) (e.g., between 1% (w/w) and 40% (w/w), 1% (w/w) and 30% (w/w), 1% (w/w) and 20% (w/w), 1% (w/w) and 10% (w/w), 10% (w/w) and 50% (w/w), 20% (w/w
  • the polysaccharide concentration is between 2% and 35% of the peptide extract (e.g., between 2% (w/w) and 30% (w/w), 2% (w/w) and 25% (w/w), 2% (w/w) and 20% (w/w), 2% (w/w) and 15% (w/w), 2% (w/w) and 10% (w/w), 2% (w/w) and 5% (w/w), 5% (w/w) and 35% (w/w), 10% (w/w) and 35% (w/w), 15% (w/w) and 35% (w/w), 20% (w/w) and 35% (w/w), 25% (w/w) and 35% (w/w), or 30% (w/w) and 35% (w/w)).
  • the peptide extract e.g., between 2% (w/w) and 30% (w/w), 2% (w/w) and 25% (w/w), 2% (w/w) and 20% (w/w
  • the peptide extract includes minerals.
  • the minerals are selected from one or more of phosphorus, magnesium, calcium, sodium, and potassium.
  • peptide extract has a mineral concentration of between 5 and 150 ng/g of the peptide extract (e.g., between 5 ng/g and 125 ng/g, 5 ng/g and 100 ng/g, 5 ng/g and 75 ng/g, 5 ng/g and 50 ng/g, 5 ng/g and 25 ng/g, 5 ng/g and 10 ng/g, 10 ng/g and 150 ng/g, 25 ng/g and 150 ng/g, 50 ng/g and 150 ng/g, 75 ng/g and 150 ng/g, 100 ng/g and 150 ng/g, or 125 ng/g and 150 ng/g).
  • the concentration of the peptide extract in the culture medium is from about 0.1 g/L to about 1 g/L (e.g., about 0.1 g/L to about 0.8 g/L, about 0.1 g/L to about 0.6 g/L, about 0.1 g/L to about 0.4 g/L, about 0.1 g/L to about 0.2 g/L, about 0.2 g/L to about 1 g/L, about 0.4 g/L to about 1 g/L, about 0.6 g/L to about 1 g/L, or about 0.8 g/L to about 1 g/L).
  • the concentration of the peptide extract in the culture medium is from about 0.5 g/L to about 0.7 g/L (e.g., 4 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT about 0.5 g/L, about 0.6 g/L, or about 0.7 g/L). In some embodiments, the concentration of peptide extract in the culture medium is about 0.7 g/L. In some embodiments, the peptide extract is isolated from the previously cultured cells by lysing and centrifuging the previously cultured cells, resulting in a supernatant, and obtaining the peptide extract from the supernatant.
  • the peptide extract is isolated from the supernatant by way of filtration.
  • the filtration is selected from one or more of membrane filtration, fast protein liquid chromatograph-gel filtration diafiltration, and ultrafiltration.
  • the filtration is membrane filtration.
  • the peptide extract has a yield of 0.4 g/L to 2.2 g/L (e.g., 0.4 g/L, 0.5 g/L, 0.6 g/L, 0.7 g/L, 0.8 g/L, 0.9 g/L, 1 g/L , 1.1 g/L, 1.2 g/L, 1.3 g/L, 1.4 g/L, 1.5 g/L, 1.6 g/L, 1.7 g/L, 1.8 g/L, 1.9 g/L, 2 g/L, 2.1 g/L, or 2.2 g/) in the supernatant.
  • 0.4 g/L 0.5 g/L, 0.6 g/L, 0.7 g/L, 0.8 g/L, 0.9 g/L, 1 g/L , 1.1 g/L, 1.2 g/L, 1.3 g/L, 1.4 g/L, 1.5 g/L, 1.6
  • the previously cultured cells have an optical density of between about 20 to about 150 (e.g., between about 20 and about 100, about 20 and about 80, about 20 and about 60, about 20 and about 40, about 40 and about 150, about 60 and about 150, about 80 and about 150, about 100 and about 150, or about 120 and about 150) immediately prior to obtaining the peptide extract and culturing it with the population of host cells.
  • optical density between about 20 to about 150 (e.g., between about 20 and about 100, about 20 and about 80, about 20 and about 60, about 20 and about 40, about 40 and about 150, about 60 and about 150, about 80 and about 150, about 100 and about 150, or about 120 and about 150) immediately prior to obtaining the peptide extract and culturing it with the population of host cells.
  • the previously cultured cells have an optical density of between about 20 to about 50 (e.g., between about 20 to about 45, about 20 to about 40, about 20 to about 35, about 20 to about 30, about 20 to about 25, about 25 to about 50, about 30 to about 50, about 35 to about 50, about 40 to about 50, or about 45 to about 50) immediately prior to obtaining the peptide extract and culturing it with the population of host cells.
  • the previously cultured cells have an optical density of about 50 immediately prior to obtaining the peptide extract and culturing it with the population of host cells.
  • the method results in an increase in host cell density of at least 1.1 fold in comparison to a reference method in which the peptide extract is not present in the culture medium.
  • the method results in an increase in host cell density of from about 1.1 fold to about 5 fold (e.g., about 1.1 fold to about 4 fold, about 1.1 fold to about 3 fold, about 1.1 fold to about 2 fold, about 2 fold to about 5 fold, about 3 fold to about 5 fold, or about 4 fold to about 5 fold) in comparison to a reference method in which the peptide extract is not present in the culture medium.
  • the method results in a decrease in the formation of reactive oxygen species by at least 10% in comparison to a reference method in which the peptide extract is not present in the culture medium.
  • the method results in a decrease in the formation of reactive oxygen species by from about 10% to about 90% (e.g., about 10% to about 70%, about 10% to about 50%, about 10% to about 30%, about 30% to about 90%, about 50% to about 90%, or about 70% to about 90%) in comparison to a reference method in which the peptide extract is not present in the culture medium.
  • after culturing the host cells for from about 2 hours to about 50 hours e.g., about 2 hours to about 5 hours, about 5 hours to about 10 hours, about 10 hours to about 15 hours, about 15 hours to about 20 hours, about 20 hours to about 25 hours, about 25 hours to about 30 hours, about 30 hours to about 35 hours, about 35 hours to about 40 hours, about 40 hours to about formation of reactive oxygen species by from about 25% to about 75% (e.g., about 25% to about 65%, about 25% to about 55%, about 25% to about 35%, about 35% to about 75%, about 45% to about 75%, about 55% to about 75%, or about 65% to about 75%) in comparison to a reference method in which the peptide extract is not present in the culture medium.
  • about 25% to about 75% e.g., about 25% to about 65%, about 25% to about 55%, about 25% to about 35%, about 35% to about 75%, about 45% to about 75%, about 55% to about 75%, or about 65% to about 75
  • the method results in an increase in host cell density of at least 1.1-fold (e.g., between 1.1 fold and 20 fold, 1.1 fold and 15 fold, 1.1 fold and 10 fold, 1.1 fold and 5 fold, 1.1 fold and 2 fold, 2 fold and 20 fold, 5 fold and 20 fold, 10 fold and 20 fold, or 15 fold and 20 fold) in comparison to a reference method in which the peptide extract is not present in the culture medium.
  • 1.1-fold e.g., between 1.1 fold and 20 fold, 1.1 fold and 15 fold, 1.1 fold and 10 fold, 1.1 fold and 5 fold, 1.1 fold and 2 fold, 2 fold and 20 fold, 5 fold and 20 fold, 10 fold and 20 fold, or 15 fold and 20 fold
  • the method results in an increase in the concentration of the biological product in comparison to a reference method in which the peptide extract is not present in the culture medium.
  • the method results in an increase in the concentration of the biological product of from about 1.1 fold to about 5 fold (e.g., about 1.1 fold to about 4 fold, about 1.1 fold to about 3 fold, about 1.1 fold to about 2 fold, about 2 fold to about 5 fold, about 3 fold to about 5 fold, or about 4 fold to about 5 fold) in comparison to a reference method in which the peptide extract is not present in the culture medium.
  • the previously cultured cells are yeast cells.
  • the yeast cells are Saccharomyces sp. cells or Kluveromyces sp. cells.
  • the yeast cells are Saccharomyces cerevisiae cells.
  • the yeast cells are Kluveromyces marxianus cells.
  • the host cells are genetically modified host cells.
  • the host cells are yeast cells.
  • the yeast cells are Saccharomyces sp. cells or Kluveromyces sp. cells.
  • the yeast cells are Saccharomyces cerevisiae cells.
  • the yeast cells are Kluveromyces marxianus cells.
  • the population of host cells is cultured for at least 10 hours. In some embodiments, the population of host cells is cultured for from about 10 hours to about 100 hours (e.g., about 10 hours to about 80 hours, about 10 hours to about 60 hours, about 10 hours to about 40 hours, about 10 hours to about 20 hours, about 20 hours to about 100 hours, about 40 hours to about 100 hours, about 60 hours to about 100 hours, or 80 hours to about 100 hours.
  • about 10 hours to about 100 hours e.g., about 10 hours to about 80 hours, about 10 hours to about 60 hours, about 10 hours to about 40 hours, about 10 hours to about 20 hours, about 20 hours to about 100 hours, about 40 hours to about 100 hours, about 60 hours to about 100 hours, or 80 hours to about 100 hours.
  • the host cells are cultured at a temperature of from about 20 o C to about 40 o C (e.g., about 20 o C to about 35 o C, about 20 o C to about 30 o C, about 20 o C to about 25 o C, about 25 o C to about 40 o C, about 30 o C to about 40 o C, or about 35 o C to about 40 o C).
  • the host cells are cultured at a temperature of about 30 o C.
  • the biological product is a fermentation product.
  • the biological product is an isoprene.
  • the biological product is an isoprenoid.
  • the biological product is a steviol glycoside.
  • the biological product is a human milk oligosaccharide. In some embodiments, the biological product is a cannabinoid. In another aspect, the disclosure provides a composition produced by any one of the methods described herein. In another aspect, the disclosure provides a composition including a population of host cells capable of producing a biological product; and a culture medium including a peptide extract obtained from a population of previously cultured cells. 7 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT In some embodiments, the peptide extract includes proteins.
  • the peptide extract has a protein concentration of between 45% (w/w) and 90% (w/w) (e.g., between 50% (w/w) and 90% (w/w), 60% (w/w) and 90% (w/w), 70% (w/w) and 90% (w/w), 80% (w/w) and 90% (w/w), 45% (w/w) and 80% (w/w), 45% (w/w) and 70% (w/w), 45% (w/w) and 60% (w/w), or 45% (w/w) and 50% (w/w)).
  • the proteins comprise one or more amino acid residues selected from His, Thr, Arg, Val, Phe, Tyr, Ile, Leu, and Lys.
  • the proteins comprise an Arg residue, optionally wherein proteins comprising Arg are at a concentration of between about 25 mg/g protein and about 40 mg/g protein (e.g., between about 25 mg/g protein and about 35 mg/g protein, about 25 mg/g protein and about 30 mg/g protein, about 30 mg/g protein and about 40 mg/g protein, or about 35 mg/g protein and about 40 mg/g protein).
  • proteins comprising Arg are at a concentration of between about 25 mg/g protein and about 40 mg/g protein (e.g., between about 25 mg/g protein and about 35 mg/g protein, about 25 mg/g protein and about 30 mg/g protein, about 30 mg/g protein and about 40 mg/g protein, or about 35 mg/g protein and about 40 mg/g protein).
  • the proteins comprise a Val residue, optionally wherein proteins comprising Val are at a concentration of between about 15 mg/g protein and about 55 mg/g protein (e.g., between about 15 mg/g protein and about 45 mg/g protein, about 15 mg/g protein and about 35 mg/g protein, about 15 mg/g protein and about 25 mg/g protein, about 25 mg/g protein and about 55 mg/g protein, about 35 mg/g protein and about 55 mg/g protein, or about 45 mg/g protein and about 55 mg/g protein)).
  • proteins comprising Val are at a concentration of between about 15 mg/g protein and about 55 mg/g protein (e.g., between about 15 mg/g protein and about 45 mg/g protein, about 15 mg/g protein and about 35 mg/g protein, about 15 mg/g protein and about 25 mg/g protein, about 25 mg/g protein and about 55 mg/g protein, about 35 mg/g protein and about 55 mg/g protein, or about 45 mg/g protein and about 55 mg/g protein)).
  • the proteins comprise a Tyr residue, optionally wherein proteins comprising Tyr are at a concentration of between about 10 mg/g protein and about 35 mg/g protein (e.g., between about 10 mg/g protein and about 30 mg/g protein, about 10 mg/g protein and about 25 mg/g, about 10 mg/g protein and about 20 mg/g protein, about 10 mg/g protein and about 15 mg/g about 15 mg/g protein and about 35 mg/g, about 20 mg/g protein and about 35 mg/g protein, about 25 mg/g protein and about 35 mg/g protein, or about 30 mg/g protein and about 35 mg/g protein).
  • proteins comprising Tyr are at a concentration of between about 10 mg/g protein and about 35 mg/g protein (e.g., between about 10 mg/g protein and about 30 mg/g protein, about 10 mg/g protein and about 25 mg/g, about 10 mg/g protein and about 20 mg/g protein, about 10 mg/g protein and about 15 mg/g about 15 mg/g protein and about 35 mg/g, about 20 mg
  • the proteins comprise an Ile residue, optionally wherein 8 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT proteins comprising Ile are at a concentration of between about 20 mg/g protein and about 50 mg/g protein (e.g., between about 30 mg/g protein and about 50 mg/g protein, about 40 mg/g protein and about 50 mg/g protein, about 20 mg/g protein and about 40 mg/g protein, or about 20 mg/g protein and about 30 mg/g protein).
  • PT-800 PCT proteins comprising Ile are at a concentration of between about 20 mg/g protein and about 50 mg/g protein (e.g., between about 30 mg/g protein and about 50 mg/g protein, about 40 mg/g protein and about 50 mg/g protein, about 20 mg/g protein and about 40 mg/g protein, or about 20 mg/g protein and about 30 mg/g protein).
  • the proteins comprise a Leu residue, optionally wherein proteins comprising Leu are at a concentration of between about 25 mg/g protein and about 60 mg/g protein (e.g., between about 35 mg/g protein and about 60 mg/g protein, about 45 mg/g protein and about 60 mg/g protein, about 55 mg/g protein and about 60 mg/g protein, about 25 mg/g protein and about 55 mg/g protein, about 25 mg/g protein and about 45 mg/g protein, or about 25 mg/g protein and about 35 mg/g protein).
  • proteins comprising Leu are at a concentration of between about 25 mg/g protein and about 60 mg/g protein (e.g., between about 35 mg/g protein and about 60 mg/g protein, about 45 mg/g protein and about 60 mg/g protein, about 55 mg/g protein and about 60 mg/g protein, about 25 mg/g protein and about 55 mg/g protein, about 25 mg/g protein and about 45 mg/g protein, or about 25 mg/g protein and about 35 mg/g protein).
  • the proteins comprise a Lys residue, optionally wherein proteins comprising Lys are at a concentration of between about 45 mg/g protein and about 65 mg/g protein (e.g., between about 50 mg/g protein and about 65 mg/g protein, about 55 mg/g protein and about 65 mg/g protein, about 60 mg/g protein and about 65 mg/g protein, about 45 mg/g protein and about 60 mg/g protein, about 45 mg/g protein and about 55 mg/g protein, or about 45 mg/g protein and about 50 mg/g protein).
  • the proteins have a molecular weight of less than 1 kDa.
  • the proteins comprise a Thr residue, optionally wherein proteins comprising Thr are at a concentration of between about 10 mg/g protein and about 30 mg/g protein (e.g., between about 10 mg/g protein and about 25 mg/g protein, about 10 mg/g protein and about 20 mg/g protein, about 10 mg/g protein and about 15 mg/g protein, about 15 mg/g protein and about 30 mg/g protein, about 20 mg/g protein and about 30 mg/g protein, or about 25 mg/g protein and about 30 mg/g protein).
  • proteins comprising Thr are at a concentration of between about 10 mg/g protein and about 30 mg/g protein (e.g., between about 10 mg/g protein and about 25 mg/g protein, about 10 mg/g protein and about 20 mg/g protein, about 10 mg/g protein and about 15 mg/g protein, about 15 mg/g protein and about 30 mg/g protein, about 20 mg/g protein and about 30 mg/g protein, or about 25 mg/g protein and about 30 mg/g protein).
  • the proteins comprise an Arg residue, optionally wherein proteins comprising Arg are at a concentration of about between 10 mg/g protein and about 20 mg/g (e.g., between about 10 mg/g protein and about 17 mg/g protein, about 10 mg/g protein and about 12 mg/g protein, about 12 mg/g protein and about 20 mg/g protein, about 15 mg/g protein and about 20 mg/g protein, or about 17 mg/g protein and about 20 mg/g protein).
  • the proteins comprise a Val residue, optionally wherein proteins comprising Val are at a concentration of about between 15 mg/g protein and about 55 mg/g protein (e.g., between about 15 mg/g protein and about 45 mg/g protein, about 15 mg/g protein and about 35 mg/g protein, about 15 mg/g protein and about 25 mg/g protein, about 25 mg/g protein and about 55 mg/g protein, about 35 mg/g protein and about 55 mg/g protein, or about 45 mg/g protein and about 55 mg/g protein)).
  • proteins comprising Val are at a concentration of about between 15 mg/g protein and about 55 mg/g protein (e.g., between about 15 mg/g protein and about 45 mg/g protein, about 15 mg/g protein and about 35 mg/g protein, about 15 mg/g protein and about 25 mg/g protein, about 25 mg/g protein and about 55 mg/g protein, about 35 mg/g protein and about 55 mg/g protein, or about 45 mg/g protein and about 55 mg/g protein)).
  • the previously cultured cells are cells that have been previously cultured for at least 10 hours. In some embodiments, the previously cultured cells are cells that have been previously cultured for from about 10 hours to about 100 hours (e.g., about 10 hours to about 80 hours, about 10 hours to about 60 hours, about 10 hours to about 40 hours, about 10 hours to about 20 hours, about 20 hours to about 100 hours, about 40 hours to about 100 hours, about 60 hours to about 100 hours, or about 80 hours to about 100 hours). In some embodiments, the previously cultured cells no longer produce a detectable quantity of the fermentation product. In some embodiments, the previously cultured cells continue to produce a detectable quantity of the fermentation product. In some embodiments, the previously cultured are yeast cells.
  • the yeast cells are Saccharomyces sp. cells or Kluveromyces sp. cells. In some embodiments, the yeast cells are Saccharomyces cerevisiae cells. In some embodiments, the yeast cells are Kluveromyces marxianus cells. In some embodiments, the host cells are genetically modified host cells. In some embodiments, the host cells are yeast cells. In some embodiments, the yeast cells are Saccharomyces sp. cells or Kluveromyces sp. cells. In some embodiments, the yeast cells are Saccharomyces cerevisiae cells. In some embodiments, the yeast cells are Kluveromyces marxianus cells. In some embodiments, the biological product is a fermentation product.
  • the biological product is an isoprene. In some embodiments, the biological product is an isoprenoid. In some embodiments, the biological product is a steviol glycoside. In some 11 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT embodiments, the biological product is a human milk oligosaccharide. In some embodiments, the biological product is a cannabinoid.
  • a numerical value or range herein includes normal variation encountered in the field, and includes plus or minus 1-10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%) of the numerical value or end points of the numerical range.
  • a value of 10 includes all numerical values from 9 to 11.
  • All numerical ranges described herein include the endpoints of the range unless otherwise noted, and all numerical values in-between the end points, to the first significant digit.
  • biological product refers to a compound, molecule, or complex of molecules that is produced by a host cell (e.g., produced enzymatically by a host cell).
  • the host cell may be one that expresses the enzyme(s) of the biosynthetic pathway that is used in the production of the biological product.
  • a biological product may be naturally produced by the host cell or may be produced by a host cell that has been genetically modified to produce the biological product (e.g., genetically modified so as to express one or more heterologous enzymes of the biosynthetic pathway corresponding to the biological product).
  • Exemplary biological products include, without limitation, carbohydrates, peptide fragments, full-length proteins, nucleic acids, and small molecules.
  • glycosides and oligosaccharides e.g., steviol glycosides and human milk oligosaccharides
  • isoprenes e.g., isoprenoids
  • cannabinoids e.g., antibodies and antigen-binding fragments thereof
  • enzymes e.g., therapeutic proteins, antigen receptors (e.g., chimeric antigen receptors (CARs)), among other biological products described herein and known in the art.
  • CARs chimeric antigen receptors
  • the term “capable of producing” refers to a host cell which includes the enzymes necessary for the production of a given compound in accordance with a biochemical pathway that produces the compound.
  • the term “express” refers to any one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a 12 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT polypeptide or protein.
  • the genetic pathway includes 3 or more members (e.g., 3, 4, 5, 6, 7, 8, 9, etc.), wherein the product of one encoded enzyme is the substrate for the next enzyme in the synthetic pathway.
  • 13 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT As used herein, the term “genetically modified” denotes a host cell that contains a heterologous nucleotide sequence. The genetically modified host cells described herein typically do not exist in nature.
  • heterologous refers to what is not normally found in nature.
  • heterologous nucleic acid refers to a nucleic acid not normally found in a given cell in nature.
  • the compounds described herein may have ionizable groups so as to be capable of preparation as pharmaceutically acceptable salts. These salts may be acid addition salts involving inorganic or organic acids or the salts may, in the case of acidic forms of the compounds described herein, be prepared from inorganic or organic bases.
  • the compounds may be prepared or used as pharmaceutically acceptable salts synthesized as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable salts may be prepared from pharmaceutically acceptable non-toxic acids and bases including inorganic and organic acids and bases.
  • Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2- hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate,
  • the cells may have been previously cultured for from 1-312 hours, such as, e.g., 1 hour, 5 hours, 10 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours, 192 hours, 216 hours, 240 hours, 264 hours, 288 hours, 312 hours, or more.
  • the cells may no longer produce a significant quantity of a biological product.
  • the previously cultured cells may be used for obtaining a peptide extract.
  • the previously cultured cells may be, for example, yeast cells (e.g., spent yeast) which have previously been cultured to produce a biological product.
  • FIG.1 is a diagram showing the process by which a peptide extract may be isolated from a population of previously cultured yeast cells.
  • FIG.2 shows a schematic representation of the molecular weight (MW) distribution of an exemplary peptide extract of the disclosure.
  • FIG.3A and FIG 3B are graphs showing the washed optical density (wOD, 600 nm) and sucrose concentration (g/L) of flask cultures over the course of fermentation for the conditions: control, when no peptide extract and no H2O2 is added (outlined circles); peptide extract is present at a concentration of 0.7 g/L (black circles); 2 mM H2O2 is present (outlined boxes); and 2 mM H2O2 plus peptide extract is present at a concentration of 0.7 g/L (black boxes).
  • the data represent the average of two flasks for each condition and error bars are standard deviations.
  • FIG.4 is a graph showing the relative farnesene concentration (%) over the course of fermentation for the conditions: control, when no extract and no H2O2 added (outlined circles); peptide extract is present at a concentration of 0.7 g/L (black circles); 2 mM H2O2 is present (outlined boxes); and 2 mM H2O2 plus peptide extract is present at a concentration of 0.7 g/L (black boxes).
  • the data represent the average of two shake-flasks for each condition and error bars are standard deviations.
  • FIG.5A and FIG.5B are bar graphs showing the change in formation of reactive oxygen species (ROS) in the presence of 2 mM H2O2 plus peptide extract at a concentration of 0.7 g/L in relation to condition 2 mM H2O2 (FIG.5A) and cell viability in each condition tested (FIG.5B) over the course of fermentation.
  • ROS reactive oxygen species
  • FIG.8A and FIG.8B are bar graphs showing the change in formation of reactive oxygen species (ROS) in condition 2 mM H2O2 plus peptide extract at a concentration of 0.7 g/L in relation to condition 2 mM H2O2 (FIG.8A) and cell viability in each condition tested (FIG.8B) over the course of fermentation.
  • ROS reactive oxygen species
  • the population of previously cultured cells may no longer produce a significant quantity of a biological product as a result of being previously cultured. Additionally or alternatively, the population of previously cultured cells may have been fermented for a desired length of time, such as a duration sufficient to produce a mixture having a peptide distribution described herein. It has presently been discovered that culturing a population of host cells in the presence of a peptide extract isolated from previously cultured yeast cells results in the reduction oxidative stress during the biological process by the peptide extract acting as an antioxidant.
  • the presence of the peptide extract reduced ROS generation, and thus, improve yeast tolerance to oxidative stress and production of the biological product, while contributing to a repurposed use of a population of previously cultured cells previously viewed as a waste product.
  • the following sections provide a detailed description of the methods of using previously cultured cells to reduce oxidative stress and increase production of a biological product.
  • ⁇ Methods of Producing a Biological Product Provided herein are methods for producing one or more biological products in a population of host cells capable of producing a biological product.
  • the disclosure provides methods of producing a biological product including providing a population of host cells capable of producing the biological product and culturing the population of host cells in a culture medium that includes a peptide extract obtained from a population of previously cultured cells.
  • the host cells are cultured in a culture medium including a peptide extract obtained from a population of previously cultured cells.
  • the peptide extract may include, for example, one or more proteins.
  • the proteins may have a molecular weight of between 100 Da and 5 kDa (e.g., between 100 Da and 4500 Da, 100 Da and 4000 Da, 100 Da and 3500 Da, 100 Da and 3000 Da, 100 Da and 2500 Da, 100 Da and 2000 Da, 100 Da and 1500 Da, 100 Da and 1000 Da, 100 Da and 500 Da, 500 Da and 5 kDa, 1000 Da and 5 kDa, 1500 Da and 5 kDa, 2000 Da and 5 kDa, 2500 Da and 5 kDa, 3000 Da and 5 kDa, 3500 Da and 5 kDa, 4000 kDa and 5 kDa, or 4500 kDa and 5 kDa).
  • the peptide extract may further include one or more polysaccharides.
  • the one or more polysaccharides may be a biological product from the previously cultured cell from which the peptide 18 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT extract was isolated.
  • the peptide extract may be isolated from a population of previously cultured cells that were, themselves, capable of producing a biological product (e.g., a fermentation product).
  • the previously cultured cells may be capable of producing an isoprene, and isoprenoid, a human milk oligosaccharide, a steviol glycoside, or a cannabinoid.
  • the previously cultured cells are capable of producing ⁇ -farnesene.
  • the population of previously cultured cells may have previously been extracted for the biological product it is capable of producing.
  • the previously cultured cells may undergo centrifugation to separate the cells from a supernatant.
  • the peptide extract is isolated from the supernatant by way of filtration.
  • the peptide extract may be obtained by membrane filtration, fast protein liquid chromatograph-gel filtration, diafiltration, and ultrafiltration.
  • the filtration is membrane filtration.
  • Membrane filtration may be used to separate the proteins of the peptide extract by size. For example, a membrane having a molecular weight cut off of about 1 kDa may be used to isolate proteins from the supernatant to result in the peptide extract.
  • the peptide extract has a yield of between 0.4 g/liter and 2.2 g/liter (e.g., 0.4 g/liter, 0.5 g/liter, 0.6 g/liter, 0.7 g/liter, 0.8 g/liter, 0.9 g/liter, 1 g/liter, 1.1 g/liter, 1.2 g/liter, 1.3 g/liter, 1.4 g/liter, 1.5 g/liter, 1.6 g/liter, 1.7 g/liter, 1.8 g/liter, 1.9 g/liter, 2 g/liter, 2.1 g/liter, or 2.2 g/liter) in the supernatant.
  • 0.4 g/liter 0.5 g/liter, 0.6 g/liter, 0.7 g/liter, 0.8 g/liter, 0.9 g/liter, 1 g/liter, 1.1 g/liter, 1.2 g/liter, 1.3 g/liter, 1.4 g/liter, 1.5 g/liter, 1.6
  • the previously cultured cells have an optical density of between about 20 to about 150 (e.g., between about 20 and about 100, about 20 and about 80, about 20 and about 60, about 20 and about 40, about 40 and about 150, about 60 and about 150, about 80 and about 150, about 100 and about 150, or about 120 and about 150) immediately prior to obtaining the peptide extract and culturing it with the population of host cells.
  • optical density between about 20 to about 150 (e.g., between about 20 and about 100, about 20 and about 80, about 20 and about 60, about 20 and about 40, about 40 and about 150, about 60 and about 150, about 80 and about 150, about 100 and about 150, or about 120 and about 150) immediately prior to obtaining the peptide extract and culturing it with the population of host cells.
  • the previously cultured cells have an optical density of between about 20 to about 50 (e.g., between about 20 to about 45, about 20 to about 40, about 20 to about 35, about 20 to about 30, about 20 to about 25, about 25 to about 50, about 30 to about 50, about 35 to about 50, about 40 to about 50, or about 45 to about 50) immediately prior to obtaining the peptide extract and culturing it with the population of host 19 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT cells.
  • the previously cultured cells have an optical density of about 50 immediately prior to obtaining the peptide extract and culturing it with the population of host cells.
  • the previously cultured cells may have been previously cultured for at least 10 hours.
  • the previously cultured cells may have been previously culture for from about 10 hours to about 100 hours (e.g., about 10 hours to about 75 hours, about 10 hours to about 50 hours, about 10 hours to about 25 hours, about 25 hours to about 100 hours, about 50 hours to about 100 hours, or about 75 hours to about 100 hours).
  • the previously cultured cells were previously capable of producing a biological product, but no longer produce a detectable quantity of the biological product by virtue of having been extensively cultured.
  • the method results in an increase in host cell density of at least 1.1-fold in comparison to a reference method in which the peptide extract is not present in the culture medium.
  • the method may result in an increase in host cell density of from about 1.1 fold to about 5 fold (e.g., from about 1.1 fold to about 4 fold, about 1.1 fold to about 3 fold, about 1.1 fold to about 2 fold, about 2 fold to about 5 fold, about 3 fold to about 5 fold, or about 4 fold to about 5 fold) in comparison to a reference method in which the peptide extract is not present in the culture medium.
  • the method results in a decrease in the formation of reactive oxygen species by at least 10% in comparison to a reference method in which the peptide extract is not present in the culture medium.
  • the method may result in a decrease in the formation of reactive oxygen species by from about 10% to about 90% (e.g., from about 10% to about 70%, about 10% to about 50%, about 10% to about 25%, about 25% to about 90%, about 50% to about 90%, or about 75% to about 90%) in comparison to a reference method in which the peptide extract is not present in the culture medium.
  • the method results in a decrease in the formation of reactive oxygen species by from about 25% to about 75% (e.g., from about 25% to about 65%, about 25% to about 55%, about 25% to about 45%, about 25% to about 35%, about 35% to about 75%, about 45% to about 75%, about 55% to about 75%, or about 65% to about 75%) in comparison to a reference method in which the peptide extract is not present in the culture medium.
  • the method may result in an increase in sugar consumption in comparison to a reference method in which the peptide extract is not present in the culture medium.
  • the method may result in an increase in the concentration of the biological product of from about 1.1 fold to about 10 fold (e.g., about 1.1 fold to about 8 fold, about 1.1 fold to about 6 fold, about 1.1 fold to about 4 fold, about 1.1 fold to about 2 fold, about 2 fold to about 10 fold, about 4 fold 20 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT to about 10 fold, about 6 fold to about 10 fold, or about 8 fold to about 10 fold) in comparison to a reference method in which the peptide extract is not present in the culture medium.
  • a reference method in which the peptide extract is not present in the culture medium.
  • the method results in an increase in the concentration of the biological product of from about 1.1 fold to about 5 fold (e.g., about 1.1 fold to about 4 fold, about 1.1 fold to about 3 fold, about 1.1 fold to about 2 fold, about 2 fold to about 5 fold, about 3 fold to about 5 fold, or about 4 fold to about 5 fold) in comparison to a reference method in which the peptide extract is not present in the culture medium.
  • Enzymes of Exemplary Biosynthetic Pathways The host cells (and/or the previously cultured cells) described herein may express one or more enzymes of a biosynthetic pathway capable of producing a biological product of interest.
  • the cells may be genetically modified to produce a biological product.
  • the cells may be genetically modified to produce a fermentation product.
  • the cells may produce a biological product such as, for example, an isoprene, an isoprenoid, a human milk oligosaccharide (HMO), a steviol glycoside, or a cannabinoid.
  • HMO human milk oligosaccharide
  • the cell may include one or more nucleic acids encoding one or more enzymes of a heterologous genetic pathway that produces a cannabinoid or a precursor of a cannabinoid.
  • the cannabinoid biosynthetic pathway may begin with hexanoic acid as the substrate for an acyl activating enzyme (AAE) to produce hexanoyl-CoA, which is used as the substrate of a tetraketide synthase (TKS) to produce tetraketide-CoA, which is used by an olivetolic acid cyclase (OAC) to produce olivetolic acid, which is then used to produce a cannabigerolic acid by a geranyl pyrophosphate (GPP) synthase and a cannabigerolic acid synthase (CBGaS).
  • AAE acyl activating enzyme
  • TTKS tetraketide synthase
  • OAC olivetolic acid cyclase
  • GFP geranyl pyrophosphate
  • CBGaS cannabigerolic acid synthase
  • the cannabinoid precursor that is produced is a substrate in the cannabinoid pathway (e.g., hexanoate or olivetolic acid).
  • the precursor is a substrate for an AAE, a TKS, an OAC, a CBGaS, or a GPP synthase.
  • the precursor, substrate, or intermediate in the cannabinoid pathway is hexanoate, olivetol, or olivetolic acid.
  • the precursor is hexanoate.
  • the cell does not contain the precursor, substrate or intermediate in an amount sufficient to produce the cannabinoid or a precursor of the cannabinoid.
  • the cell does not contain hexanoate at a level or in an amount sufficient to produce the cannabinoid in an amount over 10 mg/L.
  • the heterologous genetic pathway encodes at least one enzyme selected from the group consisting of an AAE, a TKS, an OAC, a CBGaS, and a GPP 21 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT synthase.
  • the genetically modified cell includes an AAE, TKS, OAC, CBGaS, and a GPP synthase.
  • the cannabinoid pathway is described in Keasling et al., U.S. Patent No.
  • the cell may include, in some embodiments, a heterologous AAE such that the cell is capable of producing a cannabinoid.
  • the AAE may be from Cannabis sativa or may be an enzyme from another plant or fungal source which has been shown to have AAE activity in the cannabinoid biosynthetic pathway, resulting in the production of the cannabinoid precursor olivetolic acid
  • the cell may include a heterologous TKS such that the cell is capable of producing a cannabinoid.
  • a TKS uses the hexanoyl-CoA precursor to generate tetraketide-CoA.
  • the TKS may be from Cannabis sativa or may be an enzyme from another plant or fungal source which has been shown to have TKS activity in the cannabinoid biosynthetic pathway, resulting in the production of the cannabinoid precursor olivetolic acid.
  • Some embodiments concern a cell that includes a heterologous CBGaS such that the cell is capable of producing a cannabinoid.
  • a CBGaS uses the olivetolic acid precursor and GPP precursor to generate cannabigerolic acid.
  • the GPP synthase may be from Cannabis sativa or may be an enzyme from another plant or bacterial source which has been shown to have GPP synthase activity in the cannabinoid biosynthetic pathway, resulting in the production of the cannabinoid cannabigerolic acid.
  • the population of cells may further express other heterologous enzymes in addition to the AAE, TKS, CBGaS, and/or GPP synthase.
  • the cell may include a heterologous nucleic acid that encodes at least one enzyme from the mevalonate biosynthetic pathway.
  • Enzymes which make up the mevalonate biosynthetic pathway may include but are not limited to an acetyl-CoA thiolase, an HMG-CoA synthase, an HMG-CoA reductase, a mevalonate kinase, a phosphomevalonate kinase, a mevalonate pyrophosphate decarboxylase, and an IPP: DMAPP isomerase.
  • the cell includes a heterologous nucleic acid that encodes the acetyl-CoA thiolase, the HMG-CoA synthase, the HMG-CoA reductase, the mevalonate kinase, the phosphomevalonate kinase, the mevalonate pyrophosphate decarboxylase, and the IPP: DMAPP isomerase of the mevalonate biosynthesis pathway.
  • the cell may include an olivetolic acid cyclase (OAC) as part of the cannabinoid biosynthetic pathway.
  • OAC olivetolic acid cyclase
  • the cell further includes one or more heterologous nucleic acids that each, independently, encode an acetyl-CoA synthase, and/or an aldehyde dehydrogenase, and/or a pyruvate decarboxylase.
  • the cell contains a heterologous nucleic acid encoding an aceto-CoA carboxylase (ACC).
  • ACC aceto-CoA carboxylase
  • the cell contains a heterologous nucleic acid encoding an ACC and an acetoacetyl-CoA synthase (AACS) instead of a heterologous nucleic acid encoding an acetyl-CoA thiolase.
  • AACS acetoacetyl-CoA synthase
  • cells of the disclosure may also be modified so as to express the enzymes of the biosynthetic pathway of a target HMO.
  • cells of the disclosure e.g., yeast cells
  • yeast cells may naturally express some of the enzymes of the biosynthetic pathway for a given HMO.
  • Such cells may be modified to express the remaining enzymes of the biosynthetic pathway.
  • a cell e.g., a yeast cell
  • the cells may naturally express many of the enzymes of the biosynthetic pathway of a desired HMO, and the cells may be modified so as to express the remaining enzymes of the biosynthetic pathway for the desired HMO by providing the cells with one or more heterologous nucleic acid molecules that, together, encode the remaining enzymes of the biosynthetic pathway.
  • cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing LNnT, including a ⁇ -1,3- N-acetylglucosaminyltransferase (LgtA), a ⁇ -1,4-galactosyltransferase (LgtB), and a UDP-N- acetylglucosamine diphosphorylase.
  • LgtA and LgtB enzymes useful in conjunction with the compositions and methods of the disclosure are described in the sections that follow.
  • cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing 2’-FL, including a lactose permease, a GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, an ⁇ -1,2-fucosyltransferase, and a fucosidase.
  • a lactose permease including a lactose permease, a GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, an ⁇ -1,2-fucosyltransferase, and a fucosidase.
  • cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing 3-fucosyllactose, including a GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, an ⁇ -1,3-fucosyltransferase, and a fucosidase.
  • enzymes of a pathway for synthesizing 3-fucosyllactose including a GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, an ⁇ -1,3-fucosyltransferase, and a fucosidase.
  • Exemplary enzymes useful in conjunction with the compositions and methods of the disclosure are described in the sections that follow.
  • cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing lacto-N-tetraose, including a ⁇ -1,3-N-acetylglucosaminyltransferase, a ⁇ -1,3-galactosyltransferase, and a UDP-N- acetylglucosamine diphosphorylase.
  • a pathway for synthesizing lacto-N-tetraose including a ⁇ -1,3-N-acetylglucosaminyltransferase, a ⁇ -1,3-galactosyltransferase, and a UDP-N- acetylglucosamine diphosphorylase.
  • cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing 3’-sialyllactose, including a CMP-Neu5Ac synthetase, a sialic acid synthase, a UDP-N-acetylglucosamine 2-epimerase, a UDP- N-acetylglucosamine diphosphorylase, and a CMP-N-acetylneuraminate- ⁇ -galactosamide- ⁇ -2,3- 23 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT sialyltransferase.
  • a pathway for synthesizing 3’-sialyllactose including a CMP-Neu5Ac synthetase, a sialic acid synthase, a UDP-N-acetylglucosamine 2-epime
  • cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing 6’-sialyllactose, including a CMP-Neu5Ac synthetase, a sialic acid synthase, a UDP-N-acetylglucosamine 2-epimerase, a UDP- N-acetylglucosamine diphosphorylase, and a ⁇ -galactoside- ⁇ -2,6-sialyltransferase.
  • a pathway for synthesizing 6’-sialyllactose including a CMP-Neu5Ac synthetase, a sialic acid synthase, a UDP-N-acetylglucosamine 2-epimerase, a UDP- N-acetylglucosamine diphosphorylase, and a ⁇ -galactoside- ⁇ -2,6-sialyltransfer
  • the cells of the disclosure express a LgtB polypeptide. In some embodiments, the cells of the disclosure express a protein that transports lactose into the cell. In some embodiments, the cells of the disclosure express a GDP-mannose 4,6-dehydratase. In some embodiments, the cells of the disclosure express a GDP-L-fucose synthase. In some embodiments, the cells of the disclosure express an ⁇ -1,2-fucosyltransferase polypeptide. Steviol Glycoside Biosynthetic Pathway In some embodiments, the cells are capable of producing one or more steviol glycosides may encode on or more enzymes of the steviol glycoside biosynthesis pathway.
  • the cell includes a heterologous nucleic acid encoding a CDPS.
  • KS (EC 4.2.3.19) catalyzes the conversion of copalyl diphosphate into kaurene and diphosphate.
  • KO catalyzes the conversion of kaurene into kaurenoic acid.
  • Illustrative examples of enzymes include those of Oryza sativa (accession no. Q5Z5R4), Gibberella fujikuroi (accession no. O94142), Arabidopsis thaliana (accession no.
  • the cell includes a heterologous nucleic acid encoding a KO. KAH (EC 1.14.13) also referred to as steviol synthases catalyze the conversion of kaurenoic acid into steviol.
  • enzymes include those of Stevia rebaudiana (accession no. ACD93722), Arabidopsis thaliana (accession no.
  • the cell includes a heterologous nucleic acid encoding a KAH.
  • a CPR (EC 1.6.2.4) is necessary for the activity of KO and/or KAH above.
  • enzymes include those of Stevia rebaudiana (accession no.
  • the cell includes a heterologous nucleic acid encoding a UGT74G1.
  • UGT76G1 is capable of functioning as a uridine 5’-diphospho glucosyltransferase to the: (1) C-3’ position of the 13-O-linked glucose on steviolbioside in a beta linkage forming RebB, (2) C-3’ position of the 19-O-linked glucose on stevioside in a beta linkage forming RebA, and (3) C-3’ position of the 19-O-linked glucose on RebD in a beta linkage forming RebM.
  • UGT76G1 has been described in Richman et al., 2005, Plant J., vol.41, pp.56-67; US2014/0329281; WO2016/038095; and accession no. AAR06912.1.
  • UGT85C2 is capable of functioning as a uridine 5’-diphospho glucosyl: steviol 13-OH transferase, and a uridine 5’-diphospho glucosyl: steviol-19-O-glucoside 13-OH transferase.
  • UGT85C2 is capable of converting steviol to steviolmonoside and is also capable of converting 19- glycoside to rubusoside.
  • Examples of UGT85C2 enzymes include those of Stevia rebaudiana: see e.g., Richman et al., (2005), Plant J., vol.41, pp.56-67; U.S. Patent Application Publication No. 2014/0329281; WO 2016/038095; and accession no.
  • the cell includes a heterologous nucleic acid encoding a UGT85C2.
  • UGT40087 is capable of transferring a glucose moiety to the C-2’ position of the 19-O- glucose of RebA to produce RebD.
  • UGT40087 is also capable of transferring a glucose moiety to the C-2’ position of the 19-O-glucose of stevioside to produce RebE. Examples of UGT40087 include those of accession no. XP_004982059.1 and WO 2018/031955.
  • the cell includes a heterologous nucleic acid encoding a UGT40087.
  • nucleotide sequences encoding such an enzyme include but are not limited to: (NC_000913 REGION: 2324131.2325315; Escherichia coli), (D49362; Paracoccus denitrifzcans), and (L20428; Saccharomyces cerevisiae).
  • the cell includes a heterologous nucleotide sequence encoding an enzyme that can condense acetoacetyl-CoA with another molecule of acetyl-CoA to form 3-hydroxy- 3-methylglutaryl-CoA (HMG-CoA), e.g., a HMG-CoA synthase.
  • HMG-CoA 3-hydroxy- 3-methylglutaryl-CoA
  • nucleotide sequences encoding such an enzyme include but are not limited to: (NC_00l 145. complement 19061.20536; Saccharomyces cerevisiae), (X96617; Saccharomyces cerevisiae), (X83882; Arabidopsis thaliana), (AB037907; Kitasatospora griseola), (BT007302; Homo sapiens), and (NC_002758, Locus tag SAV2546, GeneID 1122571; Staphylococcus aureus).
  • the cell includes a heterologous nucleotide sequence encoding an enzyme that can convert HMG-CoA into mevalonate, e.g., an HMG-CoA reductase.
  • an enzyme that can convert HMG-CoA into mevalonate e.g., an HMG-CoA reductase.
  • nucleotide sequences encoding such an enzyme include, but are not limited to: (NM_206548; Drosophila melanogaster), (NC_002758, Locus tag SAV2545, GeneID 1122570; Staphylococcus aureus), (NM_204485; Gallus gallus), (AB015627; Streptomyces sp.
  • the cells include a heterologous nucleotide sequence encoding an enzyme that can convert mevalonate into mevalonate 5-phosphate, e.g., a mevalonate kinase.
  • the cells include a heterologous nucleotide sequence encoding an enzyme that can convert mevalonate 5-pyrophosphate into isopentenyl diphosphate (IPP), e.g., a mevalonate pyrophosphate decarboxylase.
  • IPP isopentenyl diphosphate
  • nucleotide sequences encoding such an enzyme include but are not limited to: (X97557; Saccharomyces cerevisiae), (AF290095; Enterococcus faecium), and (U49260; Homo sapiens).
  • the cells described herein may be modified to express one or more enzymes of the 1-deoxy- D-xylulose 5-diphosphate (DXP) biosynthetic pathway.
  • Cells which are modified with one or more enzymes of the DXP biosynthetic pathway may be capable of an increased production of one or more isoprenoid compounds as compared to cell which is not modified with one or enzymes of the DXP biosynthetic pathway.
  • the cells include a heterologous nucleotide sequence encoding an enzyme that can condense two molecules of acetyl-coenzyme A to form acetoacetyl-CoA, e.g., an acetyl-CoA thiolase.
  • the cell includes a heterologous nucleotide sequence encoding an enzyme, e.g., l-deoxy-D-xylulose-5-phosphate reductoisomerase, which can convert l-deoxy-D- xylulose-5-phosphate to 2C-methyl-Derythritol- 4-phosphate.
  • an enzyme e.g., l-deoxy-D-xylulose-5-phosphate reductoisomerase, which can convert l-deoxy-D- xylulose-5-phosphate to 2C-methyl-Derythritol- 4-phosphate.
  • nucleotide sequences include but are not limited to: (AB013300; Escherichia coli), (AF148852; Arabidopsis thaliana), (NC_002947, locus tag PP1597; Pseudomonas putida KT2440), (AL939124, locus tag SCO5694; Streptomyces coelicolor A3(2)), (NC_007493, locus tag RSP 2709; Rhodobacter sphaeroides 2.4.1), and (NC_007492, locus tag Pfl_l 107; Pseudomonas jluorescens PfO-1).
  • the cell includes a heterologous nucleotide sequence encoding an enzyme, e.g., 4-diphosphocytidyl-2C-methyl-D-erythritol synthase, which can convert 2C-methyl-D- erythritol-4-phosphate to 4-diphosphocytidyl-2Cmethyl-D-erythritol.
  • an enzyme e.g., 4-diphosphocytidyl-2C-methyl-D-erythritol synthase, which can convert 2C-methyl-D- erythritol-4-phosphate to 4-diphosphocytidyl-2Cmethyl-D-erythritol.
  • nucleotide sequences include but are not limited to: (AF230736; Escherichia coli), (NC_007493, locus tag RSP 28 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT 2835; Rhodobacter sphaeroides 2.4.1), (NC_003071, locus tag AT2G02500; Arabidopsis thaliana), and (NC_002947, locus tag PP1614; Pseudomonas putida KT2440).
  • the cell includes a heterologous nucleotide sequence encoding an enzyme, e.g., 4-diphosphocytidyl-2C-methyl-D-erythritol kinase, which can convert 4- diphosphocytidyl-2C-methyl-D-erythritol to 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate.
  • an enzyme e.g., 4-diphosphocytidyl-2C-methyl-D-erythritol kinase, which can convert 4- diphosphocytidyl-2C-methyl-D-erythritol to 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate.
  • nucleotide sequences include but are not limited to: (AF216300; Escherichia coli) and (NC_007493, locus tag RSP 1779; Rh
  • the cell includes a heterologous nucleotide sequence encoding an enzyme, 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, which can convert 4- diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate to 2Cmethyl-D-erythritol 2,4-cyclodiphosphate.
  • an enzyme 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, which can convert 4- diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate to 2Cmethyl-D-erythritol 2,4-cyclodiphosphate.
  • nucleotide sequences include but are not limited to: (AF230738; Escherichia coli), (NC_007493, locus tag RSP _6071; Rhodobacter sphaeroides 2.4.1), and (NC_002947, locus tag PP1618; Pseudomonas putida KT2440).
  • the cell includes a heterologous nucleotide sequence encoding an enzyme, e.g., l-hydroxy-2-methyl-2I)-butenyl-4- diphosphate synthase, which can convert 2C-methyl- D-erythritol 2,4-cyclodiphosphate to 1- hydroxy-2-methy 1-2-(E)-butenyl 1-4-di phosphate.
  • an enzyme e.g., l-hydroxy-2-methyl-2I)-butenyl-4- diphosphate synthase, which can convert 2C-methyl- D-erythritol 2,4-cyclodiphosphate to 1- hydroxy-2-methy 1-2-(E)-butenyl 1-4-di phosphate.
  • nucleotide sequences include but are not limited to: (AY033515; Escherichia coli), (NC_002947, locus tag PP0853; Pseudomonas putida KT2440), and (NC_007493, locus tag RSP 2982; Rhodobacter sphaeroides 2.4.1).
  • the cell includes a heterologous nucleotide sequence encoding an enzyme, e.g., isopentyl/dimethylallyl diphosphate synthase, which can convert l-hydroxy-2-methyI-(E)- butenyl-4-diphosphate into either IPP or its isomer, DMAPP.
  • an enzyme e.g., isopentyl/dimethylallyl diphosphate synthase, which can convert l-hydroxy-2-methyI-(E)- butenyl-4-diphosphate into either IPP or its isomer, DMAPP.
  • nucleotide sequences include but are not limited to: (AY062212; Escherichia coli) and (NC_002947, locus tag PP0606; Pseudomonas putida KT2440).
  • the cell includes one or more heterologous nucleotide sequences encoding more than one enzyme of the DXP pathway.
  • the cell includes one or more heterologous nucleotide sequences encoding two enzymes of the DXP pathway.
  • the cell includes one or more heterologous nucleotide sequences encoding three enzymes of the DXP pathway.
  • the cell includes one or more heterologous nucleotide sequences encoding four enzymes of the DXP pathway. In some embodiments, the cell includes one or more heterologous nucleotide sequences encoding five enzymes of the DXP pathway. In some embodiments, the cell includes one or more heterologous nucleotide sequences encoding six enzymes of the DXP pathway. In some embodiments, the cell includes one or more heterologous nucleotide sequences encoding five enzymes of the DXP pathway. In some embodiments, the cell includes one or more heterologous nucleotide sequences encoding seven enzymes of the DXP pathway.
  • crosstalk (or interference) between the cell's own metabolic processes and those processes involved with the production of IPP are minimized or eliminated entirely.
  • cross talk is minimized or eliminated entirely when the cell relies exclusively on the DXP pathway for synthesizing IPP, and a MEV pathway is introduced to provide additional IPP.
  • 29 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT Such a cell would not be equipped to alter the expression of the MEV pathway enzymes or process the intermediates associated with the MEV pathway.
  • Organisms that rely exclusively or predominately on the DXP pathway include, for example, Escherichia coli.
  • the cell produces IPP via the MEV pathway, either exclusively or in combination with the DXP pathway.
  • a cell’s DXP pathway is functionally disabled so that the cell produces IPP exclusively through a heterologously introduced MEV pathway.
  • the DXP pathway can be functionally disabled by disabling gene expression or inactivating the function of one or more of the DXP pathway enzymes.
  • the cell further includes a heterologous nucleotide sequence encoding a polyprenyl synthase that can condense IPP and/or DMAPP molecules to form polyprenyl compounds containing more than five carbons.
  • the isoprenoid producing cell further comprises a heterologous nucleotide sequence encoding an enzyme that can convert IPP generated via the MEV pathway into DMAPP, e.g., an IPP isomerase.
  • an enzyme that can convert IPP generated via the MEV pathway into DMAPP
  • IPP isomerase e.g., an IPP isomerase.
  • nucleotide sequences encoding such an enzyme include but are not limited to: (NC_000913, 3031087.3031635; Escherichia coli), and (AF082326; Haematococcus pluvialis).
  • the cell includes a heterologous nucleotide sequence encoding an enzyme that can condense one molecule of IPP with one molecule of DMAPP to form one molecule of geranyl pyrophosphate (GPP), e.g., a GPP synthase.
  • GPP geranyl pyrophosphate
  • nucleotide sequences encoding such an enzyme include, but are not limited to: (AF513lll;Abies grandis), (AF513112;Abies grandis), (AF513113;Abies grandis), (AY534686; Antirrhinum majus), (AY534687; Antirrhinum majus), (Yl 7376; Arabidopsis thaliana), (AE016877, Locus APl 1092; Bacillus cereus; ATCC 14579), (AJ243739; Citrus sinensis), (AY534745; Clarkia breweri), (AY953508; fps pini), (DQ286930; Lycopersicon esculentum), (AF182828; Mentha x piperita), (AF182827; Mentha x piperita), (MPI249453; Mentha x piperita), (PZE431697, Locus CAD24425; Paracoccus 8
  • nucleotide sequences that encode such an enzyme include, but are not limited to: (ATU80605; Arabidopsis thaliana), (ATHFPS2R; Arabidopsis thaliana), (AAU36376; Artemisia annua), (AF461050; Bos taurus), (D00694; Escherichia coli K-12), (AE009951, Locus AAL95523; Fusobacterium nucleatum subsp.
  • NC_005823 Locus YP 000273; Leptospira interrogans serovar Copenhageni str. Fiocruz Ll-130), (AB003187; Micrococcus luteus), (NC_002946, Locus YP _208768; Neisseria gonorrhoeae FA 1090), (U00090, Locus AAB91752; Rhizobium sp.
  • the cell includes a heterologous nucleotide sequence encoding an enzyme that can combine IPP and DMAPP or IPP and FPP to form geranylgeranyl pyrophosphate (GGPP).
  • GGPP geranylgeranyl pyrophosphate
  • nucleotide sequences that encode such an enzyme include, but are not limited to: (ATHGERPYRS; Arabidopsis thaliana), (BT005328; Arabidopsis thaliana), (NM_l 19845; Arabidopsis thaliana), (NZ_AAJM01000380, Locus ZP 00743052; Bacillus thuringiensis serovar israelensis, ATCC 35646 sql563), (CRGGPPS; Catharanthus roseus), (NZ_AABF02000074, Locus ZP 00144509; Fusobacterium nucleatum subsp.
  • ATHGERPYRS Arabidopsis thaliana
  • BT005328 Arabidopsis thaliana
  • NM_l 19845 Arabidopsis thaliana
  • NZ_AAJM01000380 Locus ZP 00743052
  • Bacillus thuringiensis serovar israelensis ATCC 35646
  • lusitanicus (AB016044; Mus musculus), (AABX01000298, Locus NCU01427; Neurospora crassa), (NCU20940; Neurospora crassa), (NZ_AAKL01000008, Locus ZP 00943566; Ralstonia solanacearum UW551), (ABl 18238; Rattus norvegicus), (SCU31632; Saccharomyces cerevisiae), (AB016095; Synechococcus elongates), (SAGGPS; Sinapis alba), (SSOGDS; Sulfolobus acidocaldarius), (NC_007759, Locus YP 461832; Syntrophus aciditrophicus SB), (NC_006840, Locus YP 204095; Vibrio jischeri ESl 14), (NM_ 112315; Arabidopsis thaliana), (ERWCR TE;
  • the cell further includes a heterologous nucleotide sequence encoding an enzyme that can modify a polyprenyl to form a hemiterpene, a monoterpene, a sesquiterpene, a diterpene, a triterpene, a tetraterpene, a polyterpene, a steroid compound, a carotenoid, or a modified isoprenoid compound.
  • the heterologous nucleotide encodes a carene synthase.
  • nucleotide sequences include but are not limited to: (Af 457070; Cinnamomum tenuipilum), (A Y362553; Ocimum basilicum), (DQ234300; Perilla frutescens strain 1864), 31 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT (DQ234299; Perilla citriodora strain 1861), (DQ234298; Perilla citriodora strain 4935), and (DQ088667; Perilla citriodora).
  • the heterologous nucleotide encodes a linalool synthase.
  • a suitable nucleotide sequence include, but are not limited to: (AF497485; Arabidopsis thaliana), (AC002294, Locus AAB71482; Arabidopsis thaliana), (AY059757; Arabidopsis thaliana), (NM_104793; Arabidopsis thaliana), (AF154124; Artemisia annua), (AF067603; Clarkia breweri), (AF067602; Clarkia concinna), (AF067601; Clarkia breweri), (U58314; Clarkia breweri), (AY840091; Lycopersicon esculentum), (DQ263741; Lavandula angustifolia), (AY083653;Mentha citrate), (AY693647; Ocimum basilicum), (XM_ 463918; Oryza sativa), (AP004078, Locus BAD07605; Or
  • the heterologous nucleotide encodes a limonene synthase.
  • suitable nucleotide sequences include but are not limited to:(+) limonene synthases (AF514287, REGION: 47.1867; Citrus limon) and (AY055214, REGION: 48.1889; Agastache rugosa) and (-)-limonene synthases (DQ195275, REGION: 1.1905; Picea sitchensis), (AF006193, REGION: 73.1986;Abies grandis), and (MHC4SLSP, REGION: 29.1828; Mentha spicata).
  • the heterologous nucleotide encodes a myrcene synthase.
  • suitable nucleotide sequences include, but are not limited to: (U87908; Abies grandis), (A Yl 95609; Antirrhinum majus), (A Yl 95608; Antirrhinum majus), (NM_l27982; Arabidopsis thaliana TPSlO), (NM_ll3485; Arabidopsis thaliana ATTPS-CIN), (NM_ 113483; Arabidopsis thaliana ATTPS- CIN), (AF271259; Perilla frutescens), (AY473626; Picea abies), (AF369919; Picea abies), and (AJ304839; Quercus ilex).
  • the heterologous nucleotide encodes an a ⁇ -pinene synthase.
  • suitable nucleotide sequences include but are not limited to: (+) ⁇ -pinene synthase (AF543530, REGION: 1.1887; Pinus taeda), (-) ⁇ -pinene synthase (AF543527, REGION: 32.1921; Pinus taeda), and (+)/ (-)a-pinene synthase (AGU87909, REGION: 6111892;Abies grandis).
  • the heterologous nucleotide encodes a P-pinene synthase.
  • suitable nucleotide sequences include but are not limited to: (-) Ppinene synthases (AF276072, REGION: 1.1749; Artemisia annua) and (AF514288, REGION: 26.1834; Citrus limon).
  • the heterologous nucleotide encodes a sabinene synthase.
  • An illustrative example of a suitable nucleotide sequence includes but is not limited to AF05 l 901, REGION: 26.1798 from Salvia ofjicinalis.
  • the heterologous nucleotide encodes a y-terpinene synthase.
  • suitable nucleotide sequences include, but are not limited to: (AF514286, REGION: 30.1832 from Citrus limon) and (ABl 10640, REGION 1.1803 from Citrus unshiu).
  • the heterologous nucleotide encodes a terpinolene synthase.
  • a suitable nucleotide sequence include but are not limited to: (AY693650 from Ocimum basilicum) and (AY906866, REGION: 10.1887 from Pseudotsuga menziesii).
  • the heterologous nucleotide encodes an amorphadiene synthase.
  • An illustrative example of a suitable nucleotide sequence is SEQ ID NO.37 of U.S. Patent Publication No.2004/0005678.
  • the heterologous nucleotide encodes an ⁇ -farnesene synthase.
  • nucleotide sequences include but are not limited to DQ309034 from Pyrus communis cultivar d'Anjou (pear; gene name AFSl) and AY182241 from Malus domestica (apple; gene AFSl). Pechouus et al., Planta 219(1):84-94 (2004).
  • the heterologous nucleotide encodes a ⁇ -farnesene synthase.
  • suitable nucleotide sequences include but are not limited to GenBank accession number AF024615 from Mentha x piperita (peppermint; gene Tspal 1), and A Y835398 from Artemisia annua.
  • the heterologous nucleotide encodes a farnesol synthase.
  • suitable nucleotide sequences include but are not limited to GenBank accession number AF529266 from Zea mays and YDR481C from Saccharomyces cerevisiae (gene Pho8). Song, L., Applied Biochemistry and Biotechnology 128: 149-158 (2006).
  • the heterologous nucleotide encodes a nerolidol synthase.
  • a suitable nucleotide sequence includes but is not limited to AF529266 from Zea mays (maize; gene tpsl).
  • the heterologous nucleotide encodes a patchoulol synthase.
  • suitable nucleotide sequences include but are not limited to AY508730 REGION: 1.1659 from Pogostemon cablin.
  • the heterologous nucleotide encodes a nootkatone synthase.
  • a suitable nucleotide sequence include but are not limited to AF441124 REGION: 1.1647 from Citrus sinensis and AY917195 REGION: 1.1653 from Perilla frutescens.
  • the heterologous nucleotide encodes an abietadiene synthase.
  • suitable nucleotide sequences one or more heterologous nucleic acids encoding one or more enzymes are integrated into the genome of the cell.
  • one or more heterologous nucleic acids encoding one or more enzymes are present within one or more plasmids.
  • a heterologous nucleic acid of the disclosure is introduced into a host cell (e.g., yeast cell) by way of a gap repair molecular biology technique.
  • the host cell may be capable of producing a biological product or a previously cultured cell.
  • the host 33 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT cell has non-homologous end joining (NHEJ) activity, as is the case for Kluyveromyces marxianus, then the NHEJ activity in the host cell can be first disrupted in any of a number of ways.
  • NHEJ non-homologous end joining
  • a heterologous nucleic acid of the disclosure is introduced into the host cell by way of one or more site-specific nucleases capable of causing breaks at designated regions within selected nucleic acid target sites.
  • nucleases examples include, but are not limited to, endonucleases, site-specific recombinases, transposases, topoisomerases, zinc finger nucleases, TAL-effector DNA binding domain-nuclease fusion proteins (TALENs), CRISPR/Cas- associated RNA-guided endonucleases, and meganucleases. Further details related to genetic modification of host cells through site specific nuclease activity can be found in U.S. Patent No. 9,476,065, the disclosure of which is incorporated herein by reference in its entirety.
  • Nucleic Acid and Amino Acid Sequence Optimization Described herein are specific genes and proteins useful in the methods, compositions, and organisms of the disclosure; however, it will be recognized that absolute identity to such genes is not necessary. For example, changes in a particular gene or polynucleotide including a sequence encoding a polypeptide or enzyme can be performed and screened for activity. Typically, such changes include conservative mutations and silent mutations. Such modified or mutated polynucleotides and polypeptides can be screened for expression of a functional enzyme using methods known in the art.
  • Codons can be substituted to reflect the preferred codon usage of the host, in a process sometimes called "codon optimization” or "controlling for species codon bias.”
  • Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence.
  • Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively.
  • a native DNA sequence encoding the biosynthetic enzymes described above is referenced herein merely to illustrate an embodiment of the disclosure, and the disclosure includes DNA molecules of any sequence that encode the amino acid sequences of the polypeptides and proteins of the enzymes utilized in the methods of the disclosure.
  • a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity.
  • the disclosure includes such polypeptides with different amino acid sequences than the specific proteins described herein so long as the modified or variant polypeptides have the enzymatic anabolic or catabolic activity of the reference polypeptide.
  • amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the disclosure“
  • a “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties, e.g., charge or hydrophobicity.
  • R group side chain
  • a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution.
  • any of the genes encoding an enzyme described herein can be optimized by genetic/protein engineering techniques, such as directed evolution or rational mutagenesis, which are known to those of ordinary skill in the art. Such action allows those of ordinary skill in the art to optimize the enzymes for expression and activity in yeast.
  • genes encoding these enzymes can be identified from other fungal and bacterial species and can be expressed for the modulation of this pathway.
  • Saccharomyces spp. including S. cerevisiae and S. uvarum, Kluyveromyces spp., including K. thermotolerans, K. lactis, and K. marxianus
  • Pichia spp. Hansenula spp., including H. polymorpha
  • Candida spp. Trichosporon spp.
  • Yamadazyma spp. including Y. spp. stipitis
  • Torulaspora pretoriensis Issatchenkia orientalis
  • Schizosaccharomyces spp. including S.
  • Sources of genes from anaerobic fungi include, but are not limited to, Piromyces spp., Orpinomyces spp., or Neocallimastix spp.
  • Sources of prokaryotic enzymes that are useful include, but are not limited to, Escherichia.
  • Techniques known to those skilled in the art can be suitable to identify analogous genes and analogous enzymes. Techniques include, but are not limited to, cloning a gene by PCR using primers based on a published sequence of a gene/enzyme of interest, or by degenerate PCR using degenerate primers designed to amplify a conserved region among a gene of interest. Further, one skilled in the art can use techniques to identify homologous or analogous genes, proteins, or enzymes with functional homology or similarity.
  • Techniques include examining a cell or cell culture for the catalytic activity of an enzyme through in vitro enzyme assays for said activity, e.g., as described herein or in Kiritani, K., Branched-Chain Amino Acids Methods Enzymology, 1970; then isolating the enzyme with said activity through purification; determining the protein sequence of the enzyme through techniques such as Edman degradation; design of PCR primers to the likely nucleic acid sequence; amplification of said DNA sequence through PCR; and cloning of said nucleic acid sequence.
  • suitable techniques also include comparison of data concerning a candidate gene or enzyme with databases such as BRENDA, KEGG, or MetaCYC.
  • the candidate gene or enzyme can be identified within the above-mentioned databases in accordance with the teachings herein.
  • Culture and Fermentation Conditions Materials and methods for the maintenance and growth of microbial cultures are well known to those skilled in the art of microbiology or fermentation science (see, for example, Bailey et al., Biochemical Engineering Fundamentals, second edition, McGraw Hill, New York, 1986). Consideration must be given to appropriate culture medium, pH, temperature, and requirements for aerobic, microaerobic, or anaerobic conditions, depending on the specific requirements of the host cell, the fermentation, and the process.
  • the methods of producing a biological product provided herein may be performed in a suitable culture medium in a suitable container, including but not limited to a cell culture plate, a flask, or a fermentor. Further, the methods can be performed at any scale of fermentation known in the art to support industrial production of microbial products. Any suitable fermentor may be used including a stirred tank fermentor, an airlift fermentor, a bubble fermentor, or any combination thereof. In particular embodiments utilizing Saccharomyces cerevisiae as the host cell, strains can be grown in a fermentor as described in detail by Kosaric, et al, Ullmann's Encyclopedia of Industrial Chemistry, Sixth Edition, Volume 12, pages 398-473, Wiley-VCH Verlag GmbH & Co.
  • the culture medium is any culture medium in which a microorganism capable of producing a biological product can subsist, i.e., maintain growth and viability.
  • the culture medium is an aqueous medium comprising assimilable carbon, nitrogen, and phosphate sources. Such a medium can also include appropriate salts, minerals, metals, and other nutrients.
  • the carbon source and each of the essential cell nutrients are 36 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT added incrementally or continuously to the fermentation medium, and each required nutrient is maintained at essentially the minimum level needed for efficient assimilation by growing cells, for example, in accordance with a predetermined cell growth curve based on the metabolic or respiratory function of the cells which convert the carbon source to a biomass.
  • Suitable conditions and suitable medium for culturing microorganisms are well known in the art.
  • the suitable medium is supplemented with one or more additional agents, such as, for example, an inducer (e.g., when one or more nucleotide sequences encoding a gene product are under the control of an inducible promoter), a repressor (e.g., when one or more nucleotide sequences encoding a gene product are under the control of a repressible promoter), or a selection agent (e.g., an antibiotic to select for microorganisms comprising the genetic modifications).
  • the carbon source is a monosaccharide (simple sugar), a disaccharide, a polysaccharide, a non-fermentable carbon source, or one or more combinations thereof.
  • ammonia when used to control pH, it also conveniently serves as a nitrogen source in the culture medium.
  • the pH is maintained from about 3.0 to about 8.0, more preferably from about 3.5 to about 7.0, and most preferably from about 4.0 to about 6.5.
  • the carbon source concentration, such as the glucose concentration, of the culture medium is monitored during culture.
  • Glucose or sucrose concentration of the culture medium can be monitored using known techniques, such as, for example, use of the glucose oxidase enzyme test or high-pressure liquid chromatography, which can be used to monitor glucose concentration in the supernatant, e.g., a cell-free component of the culture medium.
  • the carbon source concentration should be kept below the level at which cell growth inhibition occurs. Although such concentration may vary from organism to organism, for glucose as a carbon source, cell growth inhibition occurs at glucose concentrations greater than at about 60 g/L and can be determined readily by trial. Accordingly, when glucose is used as a carbon source the glucose is preferably fed to the fermenter and maintained below detection limits.
  • the glucose concentration in the culture medium is maintained in the range of from about 1 g/L to about 40 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT 100 g/L, more preferably in the range of from about 2 g/L to about 50 g/L, and yet more preferably in the range of from about 5 g/L to about 20 g/L.
  • the carbon source concentration can be maintained within desired levels by addition of, for example, a substantially pure glucose solution, it is acceptable, and may be preferred, to maintain the carbon source concentration of the culture medium by addition of aliquots of the original culture medium.
  • Examples of a prokaryotic cell include, but are not limited to those belonging to the genera: Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter, Azobacter, Bacillus, Brevibacterium, Chromatium, Clostridium, Corynebacterium, Enterobacter, Erwinia, Escherichia, Lactobacillus, Lactococcus, Mesorhizobium, Methylobacterium, Microbacterium, Phormidium, Pseudomonas, Rhodobacter, Rhodopseudomonas, Rhodospirillum, Rhodococcus, Salmonella, Scenedesmun, Serratia, Shigella, Staphlococcus, Strepromyces, Synnecoccus, and Zymomonas.
  • prokaryotic bacterial strains include but are not limited to: Bacillus subtilis, Bacillus amyloliquefacines, Brevibacterium ammoniagenes, Brevibacterium immariophilum, Clostridium beigerinckii, Enterobacter sakazakii, Escherichia coli, Lactococcus lactis, Mesorhizobium loti, Pseudomonas aeruginosa, Pseudomonas mevalonii, Pseudomonas pudica, Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodospirillum rubrum, Salmonella enterica, Salmonella typhi, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, and the like.
  • fungal cell examples include but are not limited to those belonging to the genera: Aspergillus, Candida, Chrysosporium, 41 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT Cryotococcus, Fusarium, Kluyveromyces, Neotyphodium, Neurospora, Penicillium, Pichia, Saccharomyces, Trichoderma and Xanthophyllomyces (formerly Phaffia).
  • Illustrative examples of eukaryotic strains include but are not limited to: Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Candida albicans, Chrysosporium lucknowense, Fusarium graminearum, Fusarium venenatum, Kluyveromyces lactis, Neurospora crassa, Pichia angusta, Pichia finlandica, Pichia kodamae, Pichia membranaefaciens, Pichia methanolica, Pichia opuntiae, Pichia pastoris, Pichia pijperi, Pichia quercuum, Pichia salictaria, Pichia thermotolerans, Pichia trehalophila, Pichia stipitis, Streptomyces ambofaciens, Streptomyces aureofaciens, Streptomyces aureus, Saccaromy
  • the host cell is a yeast cell.
  • the previously cultured cell is a yeast cell.
  • yeast cells useful in conjunction with the compositions and methods described herein include yeast that have been deposited with microorganism depositories (e.g.
  • IFO, ATCC, etc. such as those that belong to the genera Aciculoconidium, Ambrosiozyma, Arthroascus, Arxiozyma, Ashbya, Babjevia, Bensingtonia, Botryoascus, Botryozyma, Brettanomyces, Bullera, Bulleromyces, Candida, Citeromyces, Clavispora, Cryptococcus, Cystofilobasidium, Debaryomyces, Dekkara, Dipodascopsis, Dipodascus, Eeniella, Endomycopsella, Eremascus, Eremothecium, Erythrobasidium, Fellomyces, Filobasidium, Galactomyces, Geotrichum, Guilliermondella, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hyphopichia, Issatchenkia, Kloeckera, Kloe
  • the strain is Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe, Dekkera bruxellensis, Kluyveromyces lactis (previously called Saccharomyces lactis), Kluveromyces marxianus, Arxula adeninivorans, or Hansenula polymorphs (now known as Pichia angusta).
  • the host microbe is a strain of the genus Candida, such as Candida lipolytica, Candida guilliermondii, Candida krusei, Candida pseudotropicalis, or Candida utilis.
  • the strain is Saccharomyces cerevisiae.
  • the host is a strain of Saccharomyces cerevisiae selected from the group consisting of Baker's yeast, CEN.PK, CEN.PK2, CBS 7959, CBS 7960, CBS 7961, CBS 7962, CBS 7963, CBS 7964, IZ-1904, 42 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT TA, BG-1, CR-1, SA-1, M-26, Y-904, PE-2, PE-5, VR-1, BR-1, BR-2, ME-2, VR-2, MA-3, MA-4, CAT- 1, CB-1, NR-1, BT-1, and AL-1.
  • Saccharomyces cerevisiae selected from the group consisting of Baker's yeast, CEN.PK, CEN.PK2, CBS 7959, CBS 7960, CBS 7961, CBS 7962, CBS 7963, CBS 7964, IZ-1904, 42 ⁇ ⁇ ATTORNEY
  • the strain of Saccharomyces cerevisiae is CEN.PK. In some embodiments, the yeast strain used is Y21900. In some embodiments, the yeast strain used is Y23508. In some embodiments, the strain is a microbe that is suitable for industrial fermentation. In particular embodiments, the microbe is conditioned to subsist under high solvent concentration, high temperature, expanded substrate utilization, nutrient limitation, osmotic stress due to sugar and salts, acidity, sulfite and bacterial contamination, or combinations thereof, which are recognized stress conditions of the industrial fermentation environment.
  • the method is based on reduction of Cu2+ to Cu1+ by protein in alkaline medium, followed by Cu1+ reaction with bicinchoninic acid (BCA), which produces a purple-colored product that can be read at 562 nm after 30 min of reaction (Synergy H1, Biotek Instruments, Winooski, USA). Dry weight was determined at 105 oC for 24 h according to standard procedures of the Association of Official Analytical Chemists (AOAC, 2005). Free and total amino acids quantification: The amino acids profile was analyzed according to Wang et al.
  • Nitrogen was injected through an inlet needle to substitute samples atmosphere and another needle was used as gas outlet. Flasks were sealed with tape and placed 1 in an oven at 115 oC for 20 h. Then, pH was adjusted to 3.2 and the solution diluted rigorously to a final volume of 10 mL. For quantification of free amino acids, peptide fractions were prepared in HCl 0.1M at concentration of 10 mg/mL. According to procedure of Pripis-Nicolau et al. (2001), 20 ⁇ L of peptide fraction was derivatized and 10 ⁇ L was injected.
  • the analysis was done in triplicate and the minerals quantified according to calibration curves of a commercial mix standards for ICP analysis (Inorganic Ventures, Christiansburg, USA) (molybdenum, zinc, cadmium, phosphorus, lead, nickel, cobalt, boron, manganese, iron, magnesium, calcium, copper, aluminum, sodium, and potassium) from 0.05 to 10 mg/L.
  • ICP analysis a microwave digestion of samples (2 mL) was performed in a speedwave XPERT (Berghof Products + Instruments GmbH, Eningen, Germany) using 5 mL of Suprapur® HNO3 and 2 mL of 35% H2O2 (Merck KGaA, Darmstadt, Germany).
  • MW peptides distribution was performed on an ultra-high-performance liquid chromatography from Bruker Elute series, coupled to an ultrahigh- resolution quadrupole ⁇ quadrupole time-of-flight (UHR ⁇ QqTOF) mass spectrometer (Impact II; Bruke Daltonik GmbH, Bremen, Germany) using an Intensity Solo 2 C18 (100 ⁇ 2.1 mm, 2.2 ⁇ M, Bruker Daltonik GmbH, Bremen, Germany) (BRHSC18022100) set for 60oC.
  • UHR ⁇ QqTOF ultrahigh- resolution quadrupole ⁇ quadrupole time-of-flight
  • the mobile phases used were 0.1% formic acid water (A) and 0.1% formic acid acetonitrile (B) at 0.250 mL/min flow rate in gradient mode: 95% A and 5% B until 14 min, 5% A and 95% B from 14 to 22 min, and 95% A and 5% 1 B until reaching the run end (25 min).
  • Instrument was operated in MS positive mode and data was collected in the range of 150 to 2200 m/z.
  • the selected parameters were as follows: capillary voltage, 4.5 kV; drying gas temperature, 220 °C; drying gas flow, 9.0 L/min; nebulizing gas pressure, 0.6 bar; collision radio frequency, 2000 Vpp; transfer time, 90 ⁇ s; and pre-pulse storage, 10 ⁇ s.
  • Membrane filtration technology has been described as an important tool for separation of soluble intracellular proteins from cell lysates based on sieving and charge-based mechanisms (Vollet Marson et al., 2020). High selectively and efficiency combined with low energy consumption are the main reasons for its use in the food processing industry, being specially chosen for peptides and proteins because of gentle treatment characteristics (Mohammad et al., 2012). In fact, it is very important to preserve a peptide’s structure and physicochemical attributes, since their bioactivities are described as being highly dependent thereon (Marson, de Castro, Belleville, et al., 2020).
  • ultrafiltration is the main pressure-driven process used because of its range of membrane MW cut-offs (Vollet Marson et al., 2020).
  • an ultrafiltration process was applied to Gpep and Mpep by-products to increase the protein concentration, since the original supernatants (non-treated) amounted about to 40 and 50% of protein purity (w/w) (Gpep and Mpep, respectively).
  • a preliminary MW evaluation was performed by size exclusion fast protein liquid chromatography-gel filtration (FPLC), where it can be observed that the majority of peptides obtained were nearby 1000 Da.
  • FPLC size exclusion fast protein liquid chromatography-gel filtration
  • the high amount of aspartic (18.8 to 105 mg/g protein) and glutamic acid (58.4 to 111 mg/g protein) increase the potential application of these extracts in food 46 ⁇ ⁇ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT market since aspartic acid is related to the synthesis of artificial sweeteners, such as aspartame (O’Mullane et al., 2014), and glutamic acid lead to strong flavor-enhancing properties.
  • Monosodium glutamate, a salt form of glutamic acid is one of the well-known flavor ingredients used by the food industry since it provides the typical “umami” flavor.
  • ultrafiltration membranes (1-50 kDa) are used to recover bioactive peptides and amino acids, and nanofiltration membranes (100-1000 Da) are widely used in low MW peptides purification processes by industry (Vollet Marson et al., 2020).
  • peptides with biological activity are made up of 3-20 amino acids and their amino acid composition and sequence is responsible for their bioactivity (Amorim, Marques, et al., 2019).
  • an ultrafiltration with 1 kDa cut-off membrane was performed, aiming to increase the protein concentration of initial waste streams supernatants, being the 1 kDa cut-off membrane chosen since this initial MW profile was nearby 1000 Da.
  • Samples were collected during fermentations to determine cell growth by optical density (600 nm), cell viability and reactive oxygen species (ROS) by flow cytometry, sugars concentration by HPLC-RID and farnesene concentration by GC-FID.
  • the batch fermentations were performed in a reactor with 2.7 L of working volume at a temperature of 30 °C, pH of 5.0, which was regulated by using a solution of 12.5 % ammonium hydroxide, aeration of 0.5 L/min and dissolved oxygen (DO) of at least 30 %, which was regulated by an agitation ramp ranging between 300 and 1200 rpm.
  • Batch reactors had an initial volume of 1-L after inoculation (10% inoculum, initial wOD600 of ⁇ 1). Cultivation time was ⁇ 47 hrs.
  • the culture base medium of seed flasks and batch bioreactor steps was the same, containing trace metals, vitamins, ammonium phosphate monobasic (7 g/L), potassium phosphate monobasic (1 g/L), magnesium sulphate heptahydrate (0.5 g/L), yeast extract (5 g/L), succinate buffer at pH 5.0 (6 g/L) and sucrose (70 g/L).
  • antifoam TERGITOL® L-81 0.1 mL/L
  • peptides extract 0.7 g/L
  • H2O2 H2O2

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Botany (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The present disclosure provides compositions and methods for producing a biological product by culturing a population of host cells in a culture medium that includes peptide extract obtained from previously cultured cells.

Description

ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT COMPOSITIONS AND METHODS FOR USING PREVIOUSLY CULTURED CELLS Background of the Invention During industrial cell cultures, host cells are exposed to diverse, harsh conditions that may reduce the overall yield of a desired biological product. The conditions to which these host cells are exposed may trigger, for example, the accumulation of intracellular reactive oxygen species (ROS), which can promote oxidative stress and lead to a premature termination of the culture process. This may have the end result of diminishing yield and productivity. These reductions in process performance have the consequence of less material efficiency, as greater quantities of precursors and host cells are ultimately needed in these instances. Accordingly, there remains a need for compositions and methods that are capable of improving the efficiency with which desired biological products are synthesized in, and isolated from, a corresponding host cell. Summary of the Invention The present disclosure provides methods and compositions for producing a desired biological product. Using the compositions and methods of the disclosure, a biological product may be obtained by culturing a population of host cells capable of producing the biological product in a culture medium. The culture medium may include a peptide extract that has been obtained from a population of previously cultured cells (e.g., previously cultured yeast cells). The previously cultured cells may, for example, no longer produce a significant quantity of a biological product as a result of being previously cultured. Additionally or alternatively, the previously cultured cells may have been fermented for a desired period of time, such as a period of time sufficient to produce a peptide composition described herein. The present disclosure is based, in part, on the surprising discovery that peptide extracts isolated from such previously cultured cells may confer antioxidant activity to a population of host cells that are presently being fermented with the aim of biosynthesizing a biological product of interest. This antioxidant activity may, in turn, have the beneficial effect of improving the amount of the desired biological product that is ultimately biosynthesized by the host cells. Without being limited by mechanism, peptide extracts obtained from previously cultured cells may exert this advantageous effect on host cells during a biological process by, for example, elevating the tolerance of the host cells to oxidative stress and reducing the amount of reactive oxygen species that are generated during the biological process. In a first aspect, the disclosure provides a method of producing a biological product including: providing a population of host cells capable of producing the biological product, and culturing the population of host cells in a culture medium that includes a peptide extract obtained from a population of previously cultured cells. In another aspect, the disclosure provides a method of culturing a population of host cells including: providing a population of host cells capable of producing a biological product, and culturing 1^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT the population of host cells in a culture medium that includes a peptide extract obtained from a population of previously cultured cells. In some embodiments, the peptide extract includes proteins. In some embodiments, the peptide extract has a protein concentration of between 20% (w/w) and 95% (w/w) (e.g., between 20% (w/w) and 75% (w/w), 20% (w/w) and 50% (w/w), 20% (w/w) and 30% (w/w), 30% (w/w) and 95% (w/w), 50% (w/w) and 95% (w/w), 70% (w/w) and 95% (w/w), or 80% (w/w) and 95% (w/w)). In some embodiments, the peptide extract has a protein concentration of between 45% (w/w) and 90% (w/w) (e.g., between 50% (w/w) and 90% (w/w), 60% (w/w) and 90% (w/w), 70% (w/w) and 90% (w/w), 80% (w/w) and 90% (w/w), 45% (w/w) and 80% (w/w), 45% (w/w) and 70% (w/w), 45% (w/w) and 60% (w/w), or 45% (w/w) and 50% (w/w)). In some embodiments, the proteins comprise one or more amino acid amino acid residues selected from histidine (His), threonine (Thr), arginine (Arg), valine (Val), phenylalanine (Phe), tyrosine (Tyr), Isoleucine (Ile), leucine (Leu), and lysine (Lys). In some embodiments, the proteins have a molecular weight of greater than 1 kDa. In some embodiments, the proteins comprise a His residue, optionally wherein proteins comprising His are at a concentration of between about 20 mg/g protein and 30 mg/g protein (e.g., between about 20 mg/g protein and about 26 mg/g protein, about 20 mg/g protein and about 24 mg/g protein, about 24 mg/g protein and about 30 mg/g protein, or about 28 mg/g protein and about 30 mg/g protein). In some embodiments, the proteins comprise a Thr residue, optionally wherein proteins comprising Thr are at a concentration of between about 25 mg/g protein and about 55 mg/g protein (e.g., between about 35 mg/g protein and about 55 mg/g protein, about 45 mg/g protein and about 55 mg/g protein, about 25 mg/g protein and about 45 mg/g protein, or about 25 mg/g protein and about 35 mg/g protein). In some embodiments, the proteins comprise an Arg residue, optionally wherein proteins comprising Arg are at a concentration of between about 25 mg/g protein and about 40 mg/g protein (e.g., between about 25 mg/g protein and about 35 mg/g protein, about 25 mg/g protein and about 30 mg/g protein, about 30 mg/g protein and about 40 mg/g protein, or about 35 mg/g protein and about 40 mg/g protein). In some embodiments, the proteins comprise a Val residue, optionally wherein proteins comprising Val are at a concentration of between about 30 mg/g protein and about 70 mg/g protein (e.g., between about 30 mg/g protein and about 60 mg/g protein, about 30 mg/g protein and about 50 mg/g protein, about 30 mg/g protein and about 40 mg/g protein, about 40 mg/g protein and about 70 mg/g protein, about 50 mg/g protein and about 70 mg/g protein, or about 60 mg/g protein and about 70 mg/g protein). In some embodiments, the proteins comprise a Phe residue, optionally wherein proteins comprising Phe are at a concentration of between about 10 mg/g protein and about 35 mg/g protein (e.g., between about 15 mg/g protein and about 35 mg/g protein, about 20 mg/g protein and about 35 mg/g, about 25 mg/g protein and about 35 mg/g, about 30 mg/g protein and about 35 mg/g, about 10 mg/g protein and about 30 mg/g, about 10 mg/g protein and about 25 mg/g, about 10 mg/g protein and about 20 mg/g, or about 10 mg/g protein and about 15 mg/g protein). In some embodiments, the proteins comprise a Tyr residue, optionally wherein proteins comprising Tyr are at a concentration of between about 10 mg/g protein and about 35 mg/g protein (e.g., between about 10 mg/g protein and about 30 mg/g protein, about 10 mg/g protein and about 25 mg/g, about 10 mg/g protein and about 20 mg/g protein, 2^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT about 10 mg/g protein and about 15 mg/g about 15 mg/g protein and about 35 mg/g, about 20 mg/g protein and about 35 mg/g protein, about 25 mg/g protein and about 35 mg/g protein, or about 30 mg/g protein and about 35 mg/g protein). In some embodiments, the proteins comprise an Ile residue, optionally wherein proteins comprising Ile are at a concentration of between about 20 mg/g protein and about 50 mg/g protein (e.g., between about 30 mg/g protein and about 50 mg/g protein, about 40 mg/g protein and about 50 mg/g protein, about 20 mg/g protein and about 40 mg/g protein, or about 20 mg/g protein and about 30 mg/g protein). In some embodiments, the proteins comprise a Leu residue, optionally wherein proteins comprising Leu are at a concentration of between about 25 mg/g protein and about 60 mg/g protein (e.g., between about 35 mg/g protein and about 60 mg/g protein, about 45 mg/g protein and about 60 mg/g protein, about 55 mg/g protein and about 60 mg/g protein, about 25 mg/g protein and about 55 mg/g protein, about 25 mg/g protein and about 45 mg/g protein, or about 25 mg/g protein and about 35 mg/g protein). In some embodiments, the proteins comprise a Lys residue, optionally wherein proteins comprising Lys are at a concentration of between about 45 mg/g protein and about 65 mg/g protein (e.g., between about 50 mg/g protein and about 65 mg/g protein, about 55 mg/g protein and about 65 mg/g protein, about 60 mg/g protein and about 65 mg/g protein, about 45 mg/g protein and about 60 mg/g protein, about 45 mg/g protein and about 55 mg/g protein, or about 45 mg/g protein and about 50 mg/g protein). In some embodiments, the proteins have a molecular weight of less than 1 kDa. In some embodiments, the proteins comprise a His residue, optionally wherein proteins comprising His are at a concentration of about between 10 mg/g protein and about 25 mg/g protein (e.g., between about 10 mg/g protein and about 20 mg/g protein, about 10 mg/g protein and about 15 mg/g protein, about 15 mg/g protein and about 25 mg/g protein, or about 20 mg/g protein and about 25 mg/g protein). In some embodiments, the proteins comprise a Thr residue, optionally wherein proteins comprising Thr are at a concentration of between about 10 mg/g protein and about 30 mg/g protein (e.g., between about 10 mg/g protein and about 25 mg/g protein, about 10 mg/g protein and about 20 mg/g protein, about 10 mg/g protein and about 15 mg/g protein, about 15 mg/g protein and about 30 mg/g protein, about 20 mg/g protein and about 30 mg/g protein, or about 25 mg/g protein and about 30 mg/g protein). In some embodiments, the proteins comprise an Arg residue, optionally wherein proteins comprising Arg are at a concentration of about between 10 mg/g protein and about 20 mg/g (e.g., between about 10 mg/g protein and about 17 mg/g protein, about 10 mg/g protein and about 12 mg/g protein, about 12 mg/g protein and about 20 mg/g protein, about 15 mg/g protein and about 20 mg/g protein, or about 17 mg/g protein and about 20 mg/g protein). In some embodiments, the proteins comprise a Val residue, optionally wherein proteins comprising Val are at a concentration of about between 15 mg/g protein and about 55 mg/g protein (e.g., between about 15 mg/g protein and about 45 mg/g protein, about 15 mg/g protein and about 35 mg/g protein, about 15 mg/g protein and about 25 mg/g protein, about 25 mg/g protein and about 55 mg/g protein, about 35 mg/g protein and about 55 mg/g protein, or about 45 mg/g protein and about 55 mg/g protein). In some embodiments, the proteins comprise a Phe residue, optionally wherein proteins comprising Phe are at a concentration of between about 5 mg/g protein and about 25 mg/g protein (e.g., between about 10 mg/g protein and about 25 mg/g protein, about 15 mg/g protein and about 25 mg/g protein, about 20 mg/g protein and 3^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT about 25 mg/g protein, about 5 mg/g protein and about 20 mg/g protein, about 5 mg/g protein and about 15 mg/g protein, or about 5 mg/g protein and about 10 mg/g^protein). In some embodiments, the proteins comprise a Tyr residue, optionally wherein proteins comprising Tyr are at a concentration of between about 10 mg/g protein and about 30 mg/g protein (e.g., between about 10 mg/g protein and about 25 mg/g protein, about 10 mg/g protein and about 20 mg/g protein, about 10 mg/g protein and about 15 mg/g protein, about 15 mg/g protein and about 25 mg/g protein, or about 20 mg/g protein and about 25 mg/g protein). In some embodiments, the proteins comprise an Ile residue, optionally wherein proteins comprising Ile are at a concentration of between about 10 mg/g protein and 35 mg/g (e.g., between about 15 mg/g protein and 35 mg/g protein, about 20 mg/g protein and 35 mg/g protein, about 25 mg/g protein and 35 mg/g protein, about 30 mg/g and 35 mg/g protein, about 10 mg/g protein and 30 mg/g protein, about 10 mg/g protein and 25 mg/g protein, about 10 mg/g protein and 20 mg/g protein, or about 10 mg/g protein and 15 mg/g protein). In some embodiments, the proteins comprise a Leu residue, optionally wherein proteins comprising Leu are at a concentration of between about 15 mg/g protein and about 45 mg/g protein (e.g., between about 25 mg/g protein and about 45 mg/^protein g, about 35 mg/g protein and about 45 mg/g^protein, about 15 mg/g protein and about 35 mg/g protein, or about 15 mg/g protein and about 25 mg/g protein). In some embodiments, the proteins comprise a Lys residue, optionally wherein proteins comprising Lys are at a concentration of between about 15 mg/g protein and 45 mg/g protein (e.g., between about 25 mg/g protein and about 45 mg/g protein, about 35 mg/g protein and about 45 mg/g protein, about 15 mg/g protein and about 35 mg/g protein, or about 15 mg/g protein and about 25 mg/g^protein). In some embodiments, the peptide extract includes polysaccharides. In some embodiments, the peptide extract has a polysaccharide concentration of between 1% (w/w) and 50% (w/w) (e.g., between 1% (w/w) and 40% (w/w), 1% (w/w) and 30% (w/w), 1% (w/w) and 20% (w/w), 1% (w/w) and 10% (w/w), 10% (w/w) and 50% (w/w), 20% (w/w) and 50% (w/w), 30% (w/w) and 50% (w/w), or 40% (w/w) and 50% (w/w)). In some embodiments, the polysaccharide concentration is between 2% and 35% of the peptide extract (e.g., between 2% (w/w) and 30% (w/w), 2% (w/w) and 25% (w/w), 2% (w/w) and 20% (w/w), 2% (w/w) and 15% (w/w), 2% (w/w) and 10% (w/w), 2% (w/w) and 5% (w/w), 5% (w/w) and 35% (w/w), 10% (w/w) and 35% (w/w), 15% (w/w) and 35% (w/w), 20% (w/w) and 35% (w/w), 25% (w/w) and 35% (w/w), or 30% (w/w) and 35% (w/w)). In some embodiments, the peptide extract includes minerals. In some embodiments, the minerals are selected from one or more of phosphorus, magnesium, calcium, sodium, and potassium. In some embodiments, peptide extract has a mineral concentration of between 5 and 150 ng/g of the peptide extract (e.g., between 5 ng/g and 125 ng/g, 5 ng/g and 100 ng/g, 5 ng/g and 75 ng/g, 5 ng/g and 50 ng/g, 5 ng/g and 25 ng/g, 5 ng/g and 10 ng/g, 10 ng/g and 150 ng/g, 25 ng/g and 150 ng/g, 50 ng/g and 150 ng/g, 75 ng/g and 150 ng/g, 100 ng/g and 150 ng/g, or 125 ng/g and 150 ng/g). In some embodiments, the concentration of the peptide extract in the culture medium is from about 0.1 g/L to about 1 g/L (e.g., about 0.1 g/L to about 0.8 g/L, about 0.1 g/L to about 0.6 g/L, about 0.1 g/L to about 0.4 g/L, about 0.1 g/L to about 0.2 g/L, about 0.2 g/L to about 1 g/L, about 0.4 g/L to about 1 g/L, about 0.6 g/L to about 1 g/L, or about 0.8 g/L to about 1 g/L). In some embodiments, the concentration of the peptide extract in the culture medium is from about 0.5 g/L to about 0.7 g/L (e.g., 4^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT about 0.5 g/L, about 0.6 g/L, or about 0.7 g/L). In some embodiments, the concentration of peptide extract in the culture medium is about 0.7 g/L. In some embodiments, the peptide extract is isolated from the previously cultured cells by lysing and centrifuging the previously cultured cells, resulting in a supernatant, and obtaining the peptide extract from the supernatant. In some embodiments, the peptide extract is isolated from the supernatant by way of filtration. In some embodiments, the filtration is selected from one or more of membrane filtration, fast protein liquid chromatograph-gel filtration diafiltration, and ultrafiltration. In some embodiments, the filtration is membrane filtration. In some embodiments, the peptide extract has a yield of 0.4 g/L to 2.2 g/L (e.g., 0.4 g/L, 0.5 g/L, 0.6 g/L, 0.7 g/L, 0.8 g/L, 0.9 g/L, 1 g/L , 1.1 g/L, 1.2 g/L, 1.3 g/L, 1.4 g/L, 1.5 g/L, 1.6 g/L, 1.7 g/L, 1.8 g/L, 1.9 g/L, 2 g/L, 2.1 g/L, or 2.2 g/) in the supernatant. In some embodiments, the previously cultured cells have an optical density of between about 20 to about 150 (e.g., between about 20 and about 100, about 20 and about 80, about 20 and about 60, about 20 and about 40, about 40 and about 150, about 60 and about 150, about 80 and about 150, about 100 and about 150, or about 120 and about 150) immediately prior to obtaining the peptide extract and culturing it with the population of host cells. In some embodiments, the previously cultured cells have an optical density of between about 20 to about 50 (e.g., between about 20 to about 45, about 20 to about 40, about 20 to about 35, about 20 to about 30, about 20 to about 25, about 25 to about 50, about 30 to about 50, about 35 to about 50, about 40 to about 50, or about 45 to about 50) immediately prior to obtaining the peptide extract and culturing it with the population of host cells. In some embodiments, the previously cultured cells have an optical density of about 50 immediately prior to obtaining the peptide extract and culturing it with the population of host cells. In some embodiments, after culturing the host cells for from about 2 hours to about 50 hours (e.g., about 2 hours to about 5 hours, about 5 hours to about 10 hours, about 10 hours to about 15 hours, about 15 hours to about 20 hours, about 20 hours to about 25 hours, about 25 hours to about 30 hours, about 30 hours to about 35 hours, about 35 hours to about 40 hours, about 40 hours to about 45 hours, or about 40 hours to about 50 hours), the method results in an increase in host cell density of at least 1.1 fold in comparison to a reference method in which the peptide extract is not present in the culture medium. In some embodiments, after culturing the host cells for from about 2 hours to about 50 hours (e.g., about 2 hours to about 5 hours, about 5 hours to about 10 hours, about 10 hours to about 15 hours, about 15 hours to about 20 hours, about 20 hours to about 25 hours, about 25 hours to about 30 hours, about 30 hours to about 35 hours, about 35 hours to about 40 hours, about 40 hours to about 45 hours, or about 40 hours to about 50 hours), the method results in an increase in host cell density of from about 1.1 fold to about 5 fold (e.g., about 1.1 fold to about 4 fold, about 1.1 fold to about 3 fold, about 1.1 fold to about 2 fold, about 2 fold to about 5 fold, about 3 fold to about 5 fold, or about 4 fold to about 5 fold) in comparison to a reference method in which the peptide extract is not present in the culture medium. In some embodiments, after culturing the host cells for from about 2 hours to about 50 hours (e.g., about 2 hours to about 5 hours, about 5 hours to about 10 hours, about 10 hours to about 15 hours, about 15 hours to about 20 hours, about 20 hours to about 25 hours, about 25 hours to about 30 hours, about 30 hours to about 35 hours, about 35 hours to about 40 hours, about 40 hours to 5^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT about 45 hours, or about 40 hours to about 50 hours), the method results in a decrease in the formation of reactive oxygen species by at least 10% in comparison to a reference method in which the peptide extract is not present in the culture medium. In some embodiments, after culturing the host cells for from about 2 hours to about 50 hours (e.g., about 2 hours to about 5 hours, about 5 hours to about 10 hours, about 10 hours to about 15 hours, about 15 hours to about 20 hours, about 20 hours to about 25 hours, about 25 hours to about 30 hours, about 30 hours to about 35 hours, about 35 hours to about 40 hours, about 40 hours to about 45 hours, or about 40 hours to about 50 hours), the method results in a decrease in the formation of reactive oxygen species by from about 10% to about 90% (e.g., about 10% to about 70%, about 10% to about 50%, about 10% to about 30%, about 30% to about 90%, about 50% to about 90%, or about 70% to about 90%) in comparison to a reference method in which the peptide extract is not present in the culture medium. In some embodiments, after culturing the host cells for from about 2 hours to about 50 hours (e.g., about 2 hours to about 5 hours, about 5 hours to about 10 hours, about 10 hours to about 15 hours, about 15 hours to about 20 hours, about 20 hours to about 25 hours, about 25 hours to about 30 hours, about 30 hours to about 35 hours, about 35 hours to about 40 hours, about 40 hours to about formation of reactive oxygen species by from about 25% to about 75% (e.g., about 25% to about 65%, about 25% to about 55%, about 25% to about 35%, about 35% to about 75%, about 45% to about 75%, about 55% to about 75%, or about 65% to about 75%) in comparison to a reference method in which the peptide extract is not present in the culture medium. In some embodiments, after culturing the host cells for from about 2 hours to about 50 hours (e.g., about 2 hours to about 5 hours, about 5 hours to about 10 hours, about 10 hours to about 15 hours, about 15 hours to about 20 hours, about 20 hours to about 25 hours, about 25 hours to about 30 hours, about 30 hours to about 35 hours, about 35 hours to about 40 hours, about 40 hours to about 45 hours, or about 40 hours to about 50 hours), the method results in an increase in host cell density of at least 1.1-fold (e.g., between 1.1 fold and 20 fold, 1.1 fold and 15 fold, 1.1 fold and 10 fold, 1.1 fold and 5 fold, 1.1 fold and 2 fold, 2 fold and 20 fold, 5 fold and 20 fold, 10 fold and 20 fold, or 15 fold and 20 fold) in comparison to a reference method in which the peptide extract is not present in the culture medium. In some embodiments, after culturing the host cells for from about 2 hours to about 50 hours (e.g., about 2 hours to about 5 hours, about 5 hours to about 10 hours, about 10 hours to about 15 hours, about 15 hours to about 20 hours, about 20 hours to about 25 hours, about 25 hours to about 30 hours, about 30 hours to about 35 hours, about 35 hours to about 40 hours, about 40 hours to about 45 hours, or about 40 hours to about 50 hours), the method results in an increase in the concentration of the biological product in comparison to a reference method in which the peptide extract is not present in the culture medium. In some embodiments, after culturing the host cells for from about 2 hours to about 50 hours (e.g., about 2 hours to about 5 hours, about 5 hours to about 10 hours, about 10 hours to about 15 hours, about 15 hours to about 20 hours, about 20 hours to about 25 hours, about 25 hours to about 30 hours, about 30 hours to about 35 hours, about 35 hours to about 40 hours, about 40 hours to about 45 hours, or about 40 hours to about 50 hours), the method results in an increase in the concentration of the biological product of from about 1.1 fold to about 10 6^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT fold (e.g., about 1.1 fold to about 8 fold, about 1.1 fold to about 6 fold, about 1.1 fold to about 4 fold, about 1.1 fold to about 2 fold, about 2 fold to about 10 fold, about 4 fold to about 10 fold, about 6 fold to about 10 fold, or about 8 fold to about 10 fold) in comparison to a reference method in which the peptide extract is not present in the culture medium. In some embodiments, after culturing the host cells for from about 2 hours to about 50 hours (e.g., about 2 hours to about 5 hours, about 5 hours to about 10 hours, about 10 hours to about 15 hours, about 15 hours to about 20 hours, about 20 hours to about 25 hours, about 25 hours to about 30 hours, about 30 hours to about 35 hours, about 35 hours to about 40 hours, about 40 hours to about 45 hours, or about 40 hours to about 50 hours), the method results in an increase in the concentration of the biological product of from about 1.1 fold to about 5 fold (e.g., about 1.1 fold to about 4 fold, about 1.1 fold to about 3 fold, about 1.1 fold to about 2 fold, about 2 fold to about 5 fold, about 3 fold to about 5 fold, or about 4 fold to about 5 fold) in comparison to a reference method in which the peptide extract is not present in the culture medium. In some embodiments, the previously cultured cells are yeast cells. In some embodiments, the yeast cells are Saccharomyces sp. cells or Kluveromyces sp. cells. In some embodiments, the yeast cells are Saccharomyces cerevisiae cells. In some embodiments, the yeast cells are Kluveromyces marxianus cells. In some embodiments, the host cells are genetically modified host cells. In some embodiments, the host cells are yeast cells. In some embodiments, the yeast cells are Saccharomyces sp. cells or Kluveromyces sp. cells. In some embodiments, the yeast cells are Saccharomyces cerevisiae cells. In some embodiments, the yeast cells are Kluveromyces marxianus cells. In some embodiments, the population of host cells is cultured for at least 10 hours. In some embodiments, the population of host cells is cultured for from about 10 hours to about 100 hours (e.g., about 10 hours to about 80 hours, about 10 hours to about 60 hours, about 10 hours to about 40 hours, about 10 hours to about 20 hours, about 20 hours to about 100 hours, about 40 hours to about 100 hours, about 60 hours to about 100 hours, or 80 hours to about 100 hours. In some embodiments, the host cells are cultured at a temperature of from about 20 oC to about 40 oC (e.g., about 20 oC to about 35 oC, about 20 oC to about 30 oC, about 20 oC to about 25 oC, about 25 oC to about 40 oC, about 30 oC to about 40 oC, or about 35 oC to about 40 oC). In some embodiments, the host cells are cultured at a temperature of about 30 oC. In some embodiments, the biological product is a fermentation product. In some embodiments, the biological product is an isoprene. In some embodiments, the biological product is an isoprenoid. In some embodiments, the biological product is a steviol glycoside. In some embodiments, the biological product is a human milk oligosaccharide. In some embodiments, the biological product is a cannabinoid. In another aspect, the disclosure provides a composition produced by any one of the methods described herein. In another aspect, the disclosure provides a composition including a population of host cells capable of producing a biological product; and a culture medium including a peptide extract obtained from a population of previously cultured cells. 7^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT In some embodiments, the peptide extract includes proteins. In some embodiments, the peptide extract has a protein concentration of between 20% (w/w) and 95% (w/w) (e.g., between 20% (w/w) and 75% (w/w), 20% (w/w) and 50% (w/w), 20% (w/w) and 30% (w/w), 30% (w/w) and 95% (w/w), 50% (w/w) and 95% (w/w), 70% (w/w) and 95% (w/w), or 80% (w/w) and 95% (w/w)). In some embodiments, the peptide extract has a protein concentration of between 45% (w/w) and 90% (w/w) (e.g., between 50% (w/w) and 90% (w/w), 60% (w/w) and 90% (w/w), 70% (w/w) and 90% (w/w), 80% (w/w) and 90% (w/w), 45% (w/w) and 80% (w/w), 45% (w/w) and 70% (w/w), 45% (w/w) and 60% (w/w), or 45% (w/w) and 50% (w/w)). In some embodiments, the proteins comprise one or more amino acid residues selected from His, Thr, Arg, Val, Phe, Tyr, Ile, Leu, and Lys. In some embodiments, the proteins have a molecular weight of greater than 1 kDa. In some embodiments, the proteins comprise a His residue, optionally wherein proteins comprising His are at a concentration of between about 20 mg/g protein and 30 mg/g protein (e.g., between about 20 mg/g protein and about 26 mg/g protein, about 20 mg/g protein and about 24 mg/g protein, about 24 mg/g protein and about 30 mg/g protein, or about 28 mg/g protein and about 30 mg/g protein). In some embodiments, the proteins comprise a Thr residue, optionally wherein proteins comprising Thr are at a concentration of between about 25 mg/g protein and about 55 mg/g protein (e.g., between about 35 mg/g protein and about 55 mg/g protein, about 45 mg/g protein and about 55 mg/g protein, about 25 mg/g protein and about 45 mg/g protein, or about 25 mg/g protein and about 35 mg/g protein). In some embodiments, the proteins comprise an Arg residue, optionally wherein proteins comprising Arg are at a concentration of between about 25 mg/g protein and about 40 mg/g protein (e.g., between about 25 mg/g protein and about 35 mg/g protein, about 25 mg/g protein and about 30 mg/g protein, about 30 mg/g protein and about 40 mg/g protein, or about 35 mg/g protein and about 40 mg/g protein). In some embodiments, the proteins comprise a Val residue, optionally wherein proteins comprising Val are at a concentration of between about 15 mg/g protein and about 55 mg/g protein (e.g., between about 15 mg/g protein and about 45 mg/g protein, about 15 mg/g protein and about 35 mg/g protein, about 15 mg/g protein and about 25 mg/g protein, about 25 mg/g protein and about 55 mg/g protein, about 35 mg/g protein and about 55 mg/g protein, or about 45 mg/g protein and about 55 mg/g protein)). In some embodiments, the proteins comprise a Phe residue, optionally wherein proteins comprising Phe are at a concentration of between about 10 mg/g protein and about 35 mg/g protein (e.g., between about 15 mg/g protein and about 35 mg/g protein, about 20 mg/g protein and about 35 mg/g, about 25 mg/g protein and about 35 mg/g, about 30 mg/g protein and about 35 mg/g, about 10 mg/g protein and about 30 mg/g, about 10 mg/g protein and about 25 mg/g, about 10 mg/g protein and about 20 mg/g, or about 10 mg/g protein and about 15 mg/g protein). In some embodiments, the proteins comprise a Tyr residue, optionally wherein proteins comprising Tyr are at a concentration of between about 10 mg/g protein and about 35 mg/g protein (e.g., between about 10 mg/g protein and about 30 mg/g protein, about 10 mg/g protein and about 25 mg/g, about 10 mg/g protein and about 20 mg/g protein, about 10 mg/g protein and about 15 mg/g about 15 mg/g protein and about 35 mg/g, about 20 mg/g protein and about 35 mg/g protein, about 25 mg/g protein and about 35 mg/g protein, or about 30 mg/g protein and about 35 mg/g protein). In some embodiments, the proteins comprise an Ile residue, optionally wherein 8^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT proteins comprising Ile are at a concentration of between about 20 mg/g protein and about 50 mg/g protein (e.g., between about 30 mg/g protein and about 50 mg/g protein, about 40 mg/g protein and about 50 mg/g protein, about 20 mg/g protein and about 40 mg/g protein, or about 20 mg/g protein and about 30 mg/g protein). In some embodiments, the proteins comprise a Leu residue, optionally wherein proteins comprising Leu are at a concentration of between about 25 mg/g protein and about 60 mg/g protein (e.g., between about 35 mg/g protein and about 60 mg/g protein, about 45 mg/g protein and about 60 mg/g protein, about 55 mg/g protein and about 60 mg/g protein, about 25 mg/g protein and about 55 mg/g protein, about 25 mg/g protein and about 45 mg/g protein, or about 25 mg/g protein and about 35 mg/g protein). In some embodiments, the proteins comprise a Lys residue, optionally wherein proteins comprising Lys are at a concentration of between about 45 mg/g protein and about 65 mg/g protein (e.g., between about 50 mg/g protein and about 65 mg/g protein, about 55 mg/g protein and about 65 mg/g protein, about 60 mg/g protein and about 65 mg/g protein, about 45 mg/g protein and about 60 mg/g protein, about 45 mg/g protein and about 55 mg/g protein, or about 45 mg/g protein and about 50 mg/g protein). In some embodiments, the proteins have a molecular weight of less than 1 kDa. In some embodiments, the proteins comprise a His residue, optionally wherein proteins comprising His are at a concentration of about between 10 mg/g protein and about 25 mg/g protein (e.g., between about 10 mg/g protein and about 20 mg/g protein, about 10 mg/g protein and about 15 mg/g protein, about 15 mg/g protein and about 25 mg/g protein, or about 20 mg/g protein and about 25 mg/g protein). In some embodiments, the proteins comprise a Thr residue, optionally wherein proteins comprising Thr are at a concentration of between about 10 mg/g protein and about 30 mg/g protein (e.g., between about 10 mg/g protein and about 25 mg/g protein, about 10 mg/g protein and about 20 mg/g protein, about 10 mg/g protein and about 15 mg/g protein, about 15 mg/g protein and about 30 mg/g protein, about 20 mg/g protein and about 30 mg/g protein, or about 25 mg/g protein and about 30 mg/g protein). In some embodiments, the proteins comprise an Arg residue, optionally wherein proteins comprising Arg are at a concentration of about between 10 mg/g protein and about 20 mg/g (e.g., between about 10 mg/g protein and about 17 mg/g protein, about 10 mg/g protein and about 12 mg/g protein, about 12 mg/g protein and about 20 mg/g protein, about 15 mg/g protein and about 20 mg/g protein, or about 17 mg/g protein and about 20 mg/g protein). In some embodiments, the proteins comprise a Val residue, optionally wherein proteins comprising Val are at a concentration of about between 15 mg/g protein and about 55 mg/g protein (e.g., between about 15 mg/g protein and about 45 mg/g protein, about 15 mg/g protein and about 35 mg/g protein, about 15 mg/g protein and about 25 mg/g protein, about 25 mg/g protein and about 55 mg/g protein, about 35 mg/g protein and about 55 mg/g protein, or about 45 mg/g protein and about 55 mg/g protein)). In some embodiments, the proteins comprise a Phe residue, optionally wherein proteins comprising Phe are at a concentration of between about 5 mg/g protein and about 25 mg/g protein (e.g., between about 10 mg/g protein and about 25 mg/g protein, about 15 mg/g protein and about 25 mg/g protein, about 20 mg/g protein and about 25 mg/g protein, about 5 mg/g protein and about 20 mg/g protein, about 5 mg/g protein and about 15 mg/g protein, or about 5 mg/g protein and about 10 mg/g^protein). In some embodiments, the proteins comprise a Tyr residue, optionally wherein proteins comprising Tyr are at a concentration 9^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT of between about 10 mg/g protein and about 30 mg/g protein (e.g., between about 10 mg/g protein and about 25 mg/g protein, about 10 mg/g protein and about 20 mg/g protein, about 10 mg/g protein and about 15 mg/g protein, about 15 mg/g protein and about 25 mg/g protein, or about 20 mg/g protein and about 25 mg/g protein). In some embodiments, the proteins comprise an Ile residue, optionally wherein proteins comprising Ile are at a concentration of between about 10 mg/g protein and 35 mg/g (e.g., between about 15 mg/g protein and 35 mg/g protein, about 20 mg/g protein and 35 mg/g protein, about 25 mg/g protein and 35 mg/g protein, about 30 mg/g and 35 mg/g protein, about 10 mg/g protein and 30 mg/g protein, about 10 mg/g protein and 25 mg/g protein, about 10 mg/g protein and 20 mg/g protein, or about 10 mg/g protein and 15 mg/g protein). In some embodiments, the proteins comprise a Leu residue, optionally wherein proteins comprising Leu are at a concentration of between about 15 mg/g protein and about 45 mg/g protein (e.g., between about 25 mg/g protein and about 45 mg/^protein g, about 35 mg/g protein and about 45 mg/g^protein, about 15 mg/g protein and about 35 mg/g protein, or about 15 mg/g protein and about 25 mg/g protein). In some embodiments, the proteins comprise a Lys residue, optionally wherein proteins comprising Lys are at a concentration of between about 15 mg/g protein and 45 mg/g protein (e.g., between about 25 mg/g protein and about 45 mg/g protein, about 35 mg/g protein and about 45 mg/g protein, about 15 mg/g protein and about 35 mg/g protein, or about 15 mg/g protein and about 25 mg/g^protein). In some embodiments, the peptide extract includes polysaccharides. In some embodiments, the peptide extract has a polysaccharide concentration of between 1% (w/w) and 50% (w/w) (e.g., between 1% (w/w) and 40% (w/w), 1% (w/w) and 30% (w/w), 1% (w/w) and 20% (w/w), 1% (w/w) and 10% (w/w), 10% (w/w) and 50% (w/w), 20% (w/w) and 50% (w/w), 30% (w/w) and 50% (w/w), or 40% (w/w) and 50% (w/w)). In some embodiments, the polysaccharide concentration is between 2% and 35% of the peptide extract (e.g., between 2% (w/w) and 30% (w/w), 2% (w/w) and 25% (w/w), 2% (w/w) and 20% (w/w), 2% (w/w) and 15% (w/w), 2% (w/w) and 10% (w/w), 2% (w/w) and 5% (w/w), 5% (w/w) and 35% (w/w), 10% (w/w) and 35% (w/w), 15% (w/w) and 35% (w/w), 20% (w/w) and 35% (w/w), 25% (w/w) and 35% (w/w), or 30% (w/w) and 35% (w/w)). In some embodiments, the peptide extract includes minerals. In some embodiments, the minerals are selected from one or more of phosphorus, magnesium, calcium, sodium, and potassium. In some embodiments, peptide extract has a mineral concentration of between 5 and 150 ng/g of the peptide extract (e.g., between 5 ng/g and 125 ng/g, 5 ng/g and 100 ng/g, 5 ng/g and 75 ng/g, 5 ng/g and 50 ng/g, 5 ng/g and 25 ng/g, 5 ng/g and 10 ng/g, 10 ng/g and 150 ng/g, 25 ng/g and 150 ng/g, 50 ng/g and 150 ng/g, 75 ng/g and 150 ng/g, 100 ng/g and 150 ng/g, or 125 ng/g and 150 ng/g). In some embodiments, the concentration of the peptide extract in the culture medium is from about 0.1 g/L to about 1 g/L (e.g., about 0.1 g/L to about 0.8 g/L, about 0.1 g/L to about 0.6 g/L, about 0.1 g/L to about 0.4 g/L, about 0.1 g/L to about 0.2 g/L, about 0.2 g/L to about 1 g/L, about 0.4 g/L to about 1 g/L, about 0.6 g/L to about 1 g/L, or about 0.8 g/L to about 1 g/L). In some embodiments, the concentration of the peptide extract in the culture medium is from about 0.5 g/L to about 0.7 g/L (e.g., about 0.5 g/L, about 0.6 g/L, or about 0.7 g/L). In some embodiments, the concentration of peptide extract in the culture medium is about 0.7 g/L. In some embodiments, the peptide extract is isolated from a population of previously cultured cells by centrifuging the previously cultured cells, resulting in 10^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT a supernatant, and obtaining the peptide extract from the supernatant. In some embodiments, the peptide extract is isolated from the supernatant by way of filtration. In some embodiments, the filtration is selected from one or more of membrane filtration, fast protein liquid chromatograph-gel filtration diafiltration, and ultrafiltration. In some embodiments, the filtration is membrane filtration. In some embodiments, the peptide extract has a yield of 0.4 g/L to 2.2 g/L (e.g., 0.4 g/L, 0.5 g/L, 0.6 g/L, 0.7 g/L, 0.8 g/L, 0.9 g/L, 1 g/L , 1.1 g/L, 1.2 g/L, 1.3 g/L, 1.4 g/L, 1.5 g/L, 1.6 g/L, 1.7 g/L, 1.8 g/L, 1.9 g/L, 2 g/L, 2.1 g/L, or 2.2 g/) the supernatant. In some embodiments, the previously cultured cells have an optical density of between about 20 to about 150 (e.g., between about 20 and about 100, about 20 and about 80, about 20 and about 60, about 20 and about 40, about 40 and about 150, about 60 and about 150, about 80 and about 150, about 100 and about 150, or about 120 and about 150) immediately prior to obtaining the peptide extract and culturing it with the population of host cells. In some embodiments, the previously cultured cells have an optical density of between about 20 to about 50 (e.g., between about 20 to about 45, about 20 to about 40, about 20 to about 35, about 20 to about 30, about 20 to about 25, about 25 to about 50, about 30 to about 50, about 35 to about 50, about 40 to about 50, or about 45 to about 50) immediately prior to obtaining the peptide extract and culturing it with the population of host cells. In some embodiments, the previously cultured cells have an optical density of about 50 immediately prior to obtaining the peptide extract and culturing it with the population of host cells. In some embodiments, the previously cultured cells are cells that, as a result of having previously been cultured, no longer produce a significant quantity of the biological product. In some embodiments, the previously cultured cells are cells that have been previously cultured for at least 10 hours. In some embodiments, the previously cultured cells are cells that have been previously cultured for from about 10 hours to about 100 hours (e.g., about 10 hours to about 80 hours, about 10 hours to about 60 hours, about 10 hours to about 40 hours, about 10 hours to about 20 hours, about 20 hours to about 100 hours, about 40 hours to about 100 hours, about 60 hours to about 100 hours, or about 80 hours to about 100 hours). In some embodiments, the previously cultured cells no longer produce a detectable quantity of the fermentation product. In some embodiments, the previously cultured cells continue to produce a detectable quantity of the fermentation product. In some embodiments, the previously cultured are yeast cells. In some embodiments, the yeast cells are Saccharomyces sp. cells or Kluveromyces sp. cells. In some embodiments, the yeast cells are Saccharomyces cerevisiae cells. In some embodiments, the yeast cells are Kluveromyces marxianus cells. In some embodiments, the host cells are genetically modified host cells. In some embodiments, the host cells are yeast cells. In some embodiments, the yeast cells are Saccharomyces sp. cells or Kluveromyces sp. cells. In some embodiments, the yeast cells are Saccharomyces cerevisiae cells. In some embodiments, the yeast cells are Kluveromyces marxianus cells. In some embodiments, the biological product is a fermentation product. In some embodiments, the biological product is an isoprene. In some embodiments, the biological product is an isoprenoid. In some embodiments, the biological product is a steviol glycoside. In some 11^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT embodiments, the biological product is a human milk oligosaccharide. In some embodiments, the biological product is a cannabinoid. Definitions As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. The term “about” when modifying a numerical value or range herein includes normal variation encountered in the field, and includes plus or minus 1-10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%) of the numerical value or end points of the numerical range. Thus, a value of 10 includes all numerical values from 9 to 11. All numerical ranges described herein include the endpoints of the range unless otherwise noted, and all numerical values in-between the end points, to the first significant digit. As used herein, the term “biological product” refers to a compound, molecule, or complex of molecules that is produced by a host cell (e.g., produced enzymatically by a host cell). The host cell may be one that expresses the enzyme(s) of the biosynthetic pathway that is used in the production of the biological product. A biological product may be naturally produced by the host cell or may be produced by a host cell that has been genetically modified to produce the biological product (e.g., genetically modified so as to express one or more heterologous enzymes of the biosynthetic pathway corresponding to the biological product). Exemplary biological products include, without limitation, carbohydrates, peptide fragments, full-length proteins, nucleic acids, and small molecules. Particular examples include glycosides and oligosaccharides (e.g., steviol glycosides and human milk oligosaccharides), isoprenes, isoprenoids, cannabinoids, antibodies and antigen-binding fragments thereof, enzymes, therapeutic proteins, antigen receptors (e.g., chimeric antigen receptors (CARs)), among other biological products described herein and known in the art. As used herein, the term “capable of producing” refers to a host cell which includes the enzymes necessary for the production of a given compound in accordance with a biochemical pathway that produces the compound. For example, a cell (e.g., a yeast cell) that is “capable of producing” squalene is one that contains the enzymes necessary for production of the squalene according to the squalene biosynthetic pathway. As used herein, the term “endogenous” describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell). As used herein, the term “exogenous” describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell). Exogenous materials include those that are provided from an external source to an organism or to cultured matter extracted therefrom. As used herein in the context of a gene, the term “express” refers to any one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a 12^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT polypeptide or protein. Expression of a gene of interest in a cell, tissue sample, or subject can manifest, for example, as: an increase in the quantity or concentration of mRNA encoding a corresponding protein (as assessed, e.g., using RNA detection procedures described herein or known in the art, such as quantitative polymerase chain reaction (qPCR) and RNA seq techniques), an increase in the quantity or concentration of a corresponding protein (as assessed, e.g., using protein detection methods described herein or known in the art, such as enzyme-linked immunosorbent assays (ELISA), among others), and/or an increase in the activity of a corresponding protein (e.g., in the case of an enzyme, as assessed using an enzymatic activity assay described herein or known in the art). The term "expression cassette" or “expression construct” refers to a nucleic acid construct that, when introduced into a host cell, results in transcription and/or translation of an RNA or polypeptide, respectively. In the case of expression of transgenes, one of skill will recognize that the inserted polynucleotide sequence need not be identical but may be only substantially identical to a sequence of the gene from which it was derived. As explained herein, these substantially identical variants are specifically covered by reference to a specific nucleic acid sequence. One example of an expression cassette is a polynucleotide construct that includes a polynucleotide sequence encoding a polypeptide for use in the invention operably linked to a promoter, e.g., its native promoter, where the expression cassette is introduced into a heterologous microorganism. In some embodiments, an expression cassette includes a polynucleotide sequence encoding a polypeptide of the invention where the polynucleotide that is targeted to a position in the genome of a microorganism such that expression of the polynucleotide sequence is driven by a promoter that is present in the microorganism. As used herein, the term “fermentation product” refers to a compound that is produced by a host cell (e.g., yeast cell), which is cultured in a medium and under conditions suitable for the host cells to produce the fermentation product. The fermentation product may be naturally produced by the host cells or may be produced by host cells that have been genetically modified to produce the fermentation product. As used herein, the term “gene” refers to the segment of DNA involved in producing or encoding a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). Alternatively, the term “gene” can refer to the segment of DNA involved in producing or encoding a non-translated RNA, such as an rRNA, tRNA, gRNA, or micro RNA. A “genetic pathway” or “biosynthetic pathway” as used herein refers to a set of at least two different coding sequences, where the coding sequences encode enzymes that catalyze different parts of a synthetic pathway to form a desired product (e.g., squalene). In a genetic pathway, a first encoded enzyme uses a substrate to make a first product which in turn is used as a substrate for a second encoded enzyme to make a second product. In some embodiments, the genetic pathway includes 3 or more members (e.g., 3, 4, 5, 6, 7, 8, 9, etc.), wherein the product of one encoded enzyme is the substrate for the next enzyme in the synthetic pathway. 13^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT As used herein, the term “genetically modified” denotes a host cell that contains a heterologous nucleotide sequence. The genetically modified host cells described herein typically do not exist in nature. As used herein, the term “heterologous” refers to what is not normally found in nature. The term “heterologous nucleic acid” refers to a nucleic acid not normally found in a given cell in nature. A heterologous nucleic acid can be: (a) foreign to its host cell, i.e., exogenous to the host cell such that a host cell does not naturally contain the nucleic acid; (b) naturally found in the host cell, i.e., endogenous or native to the host cell, but present at an unnatural quantity in the cell (i.e., greater or lesser quantity than naturally found in the host cell); (c) be naturally found in the host cell but positioned outside of its natural locus. A “heterologous” polypeptide refers to a polypeptide that is encoded by a “heterologous nucleic acid”. Thus, for example, a “heterologous” polypeptide may be naturally produced by a host cell but is encoded by a heterologous nucleic acid that has been introduced into the host cell by genetic engineering. For example, a “heterologous” polypeptide can include embodiments in which an endogenous polypeptide is produced by an expression construct and is overexpressed in the host cell compared to native levels of the polypeptide produced by the host cell. A “heterologous genetic pathway” or a “heterologous biosynthetic pathway” as used herein refer to a genetic pathway that does not normally or naturally exist in an organism or cell. The term “host cell” as used in the context of this disclosure refers to a microorganism, such as yeast, and includes an individual cell or cell culture. In some examples, the host cell includes a heterologous vector or heterologous polynucleotide as described herein. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells into which a recombinant vector or a heterologous polynucleotide has been introduced, including by transformation, transfection, and the like. The terms “human milk oligosaccharide” and “HMO” are used Interchangeably herein to refer to a group of nearly 200 identified sugar molecules that are found as the third most abundant component in human breast milk. HMOs in human breast milk are a complex mixture of free, indigestible carbohydrates with many different biological roles, including promoting the development of a functional infant immune system. HMOs include, without limitation, lacto-N-neotetraose (LNnT), 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), lacto-N-fucopentaose (LNFP) I, LNFP II, LNFP III, LNFP V, LNFP VI, lacto-N-difucohexaose (LNDFH) I, LNDFH II, lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), fucosyllacto-N-hexaose (F-LNH) I, F-LNH II, difucosyllacto-N-hexaose (DFLNH) I, DFLNH II, difucosyllacto-N-neohexaose (DFLNnH), difucosyl-para-lacto-N-hexaose (DF-para-LNH), difucosyl-para-lacto-N-neohexaose (DF-para-LNnH), trifucosyllacto-N-hexaose (TF-LNH), 3’-siallylactose (3’-SL), 6’-siallylactose (6’-SL), sialyllacto-N- tetraose (LST) a, LST b, LST c, disialyllacto-N-tetraose (DS-LNT), fucosyl-sialyllacto-N-tetraose (F- LST) a, F-LST b, fucosyl-sialyllacto-N-hexaose (FS-LNH), fucosyl-sialyllacto-N-neohexaose (FS- LNnH) I, and fucosyl-disialyllacto-N-hexaose (FDS-LNH II), among others. As used herein, the term “medium” refers to culture medium and/or fermentation medium. 14^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT The terms “modified,” “recombinant” and “engineered,” when used to modify a host cell described herein, refer to host cells or organisms that do not exist in nature, or express compounds, nucleic acids or proteins at levels that are not expressed by naturally occurring cells or organisms. As used herein, the phrase “operably linked” refers to a functional linkage between nucleic acid sequences such that the linked promoter and/or regulatory region functionally controls expression of the coding sequence. "Percent (%) sequence identity" with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows: 100 multiplied by (the fraction X/Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid. As used herein, the term “pharmaceutical composition” refers to a mixture containing a therapeutic compound or prophylactic compound to be administered to a subject, such as a mammal, e.g., a human, in order to prevent, treat or control a particular disease or condition affecting or that may affect the mammal. As used herein, the term “pharmaceutically acceptable salt” means any pharmaceutically acceptable salt of a compound described herein. For example, pharmaceutically acceptable salts of any of the compounds described herein include those that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, or allergic response, and are commensurate with a reasonable benefit/risk ratio. Examples of pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1- 19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. Such salts can be prepared, for example, in situ during the final isolation and purification of a compound described herein or separately by reacting a free base group with a suitable organic acid. 15^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT The compounds described herein may have ionizable groups so as to be capable of preparation as pharmaceutically acceptable salts. These salts may be acid addition salts involving inorganic or organic acids or the salts may, in the case of acidic forms of the compounds described herein, be prepared from inorganic or organic bases. The compounds may be prepared or used as pharmaceutically acceptable salts synthesized as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable salts may be prepared from pharmaceutically acceptable non-toxic acids and bases including inorganic and organic acids and bases. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2- hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine. As used herein, the term “previously cultured cells” refers to cells that have been previously cultured. For example, the cells may have been previously cultured for from 1-312 hours, such as, e.g., 1 hour, 5 hours, 10 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours, 192 hours, 216 hours, 240 hours, 264 hours, 288 hours, 312 hours, or more. Alternatively, as a result of having previously been cultured, the cells may no longer produce a significant quantity of a biological product. In some instances, the previously cultured cells may be used for obtaining a peptide extract. The previously cultured cells may be, for example, yeast cells (e.g., spent yeast) which have previously been cultured to produce a biological product. In some instances, the previously cultured cells are Saccharomyces cerevisiae. As used herein, the term “production” generally refers to an amount of compound produced by a host cell provided herein. In some embodiments, production is expressed as a yield of the compound by the host cell. In other embodiments, production is expressed as a productivity of the host cell in producing the compound. As used herein, the term “protein concentration” refers to the sum total of polypeptides present in a peptide extract mixture. As used herein, the term “steviol glycoside” refers to a glycoside of steviol including but not limited to 19-glycoside, steviolmonoside, steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside A (RebA), rebaudioside B (RebB), rebaudioside C (RebC), rebaudioside D (RebD), rebaudioside E (RebE), rebaudioside F (RebF), rebaudioside G (RebG), rebaudioside H (RebH), rebaudioside I (RebI), rebaudioside J (RebJ), rebaudioside K (RebK), rebaudioside L (RebL), 16^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT rebaudioside M (RebM), rebaudioside N (RebN), rebaudioside O (RebO), rebaudioside D2, and rebaudioside M2. Brief Description of the Drawings FIG.1 is a diagram showing the process by which a peptide extract may be isolated from a population of previously cultured yeast cells. FIG.2 shows a schematic representation of the molecular weight (MW) distribution of an exemplary peptide extract of the disclosure. FIG.3A and FIG 3B are graphs showing the washed optical density (wOD, 600 nm) and sucrose concentration (g/L) of flask cultures over the course of fermentation for the conditions: control, when no peptide extract and no H2O2 is added (outlined circles); peptide extract is present at a concentration of 0.7 g/L (black circles); 2 mM H2O2 is present (outlined boxes); and 2 mM H2O2 plus peptide extract is present at a concentration of 0.7 g/L (black boxes). The data represent the average of two flasks for each condition and error bars are standard deviations. FIG.4 is a graph showing the relative farnesene concentration (%) over the course of fermentation for the conditions: control, when no extract and no H2O2 added (outlined circles); peptide extract is present at a concentration of 0.7 g/L (black circles); 2 mM H2O2 is present (outlined boxes); and 2 mM H2O2 plus peptide extract is present at a concentration of 0.7 g/L (black boxes). The data represent the average of two shake-flasks for each condition and error bars are standard deviations. FIG.5A and FIG.5B are bar graphs showing the change in formation of reactive oxygen species (ROS) in the presence of 2 mM H2O2 plus peptide extract at a concentration of 0.7 g/L in relation to condition 2 mM H2O2 (FIG.5A) and cell viability in each condition tested (FIG.5B) over the course of fermentation. The data represent the average of two flasks for each condition and error bars are standard deviations. FIG.6A and FIG.6B are bar graphs showing the wOD, 600 nm (FIG.6A) and sucrose concentration (g/L) (FIG.6B) of bioreactor cultures over the course of fermentation for the conditions: control, with no extract and no H2O2 added (outlined circle); peptide extract at a concentration of 0.7 g/L (black circle); 2 mM H2O2 (outlined square); and 2 mM H2O2 plus peptide extract at a concentration of 0.7 g/L (black square). FIG.7 is a graph showing the relative farnesene concentration (%) over the course of fermentation for the conditions: control, with no extract and no H2O2 added (outlined circle); peptide extract at a concentration of 0.7 g/L (black circle); 2 mM H2O2 (outlined square); and 2 mM H2O2 plus peptide extract at a concentration of 0.7 g/L (black square). FIG.8A and FIG.8B are bar graphs showing the change in formation of reactive oxygen species (ROS) in condition 2 mM H2O2 plus peptide extract at a concentration of 0.7 g/L in relation to condition 2 mM H2O2 (FIG.8A) and cell viability in each condition tested (FIG.8B) over the course of fermentation. 17^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT Detailed Description The present disclosure provides compositions and methods for producing a biological product by culturing a population of host cells capable of producing the biological product in a culture medium including a peptide extract obtained from a population of previously cultured cells. The population of previously cultured cells may no longer produce a significant quantity of a biological product as a result of being previously cultured. Additionally or alternatively, the population of previously cultured cells may have been fermented for a desired length of time, such as a duration sufficient to produce a mixture having a peptide distribution described herein. It has presently been discovered that culturing a population of host cells in the presence of a peptide extract isolated from previously cultured yeast cells results in the reduction oxidative stress during the biological process by the peptide extract acting as an antioxidant. The presence of the peptide extract reduced ROS generation, and thus, improve yeast tolerance to oxidative stress and production of the biological product, while contributing to a repurposed use of a population of previously cultured cells previously viewed as a waste product. The following sections provide a detailed description of the methods of using previously cultured cells to reduce oxidative stress and increase production of a biological product. ^ Methods of Producing a Biological Product ^ Provided herein are methods for producing one or more biological products in a population of host cells capable of producing a biological product. The disclosure provides methods of producing a biological product including providing a population of host cells capable of producing the biological product and culturing the population of host cells in a culture medium that includes a peptide extract obtained from a population of previously cultured cells. In some embodiments, the host cells are cultured in a culture medium including a peptide extract obtained from a population of previously cultured cells. The peptide extract may include, for example, one or more proteins. The proteins may have a molecular weight of between 100 Da and 5 kDa (e.g., between 100 Da and 4500 Da, 100 Da and 4000 Da, 100 Da and 3500 Da, 100 Da and 3000 Da, 100 Da and 2500 Da, 100 Da and 2000 Da, 100 Da and 1500 Da, 100 Da and 1000 Da, 100 Da and 500 Da, 500 Da and 5 kDa, 1000 Da and 5 kDa, 1500 Da and 5 kDa, 2000 Da and 5 kDa, 2500 Da and 5 kDa, 3000 Da and 5 kDa, 3500 Da and 5 kDa, 4000 kDa and 5 kDa, or 4500 kDa and 5 kDa). The proteins of the peptide extract may include polypeptides and/or free amino acids. The peptide extract may have a protein concentration of between 20% (w/w) and 95% (w/w) (e.g., between 20% (w/w) and 80% (w/w), 20% (w/w) and 50% (w/w), 20% (w/w) and 30% (w/w), 30% (w/w) and 95% (w/w), 50% (w/w) and 95% (w/w), or 80% (w/w) and 95% (w/w)); for example, the peptide extract has a protein concentration of between 45% (w/w) and 90% (w/w) (e.g., between 45% (w/w) and 80% (w/w), 45% (w/w) and 70% (w/w), 45% (w/w) and 60% (w/w), 45% (w/w) and 50% (w/w), 50% (w/w) and 90% (w/w), 60% (w/w) and 90% (w/w), 70% (w/w) and 90% (w/w), or 80% (w/w) and 90% (w/w)). The peptide extract may further include one or more polysaccharides. The one or more polysaccharides may be a biological product from the previously cultured cell from which the peptide 18^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT extract was isolated. The concentration of the one or more polysaccharides in the peptide extract may be between 1% (w/w) and 50% (w/w) (e.g., between 1% (w/w) and 40% (w/w), 1% (w/w) and 30% (w/w), 1% (w/w) and 20% (w/w), 1% (w/w) and 10% (w/w), 10% (w/w) and 50% (w/w), 20% (w/w) and 50% (w/w), 30% (w/w) and 50% (w/w), or 40% (w/w) and 500% (w/w)); for example, the polysaccharide concentration is between 2% (w/w) and 35% (w/w) (e.g., between 2% (w/w) and 30% (w/w), 2% (w/w) and 25% (w/w), 2% (w/w) and 20% (w/w), 2% (w/w) and 15% (w/w), 2% (w/w) and 10% (w/w), 10% (w/w) and 35% (w/w), 15% (w/w) and 35% (w/w), 20% (w/w) and 35% (w/w), 25% (w/w) and 35% (w/w), or 30% (w/w) and 35% (w/w)) of the peptide extract. The peptide extract may further include one or more minerals. For example, the peptide extract may include phosphorus, magnesium, calcium, sodium, or potassium. In some embodiments, the peptide extract has a mineral concentration of between 5 ng/g and 150 ng/g (e.g., between 5 ng/g and 125 ng/g, 5 ng/g and 100 ng/g, 5 ng/g and 75 ng/g, 5 ng/g and 50 ng/g, 5 ng/g and 25 ng/g, 5 ng/ and 10 ng/g, 10 ng/g and 150 ng/g, 25 ng/g and 150 ng/g, 50 ng/g and 150 ng/g, 75 ng/g and 150 ng/g, 100 ng/g and 150 ng/g, or 125 ng/g and 150 ng/g) of the peptide extract. The peptide extract may be isolated from a population of previously cultured cells that were, themselves, capable of producing a biological product (e.g., a fermentation product). For example, the previously cultured cells may be capable of producing an isoprene, and isoprenoid, a human milk oligosaccharide, a steviol glycoside, or a cannabinoid. In some embodiments, the previously cultured cells are capable of producing ^-farnesene. The population of previously cultured cells may have previously been extracted for the biological product it is capable of producing. The previously cultured cells may undergo centrifugation to separate the cells from a supernatant. In some embodiments, the peptide extract is isolated from the supernatant by way of filtration. For example, the peptide extract may be obtained by membrane filtration, fast protein liquid chromatograph-gel filtration, diafiltration, and ultrafiltration. In some embodiments, the filtration is membrane filtration. Membrane filtration may be used to separate the proteins of the peptide extract by size. For example, a membrane having a molecular weight cut off of about 1 kDa may be used to isolate proteins from the supernatant to result in the peptide extract. In some embodiments, the peptide extract has a yield of between 0.4 g/liter and 2.2 g/liter (e.g., 0.4 g/liter, 0.5 g/liter, 0.6 g/liter, 0.7 g/liter, 0.8 g/liter, 0.9 g/liter, 1 g/liter, 1.1 g/liter, 1.2 g/liter, 1.3 g/liter, 1.4 g/liter, 1.5 g/liter, 1.6 g/liter, 1.7 g/liter, 1.8 g/liter, 1.9 g/liter, 2 g/liter, 2.1 g/liter, or 2.2 g/liter) in the supernatant. In some embodiments, the previously cultured cells have an optical density of between about 20 to about 150 (e.g., between about 20 and about 100, about 20 and about 80, about 20 and about 60, about 20 and about 40, about 40 and about 150, about 60 and about 150, about 80 and about 150, about 100 and about 150, or about 120 and about 150) immediately prior to obtaining the peptide extract and culturing it with the population of host cells. In some embodiments, the previously cultured cells have an optical density of between about 20 to about 50 (e.g., between about 20 to about 45, about 20 to about 40, about 20 to about 35, about 20 to about 30, about 20 to about 25, about 25 to about 50, about 30 to about 50, about 35 to about 50, about 40 to about 50, or about 45 to about 50) immediately prior to obtaining the peptide extract and culturing it with the population of host 19^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT cells. In some embodiments, the previously cultured cells have an optical density of about 50 immediately prior to obtaining the peptide extract and culturing it with the population of host cells. The previously cultured cells may have been previously cultured for at least 10 hours. For example, the previously cultured cells may have been previously culture for from about 10 hours to about 100 hours (e.g., about 10 hours to about 75 hours, about 10 hours to about 50 hours, about 10 hours to about 25 hours, about 25 hours to about 100 hours, about 50 hours to about 100 hours, or about 75 hours to about 100 hours). In some embodiments, the previously cultured cells were previously capable of producing a biological product, but no longer produce a detectable quantity of the biological product by virtue of having been extensively cultured. In some embodiments, after culturing the host cells for from about 2 hours to about 50 hours in the presence of the peptide extract, the method results in an increase in host cell density of at least 1.1-fold in comparison to a reference method in which the peptide extract is not present in the culture medium. For example, the method may result in an increase in host cell density of from about 1.1 fold to about 5 fold (e.g., from about 1.1 fold to about 4 fold, about 1.1 fold to about 3 fold, about 1.1 fold to about 2 fold, about 2 fold to about 5 fold, about 3 fold to about 5 fold, or about 4 fold to about 5 fold) in comparison to a reference method in which the peptide extract is not present in the culture medium. In some embodiments, after culturing the host cells for from about 2 hours to about 50 hours, the method results in a decrease in the formation of reactive oxygen species by at least 10% in comparison to a reference method in which the peptide extract is not present in the culture medium. For example, the method may result in a decrease in the formation of reactive oxygen species by from about 10% to about 90% (e.g., from about 10% to about 70%, about 10% to about 50%, about 10% to about 25%, about 25% to about 90%, about 50% to about 90%, or about 75% to about 90%) in comparison to a reference method in which the peptide extract is not present in the culture medium. In some embodiments, the method results in a decrease in the formation of reactive oxygen species by from about 25% to about 75% (e.g., from about 25% to about 65%, about 25% to about 55%, about 25% to about 45%, about 25% to about 35%, about 35% to about 75%, about 45% to about 75%, about 55% to about 75%, or about 65% to about 75%) in comparison to a reference method in which the peptide extract is not present in the culture medium. After culturing the host cells for from about 2 hours to about 50 hours, the method may result in an increase in sugar consumption in comparison to a reference method in which the peptide extract is not present in the culture medium. For example, the sugar consumption may increase by at least 1.1 fold in comparison to when the peptide extract is not present in the composition. In some embodiments, the sugar consumption increases 1.1 fold to about 10 fold in comparison to a reference method in which the peptide extract is not present in the culture medium After culturing the host cells for from about 2 hours to about 50 hours in presence of the peptide extract, the method may result in an increase in the concentration of the biological product in comparison to a reference method in which the peptide extract is not present in the culture medium. For example, the method may result in an increase in the concentration of the biological product of from about 1.1 fold to about 10 fold (e.g., about 1.1 fold to about 8 fold, about 1.1 fold to about 6 fold, about 1.1 fold to about 4 fold, about 1.1 fold to about 2 fold, about 2 fold to about 10 fold, about 4 fold 20^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT to about 10 fold, about 6 fold to about 10 fold, or about 8 fold to about 10 fold) in comparison to a reference method in which the peptide extract is not present in the culture medium. In some embodiments, the method results in an increase in the concentration of the biological product of from about 1.1 fold to about 5 fold (e.g., about 1.1 fold to about 4 fold, about 1.1 fold to about 3 fold, about 1.1 fold to about 2 fold, about 2 fold to about 5 fold, about 3 fold to about 5 fold, or about 4 fold to about 5 fold) in comparison to a reference method in which the peptide extract is not present in the culture medium. Enzymes of Exemplary Biosynthetic Pathways The host cells (and/or the previously cultured cells) described herein may express one or more enzymes of a biosynthetic pathway capable of producing a biological product of interest. In some embodiments, for example, host cells and/or previously cultured cells of the disclosure (e.g., yeast cells) may naturally express some of the enzymes of the biosynthetic pathway for a given biological product. Such cells may be modified to express the remaining or heterologous enzymes of the biosynthetic pathway. In some embodiments, for instance, a cell (e.g., a yeast cell) may naturally express many of the enzymes of the biosynthetic pathway of a desired biological product, and the cells may be modified so as to express the remaining enzymes of the biosynthetic pathway for the desired biological product by providing the cells with one or more heterologous nucleic acid molecules that, together, encode the remaining enzymes of the biosynthetic pathway. In some embodiments, the cells may be genetically modified to produce a biological product. For example, the cells may be genetically modified to produce a fermentation product. The cells may produce a biological product such as, for example, an isoprene, an isoprenoid, a human milk oligosaccharide (HMO), a steviol glycoside, or a cannabinoid. Cannabinoid Biosynthetic Pathway The cell may include one or more nucleic acids encoding one or more enzymes of a heterologous genetic pathway that produces a cannabinoid or a precursor of a cannabinoid. The cannabinoid biosynthetic pathway may begin with hexanoic acid as the substrate for an acyl activating enzyme (AAE) to produce hexanoyl-CoA, which is used as the substrate of a tetraketide synthase (TKS) to produce tetraketide-CoA, which is used by an olivetolic acid cyclase (OAC) to produce olivetolic acid, which is then used to produce a cannabigerolic acid by a geranyl pyrophosphate (GPP) synthase and a cannabigerolic acid synthase (CBGaS). In some embodiments, the cannabinoid precursor that is produced is a substrate in the cannabinoid pathway (e.g., hexanoate or olivetolic acid). In some embodiments, the precursor is a substrate for an AAE, a TKS, an OAC, a CBGaS, or a GPP synthase. In some embodiments, the precursor, substrate, or intermediate in the cannabinoid pathway is hexanoate, olivetol, or olivetolic acid. In some embodiments, the precursor is hexanoate. In some embodiments, the cell does not contain the precursor, substrate or intermediate in an amount sufficient to produce the cannabinoid or a precursor of the cannabinoid. In some embodiments, the cell does not contain hexanoate at a level or in an amount sufficient to produce the cannabinoid in an amount over 10 mg/L. In some embodiments, the heterologous genetic pathway encodes at least one enzyme selected from the group consisting of an AAE, a TKS, an OAC, a CBGaS, and a GPP 21^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT synthase. In some embodiments, the genetically modified cell includes an AAE, TKS, OAC, CBGaS, and a GPP synthase. The cannabinoid pathway is described in Keasling et al., U.S. Patent No. 10,563,211, the disclosure of which is incorporated herein by reference. The cell may include, in some embodiments, a heterologous AAE such that the cell is capable of producing a cannabinoid. The AAE may be from Cannabis sativa or may be an enzyme from another plant or fungal source which has been shown to have AAE activity in the cannabinoid biosynthetic pathway, resulting in the production of the cannabinoid precursor olivetolic acid In some embodiments, the cell may include a heterologous TKS such that the cell is capable of producing a cannabinoid. A TKS uses the hexanoyl-CoA precursor to generate tetraketide-CoA. The TKS may be from Cannabis sativa or may be an enzyme from another plant or fungal source which has been shown to have TKS activity in the cannabinoid biosynthetic pathway, resulting in the production of the cannabinoid precursor olivetolic acid. Some embodiments concern a cell that includes a heterologous CBGaS such that the cell is capable of producing a cannabinoid. A CBGaS uses the olivetolic acid precursor and GPP precursor to generate cannabigerolic acid. The CBGaS may be from Cannabis sativa or may be an enzyme from another plant or fungal source which has been shown to have CBGaS activity in the cannabinoid biosynthetic pathway, resulting in the production of the cannabinoid cannabigerolic acid. Some embodiments concern a cell that includes a heterologous GPP synthase such that the cell is capable of producing a cannabinoid. A GPP synthase uses the product of the isoprenoid biosynthesis pathway precursor to generate cannabigerolic acid together with a prenyltransferase enzyme. The GPP synthase may be from Cannabis sativa or may be an enzyme from another plant or bacterial source which has been shown to have GPP synthase activity in the cannabinoid biosynthetic pathway, resulting in the production of the cannabinoid cannabigerolic acid. The population of cells may further express other heterologous enzymes in addition to the AAE, TKS, CBGaS, and/or GPP synthase. For example, in some embodiments, the cell may include a heterologous nucleic acid that encodes at least one enzyme from the mevalonate biosynthetic pathway. Enzymes which make up the mevalonate biosynthetic pathway may include but are not limited to an acetyl-CoA thiolase, an HMG-CoA synthase, an HMG-CoA reductase, a mevalonate kinase, a phosphomevalonate kinase, a mevalonate pyrophosphate decarboxylase, and an IPP: DMAPP isomerase. In some embodiments, the cell includes a heterologous nucleic acid that encodes the acetyl-CoA thiolase, the HMG-CoA synthase, the HMG-CoA reductase, the mevalonate kinase, the phosphomevalonate kinase, the mevalonate pyrophosphate decarboxylase, and the IPP: DMAPP isomerase of the mevalonate biosynthesis pathway. In some embodiments, the cell may include an olivetolic acid cyclase (OAC) as part of the cannabinoid biosynthetic pathway. In some embodiments, the cell further includes one or more heterologous nucleic acids that each, independently, encode an acetyl-CoA synthase, and/or an aldehyde dehydrogenase, and/or a pyruvate decarboxylase. In some embodiments, the cell contains a heterologous nucleic acid encoding an aceto-CoA carboxylase (ACC). 22^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT In some embodiments, the cell contains a heterologous nucleic acid encoding an ACC and an acetoacetyl-CoA synthase (AACS) instead of a heterologous nucleic acid encoding an acetyl-CoA thiolase. Human Milk Oligosaccharide Biosynthetic Pathway In addition to being modified so as to be deficient in expression and/or activity of one or more endogenous oxidoreductases (e.g., one or more endogenous aldose reductases described herein), cells of the disclosure may also be modified so as to express the enzymes of the biosynthetic pathway of a target HMO. In some embodiments, for example, cells of the disclosure (e.g., yeast cells) may naturally express some of the enzymes of the biosynthetic pathway for a given HMO. Such cells may be modified to express the remaining enzymes of the biosynthetic pathway. In some embodiments, for instance, a cell (e.g., a yeast cell) may naturally express many of the enzymes of the biosynthetic pathway of a desired HMO, and the cells may be modified so as to express the remaining enzymes of the biosynthetic pathway for the desired HMO by providing the cells with one or more heterologous nucleic acid molecules that, together, encode the remaining enzymes of the biosynthetic pathway. In some embodiments, cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing LNnT, including a ^-1,3- N-acetylglucosaminyltransferase (LgtA), a ^-1,4-galactosyltransferase (LgtB), and a UDP-N- acetylglucosamine diphosphorylase. Exemplary LgtA and LgtB enzymes useful in conjunction with the compositions and methods of the disclosure are described in the sections that follow. In some embodiments, cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing 2’-FL, including a lactose permease, a GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, an ^-1,2-fucosyltransferase, and a fucosidase. Exemplary enzymes useful in conjunction with the compositions and methods of the disclosure are described in the sections that follow. In some embodiments, cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing 3-fucosyllactose, including a GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, an ^-1,3-fucosyltransferase, and a fucosidase. Exemplary enzymes useful in conjunction with the compositions and methods of the disclosure are described in the sections that follow. In some embodiments, cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing lacto-N-tetraose, including a ^-1,3-N-acetylglucosaminyltransferase, a ^-1,3-galactosyltransferase, and a UDP-N- acetylglucosamine diphosphorylase. Exemplary enzymes useful in conjunction with the compositions and methods of the disclosure are described in the sections that follow. In some embodiments, cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing 3’-sialyllactose, including a CMP-Neu5Ac synthetase, a sialic acid synthase, a UDP-N-acetylglucosamine 2-epimerase, a UDP- N-acetylglucosamine diphosphorylase, and a CMP-N-acetylneuraminate-^-galactosamide-^-2,3- 23^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT sialyltransferase. Exemplary enzymes useful in conjunction with the compositions and methods of the disclosure are described in the sections that follow. In some embodiments, cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing 6’-sialyllactose, including a CMP-Neu5Ac synthetase, a sialic acid synthase, a UDP-N-acetylglucosamine 2-epimerase, a UDP- N-acetylglucosamine diphosphorylase, and a ^-galactoside-^-2,6-sialyltransferase. Exemplary enzymes useful in conjunction with the compositions and methods of the disclosure are described in the sections that follow. In some embodiments, cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing difucosyllactose, including a GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, an ^-1,2-fucosyltransferase, and an ^- 1,3-fucosyltransferase. Exemplary enzymes useful in conjunction with the compositions and methods of the disclosure are described in the sections that follow. In some embodiments, the cells of the disclosure express an LgtA polypeptide. The LgtA polypeptides of the disclosure can be used to produce one or more of a variety of HMOs, including, without limitation, LNnT, LNT, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, LNDFH I, LNDFH II, LNH, LNnH, F-LNH I, F-LNH II, DFLNH I, DFLNH II, DFLNnH, DF-para-LNH, DF-para-LNnH, TF-LNH, LST a, LST b, LST c, DS-LNT, F-LST a, F-LST b, FS-LNH, FS-LNnH I, and FDS-LNH II. In some embodiments, the cells of the disclosure express a LgtB polypeptide. In some embodiments, the cells of the disclosure express a protein that transports lactose into the cell. In some embodiments, the cells of the disclosure express a GDP-mannose 4,6-dehydratase. In some embodiments, the cells of the disclosure express a GDP-L-fucose synthase. In some embodiments, the cells of the disclosure express an ^-1,2-fucosyltransferase polypeptide. Steviol Glycoside Biosynthetic Pathway In some embodiments, the cells are capable of producing one or more steviol glycosides may encode on or more enzymes of the steviol glycoside biosynthesis pathway. In some embodiments, the steviol glycoside biosynthesis pathway is activated in the genetically modified cells by engineering the cells to express polynucleotides encoding enzymes capable of catalyzing the biosynthesis of steviol glycosides. In some embodiments, the genetically modified cells contain one or more heterologous polynucleotides encoding a geranylgeranyl diphosphate synthase (GGPPS), a copalyl diphosphate synthase (CDPS), a kaurene synthase (KS), a kaurene oxidase (KO), a kaurene acid hydroxylase (KAH), a cytochrome P450 reductase (CPR), and/or one or more additional UDP- glycosyltransferases, such as UGT74G1, UGT76G1, UGT85C2, UGT91D, EUGT11, and/or UGT40087. In some embodiments, the genetically modified cells contain one or more heterologous polynucleotides encoding a variant GGPPS, CDPS, KS, KO, KAH, CPR, UDP-glycosyltransferase, UGT74G1, UGT76G1, UGT85C2, UGT91D, EUGT11, and/or UGT40087. In certain embodiments, the variant enzyme may have from 1 up to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 1313, 15, 16, 24^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT 17, 18, 19, or 20) amino acid substitutions relative to a reference enzyme. In certain embodiments, the coding sequence of the polynucleotide is codon optimized for the particular cell. GGPPS (EC 2.5.1.29) catalyzes the conversion of farnesyl pyrophosphate into geranylgeranyl diphosphate. Examples of GGPPS include those of Stevia rebaudiana (accession no. ABD92926), Gibberella fujikuroi (accession no. CAA75568), Mus musculus (accession no. AAH69913), Thalassiosira pseudonana (accession no. XP_002288339), Streptomyces clavuligerus (accession no. ZP-05004570), Sulfulobus acidocaldarius (accession no. BAA43200), Synechococcus sp. (accession no. ABC98596), Arabidopsis thaliana (accession no. MP_195399), and Blakeslea trispora (accession no. AFC92798.1), and those described in U.S. Patent No.9,631,215. CDPS (EC 5.5.1.13) catalyzes the conversion of geranylgeranyl diphosphate into copalyl diphosphate. Examples of copalyl diphosphate synthases include those from Stevia rebaudiana (accession no. AAB87091), Streptomyces clavuligerus (accession no. EDY51667), Bradyrhizobioum japonicum (accession no. AAC28895.1), Zea mays (accession no. AY562490), Arabidopsis thaliana (accession no. NM_116512), and Oryza sativa (accession no. Q5MQ85.1), and those described in U.S. Patent No.9,631,215. In some embodiments, the cell includes a heterologous nucleic acid encoding a CDPS. KS (EC 4.2.3.19) catalyzes the conversion of copalyl diphosphate into kaurene and diphosphate. Examples of enzymes include those of Bradyrhizobium japonicum (accession no. AAC28895.1), Arabidopsis thaliana (accession no. Q9SAK2), and Picea glauca (accession no. ADB55711.1), and those described in U.S. Patent No.9,631,215. In some embodiments, the cell includes a heterologous nucleic acid encoding a KS. CDPS-KS bifunctional enzymes (EC 5.5.1.13 and EC 4.2.3.19) may also be used in the cells of the invention. Examples include those of Phomopsis amygdali (accession no. BAG30962), Phaeosphaeria sp. (accession no. O13284), Physcomitrella patens (accession no. BAF61135), and Gibberella fujikuroi (accession no. Q9UVY5.1), and those described in U.S. Patent Application Publication Nos.2014/032928 A1, 2014/0357588 A1, 2015/0159188, and WO 2016/038095. KO (EC 1.14.13.88) catalyzes the conversion of kaurene into kaurenoic acid. Illustrative examples of enzymes include those of Oryza sativa (accession no. Q5Z5R4), Gibberella fujikuroi (accession no. O94142), Arabidopsis thaliana (accession no. Q93ZB2), Stevia rebaudiana (accession no. AAQ63464.1), and Pisum sativum (Uniprot no. Q6XAF4), and those described in U.S. Patent Application Publication Nos.2014/0329281 A1, 2014/0357588 A1, 2015/0159188, and WO 2016/038095. In some embodiments, the cell includes a heterologous nucleic acid encoding a KO. KAH (EC 1.14.13) also referred to as steviol synthases catalyze the conversion of kaurenoic acid into steviol. Examples of enzymes include those of Stevia rebaudiana (accession no. ACD93722), Arabidopsis thaliana (accession no. NP_197872), Vitis vinifera (accession no. XP_002282091), and Medicago trunculata (accession no. ABC59076), and those described in U.S. Patent Application Publication Nos.2014/0329281, 2014/0357588, 2015/0159188, and WO 2016/038095. In some embodiments, the cell includes a heterologous nucleic acid encoding a KAH. A CPR (EC 1.6.2.4) is necessary for the activity of KO and/or KAH above. Examples of enzymes include those of Stevia rebaudiana (accession no. ABB88839), Arabidopsis thaliana 25^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT (accession no. NP_194183), Gibberella fujikuroi (accession no. CAE09055), and Artemisia annua (accession no. ABC47946.1), and those described in U.S. Patent Application Publication Nos. 2014/0329281, 2014/0357588, 2015/0159188, and WO 2016/038095. In some embodiments, the cell includes a heterologous nucleic acid encoding a CPR. UGT74G1 is capable of functioning as a uridine 5’-diphospho glucosyl: steviol 19-COOH transferase and as a uridine 5’-diphospho glucosyl: steviol-13-O-glucoside 19-COOH transferase. Accordingly, UGT74G1 is capable of converting steviol to 19-glycoside; converting steviol to 19- glycoside, steviolmonoside to rubusoside; and steviolbioside to stevioside. UGT74G1 has been described in Richman et al., 2005, Plant J., vol.41, pp.56-67; U.S. Patent Application Publication No. 2014/0329281; WO 2016/038095; and accession no. AAR06920.1. In some embodiments, the cell includes a heterologous nucleic acid encoding a UGT74G1. UGT76G1 is capable of transferring a glucose moiety to the C-3’ position of an acceptor molecule a steviol glycoside (where glycoside = Glcb(1^2)Glc). This chemistry can occur at either the C-13-O-linked glucose of the acceptor molecule, or the C-19-O-linked glucose acceptor molecule. Accordingly, UGT76G1 is capable of functioning as a uridine 5’-diphospho glucosyltransferase to the: (1) C-3’ position of the 13-O-linked glucose on steviolbioside in a beta linkage forming RebB, (2) C-3’ position of the 19-O-linked glucose on stevioside in a beta linkage forming RebA, and (3) C-3’ position of the 19-O-linked glucose on RebD in a beta linkage forming RebM. UGT76G1 has been described in Richman et al., 2005, Plant J., vol.41, pp.56-67; US2014/0329281; WO2016/038095; and accession no. AAR06912.1. UGT85C2 is capable of functioning as a uridine 5’-diphospho glucosyl: steviol 13-OH transferase, and a uridine 5’-diphospho glucosyl: steviol-19-O-glucoside 13-OH transferase. UGT85C2 is capable of converting steviol to steviolmonoside and is also capable of converting 19- glycoside to rubusoside. Examples of UGT85C2 enzymes include those of Stevia rebaudiana: see e.g., Richman et al., (2005), Plant J., vol.41, pp.56-67; U.S. Patent Application Publication No. 2014/0329281; WO 2016/038095; and accession no. AAR06916.1. In some embodiments, the cell includes a heterologous nucleic acid encoding a UGT85C2. UGT40087 is capable of transferring a glucose moiety to the C-2’ position of the 19-O- glucose of RebA to produce RebD. UGT40087 is also capable of transferring a glucose moiety to the C-2’ position of the 19-O-glucose of stevioside to produce RebE. Examples of UGT40087 include those of accession no. XP_004982059.1 and WO 2018/031955. In some embodiments, the cell includes a heterologous nucleic acid encoding a UGT40087. Isoprenoid Biosynthetic Pathway The cells described herein may be modified to express one or more enzymes of the mevalonate-dependent (MEV) biosynthetic pathway. Cells which are modified with one or more enzymes of the MEV biosynthetic pathway may be capable of an increased production of one or more isoprenoid compounds as compared to cell which is not modified with one or enzymes of the MEV biosynthetic pathway. 26^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT In some embodiments, the isoprenoid producing cell comprises a heterologous nucleotide sequence encoding an enzyme that can condense two molecules of acetyl-coenzyme A to form acetoacetyl-CoA, e.g., an acetyl-CoA thiolase. Illustrative examples of nucleotide sequences encoding such an enzyme include but are not limited to: (NC_000913 REGION: 2324131.2325315; Escherichia coli), (D49362; Paracoccus denitrifzcans), and (L20428; Saccharomyces cerevisiae). In some embodiments, the cell includes a heterologous nucleotide sequence encoding an enzyme that can condense acetoacetyl-CoA with another molecule of acetyl-CoA to form 3-hydroxy- 3-methylglutaryl-CoA (HMG-CoA), e.g., a HMG-CoA synthase. Illustrative examples of nucleotide sequences encoding such an enzyme include but are not limited to: (NC_00l 145. complement 19061.20536; Saccharomyces cerevisiae), (X96617; Saccharomyces cerevisiae), (X83882; Arabidopsis thaliana), (AB037907; Kitasatospora griseola), (BT007302; Homo sapiens), and (NC_002758, Locus tag SAV2546, GeneID 1122571; Staphylococcus aureus). In some embodiments, the cell includes a heterologous nucleotide sequence encoding an enzyme that can convert HMG-CoA into mevalonate, e.g., an HMG-CoA reductase. Illustrative examples of nucleotide sequences encoding such an enzyme include, but are not limited to: (NM_206548; Drosophila melanogaster), (NC_002758, Locus tag SAV2545, GeneID 1122570; Staphylococcus aureus), (NM_204485; Gallus gallus), (AB015627; Streptomyces sp. KO 3988), (AF542543; Nicotiana attenuata), (AB037907; Kitasatospora griseola), (AX128213, providing the sequence encoding a truncated HMGR; Saccharomyces cerevisiae), and (NC_001145: complement (115734.118898; Saccharomyces cerevisiae). In some embodiments, the cells include a heterologous nucleotide sequence encoding an enzyme that can convert mevalonate into mevalonate 5-phosphate, e.g., a mevalonate kinase. Illustrative examples of nucleotide sequences encoding such an enzyme include but are not limited to: (L77688; Arabidopsis thaliana), and (X55875; Saccharomyces cerevisiae). In some embodiments, the cells include a heterologous nucleotide sequence encoding an enzyme that can convert mevalonate 5-phosphate into mevalonate 5-pyrophosphate, e.g., a phosphomevalonate kinase. Illustrative examples of nucleotide sequences encoding such an enzyme include but are not limited to: (Af 429385; Hevea brasiliensis), (NM_006556; Homo sapiens), and (NC_00l 145. Complement 712315.713670; Saccharomyces cerevisiae). In some embodiments, the cells include a heterologous nucleotide sequence encoding an enzyme that can convert mevalonate 5-pyrophosphate into isopentenyl diphosphate (IPP), e.g., a mevalonate pyrophosphate decarboxylase. Illustrative examples of nucleotide sequences encoding such an enzyme include but are not limited to: (X97557; Saccharomyces cerevisiae), (AF290095; Enterococcus faecium), and (U49260; Homo sapiens). In some embodiments, the cells include one or more heterologous nucleotide sequences encoding more than one enzyme of the MEV pathway. In some embodiments, the cell includes one or more heterologous nucleotide sequences encoding two enzymes of the MEV pathway. In some embodiments, the cell includes one or more heterologous nucleotide sequences encoding an enzyme that can convert HMG-CoA into mevalonate and an enzyme that can convert mevalonate into 27^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT mevalonate 5-phosphate. In some embodiments, the cell includes one or more heterologous nucleotide sequences encoding three enzymes of the MEV pathway. In some embodiments, the cell includes one or more heterologous nucleotide sequences encoding four enzymes of the MEV pathway. In some embodiments, the cell includes one or more heterologous nucleotide sequences encoding five enzymes of the MEV pathway. In some embodiments, the cell includes one or more heterologous nucleotide sequences encoding six enzymes of the MEV pathway. In some embodiments, the cell further includes a heterologous nucleotide sequence encoding an enzyme that can convert IPP generated via the MEV pathway into its isomer, dimethylallyl pyrophosphate (DMAPP). DMAPP can be condensed and modified through the action of various additional enzymes to form simple and more complex isoprenoids. The cells described herein may be modified to express one or more enzymes of the 1-deoxy- D-xylulose 5-diphosphate (DXP) biosynthetic pathway. Cells which are modified with one or more enzymes of the DXP biosynthetic pathway may be capable of an increased production of one or more isoprenoid compounds as compared to cell which is not modified with one or enzymes of the DXP biosynthetic pathway. In some embodiments, the cells include a heterologous nucleotide sequence encoding an enzyme that can condense two molecules of acetyl-coenzyme A to form acetoacetyl-CoA, e.g., an acetyl-CoA thiolase. Illustrative examples of nucleotide sequences encoding such an enzyme include but are not limited to: (NC_000913 REGION: 2324131.2325315; Escherichia coli), (D49362; Paracoccus denitrifzcans), and (L20428; Saccharomyces cerevisiae). In some embodiments, the cell includes a heterologous nucleotide sequence encoding an enzyme, e.g., l-deoxy-D-xylulose-5-phosphate synthase, which can condense pyruvate with D- glyceraldehyde 3-phosphate to make l-deoxy-D-xylulose- 5-phosphate. Illustrative examples of nucleotide sequences encoding such an enzyme include but are not limited to: (AF035440; Escherichia coli), (NC_002947, locus tag PP0527; Pseudomonas putida KT2440), (CP000026, locus tag SPA2301; Salmonella enterica Paratyphi, see ATCC 9150), (NC_007493, locus tag RSP _0254; Rhodobacter sphaeroides 2.4.1 ), (NC_ 005296, locus tag RP A0952; Rhodopseudomonas palustris CGA009), (NC_004556, locus tag PD1293; Xylellafastidiosa Temecula]), and (NC_003076, locus tag AT5Gl 1380; Arabidopsis thaliana). In some embodiments, the cell includes a heterologous nucleotide sequence encoding an enzyme, e.g., l-deoxy-D-xylulose-5-phosphate reductoisomerase, which can convert l-deoxy-D- xylulose-5-phosphate to 2C-methyl-Derythritol- 4-phosphate. Illustrative examples of nucleotide sequences include but are not limited to: (AB013300; Escherichia coli), (AF148852; Arabidopsis thaliana), (NC_002947, locus tag PP1597; Pseudomonas putida KT2440), (AL939124, locus tag SCO5694; Streptomyces coelicolor A3(2)), (NC_007493, locus tag RSP 2709; Rhodobacter sphaeroides 2.4.1), and (NC_007492, locus tag Pfl_l 107; Pseudomonas jluorescens PfO-1). In some embodiments, the cell includes a heterologous nucleotide sequence encoding an enzyme, e.g., 4-diphosphocytidyl-2C-methyl-D-erythritol synthase, which can convert 2C-methyl-D- erythritol-4-phosphate to 4-diphosphocytidyl-2Cmethyl-D-erythritol. Illustrative examples of nucleotide sequences include but are not limited to: (AF230736; Escherichia coli), (NC_007493, locus tag RSP 28^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT 2835; Rhodobacter sphaeroides 2.4.1), (NC_003071, locus tag AT2G02500; Arabidopsis thaliana), and (NC_002947, locus tag PP1614; Pseudomonas putida KT2440). In some embodiments, the cell includes a heterologous nucleotide sequence encoding an enzyme, e.g., 4-diphosphocytidyl-2C-methyl-D-erythritol kinase, which can convert 4- diphosphocytidyl-2C-methyl-D-erythritol to 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate. Illustrative examples of nucleotide sequences include but are not limited to: (AF216300; Escherichia coli) and (NC_007493, locus tag RSP 1779; Rhodobacter sphaeroides 2.4.1). In some embodiments, the cell includes a heterologous nucleotide sequence encoding an enzyme, 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, which can convert 4- diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate to 2Cmethyl-D-erythritol 2,4-cyclodiphosphate. Illustrative examples of nucleotide sequences include but are not limited to: (AF230738; Escherichia coli), (NC_007493, locus tag RSP _6071; Rhodobacter sphaeroides 2.4.1), and (NC_002947, locus tag PP1618; Pseudomonas putida KT2440). In some embodiments, the cell includes a heterologous nucleotide sequence encoding an enzyme, e.g., l-hydroxy-2-methyl-2I)-butenyl-4- diphosphate synthase, which can convert 2C-methyl- D-erythritol 2,4-cyclodiphosphate to 1- hydroxy-2-methy 1-2-(E)-butenyl 1-4-di phosphate. Illustrative examples of nucleotide sequences include but are not limited to: (AY033515; Escherichia coli), (NC_002947, locus tag PP0853; Pseudomonas putida KT2440), and (NC_007493, locus tag RSP 2982; Rhodobacter sphaeroides 2.4.1). In some embodiments, the cell includes a heterologous nucleotide sequence encoding an enzyme, e.g., isopentyl/dimethylallyl diphosphate synthase, which can convert l-hydroxy-2-methyI-(E)- butenyl-4-diphosphate into either IPP or its isomer, DMAPP. Illustrative examples of nucleotide sequences include but are not limited to: (AY062212; Escherichia coli) and (NC_002947, locus tag PP0606; Pseudomonas putida KT2440). In some embodiments, the cell includes one or more heterologous nucleotide sequences encoding more than one enzyme of the DXP pathway. In some embodiments, the cell includes one or more heterologous nucleotide sequences encoding two enzymes of the DXP pathway. In some embodiments, the cell includes one or more heterologous nucleotide sequences encoding three enzymes of the DXP pathway. In some embodiments, the cell includes one or more heterologous nucleotide sequences encoding four enzymes of the DXP pathway. In some embodiments, the cell includes one or more heterologous nucleotide sequences encoding five enzymes of the DXP pathway. In some embodiments, the cell includes one or more heterologous nucleotide sequences encoding six enzymes of the DXP pathway. In some embodiments, the cell includes one or more heterologous nucleotide sequences encoding five enzymes of the DXP pathway. In some embodiments, the cell includes one or more heterologous nucleotide sequences encoding seven enzymes of the DXP pathway. In some embodiments, “crosstalk” (or interference) between the cell's own metabolic processes and those processes involved with the production of IPP are minimized or eliminated entirely. For example, cross talk is minimized or eliminated entirely when the cell relies exclusively on the DXP pathway for synthesizing IPP, and a MEV pathway is introduced to provide additional IPP. 29^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT Such a cell would not be equipped to alter the expression of the MEV pathway enzymes or process the intermediates associated with the MEV pathway. Organisms that rely exclusively or predominately on the DXP pathway include, for example, Escherichia coli. In some embodiments, the cell produces IPP via the MEV pathway, either exclusively or in combination with the DXP pathway. In other embodiments, a cell’s DXP pathway is functionally disabled so that the cell produces IPP exclusively through a heterologously introduced MEV pathway. The DXP pathway can be functionally disabled by disabling gene expression or inactivating the function of one or more of the DXP pathway enzymes. In some embodiments, the cell further includes a heterologous nucleotide sequence encoding a polyprenyl synthase that can condense IPP and/or DMAPP molecules to form polyprenyl compounds containing more than five carbons. In some embodiments, the isoprenoid producing cell further comprises a heterologous nucleotide sequence encoding an enzyme that can convert IPP generated via the MEV pathway into DMAPP, e.g., an IPP isomerase. Illustrative examples of nucleotide sequences encoding such an enzyme include but are not limited to: (NC_000913, 3031087.3031635; Escherichia coli), and (AF082326; Haematococcus pluvialis). In some embodiments, the cell includes a heterologous nucleotide sequence encoding an enzyme that can condense one molecule of IPP with one molecule of DMAPP to form one molecule of geranyl pyrophosphate (GPP), e.g., a GPP synthase. Illustrative examples of nucleotide sequences encoding such an enzyme include, but are not limited to: (AF513lll;Abies grandis), (AF513112;Abies grandis), (AF513113;Abies grandis), (AY534686; Antirrhinum majus), (AY534687; Antirrhinum majus), (Yl 7376; Arabidopsis thaliana), (AE016877, Locus APl 1092; Bacillus cereus; ATCC 14579), (AJ243739; Citrus sinensis), (AY534745; Clarkia breweri), (AY953508; fps pini), (DQ286930; Lycopersicon esculentum), (AF182828; Mentha x piperita), (AF182827; Mentha x piperita), (MPI249453; Mentha x piperita), (PZE431697, Locus CAD24425; Paracoccus 862; Vi tis vinifera), and (AF203881, Locus In some embodiments, the cell includes a heterologous nucleotide sequence encoding an enzyme that can condense two molecules of IPP with one molecule of DMAPP or add a molecule of IPP to a molecule of GPP, to form a molecule of farnesyl pyrophosphate (FPP), e.g., a FPP synthase. Illustrative examples of nucleotide sequences that encode such an enzyme include, but are not limited to: (ATU80605; Arabidopsis thaliana), (ATHFPS2R; Arabidopsis thaliana), (AAU36376; Artemisia annua), (AF461050; Bos taurus), (D00694; Escherichia coli K-12), (AE009951, Locus AAL95523; Fusobacterium nucleatum subsp. nucleatum ATCC 25586), (GFFPPSGEN; Gibberella Jujikuroi), (CP000009, Locus AAW60034; Gluconobacter oxydans 621H), (AF019892; Helianthus annuus ), (HUMP APS; Homo sapiens), (KLPFPSQCR; Kluyveromyces lactis ), (LAU15777; Lupinus albus), (LAU20771; Lupinus albus), (AF309508; Mus musculus), (NCFPPSGEN; Neurospora crassa), (PAFPSl; Parthenium argentatum), (PAFPS2; Parthenium argentatum), (RA TF APS; Rattus norvegicus), (YSCFPP; Saccharomyces cerevisiae), (D89104; SchizoSaccharomyces pombe), (CP000003, Locus AAT87386; Streptococcus pyogenes), (CP0000l 7, Locus AAZ51849; 30^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT Streptococcus pyogenes), (NC_ 008022, Locus YP 598856; Streptococcus pyogenes MGAS 10270), (NC_ 008023, Locus YP 600845; Streptococcus pyogenes MGAS2096), (NC_008024, Locus YP 602832; Streptococcus pyogenes MGAS10750), (MZEFPS; Zea mays), (AE000657, Locus AAC06913; Aquifex aeolicus VF5), (NM_202836; Arabidopsis thaliana), (D84432, Locus BAA12575; Bacillus subtilis), (Ul2678, Locus AAC28894; Bradyrhizobiumjaponicum USDA 110), (BACFDPS; Geobacillus stearothermophilus), (NC_002940, Locus NP 873754; Haemophilus ducreyi 35000HP), (L42023, Locus AAC23087; Haemophilus injluenzae Rd KW20), (J05262; Homo sapiens), (YP 395294; Lactobacillus sakei subsp. sakei 23K), (NC_005823, Locus YP 000273; Leptospira interrogans serovar Copenhageni str. Fiocruz Ll-130), (AB003187; Micrococcus luteus), (NC_002946, Locus YP _208768; Neisseria gonorrhoeae FA 1090), (U00090, Locus AAB91752; Rhizobium sp. NGR234), (J05091; Saccharomyces cerevisiae), (CP000031, Locus AAV93568; Silicibacter pomeroyi DSS-3), (AE008481, Locus AAK99890; Streptococcus pneumoniae R6), and (NC_ 004556, Locus NP 779706; Xylella fastidiosa Temecula1). In some embodiments, the cell includes a heterologous nucleotide sequence encoding an enzyme that can combine IPP and DMAPP or IPP and FPP to form geranylgeranyl pyrophosphate (GGPP). Illustrative examples of nucleotide sequences that encode such an enzyme include, but are not limited to: (ATHGERPYRS; Arabidopsis thaliana), (BT005328; Arabidopsis thaliana), (NM_l 19845; Arabidopsis thaliana), (NZ_AAJM01000380, Locus ZP 00743052; Bacillus thuringiensis serovar israelensis, ATCC 35646 sql563), (CRGGPPS; Catharanthus roseus), (NZ_AABF02000074, Locus ZP 00144509; Fusobacterium nucleatum subsp. vincentii, ATCC 49256), (GFGGPPSGN; Gibberellafujikuroi), (AY371321; Ginkgo biloba), (AB055496; Hevea brasiliensis), (AB0l 7971; Homo sapiens), (MCI276129; Mucor circinelloides f. lusitanicus), (AB016044; Mus musculus), (AABX01000298, Locus NCU01427; Neurospora crassa), (NCU20940; Neurospora crassa), (NZ_AAKL01000008, Locus ZP 00943566; Ralstonia solanacearum UW551), (ABl 18238; Rattus norvegicus), (SCU31632; Saccharomyces cerevisiae), (AB016095; Synechococcus elongates), (SAGGPS; Sinapis alba), (SSOGDS; Sulfolobus acidocaldarius), (NC_007759, Locus YP 461832; Syntrophus aciditrophicus SB), (NC_006840, Locus YP 204095; Vibrio jischeri ESl 14), (NM_ 112315; Arabidopsis thaliana), (ERWCR TE; Pantoea agglomerans), (D90087, Locus BAA14124; Pantoea ananatis), (X52291, Locus CAA36538; Rhodobacter capsulatus), (AF195122, Locus AAF24294; Rhodobacter sphaeroides), and (NC_004350, Locus NP 721015; Streptococcus mutans UA159). In some embodiments, the cell further includes a heterologous nucleotide sequence encoding an enzyme that can modify a polyprenyl to form a hemiterpene, a monoterpene, a sesquiterpene, a diterpene, a triterpene, a tetraterpene, a polyterpene, a steroid compound, a carotenoid, or a modified isoprenoid compound. In some embodiments, the heterologous nucleotide encodes a carene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (AF461460, REGION 43.1926; Picea abies) and (AF527416, REGION: 78.1871; Salvia stenophylla). In some embodiments, the heterologous nucleotide encodes a geraniol synthase. Illustrative examples of suitable nucleotide sequences include but are not limited to: (Af 457070; Cinnamomum tenuipilum), (A Y362553; Ocimum basilicum), (DQ234300; Perilla frutescens strain 1864), 31^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT (DQ234299; Perilla citriodora strain 1861), (DQ234298; Perilla citriodora strain 4935), and (DQ088667; Perilla citriodora). In some embodiments, the heterologous nucleotide encodes a linalool synthase. Illustrative examples of a suitable nucleotide sequence include, but are not limited to: (AF497485; Arabidopsis thaliana), (AC002294, Locus AAB71482; Arabidopsis thaliana), (AY059757; Arabidopsis thaliana), (NM_104793; Arabidopsis thaliana), (AF154124; Artemisia annua), (AF067603; Clarkia breweri), (AF067602; Clarkia concinna), (AF067601; Clarkia breweri), (U58314; Clarkia breweri), (AY840091; Lycopersicon esculentum), (DQ263741; Lavandula angustifolia), (AY083653;Mentha citrate), (AY693647; Ocimum basilicum), (XM_ 463918; Oryza sativa), (AP004078, Locus BAD07605; Oryza sativa), (XM_ 463918, Locus XP _ 463918; Oryza sativa), (AY917193; Perilla citriodora), (AF271259; Perillafrutescens), (AY473623; Picea abies), (DQ195274; Picea sitchensis), and (AF444798; Perilla frutescens var. crispa cultivar No.79). In some embodiments, the heterologous nucleotide encodes a limonene synthase. Illustrative examples of suitable nucleotide sequences include but are not limited to:(+) limonene synthases (AF514287, REGION: 47.1867; Citrus limon) and (AY055214, REGION: 48.1889; Agastache rugosa) and (-)-limonene synthases (DQ195275, REGION: 1.1905; Picea sitchensis), (AF006193, REGION: 73.1986;Abies grandis), and (MHC4SLSP, REGION: 29.1828; Mentha spicata). In some embodiments, the heterologous nucleotide encodes a myrcene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (U87908; Abies grandis), (A Yl 95609; Antirrhinum majus), (A Yl 95608; Antirrhinum majus), (NM_l27982; Arabidopsis thaliana TPSlO), (NM_ll3485; Arabidopsis thaliana ATTPS-CIN), (NM_ 113483; Arabidopsis thaliana ATTPS- CIN), (AF271259; Perilla frutescens), (AY473626; Picea abies), (AF369919; Picea abies), and (AJ304839; Quercus ilex). In some embodiments, the heterologous nucleotide encodes an ocimene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (AYl 95607; Antirrhinum majus), (A Yl 95609; Antirrhinum majus), (A Yl 95608; Antirrhinum majus), (AK221024; Arabidopsis thaliana), (NM_ 113485; Arabidopsis thaliana ATTPS-CIN), (NM_ll3483; Arabidopsis thaliana ATTPS-CIN), (NM_ll 7775; Arabidopsis thaliana ATTPS03), (NM_001036574; Arabidopsis thaliana ATTPS03), (NM_l27982; Arabidopsis thaliana TPS 10), (AB 110642; Citrus unshiu CitMTSL4), and (AY575970; Lotus corniculatus var. Japonicus ). In some embodiments, the heterologous nucleotide encodes an a^-pinene synthase. Illustrative examples of suitable nucleotide sequences include but are not limited to: (+) ^-pinene synthase (AF543530, REGION: 1.1887; Pinus taeda), (-) ^-pinene synthase (AF543527, REGION: 32.1921; Pinus taeda), and (+)/ (-)a-pinene synthase (AGU87909, REGION: 6111892;Abies grandis). In some embodiments, the heterologous nucleotide encodes a P-pinene synthase. Illustrative examples of suitable nucleotide sequences include but are not limited to: (-) Ppinene synthases (AF276072, REGION: 1.1749; Artemisia annua) and (AF514288, REGION: 26.1834; Citrus limon). In some embodiments, the heterologous nucleotide encodes a sabinene synthase. An illustrative example of a suitable nucleotide sequence includes but is not limited to AF05 l 901, REGION: 26.1798 from Salvia ofjicinalis. 32^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT In some embodiments, the heterologous nucleotide encodes a y-terpinene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (AF514286, REGION: 30.1832 from Citrus limon) and (ABl 10640, REGION 1.1803 from Citrus unshiu). In some embodiments, the heterologous nucleotide encodes a terpinolene synthase. Illustrative examples of a suitable nucleotide sequence include but are not limited to: (AY693650 from Ocimum basilicum) and (AY906866, REGION: 10.1887 from Pseudotsuga menziesii). In some embodiments, the heterologous nucleotide encodes an amorphadiene synthase. An illustrative example of a suitable nucleotide sequence is SEQ ID NO.37 of U.S. Patent Publication No.2004/0005678. In some embodiments, the heterologous nucleotide encodes an ^-farnesene synthase. Illustrative examples of suitable nucleotide sequences include but are not limited to DQ309034 from Pyrus communis cultivar d'Anjou (pear; gene name AFSl) and AY182241 from Malus domestica (apple; gene AFSl). Pechouus et al., Planta 219(1):84-94 (2004). In some embodiments, the heterologous nucleotide encodes a ^-farnesene synthase. Illustrative examples of suitable nucleotide sequences include but are not limited to GenBank accession number AF024615 from Mentha x piperita (peppermint; gene Tspal 1), and A Y835398 from Artemisia annua. Picaud et al., Phytochemistry 66(9): 961-967 (2005). In some embodiments, the heterologous nucleotide encodes a farnesol synthase. Illustrative examples of suitable nucleotide sequences include but are not limited to GenBank accession number AF529266 from Zea mays and YDR481C from Saccharomyces cerevisiae (gene Pho8). Song, L., Applied Biochemistry and Biotechnology 128: 149-158 (2006). In some embodiments, the heterologous nucleotide encodes a nerolidol synthase. An illustrative example of a suitable nucleotide sequence includes but is not limited to AF529266 from Zea mays (maize; gene tpsl). In some embodiments, the heterologous nucleotide encodes a patchoulol synthase. Illustrative examples of suitable nucleotide sequences include but are not limited to AY508730 REGION: 1.1659 from Pogostemon cablin. In some embodiments, the heterologous nucleotide encodes a nootkatone synthase. Illustrative examples of a suitable nucleotide sequence include but are not limited to AF441124 REGION: 1.1647 from Citrus sinensis and AY917195 REGION: 1.1653 from Perilla frutescens. In some embodiments, the heterologous nucleotide encodes an abietadiene synthase. Illustrative examples of suitable nucleotide sequences In some embodiments, one or more heterologous nucleic acids encoding one or more enzymes are integrated into the genome of the cell. In some embodiments, one or more heterologous nucleic acids encoding one or more enzymes are present within one or more plasmids. ^ ^^ Introduction of Heterologous Nucleic Acids into a Host Cell In some embodiments, a heterologous nucleic acid of the disclosure is introduced into a host cell (e.g., yeast cell) by way of a gap repair molecular biology technique. The host cell may be capable of producing a biological product or a previously cultured cell. In these methods, if the host 33^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT cell has non-homologous end joining (NHEJ) activity, as is the case for Kluyveromyces marxianus, then the NHEJ activity in the host cell can be first disrupted in any of a number of ways. Further details related to genetic modification of host cells (e.g., yeast cells) through gap repair can be found in U.S. Patent No.9,476,065, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, a heterologous nucleic acid of the disclosure is introduced into the host cell by way of one or more site-specific nucleases capable of causing breaks at designated regions within selected nucleic acid target sites. Examples of such nucleases include, but are not limited to, endonucleases, site-specific recombinases, transposases, topoisomerases, zinc finger nucleases, TAL-effector DNA binding domain-nuclease fusion proteins (TALENs), CRISPR/Cas- associated RNA-guided endonucleases, and meganucleases. Further details related to genetic modification of host cells through site specific nuclease activity can be found in U.S. Patent No. 9,476,065, the disclosure of which is incorporated herein by reference in its entirety. Nucleic Acid and Amino Acid Sequence Optimization Described herein are specific genes and proteins useful in the methods, compositions, and organisms of the disclosure; however, it will be recognized that absolute identity to such genes is not necessary. For example, changes in a particular gene or polynucleotide including a sequence encoding a polypeptide or enzyme can be performed and screened for activity. Typically, such changes include conservative mutations and silent mutations. Such modified or mutated polynucleotides and polypeptides can be screened for expression of a functional enzyme using methods known in the art. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or functionally equivalent polypeptides can also be used to clone and express the polynucleotides encoding such enzymes. As will be understood by those of skill in the art, it can be advantageous to modify a coding sequence to enhance its expression in a particular host. The genetic code is redundant with 64 possible codons, but most organisms typically use a subset of these codons. The codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons. Codons can be substituted to reflect the preferred codon usage of the host, in a process sometimes called "codon optimization" or "controlling for species codon bias." Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (Murray et al., 1989, Nucl Acids Res.17: 477-508) can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and E. coli commonly use UAA as the stop codon (Dalphin et al., 1996, Nucl Acids Res.24: 216-8). 34^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT Those of skill in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA molecules differing in their nucleotide sequences can be used to encode a given heterologous polypeptide of the disclosure. A native DNA sequence encoding the biosynthetic enzymes described above is referenced herein merely to illustrate an embodiment of the disclosure, and the disclosure includes DNA molecules of any sequence that encode the amino acid sequences of the polypeptides and proteins of the enzymes utilized in the methods of the disclosure. In similar fashion, a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity. The disclosure includes such polypeptides with different amino acid sequences than the specific proteins described herein so long as the modified or variant polypeptides have the enzymatic anabolic or catabolic activity of the reference polypeptide. Furthermore, the amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the disclosure“ When "homologous" is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties, e.g., charge or hydrophobicity. In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (See, e.g., Pearson W. R., 1994, Methods in Mol. Biol.25: 365-89). Furthermore, any of the genes encoding an enzyme described herein (or any of the regulatory elements that control or modulate expression thereof) can be optimized by genetic/protein engineering techniques, such as directed evolution or rational mutagenesis, which are known to those of ordinary skill in the art. Such action allows those of ordinary skill in the art to optimize the enzymes for expression and activity in yeast. In addition, genes encoding these enzymes can be identified from other fungal and bacterial species and can be expressed for the modulation of this pathway. A variety of organisms could serve as sources for these enzymes, including, but not limited to, Saccharomyces spp., including S. cerevisiae and S. uvarum, Kluyveromyces spp., including K. thermotolerans, K. lactis, and K. marxianus, Pichia spp., Hansenula spp., including H. polymorpha, Candida spp., Trichosporon spp., Yamadazyma spp., including Y. spp. stipitis, Torulaspora pretoriensis, Issatchenkia orientalis, Schizosaccharomyces spp., including S. pombe, Cryptococcus spp., Aspergillus spp., Neurospora spp., or Ustilago spp. Sources of genes from anaerobic fungi include, but are not limited to, Piromyces spp., Orpinomyces spp., or Neocallimastix spp. Sources of prokaryotic enzymes that are useful include, but are not limited to, Escherichia. coli, Zymomonas mobilis, Staphylococcus aureus, Bacillus spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Lactococcus spp., Enterobacter spp., Salmonella spp., or X. dendrorhous. 35^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT Techniques known to those skilled in the art may be suitable to identify additional homologous genes and homologous enzymes. Generally, analogous genes and/or analogous enzymes can be identified by functional analysis and will have functional similarities. Techniques known to those skilled in the art can be suitable to identify analogous genes and analogous enzymes. Techniques include, but are not limited to, cloning a gene by PCR using primers based on a published sequence of a gene/enzyme of interest, or by degenerate PCR using degenerate primers designed to amplify a conserved region among a gene of interest. Further, one skilled in the art can use techniques to identify homologous or analogous genes, proteins, or enzymes with functional homology or similarity. Techniques include examining a cell or cell culture for the catalytic activity of an enzyme through in vitro enzyme assays for said activity, e.g., as described herein or in Kiritani, K., Branched-Chain Amino Acids Methods Enzymology, 1970; then isolating the enzyme with said activity through purification; determining the protein sequence of the enzyme through techniques such as Edman degradation; design of PCR primers to the likely nucleic acid sequence; amplification of said DNA sequence through PCR; and cloning of said nucleic acid sequence. To identify homologous or similar genes and/or homologous or similar enzymes, suitable techniques also include comparison of data concerning a candidate gene or enzyme with databases such as BRENDA, KEGG, or MetaCYC. The candidate gene or enzyme can be identified within the above-mentioned databases in accordance with the teachings herein. ^ Culture and Fermentation Conditions ^ Materials and methods for the maintenance and growth of microbial cultures are well known to those skilled in the art of microbiology or fermentation science (see, for example, Bailey et al., Biochemical Engineering Fundamentals, second edition, McGraw Hill, New York, 1986). Consideration must be given to appropriate culture medium, pH, temperature, and requirements for aerobic, microaerobic, or anaerobic conditions, depending on the specific requirements of the host cell, the fermentation, and the process. ^ The methods of producing a biological product provided herein may be performed in a suitable culture medium in a suitable container, including but not limited to a cell culture plate, a flask, or a fermentor. Further, the methods can be performed at any scale of fermentation known in the art to support industrial production of microbial products. Any suitable fermentor may be used including a stirred tank fermentor, an airlift fermentor, a bubble fermentor, or any combination thereof. In particular embodiments utilizing Saccharomyces cerevisiae as the host cell, strains can be grown in a fermentor as described in detail by Kosaric, et al, Ullmann's Encyclopedia of Industrial Chemistry, Sixth Edition, Volume 12, pages 398-473, Wiley-VCH Verlag GmbH & Co. KDaA, Weinheim, Germany. In some embodiments, the culture medium is any culture medium in which a microorganism capable of producing a biological product can subsist, i.e., maintain growth and viability. In some embodiments, the culture medium is an aqueous medium comprising assimilable carbon, nitrogen, and phosphate sources. Such a medium can also include appropriate salts, minerals, metals, and other nutrients. In some embodiments, the carbon source and each of the essential cell nutrients are 36^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT added incrementally or continuously to the fermentation medium, and each required nutrient is maintained at essentially the minimum level needed for efficient assimilation by growing cells, for example, in accordance with a predetermined cell growth curve based on the metabolic or respiratory function of the cells which convert the carbon source to a biomass. ^ Suitable conditions and suitable medium for culturing microorganisms are well known in the art. In some embodiments, the suitable medium is supplemented with one or more additional agents, such as, for example, an inducer (e.g., when one or more nucleotide sequences encoding a gene product are under the control of an inducible promoter), a repressor (e.g., when one or more nucleotide sequences encoding a gene product are under the control of a repressible promoter), or a selection agent (e.g., an antibiotic to select for microorganisms comprising the genetic modifications). ^ In some embodiments, the carbon source is a monosaccharide (simple sugar), a disaccharide, a polysaccharide, a non-fermentable carbon source, or one or more combinations thereof. Non-limiting examples of suitable monosaccharides include glucose, galactose, mannose, fructose, ribose, and combinations thereof. Non-limiting examples of suitable disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof. Non-limiting examples of suitable polysaccharides include starch, glycogen, cellulose, chitin, and combinations thereof. Non- limiting examples of suitable non-fermentable carbon sources include acetate and glycerol. The concentration of a carbon source, such as glucose or sucrose, in the culture medium should promote cell growth, but not be so high as to repress growth of the microorganism used. Typically, cultures are run with a carbon source, such as glucose or sucrose, being added at levels to achieve the desired level of growth and biomass. Production of biological products, such as isoprenoids, may also occur in these culture conditions, but at undetectable levels (with detection limits being about <0.1 g/l). In other embodiments, the concentration of a carbon source, such as glucose or sucrose, in the culture medium is greater than about 1 g/L, preferably greater than about 2 g/L, and more preferably greater than about 5 g/L. In addition, the concentration of a carbon source, such as glucose or sucrose, in the culture medium is typically less than about 100 g/L, preferably less than about 50 g/L, and more preferably less than about 20 g/L. It should be noted that references to culture component concentrations can refer to both initial and/or ongoing component concentrations. In some cases, it may be desirable to allow the culture medium to become depleted of a carbon source during culture. ^ Sources of assimilable nitrogen that can be used in a suitable culture medium include, but are not limited to, simple nitrogen sources, organic nitrogen sources and complex nitrogen sources. Such nitrogen sources include anhydrous ammonia, ammonium salts and substances of animal, vegetable and/or microbial origin. Suitable nitrogen sources include, but are not limited to, protein hydrolysates, microbial biomass hydrolysates, peptone, yeast extract, ammonium sulfate, urea, and amino acids. Typically, the concentration of the nitrogen sources, in the culture medium is greater than about 0.1 g/L, preferably greater than about 0.25 g/L, and more preferably greater than about 1.0 g/L. Beyond certain concentrations, however, the addition of a nitrogen source to the culture medium is not advantageous for the growth of the microorganisms. As a result, the concentration of the nitrogen sources, in the culture medium is less than about 20 g/L, preferably less than about 10 g/L and more 37^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT preferably less than about 5 g/L. Further, in some instances it may be desirable to allow the culture medium to become depleted of the nitrogen sources during culture. ^ The effective culture medium can contain other compounds such as inorganic salts, vitamins, trace metals, or growth promoters. Such other compounds can also be present in carbon, nitrogen, or mineral sources in the effective medium or can be added specifically to the medium. The culture medium can also contain a suitable phosphate source. Such phosphate sources include both inorganic and organic phosphate sources. Preferred phosphate sources include, but are not limited to, phosphate salts such as mono or dibasic sodium and potassium phosphates, ammonium phosphate, and mixtures thereof. Typically, the concentration of phosphate in the culture medium is greater than about 1.0 g/L, preferably greater than about 2.0 g/L, and more preferably greater than about 5.0 g/L. Beyond certain concentrations, however, the addition of phosphate to the culture medium is not advantageous for the growth of the microorganisms. Accordingly, the concentration of phosphate in the culture medium is typically less than about 20 g/L, preferably less than about 15 g/L, and more preferably less than about 10 g/L. ^ A suitable culture medium can also include a source of magnesium, preferably in the form of a physiologically acceptable salt, such as magnesium sulfate heptahydrate, although other magnesium sources in concentrations that contribute similar amounts of magnesium can be used. Typically, the concentration of magnesium in the culture medium is greater than about 0.5 g/L, preferably greater than about 1.0 g/L, and more preferably greater than about 2.0 g/L. Beyond certain concentrations, however, the addition of magnesium to the culture medium is not advantageous for the growth of the microorganisms. Accordingly, the concentration of magnesium in the culture medium is typically less than about 10 g/L, preferably less than about 5 g/L, and more preferably less than about 3 g/L. Further, in some instances, it may be desirable to allow the culture medium to become depleted of a magnesium source during culture. In some embodiments, the culture medium can also include a biologically acceptable chelating agent, such as the dihydrate of trisodium citrate. In such instance, the concentration of a chelating agent in the culture medium is greater than about 0.2 g/L, preferably greater than about 0.5 g/L, and more preferably greater than about 1 g/L. Beyond certain concentrations, however, the addition of a chelating agent to the culture medium is not advantageous for the growth of the microorganisms. Accordingly, the concentration of a chelating agent in the culture medium is typically less than about 10 g/L, preferably less than about 5 g/L, and more preferably less than about 2 g/L. The culture medium can also initially include a biologically acceptable acid or base to maintain the desired pH of the culture medium. Biologically acceptable acids include, but are not limited to, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, and mixtures thereof. Biologically acceptable bases include, but are not limited to, ammonium hydroxide, sodium hydroxide, potassium hydroxide, and mixtures thereof. In some embodiments, the base used is ammonium hydroxide. The culture medium can also include a biologically acceptable calcium source, including, but not limited to, calcium chloride. Typically, the concentration of the calcium source, such as calcium chloride, dihydrate, in the culture medium is within the range of from about 5 mg/L to about 2000 38^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT mg/L, preferably within the range of from about 20 mg/L to about 1000 mg/L, and more preferably in the range of from about 50 mg/L to about 500 mg/L. The culture medium can also include sodium chloride. Typically, the concentration of sodium chloride in the culture medium is within the range of from about 0.1 g/L to about 5 g/L, preferably within the range of from about 1 g/L to about 4 g/L, and more preferably in the range of from about 2 g/L to about 4 g/L. In some embodiments, the culture medium can also include trace metals. Such trace metals can be added to the culture medium as a stock solution that, for convenience, can be prepared separately from the rest of the culture medium. Typically, the amount of such a trace metals solution added to the culture medium is greater than about 1 mL/L, preferably greater than about 5 mL/L, and more preferably greater than about 10 mL/L. Beyond certain concentrations, however, the addition of trace metals to the culture medium is not advantageous for the growth of the microorganisms. Accordingly, the amount of such a trace metals solution added to the culture medium is typically less than about 100 mL/L, preferably less than about 50 mL/L, and more preferably less than about 30 mL/L. It should be noted that, in addition to adding trace metals in a stock solution, the individual components can be added separately, each within ranges corresponding independently to the amounts of the components dictated by the above ranges of the trace metals solution. The culture medium can include other vitamins, such as pantothenate, biotin, calcium pantothenate, inositol, para-aminobenzoic acid, nicotinic acid, pyridoxine-HCl, and thiamine-HCl. Such vitamins can be added to the culture medium as a stock solution that, for convenience, can be prepared separately from the rest of the culture medium. Beyond certain concentrations, however, the addition of vitamins to the culture medium is not advantageous for the growth of the microorganisms. The culture medium may include trace metals, such as iron, copper, cobalt, zinc, selenium, chromium, iodine, and molybdenum. Such trace metals can be added to the culture medium as a stock solution that, for convenience, can be prepared separately from the rest of the culture medium. In some embodiments, the culture medium may include a surfactant. In some embodiments, the surfactant may be an anionic surfactant; for example, the surfactant may be alkyl-naphthalene sulfonate, alkyl benzene sulfonate, or the like. In some embodiments, the surfactant is a nonionic surfactant. Suitable surfactants include biocompatible nonionic surfactants such as Brij (e.g., polyoxyethylene (4) lauryl ether, also known as Brij-30; polyoxyethylene (2) oleyl ether; polyoxyethylene (2) stearyl ether; etc.); micelles; and the like. In some embodiments, the surfactant is a secondary ether polyol. In some embodiments, the surfactant is TERGITOL L-62 (Dow Chemical Company). In some embodiments, the surfactant is TERGAZYME (Alconox), which may be used in an amount of between 0% (w/v) and about 1% (w/v). The fermentation methods described herein can be performed in conventional culture modes, which include, but are not limited to, batch, fed-batch, cell recycle, continuous and semi-continuous. In some embodiments, the fermentation is carried out in fed-batch mode. In such a case, some of the components of the medium are depleted during culture, including pantothenate during the production stage of the fermentation. In some embodiments, the culture may be supplemented with relatively 39^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT high concentrations of such components at the outset, for example, of the production stage, so that growth and/or production is supported for a period of time before additions are required. The preferred ranges of these components are maintained throughout the culture by making additions as levels are depleted by culture. Levels of components in the culture medium can be monitored by, for example, sampling the culture medium periodically and assaying for concentrations. Alternatively, once a standard culture procedure is developed, additions can be made at timed intervals corresponding to known levels at particular times throughout the culture. As will be recognized by those in the art, the rate of consumption of nutrient increases during culture as the cell density of the medium increases. Moreover, to avoid introduction of foreign microorganisms into the culture medium, addition is performed using aseptic addition methods, as are known in the art. In addition, anti-foaming agent may be added during the culture. The population of host cells may be cultured for any amount of time suitable for the host cells to produce a biological product. For example, the population of host cells may be cultured for at least 10 hours. In some embodiments, the population of host cells is cultured for from about 10 hours to about 100 hours, such as from about 10 hours to about 75 hours, about 10 hours to about 50 hours, about 10 hours to about 25 hours, about 25 hours to about 100 hours, about 50 hours to about 100 hours, or about 75 hours to about 100 hours. The temperature of the culture medium can be any temperature suitable for growth of the genetically modified cells and/or production of compounds of interest. For example, prior to inoculation of the culture medium with an inoculum, the culture medium can be brought to and maintained at a temperature in the range of from about 20 oC to about 45 oC, preferably to a temperature in the range of from about 25 oC to about 40 oC and more preferably in the range of from about 28 oC to about 32 oC.41. In some embodiments, the host cells are cultured at a temperature of from about 20 oC to about 40 oC. In some embodiments, the host cells are cultured at a temperature of about 30 oC. The pH of the culture medium can be controlled by the addition of acid or base to the culture medium. In such cases when ammonia is used to control pH, it also conveniently serves as a nitrogen source in the culture medium. Preferably, the pH is maintained from about 3.0 to about 8.0, more preferably from about 3.5 to about 7.0, and most preferably from about 4.0 to about 6.5. In some embodiments, the carbon source concentration, such as the glucose concentration, of the culture medium is monitored during culture. Glucose or sucrose concentration of the culture medium can be monitored using known techniques, such as, for example, use of the glucose oxidase enzyme test or high-pressure liquid chromatography, which can be used to monitor glucose concentration in the supernatant, e.g., a cell-free component of the culture medium. As stated previously, the carbon source concentration should be kept below the level at which cell growth inhibition occurs. Although such concentration may vary from organism to organism, for glucose as a carbon source, cell growth inhibition occurs at glucose concentrations greater than at about 60 g/L and can be determined readily by trial. Accordingly, when glucose is used as a carbon source the glucose is preferably fed to the fermenter and maintained below detection limits. Alternatively, the glucose concentration in the culture medium is maintained in the range of from about 1 g/L to about 40^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT 100 g/L, more preferably in the range of from about 2 g/L to about 50 g/L, and yet more preferably in the range of from about 5 g/L to about 20 g/L. Although the carbon source concentration can be maintained within desired levels by addition of, for example, a substantially pure glucose solution, it is acceptable, and may be preferred, to maintain the carbon source concentration of the culture medium by addition of aliquots of the original culture medium. The use of aliquots of the original culture medium may be desirable because the concentrations of other nutrients in the medium (e.g., the nitrogen and phosphate sources) can be maintained simultaneously. Likewise, the trace metals concentrations can be maintained in the culture medium by addition of aliquots of the trace metals solution. ^ Cell Strains Any suitable cell may be used in the practice of the present invention as the host cell or previously cultured cell. Illustrative examples of suitable cells include any archae, prokaryotic, or eukaryotic cell. Examples of an archae cell include but are not limited to those belonging to the genera: Aeropyrum, Archaeglobus, Halobacterium, Methanococcus, Methanobacterium, Pyrococcus, Sulfolobus, and Thermoplasma. Illustrative examples of archae strains include but are not limited to: Aeropyrum pernix, Archaeoglobus fulgidus, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Pyrococcus abyssi, Pyrococcus horikoshii, Thermoplasma acidophilum, Thermoplasma volcanium. Examples of a prokaryotic cell include, but are not limited to those belonging to the genera: Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter, Azobacter, Bacillus, Brevibacterium, Chromatium, Clostridium, Corynebacterium, Enterobacter, Erwinia, Escherichia, Lactobacillus, Lactococcus, Mesorhizobium, Methylobacterium, Microbacterium, Phormidium, Pseudomonas, Rhodobacter, Rhodopseudomonas, Rhodospirillum, Rhodococcus, Salmonella, Scenedesmun, Serratia, Shigella, Staphlococcus, Strepromyces, Synnecoccus, and Zymomonas. Illustrative examples of prokaryotic bacterial strains include but are not limited to: Bacillus subtilis, Bacillus amyloliquefacines, Brevibacterium ammoniagenes, Brevibacterium immariophilum, Clostridium beigerinckii, Enterobacter sakazakii, Escherichia coli, Lactococcus lactis, Mesorhizobium loti, Pseudomonas aeruginosa, Pseudomonas mevalonii, Pseudomonas pudica, Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodospirillum rubrum, Salmonella enterica, Salmonella typhi, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, and the like. In general, if a bacterial host cell is used, a non-pathogenic strain is preferred. Illustrative examples of non-pathogenic strains include but are not limited to: Bacillus subtilis, Escherichia coli, Lactibacillus acidophilus, Lactobacillus helveticus, Pseudomonas aeruginosa, Pseudomonas mevalonii, Pseudomonas pudita, Rhodobacter sphaeroides, Rodobacter capsulatus, Rhodospirillum rubrum, and the like. Examples of eukaryotic cells include but are not limited to fungal cells. Examples of fungal cell include but are not limited to those belonging to the genera: Aspergillus, Candida, Chrysosporium, 41^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT Cryotococcus, Fusarium, Kluyveromyces, Neotyphodium, Neurospora, Penicillium, Pichia, Saccharomyces, Trichoderma and Xanthophyllomyces (formerly Phaffia). Illustrative examples of eukaryotic strains include but are not limited to: Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Candida albicans, Chrysosporium lucknowense, Fusarium graminearum, Fusarium venenatum, Kluyveromyces lactis, Neurospora crassa, Pichia angusta, Pichia finlandica, Pichia kodamae, Pichia membranaefaciens, Pichia methanolica, Pichia opuntiae, Pichia pastoris, Pichia pijperi, Pichia quercuum, Pichia salictaria, Pichia thermotolerans, Pichia trehalophila, Pichia stipitis, Streptomyces ambofaciens, Streptomyces aureofaciens, Streptomyces aureus, Saccaromyces bayanus, Saccaromyces boulardi, Saccharomyces cerevisiae, Streptomyces fungicidicus, Streptomyces griseochromogenes, Streptomyces griseus, Streptomyces lividans, Streptomyces olivogriseus, Streptomyces rameus, Streptomyces tanashiensis, Streptomyces vinaceus, Trichoderma reesei and Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma). In some embodiments of the present disclosure, the host cell is a yeast cell. In some embodiments, the previously cultured cell is a yeast cell. Yeast cells useful in conjunction with the compositions and methods described herein include yeast that have been deposited with microorganism depositories (e.g. IFO, ATCC, etc.), such as those that belong to the genera Aciculoconidium, Ambrosiozyma, Arthroascus, Arxiozyma, Ashbya, Babjevia, Bensingtonia, Botryoascus, Botryozyma, Brettanomyces, Bullera, Bulleromyces, Candida, Citeromyces, Clavispora, Cryptococcus, Cystofilobasidium, Debaryomyces, Dekkara, Dipodascopsis, Dipodascus, Eeniella, Endomycopsella, Eremascus, Eremothecium, Erythrobasidium, Fellomyces, Filobasidium, Galactomyces, Geotrichum, Guilliermondella, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hyphopichia, Issatchenkia, Kloeckera, Kloeckeraspora, Kluyveromyces, Kondoa, Kuraishia, Kurtzmanomyces, Leucosporidium, Lipomyces, Lodderomyces, Malassezia, Metschnikowia, Mrakia, Myxozyma, Nadsonia, Nakazawaea, Nematospora, Ogataea, Oosporidium, Pachysolen, Phachytichospora, Phaffia, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Saccharomycodes, Saccharomycopsis, Saitoella, Sakaguchia, Saturnospora, Schizoblastosporion, chizosaccharomyces, Schwanniomyces, Sporidiobolus, Sporobolomyces, Sporopachydermia, Stephanoascus, Sterigmatomyces, Sterigmatosporidium, Symbiotaphrina, Sympodiomyces, Sympodiomycopsis, Torulaspora, Trichosporiella, Trichosporon, Trigonopsis, Tsuchiyaea, Udeniomyces, Waltomyces, Wickerhamia, Wickerhamiella, Williopsis, Yamadazyma, Yarrowia, Zygoascus, Zygosaccharomyces, Zygowilliopsis, and Zygozyma, among others. In some embodiments, the strain is Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe, Dekkera bruxellensis, Kluyveromyces lactis (previously called Saccharomyces lactis), Kluveromyces marxianus, Arxula adeninivorans, or Hansenula polymorphs (now known as Pichia angusta). In some embodiments, the host microbe is a strain of the genus Candida, such as Candida lipolytica, Candida guilliermondii, Candida krusei, Candida pseudotropicalis, or Candida utilis. In a particular embodiment, the strain is Saccharomyces cerevisiae. In some embodiments, the host is a strain of Saccharomyces cerevisiae selected from the group consisting of Baker's yeast, CEN.PK, CEN.PK2, CBS 7959, CBS 7960, CBS 7961, CBS 7962, CBS 7963, CBS 7964, IZ-1904, 42^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT TA, BG-1, CR-1, SA-1, M-26, Y-904, PE-2, PE-5, VR-1, BR-1, BR-2, ME-2, VR-2, MA-3, MA-4, CAT- 1, CB-1, NR-1, BT-1, and AL-1. In some embodiments, the strain of Saccharomyces cerevisiae is CEN.PK. In some embodiments, the yeast strain used is Y21900. In some embodiments, the yeast strain used is Y23508. In some embodiments, the strain is a microbe that is suitable for industrial fermentation. In particular embodiments, the microbe is conditioned to subsist under high solvent concentration, high temperature, expanded substrate utilization, nutrient limitation, osmotic stress due to sugar and salts, acidity, sulfite and bacterial contamination, or combinations thereof, which are recognized stress conditions of the industrial fermentation environment. Examples The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Example 1. Isolation of peptide extracts from previously cultured yeast Peptide-rich extracts were obtained from waste streams 1 of ^-glucan (Gpep) and mannan (Mpep) extraction from spent yeast (Freimund et al., 2003; X. Y. Liu et al., 2008; Tian et al., 2019). The supernatants from these processes containing soluble protein were put through by 1 kDa cut-off membrane ultrafiltration in an Amicon® stirred cell model (Merck KGaA, Darmstadt, Germany) in order to get peptide rich fractions with different MW: Gpep > 1 kDa, Gpep < 1 kDa, Mpep > 1 kDa and Mpep < 1 kDa. As described in Figure 1, supernatant was submitted to ultrafiltration with a Ultracel® 1 kDa ultrafiltration discs (regenerated cellulose, 76 mm diameter) (Merck KGaA, Darmstadt, Germany) in order to concentrate the protein in retentate. Thereafter, the retentate was diafiltrated with 3 volumes of deionized water to further purify the obtained fractions. At the end of ultrafiltration process, samples were freeze-dried (Freeze-dryer Alpha 2-4 LSCbasic, Martin Christ, Osterode am Harz, Germany). Chemical characterization of peptide fractions Protein and dry weight quantification: Protein content of fractions was determined by Pierce™ BCA protein assay kit (Thermo Fisher Scientific Inc., Massachusetts, USA). The method is based on reduction of Cu2+ to Cu1+ by protein in alkaline medium, followed by Cu1+ reaction with bicinchoninic acid (BCA), which produces a purple-colored product that can be read at 562 nm after 30 min of reaction (Synergy H1, Biotek Instruments, Winooski, USA). Dry weight was determined at 105 ºC for 24 h according to standard procedures of the Association of Official Analytical Chemists (AOAC, 2005). Free and total amino acids quantification: The amino acids profile was analyzed according to Wang et al. (2016) by derivatization iodoacetic acid and o-phthaldialdehyde methodology, using 43^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT reverse phase high performance liquid chromatography with a Chromolith® Performance RP18 (4.6 x 100 mm) column (Merck KGaA, Darmstadt, Germany) for separation, and coupled to high resolution fluorescence detector (Agilent Technologies, Inc., California, USA). For determination of total amino acids, an acid hydrolysis during 20 h at 115ºC was performed at 10 mg of peptide fraction in 3 mL HCl 6M before HPLC analysis. Nitrogen was injected through an inlet needle to substitute samples atmosphere and another needle was used as gas outlet. Flasks were sealed with tape and placed 1 in an oven at 115 ºC for 20 h. Then, pH was adjusted to 3.2 and the solution diluted rigorously to a final volume of 10 mL. For quantification of free amino acids, peptide fractions were prepared in HCl 0.1M at concentration of 10 mg/mL. According to procedure of Pripis-Nicolau et al. (2001), 20 ^L of peptide fraction was derivatized and 10 ^L was injected. The analysis was done in triplicate and the amino acids quantified according to calibration curves of pure standards (aspartic acid (Asp), glutamic acid (Glu), cysteine (Cys), asparagine (Asn), serine (Ser), histidine (His), glycine (Gly), threonine (Thr), arginine (Arg), alanine (Ala), tyrosine (Tyr), valine (Val), methionine (Met), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), and lysine (Lys)) (Sigma-Aldrich, Inc., St. Louis, USA) from 1 to 30 mg/L, using norvaline (Sigma-Aldrich, Inc., St. Louis, USA) as internal standard. Minerals: Mineral content of peptide-rich extracts was measured in an optical emission spectrometer Model Optima 7000 DV ™ ICP-OES (Dual View, PerkinElmer Life and Analytical Sciences, Shelton, CT, USA) with radial configuration according to procedure of Chatelain et al. (2014). The analysis was done in triplicate and the minerals quantified according to calibration curves of a commercial mix standards for ICP analysis (Inorganic Ventures, Christiansburg, USA) (molybdenum, zinc, cadmium, phosphorus, lead, nickel, cobalt, boron, manganese, iron, magnesium, calcium, copper, aluminum, sodium, and potassium) from 0.05 to 10 mg/L. Before ICP analysis, a microwave digestion of samples (2 mL) was performed in a speedwave XPERT (Berghof Products + Instruments GmbH, Eningen, Germany) using 5 mL of Suprapur® HNO3 and 2 mL of 35% H2O2 (Merck KGaA, Darmstadt, Germany). Molecular weight distribution: The evaluation of MW peptides distribution was performed on an ultra-high-performance liquid chromatography from Bruker Elute series, coupled to an ultrahigh- resolution quadrupole−quadrupole time-of-flight (UHR−QqTOF) mass spectrometer (Impact II; Bruke Daltonik GmbH, Bremen, Germany) using an Intensity Solo 2 C18 (100 × 2.1 mm, 2.2 ^M, Bruker Daltonik GmbH, Bremen, Germany) (BRHSC18022100) set for 60ºC. The mobile phases used were 0.1% formic acid water (A) and 0.1% formic acid acetonitrile (B) at 0.250 mL/min flow rate in gradient mode: 95% A and 5% B until 14 min, 5% A and 95% B from 14 to 22 min, and 95% A and 5% 1 B until reaching the run end (25 min). Instrument was operated in MS positive mode and data was collected in the range of 150 to 2200 m/z. The selected parameters were as follows: capillary voltage, 4.5 kV; drying gas temperature, 220 °C; drying gas flow, 9.0 L/min; nebulizing gas pressure, 0.6 bar; collision radio frequency, 2000 Vpp; transfer time, 90 ^s; and pre-pulse storage, 10 ^s. Post- acquisition internal mass calibration used ESI-L Low Concentration Tuning Mix (Agilent Technologies Inc., CA, USA) delivered by a syringe pump at the start of each chromatographic analysis. 44^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT Results and discussion Nutritional analysis: The supernatants of extraction of ^-glucan (Gpep) and mannan (Mpep) processes from spent yeast processes were collected in order to produce peptide-rich extracts in a circular economy-based approach. Membrane filtration technology has been described as an important tool for separation of soluble intracellular proteins from cell lysates based on sieving and charge-based mechanisms (Vollet Marson et al., 2020). High selectively and efficiency combined with low energy consumption are the main reasons for its use in the food processing industry, being specially chosen for peptides and proteins because of gentle treatment characteristics (Mohammad et al., 2012). In fact, it is very important to preserve a peptide’s structure and physicochemical attributes, since their bioactivities are described as being highly dependent thereon (Marson, de Castro, Belleville, et al., 2020). According to size of proteins and their fractions of interest, ultrafiltration is the main pressure-driven process used because of its range of membrane MW cut-offs (Vollet Marson et al., 2020). In a first approach, an ultrafiltration process was applied to Gpep and Mpep by-products to increase the protein concentration, since the original supernatants (non-treated) amounted about to 40 and 50% of protein purity (w/w) (Gpep and Mpep, respectively). To select the ultrafiltration membrane MW cut-off, a preliminary MW evaluation was performed by size exclusion fast protein liquid chromatography-gel filtration (FPLC), where it can be observed that the majority of peptides obtained were nearby 1000 Da. After protein concentration at 1 kDa, a diafiltration was executed in order to purify the final extracts. At the end of membrane filtration, they were dried by freeze-drying. This process resulted in four protein rich extracts with different MW and nutritional characteristics: Gpep > 1 kDa, Gpep < 1 kDa, Mpep > 1 kDa and Mpep < 1 kDa (Table 1). The protein concentration in Gpep and Mpep fractions ranged from 48 to 86% (Table 1) with a protein yield from 0.4 to 2.2 g per litter of supernatant processed. Gpep waste stream allowed the higher amount of protein yield (Gpep > 1 kDa: 2.2 ± 0.5; Gpep < 1 kDa:1.8 ± 0.4 g/L supernatant) in comparison 1 with Mpep (both 0.4 ± 0.1 g/L supernatant). On the other hand, Mpep > 1 kDa had the highest protein concentration since both extracts from Gpep rounded the same protein amount (Table 1). A small sugar content was determined in all peptide extracts (3-7%) (except Gpep > 1 kDa) (Table 1), as expected, since we are working on waste streams from polysaccharides extraction. These processes usually intend to work with cell wall material which is composed of 25 to 35% of polysaccharides, where ^-glucan and mannan are located (Feldmann, 2012). They start with disintegration of this yeast structure in order to increase these polysaccharides accessibility and eliminate the protein linked to these compounds (Freimund et al., 2003; X. Y. Liu et al., 2008; Tian et al., 2019) which means that their waste streams are likely rich in protein and poor in polysaccharides content. The extract with the high sugar content (Gpep > 1 kDa) can contain some oligosaccharides, which are small fragments of saccharides polymers, that were possibly released to the supernatant during ^-glucan extraction and retained by our 1 kDa ultrafiltration because of their size. Table 1. Protein concentration (% w/w), sugars (% w/w) and minerals (ng/g extract) of peptide-rich extracts. 45^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT Results are expressed in average ± standard deviation (n=2). ND – Not detected (below low detection limit), P – Phosphorus, Mg – Magnesium, Ca – Calcium, Na – Sodium, K –Potassium. Concerning minerals concentrations, fractions < 1 kDa presented the higher concentrations (67-118 ng/g) (Table 1), in particular Mpep < 1 kDa with an excess of sodium (Na) and potassium (K), which can be related with the addition of saline solutions in some steps of the extraction process (Freimund et al., 2003). In our samples, the amino acid profiles were traced by quantification of total and free amino acids in the peptide-rich extracts. Despite tryptophan (Trp) being an EAA, the applied analytical method was not suitable for its quantification. The results of the other EAA determined are presented in Table 2, and a range from 119 to 235 mg/g protein of extract can be observed. The total EAA of Gpep >1 kDa, Gpep < 1 kDa and Mpep > 1 kDa exceeded the FAO/WHO recommendation (World Health Organization, 2007), as well as individual EAA in all fractions (except histidine, leucine, and lysine in Mpep < 1 kDa) which makes the obtained peptide-rich extracts of present study good candidates for dietary supplementation and functional foods. Table 2. Content of essential amino acids (mg/g protein) of peptide-rich extracts. Results are expressed in average ± standard deviation (n=2). a Non-essential, b World Health Organization (2007), c Phe + Tyr. EAA – Essential amino acids, NM – Not mentioned, His – Histidine, Thr – Threonine, Arg – Arginine, Ala – Alanine, Val – Valine, Phe – Phenylalanine, Ile – Isoleucine, Leu - Leucine, Lys – Lysine, Tyr - Tyrosine. Regarding the non-essential amino acids, the high amount of aspartic (18.8 to 105 mg/g protein) and glutamic acid (58.4 to 111 mg/g protein) increase the potential application of these extracts in food 46^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT market since aspartic acid is related to the synthesis of artificial sweeteners, such as aspartame (O’Mullane et al., 2014), and glutamic acid lead to strong flavor-enhancing properties. Monosodium glutamate, a salt form of glutamic acid, is one of the well-known flavor ingredients used by the food industry since it provides the typical “umami” flavor. Recently, its use has become a strategy for common salt substituent in foods as well (Maluly et al., 2017). For athletic performance, alanine supplementation has become a common practice among competitive athletes since its mechanism is involved in delaying fatigue during high-intensity exercise (Hoffman et al., 2018). The quantification of free amino acids showed that the fractions < 1 kDa had a higher amount (121 to 243 mg/g protein) than > 1 kDa (8 to 13 mg/g protein), since amino acids in their free form has low MW and easily concentrate in ultrafiltration permeate. The discovery of aspartic and glutamic acid, cysteine, histidine, and lysine in Gpep and Mpep extracts, especially in free form, shows potential for iron-binding to produce iron-peptide chelates as demonstrated by Hoz et al. (2014). These amino acids have functional groups capable of establishing coordinated covalent bonds and their considerable free amounts in fractions < 1 kDa can make them iron-delivery components to produce food supplements targeted for anti-anaemic market. Molecular weight profile: Generally, ultrafiltration membranes (1-50 kDa) are used to recover bioactive peptides and amino acids, and nanofiltration membranes (100-1000 Da) are widely used in low MW peptides purification processes by industry (Vollet Marson et al., 2020). In fact, peptides with biological activity are made up of 3-20 amino acids and their amino acid composition and sequence is responsible for their bioactivity (Amorim, Marques, et al., 2019). As mentioned above, an ultrafiltration with 1 kDa cut-off membrane was performed, aiming to increase the protein concentration of initial waste streams supernatants, being the 1 kDa cut-off membrane chosen since this initial MW profile was nearby 1000 Da. As expected, the fractions of Gpep and Mpep < 1 kDa had approximately 88 and 85% of peptides under 1000 Da, respectively, being about 50% of these under 500 Da. On other hand, the Gpep and Mpep fractions > 1 kDa presented 39% and 63% of peptides above 1 kDa (Figure 2). The unexpected large percentage of peptides under 1 kDa observed in Gpep > 1 kDa fraction (61%) may be related with the limited selectively of ultrafiltration cut-off membranes due to fouling phenomena and critical flux since they are directly related with transference phenom and protein interactions, which have been described as the main challenges of membrane filtration processes (Vollet Marson et al., 2020). Example 2. Shake flask fermentation in the presence of peptide extract from previously cultured yeast cells Materials and Methods: The strain used in this study was a Saccharomyces cerevisiae engineered to produce ^- farnesene constitutively (Beaker, Y21900). A pre-culture of this strain (inoculum preparation) was grown in 63 mL of culture medium in one of 250 mL Erlenmeyer flask containing four baffles, inoculated with two cryovial (containing 1 mL of culture volume each), and incubated at 30°C and 200 rpm in an orbital incubator for 42 hours. The standard culture medium of seed flasks and batch bioreactor fermentations was the same, containing trace metals, vitamins (according to van Hoek et al.2000), ammonium phosphate monobasic (7 g/L), potassium phosphate monobasic (1 g/L), 47^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT magnesium sulphate heptahydrate (0.5 g/L), yeast extract (5 g/L), succinate buffer at pH 5.0 (6 g/L) and sucrose (35g/L in shake-flask and 60 g/L in bioreactor fermentation). To study yeast tolerance to H^O^, an oxidative stress inducer, cells were incubated in different conditions: standard medium (Control), medium with 0.7 g/L peptides extract, medium with 2 mM H^O^ and medium with 0.7 g/L peptides extract plus 2 mM H^O^. The peptides extract was obtained according to Example 1. For shake-flask assays, pre-cultured cells were harvested and suspended in 100 mL of culture medium to an initial washed optical density (wOD600) of 0.5 in eight 1 L Erlenmeyer flasks with 4 baffles. Shake-flasks were incubated in an orbital incubator at 30 °C and 200 rpm until sucrose depletion. Samples were collected during fermentations to determine cell growth by optical density (600 nm), cell viability and reactive oxygen species (ROS) by flow cytometry, sugars concentration by HPLC-RID and farnesene concentration by GC-FID. The addition of 0.7 g/L peptides extract into the culture medium in the presence of the stress factor (0.7 g/L peptides + 2 mM H2O2 shake-flask) improved yeast growth (between 1.5 and 2 folds), sucrose consumption (between 1.2 and 1.7 folds), and farnesene production (between 1.6 and 2.8 folds), compared to culture without peptides extract (only stress factor, 2 mM H2O2 shake-flask) in both, shake-flask, and batch bioreactor fermentations. ROS reductions between 1.2 and 4-fold in shake-flask and between 1.4 and 1.65-fold in bioreactor fermentations were seen in most of the time points analyzed. Cell viability was also higher in the presence of the natural antioxidant under induced stress condition. Results and Discussion: The capacity of a peptide-rich extract with antioxidant activity obtained from previously cultured yeast to reduce oxidative stress in Saccharomyces cerevisiae fermentation was studied in shake-flasks assays using as a model the Beaker yeast strain, producer of ^-farnesene. The addition of 0.7 g/L peptides extract into the culture medium in the presence of the stress factor of 2 mM H2O2 improved yeast growth (Figure 3A), sucrose consumption (Figure 3B), and farnesene production (Figure 4), compared to culture without peptide extract having only the stress factor of 2 mM H2O2, at least until 30.5 hours of fermentation (last sampling point analyzed before sucrose depletion). Namely, during the exponential phase, cell density was 2-fold higher, and more sugar was consumed per time point (Figure 4). Farnesene concentration could be improved by 2.8-fold in middle of exponential phase (30.5h) (Figure 4). ROS generated in beginning of exponential phase (from 2 to 22.5 h) were kept in average at 67% to 36% lower levels by the presence of the natural antioxidant on stress condition (Figure 5A). Cell viability was also improved by addition of the peptide extract (Figure 5B). Although peptides extract slightly improved standard fermentation performance (no stress induced) (Figures 3 and 4), it promoted almost full fermentation recovery from induced oxidative stress, which can be attributed to the antioxidant capacity of the peptide extract. This work describes, for the first time, the use of peptide extract isolated from previously cultured yeast cells to reduce oxidative stress during a fermentation process. ^ 48^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT Example 3. Bioreactor fermentation in the presence of peptide extract from previously cultured yeast cells Materials and Methods: Farnesene fermentations using yeast strain Y21900, were started with activation of yeast from two cryovials, each with 1 mL of glycerol stock culture, in a 250 mL Erlenmeyer flask with four baffles. Two seed flasks passages with incubation at 30 °C and 200 rpm were performed to obtain the inoculum for the batch bioreactor fermentations. Cultivation time in the first and second seed flask was 42 hrs and 22 hrs, respectively. The batch fermentations were performed in a reactor with 2.7 L of working volume at a temperature of 30 °C, pH of 5.0, which was regulated by using a solution of 12.5 % ammonium hydroxide, aeration of 0.5 L/min and dissolved oxygen (DO) of at least 30 %, which was regulated by an agitation ramp ranging between 300 and 1200 rpm. Batch reactors had an initial volume of 1-L after inoculation (10% inoculum, initial wOD600 of ~1). Cultivation time was ~47 hrs. The culture base medium of seed flasks and batch bioreactor steps was the same, containing trace metals, vitamins, ammonium phosphate monobasic (7 g/L), potassium phosphate monobasic (1 g/L), magnesium sulphate heptahydrate (0.5 g/L), yeast extract (5 g/L), succinate buffer at pH 5.0 (6 g/L) and sucrose (70 g/L). In batch bioreactor medium, antifoam TERGITOL® L-81 (0.1 mL/L) was used, and peptides extract (0.7 g/L) and/or H2O2 (2 mM) were added according to selected conditions (Table 3). 49^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT Table 3 – Conditions tested in each batch bioreactor fermentation Temperature (°C), pH, DO, agitation, aeration, and off-gas data, such as CO2, O2, oxygen uptake rate (OUR) and ethanol, were monitored and recorded automatically. Whole cell broth samples were collected and prepared for the measurements of farnesene concentration (g/kg, by GC- FID), washed optical density (wOD600, by spectrophotometer), sugars (g/L, by HPLC-RID), reactive oxygen species (ROS) and cell viability (%, by flow cytometry). Results and Discussion: As in shake-flasks experiments, in batch bioreactor fermentations, the addition of 0.7 g/L peptides extract into the culture medium could also alleviate the negative effect of the stress inducer H2O2. Relevant positive effects were observed on yeast growth, sugar consumption and farnesene concentration. Namely, during the exponential phase, cell density was up to 1.5-fold higher (Figure 6A), and sucrose consumption was faster (Figure 6B). Farnesene concentration was also higher, especially at sucrose depletion (Figure 7). At this point, in the presence of 2 mM H2O2, the concentration of was only 45% of that under the control conditions (i.e., no peptide extract, no induced stress), while in presence of the peptide extract this value was raised to 70% (Figure 7). Thus, this supplement led to an improvement of product formation of up to 1.6-fold when cells were subjected to stress. These observations can be attributed to a reduction in oxidative stress promoted by the bioactive peptides, since the ROS were kept at lower levels when this natural antioxidant was present (Figure 8A). In fact, a ROS reduction of 30-40% on average was observed for most of the time points that were analyzed (0 hr, 8 hrs, 10 hrs, and 30 hrs) (Figure 8A). Cell viability was also higher in the presence of the peptide extract under induced stress conditions (Figure 8B). The range in cell viability in the presence of H2O2 was 96-89%, whereas in the presence of H2O2 plus peptide extract that range was 96-94% (Figure 8B). It is worth noting that the peptide extract per se did not interfere with the fermentation, since when it was added to the base medium, the resulting overall fermentation profile was close to is observed under standard fermentation conditions (i.e., control) (Figures 6 and 7). This means that the peptide extract, at the concentration of 0.7 g/L, did not introduce significant differences in yeast growth, sugar consumption and farnesene concentration over the course of the fermentation in the bioreactor. This confirmed that the positive effect of peptide extract observed under induced oxidative stress was based on the antioxidant properties of the peptide extract and not on an additive effect. The results from both fermentation experiments, shake-flasks (Example 2) and bioreactors (Example 3), indicated that the supplementation of Beaker fermentation with peptides recovered from industrial previously cultured cells is a promising strategy to reduce ROS generation, and thus, improve yeast tolerance to oxidative stress and product production, while contributing to waste valorization. 50^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT ^ Other Embodiments All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. Other embodiments are within the claims. 51^ ^

Claims

ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT CLAIMS 1. A method of producing a biological product, the method comprising: a) providing a population of host cells capable of producing the biological product, and b) culturing the population of host cells in a culture medium comprising a peptide extract obtained from a population of previously cultured cells. 2. A method of culturing a population of host cells, the method comprising: a) providing a population of host cells capable of producing a biological product, and b) culturing the population of host cells in a culture medium comprising a peptide extract obtained from a population of previously cultured cells. 3. A method of reducing oxidative stress during a cell culture, the method comprising culturing a population of host cells capable of producing a biological product in a culture medium comprising a peptide extract obtained from a population of previously cultured cells. 4. The method of any one of claims 1-3, wherein the peptide extract comprises proteins. 5. The method of claim 4, wherein the peptide extract has a protein concentration of between 20% (w/w) and 95% (w/w). 6. The method of claim 5, wherein the peptide extract has a protein concentration of between 45% (w/w) and 90% (w/w). 7. The method of any one of claims 4-6, wherein the proteins comprise one or more amino acid residues selected from His, Thr, Arg, Val, Phe, Tyr, Ile, Leu, and Lys. 8. The method of claim 7, wherein the proteins have a molecular weight of greater than 1 kDa. 9. The method of claim 7 or 8, wherein the proteins comprise a His residue, optionally wherein proteins comprising His are present at a concentration of between about 20 mg/g protein and 30 mg/g protein. 10. The method of any one of claims 7-9, wherein the proteins comprise a Thr residue, optionally wherein proteins comprising Thr are present at a concentration of between about 25 mg/g protein and 55 mg/g protein. 52^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT 11. The method of any one of claims 7-10, wherein the proteins comprise an Arg residue, optionally wherein proteins comprising Arg are present at a concentration of between about 25 mg/g protein and 40 mg/g protein. 12. The method of any one of claims 7-11, wherein the proteins comprise a Val residue, optionally wherein proteins comprising Val are present at a concentration of between about 30 mg/g protein and 70 mg/g protein. 13. The method of any one of claims 7-12, wherein the proteins comprise a Phe residue, optionally wherein proteins comprising Phe are present at a concentration of between about 10 mg/g protein and 35 mg/g protein. 14. The method of any one of claims 7-13, wherein the proteins comprise a Tyr residue, optionally wherein proteins comprising Tyr are present at a concentration of between about 10 mg/g protein and 35 mg/g protein. 15. The method of any one of claims 7-14, wherein the proteins comprise an Ile residue, optionally wherein proteins comprising Ile are present at a concentration of between about 20 mg/g protein and 50 mg/g protein. 16. The method of any one of claims 7-15, wherein the proteins comprise a Leu residue, optionally wherein proteins comprising Leu are present at a concentration of between about 25 mg/g protein and 60 mg/g protein. 17. The method of any one of claims 7-16, wherein the proteins comprise a Lys residue, optionally wherein proteins comprising Lys are present at a concentration of between about 45 mg/g protein and 65 mg/g protein. 18. The method of claim 7, wherein the proteins have a molecular weight of less than 1 kDa. 19. The method of claim 7 or 18, wherein the proteins comprise a His residue, optionally wherein proteins comprising His are present at a concentration of between about 10 mg/g protein and 25 mg/g protein. 20. The method of any one of claims 7, 18, and 19, wherein the proteins comprise a Thr residue, optionally wherein proteins comprising Thr are present at a concentration of between about 10 mg/g protein and 30 mg/g protein. 53^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT 21. The method of any one of claims 7 and 18-20, wherein the proteins comprise an Arg residue, optionally wherein proteins comprising Arg are present at a concentration of between about 10 mg/g protein and 20 mg/g protein. 22. The method of any one of claims 7 and 18-21, wherein the proteins comprise a Val residue, optionally wherein proteins comprising Val are present at a concentration of between about 40 mg/g protein and 65 mg/g protein. 23. The method of any one of claims 7 and 18-22, wherein the proteins comprise a Phe residue, optionally wherein proteins comprising Phe are present at a concentration of between about 5 mg/g protein and 25 mg/g protein. 24. The method of any one of claims 7 and 18-23, wherein the proteins comprise a Tyr residue, optionally wherein proteins comprising Tyr are present at a concentration of between about 10 mg/g protein and 30 mg/g protein. 25. The method of any one of claims 7 and 18-24, wherein the proteins comprise an Ile residue, optionally wherein proteins comprising Ile are present at a concentration of between about 10 mg/g protein and 35 mg/g protein. 26. The method of any one of claims 7 and 18-25, wherein the proteins comprise a Leu residue, optionally wherein proteins comprising Leu are present at a concentration of between about 15 mg/g protein and 45 mg/g protein. 27. The method of any one of claims 7 and 18-26, wherein the proteins comprise a Lys residue, optionally wherein proteins comprising Lys are present at a concentration of between about 15 mg/g protein and 45 mg/g protein. 28. The method of any one of claims 1-27, wherein the peptide extract comprises polysaccharides. 29. The method of claim 28, wherein the peptide extract has a polysaccharide concentration of between 1% (w/w) and 50% (w/w). 30. The method of claim 29, wherein the polysaccharide concentration is between 2% and 35% of the peptide extract. 31. The method of any one of claims 1-30, wherein the peptide extract comprises minerals. 54^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT 32. The method of claim 31, wherein the minerals are selected from one or more of phosphorus, magnesium, calcium, sodium, and potassium. 33. The method of claim 31 or 32, wherein peptide extract has a mineral concentration of between 5 and 150 ng/g of the peptide extract. 34. The method of any one of claims 1-33, wherein the concentration of the peptide extract in the culture medium is from about 0.1 g/L to about 1 g/L. 35. The method of claim 34, wherein the concentration of the peptide extract in the culture medium is from about 0.5 g/L to about 0.7 g/L. 36. The method of claim 35, wherein the concentration of peptide extract in the culture medium is about 0.7 g/L. 37. The method of any one of claims 1-36, wherein the peptide extract is isolated from a population of previously cultured cells capable of producing a biological product. 38. The method of claim 37, wherein the peptide extract is isolated from the previously cultured cells by lysing and centrifuging the previously cultured cells, thereby resulting in a supernatant, and subsequently obtaining the peptide extract from the supernatant. 39. The method of claim 38, wherein the peptide extract is isolated from the supernatant by way of filtration. 40. The method of claim 39, wherein the filtration is selected from one or more of membrane filtration, fast protein liquid chromatograph-gel filtration diafiltration, and ultrafiltration. 41. The method of claim 40, wherein the filtration is membrane filtration. 42. The method of any one of claims 38-41, wherein the peptide extract has a yield of 0.4 to 2.2 g/liter in the supernatant. 43. The method of any one of claims 1-42, wherein the previously cultured cells have an optical density of from about 20 to about 150 immediately prior to obtaining the peptide extract and culturing it with the population of host cells, optionally wherein the previously cultured cells have an optical density of from 55^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT about 20 to about 50 immediately prior to obtaining the peptide extract and culturing it with the population of host cells. 44. The method of claim 43, wherein the previously cultured cells have an optical density of about 50 immediately prior to obtaining the peptide extract and culturing it with the population of host cells. 45. The method of any one of claims 1-44, wherein after culturing the host cells for from about 2 hours to about 50 hours, the method results in an increase in host cell density of at least 1.1-fold in comparison to a reference method in which the peptide extract is not present in the culture medium. 46. The method of claim 45, wherein after culturing the host cells for from about 2 hours to about 50 hours, the method results in an increase in host cell density of from about 1.1-fold to about 5-fold in comparison to a reference method in which the peptide extract is not present in the culture medium. 47. The method of any one of claims 1-46, wherein after culturing the host cells for from about 2 hours to about 50 hours, the method results in a decrease in the formation of reactive oxygen species by at least 10% in comparison to a reference method in which the peptide extract is not present in the culture medium. 48. The method of claim 47, wherein after culturing the host cells for from about 2 hours to about 50 hours, the method results in a decrease in the in the formation of reactive oxygen species by from about 10% to about 90% in comparison to a reference method in which the peptide extract is not present in the culture medium. 49. The method of claim 48, wherein after culturing the host cells for from about 2 hours to about 50 hours, the method results in a decrease in the formation of reactive oxygen species by from about 25% to about 75% in comparison to a reference method in which the peptide extract is not present in the culture medium. 50. The method of any one of claims 1-49, wherein after culturing the host cells for from about 2 hours to about 50 hours, the method results in an increase in sugar consumption of at least 1.1-fold in comparison to a reference method in which the peptide extract is not present in the culture medium. 51. The method of any one of claims 1-50, wherein after culturing the host cells for from about 2 hours to about 50 hours, the method results in an increase in the concentration of the biological product in comparison to a reference method in which the peptide extract is not present in the culture medium. 56^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT 52. The method of claim 51, wherein after culturing the host cells for from about 2 hours to about 50 hours, the method results in an increase in the concentration of the biological product of from about 1.1- fold to about 10-fold in comparison to a reference method in which the peptide extract is not present in the culture medium. 53. The method of claim 52, wherein after culturing the host cells for from about 2 hours to about 50 hours, the method results in an increase in the concentration of the biological product of from about 1.1- fold to about 5-fold in comparison to a reference method in which the peptide extract is not present in the culture medium. 54. The method of any one of claims 1-53, wherein the previously cultured cells are cells that: i) have been previously cultured for at least 10 hours; and/or ii) were previously capable of producing a biological product, but as a result of having previously been cultured, no longer produce a significant quantity of said biological product. 55. The method of claim 54, wherein the previously cultured cells have been previously cultured for from about 10 hours to about 100 hours. 56. The method of any one of claims 1-55, wherein the previously cultured cells were previously capable of producing a biological product, but as a result of having previously been cultured, no longer produce a significant quantity of said biological product. 57. The method of any one of claims 1-56, wherein the previously cultured cells are yeast cells. 58. The method of claim 57, wherein the yeast cells are Saccharomyces sp. cells or Kluveromyces sp. cells. 59. The method of claim 58, wherein the yeast cells are Saccharomyces cerevisiae cells. 60. The method of claim 58, wherein the yeast cells are Kluveromyces marxianus cells. 61. The method of claim any one of claims 1-60, wherein the host cells are genetically modified host cells. 62. The method of any one of claims 1-61, wherein the host cells are yeast cells. 57^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT 63. The method of claim 62, wherein the yeast cells are Saccharomyces sp. cells or Kluveromyces sp. cells. 64. The method of claim 63, wherein the yeast cells are Saccharomyces cerevisiae cells. 65. The method of claim 63, wherein the yeast cells are Kluveromyces marxianus cells. 66. The method of any one of claims 1-65, wherein the population of host cells is cultured for at least 10 hours. 67. The method of claim 66, wherein the population of host cells is cultured for from about 10 hours to about 100 hours. 68. The method of any one of claims 1-67, wherein the host cells are cultured at a temperature of from about 20 oC to about 40 oC. 69. The method of claim 68, wherein the host cells are cultured at a temperature of about 30 oC. 70. The method of any one of claims 1-69, wherein the biological product is a fermentation product. 71. The method of any one of claims 1-70, wherein the biological product is an isoprene. 72. The method of any one of claims 1-70, wherein the biological product is an isoprenoid. 73. The method of any one of claims 1-70, wherein the biological product is a steviol glycoside. 74. The method of any one of claims 1-70, wherein the biological product is a human milk oligosaccharide. 75. The method of any one of claims 1-70, wherein the biological product is a cannabinoid. 76. A composition produced by the methods of any one of claims 1-75. 77. A composition comprising: (i) a population of host cells capable of producing a biological product; and (ii) a culture medium comprising a peptide extract obtained from a population of previously cultured cells. 58^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT 78. The composition of claim 76 or 77, wherein the peptide extract comprises proteins. 79. The composition of claim 78, wherein the peptide extract has a protein concentration of between 20% (w/w) and 95% (w/w). 80. The composition of claim 79, wherein the peptide extract has a protein concentration of between 45% (w/w) and 90% (w/w). 81. The composition of any one of claims 78-80, wherein the proteins comprise one or more amino acid residues selected from His, Thr, Arg, Val, Phe, Tyr, Ile, Leu, and Lys. 82. The composition of claim 81, wherein the proteins have a molecular weight of greater than 1 kDa. 83. The composition of claim 81 or 82, wherein the proteins comprise a His residue, optionally wherein proteins comprising His are present at a concentration of between about 20 mg/g protein and 30 mg/g protein. 84. The composition of any one of claims 81-83, wherein the proteins comprise a Thr residue, optionally wherein proteins comprising Thr are present at a concentration of between about 25 mg/g protein and 55 mg/g protein. 85. The composition of any one of claims 81-84, wherein the proteins comprise an Arg residue, optionally wherein proteins comprising Arg are present at a concentration of between about 25 mg/g protein and 40 mg/g protein. 86. The composition of any one of claims 81-85, wherein the proteins comprise a Val residue, optionally wherein proteins comprising Val are present at a concentration of between about 30 mg/g protein and 70 mg/g protein. 87. The composition of any one of claims 81-86, wherein the proteins comprise a Phe residue, optionally wherein proteins comprising Phe are present at a concentration of between about 10 mg/g protein and 35 mg/g protein. 88. The composition of any one of claims 81-87, wherein the proteins comprise a Tyr residue, optionally wherein proteins comprising Tyr are present at a concentration of between about 10 mg/g protein and 35 mg/g protein. 59^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT 89. The composition of any one of claims 81-88, wherein the proteins comprise an Ile residue, optionally wherein proteins comprising Ile are present at a concentration of between about 20 mg/g protein and 50 mg/g protein. 90. The composition of any one of claims 81-89, wherein the proteins comprise a Leu residue, optionally wherein proteins comprising Leu are at a concentration of between about 25 mg/g protein and 60 mg/g protein. 91. The composition of any one of claims 81-90, wherein the proteins comprise a Lys residue, optionally wherein proteins comprising Lys are present at a concentration of between about 45 mg/g protein and 65 mg/g protein. 92. The composition of claim 81, wherein the proteins have a molecular weight of less than 1 kDa. 93. The composition of claim 81 or 92, wherein the proteins comprise a His residue, optionally wherein proteins comprising His are present at a concentration of between about 10 mg/g protein and 25 mg/g protein. 94. The composition of any one of claims 81, 92, and 93, wherein the proteins comprise a Thr residue, optionally wherein proteins comprising Thr are present at a concentration of between about 10 mg/g protein and 30 mg/g protein. 95. The composition of any one of claims 81 and 92-94, wherein the proteins comprise an Arg residue, optionally wherein proteins comprising Arg are present at a concentration of between about 10 mg/g protein and 20 mg/g protein. 96. The composition of any one of claims 81 and 92-95, wherein the proteins comprise a Val residue, optionally wherein proteins comprising Val are present at a concentration of between about 15 mg/g protein and 55 mg/g protein. 97. The composition of any one of claims 81 and 92-96, wherein the proteins comprise a Phe residue, optionally wherein proteins comprising Phe are present at a concentration of between about 5 mg/g protein and 25 mg/g protein. 60^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT 98. The composition of any one of claims 81 and 92-97, wherein the proteins comprise a Tyr residue, optionally wherein proteins comprising Tyr are present at a concentration of between about 10 mg/g protein and 30 mg/g protein. 99. The composition of any one of claims 81 and 92-98, wherein the proteins comprise an Ile residue, optionally wherein proteins comprising Ile are present at a concentration of between about 10 mg/g protein and 35 mg/g protein. 100. The composition of any one of claims 81 and 92-99, wherein the proteins comprise a Leu residue, optionally wherein proteins comprising Leu are present at a concentration of between about 15 mg/g protein and 45 mg/g protein. 101. The composition of any one of claims 81 and 92-100, wherein the proteins comprise a Lys residue, optionally wherein proteins comprising Lys are present at a concentration of between about 15 mg/g protein and 45 mg/g protein. 102. The composition of any one of claims 76-101, wherein the peptide extract comprises polysaccharides. 103. The composition of claim 102, wherein the peptide extract has a polysaccharide concentration of between 1% (w/w) and 50% (w/w). 104. The composition of claim 103, wherein the polysaccharide concentration is between 2% and 35% of the peptide extract 105. The composition of any one of claims 76-104, wherein the peptide extract comprises minerals. 106. The composition of claim 105, where the minerals are selected from one or more of phosphorus, magnesium, calcium, sodium, and potassium. 107. The composition of claim 105 or 106, wherein peptide extract has a mineral concentration of between 5 and 150 ng/g of the peptide extract. 108. The composition of any one of claims 76-107, wherein the concentration of the peptide extract in the culture medium is from about 0.1 g/L to about 1 g/L. 61^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT 109. The method of claim 108, wherein the concentration of the peptide extract in the culture medium is from about 0.5 g/L to about 0.7 g/L. 110. The method of claim 109, wherein the concentration of peptide extract in the culture medium is about 0.7 g/L. 111. The composition of any one of claims 76-110, wherein the peptide extract is isolated from a population of previously cultured capable of producing a biological product. 112. The composition of claim 111, wherein the peptide extract is isolated from the previously cultured cells by centrifuging the previously cultured cells, thereby resulting in a supernatant, and subsequently obtaining the peptide extract from the supernatant. 113. The composition of claim 112, wherein the peptide extract is isolated from the supernatant by way of filtration 114. The composition of claim 113, wherein the filtration is selected from one or more of membrane filtration, fast protein liquid chromatograph-gel filtration diafiltration, and ultrafiltration. 115. The composition of claim 114, wherein the filtration is membrane filtration 116. The composition of any one of claims 112-115, wherein the peptide extract has a yield of 0.4 to 2.2 g/liter in the supernatant. 117. The composition of any one of claims 76-116, wherein the previously cultured cells have an optical density of from about 20 to about 150 immediately prior to obtaining the peptide extract and culturing it with the population of host cells, optionally wherein the previously cultured cells have an optical density of from about 20 to about 50 immediately prior to obtaining the peptide extract and culturing it with the population of host cells. 118. The composition of claim 117, wherein the previously cultured cells have an optical density of about 50 immediately prior to obtaining the peptide extract and culturing it with the population of host cells. 119. The composition of any one of claims 76-118, wherein the previously cultured cells are cells that: i) have been previously cultured for at least 10 hours; and/or ii) were previously capable of producing a biological product, but as a result of having previously been cultured, no longer produce a significant quantity of said biological product. 62^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT 120. The composition of any one of claims 76-119, wherein the previously cultured cells are cells that have been previously cultured for at least 10 hours. 121. The composition of claim 120, wherein the previously cultured cells are cells that have been previously cultured for from about 10 hours to about 100 hours. 122. The composition of any one of claims 76-121, wherein the previously cultured cells were previously capable of producing a biological product, but as a result of having previously been cultured, no longer produce a significant quantity of said biological product. 123. The composition of any one of claims 76-122, wherein the previously cultured cells are yeast cells. 124. The composition of claim 123, wherein the yeast cells are Saccharomyces sp. cells or Kluveromyces sp. cells. 125. The composition of claim 124, wherein the yeast cells are Saccharomyces cerevisiae cells. 126. The composition of claim 124, wherein the yeast cells are Kluveromyces marxianus cells. 127. The composition of claim any one of claims 76-126, wherein the host cells are genetically modified host cells. 128. The composition of any one of claims 76-127, wherein the host cells are yeast cells. 129. The composition of claim 128, wherein the yeast cells are Saccharomyces sp. cells or Kluveromyces sp. cells. 130. The composition of claim 129, wherein the yeast cells are Saccharomyces cerevisiae cells. 131. The composition of claim 129, wherein the yeast cells are Kluveromyces marxianus cells. 132. The composition of any one of claims 76-131, wherein the biological product is a fermentation product. 133. The composition of any one of claims 76-132, wherein the biological product is an isoprene. 63^ ^ ATTORNEY DOCKET: 51494-025WO2 AMYRIS REFERENCE NO.: PT-800 PCT 134. The composition of any one of claims 76-132, wherein the biological product is an isoprenoid. 135. The composition of any one of claims 55-132, wherein the biological product is a steviol glycoside. 136. The composition of any one of claims 55-132, wherein the biological product is a human milk oligosaccharide. 137. The composition of any one of claims 55-132, wherein the biological product is a cannabinoid. 64^ ^
EP23822111.3A 2022-12-02 2023-12-01 Compositions and methods for using previously cultured cells Pending EP4627098A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263429893P 2022-12-02 2022-12-02
PCT/IB2023/062144 WO2024116153A1 (en) 2022-12-02 2023-12-01 Compositions and methods for using previously cultured cells

Publications (1)

Publication Number Publication Date
EP4627098A1 true EP4627098A1 (en) 2025-10-08

Family

ID=89190860

Family Applications (1)

Application Number Title Priority Date Filing Date
EP23822111.3A Pending EP4627098A1 (en) 2022-12-02 2023-12-01 Compositions and methods for using previously cultured cells

Country Status (2)

Country Link
EP (1) EP4627098A1 (en)
WO (1) WO2024116153A1 (en)

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7192751B2 (en) 2001-12-06 2007-03-20 The Regents Of The University Of California Biosynthesis of amorpha-4,11-diene
MX318543B (en) * 2008-07-02 2014-03-18 Danisco Us Inc Compositions and methods for producing isoprene free of c5 hydrocarbons under decoupling conditions and/or safe operating ranges.
US8522016B2 (en) 2010-06-18 2013-08-27 Axis Technology Software, LLC On-the-fly data masking
CN108396044A (en) 2011-08-08 2018-08-14 埃沃尔瓦公司 The recombinant production of steviol glycoside class
US10000783B2 (en) 2012-03-16 2018-06-19 Suntory Holdings Limited Steviol glucosyltransferases and genes encoding the same
KR101487058B1 (en) * 2012-05-14 2015-01-28 지에스칼텍스 주식회사 Method of producing biochemicals using hydrolyzed fermentation waste
US9752174B2 (en) 2013-05-28 2017-09-05 Purecircle Sdn Bhd High-purity steviol glycosides
EP3083958B1 (en) 2013-12-19 2019-04-17 Amyris, Inc. Methods for genomic integration
AU2015314251A1 (en) 2014-09-09 2017-03-16 Evolva Sa Production of steviol glycosides in recombinant hosts
AU2017308143B2 (en) 2016-08-12 2022-04-07 Amyris, Inc. UDP-dependent glycosyltransferase for high efficiency production of rebaudiosides
CN110914416B (en) 2017-04-27 2023-07-21 加州大学董事会 Microorganisms and methods for producing cannabinoids and cannabinoid derivatives

Also Published As

Publication number Publication date
WO2024116153A1 (en) 2024-06-06

Similar Documents

Publication Publication Date Title
US12371677B2 (en) UDP-dependent glycosyltransferase for high efficiency production of rebaudiosides
JP5580488B2 (en) Methods for generating terpene synthase variants
AU2007267033B2 (en) Production of isoprenoids
MX2007000973A (en) Genetically modified host cells and use of same for producing isoprenoid compounds.
CN112771169B (en) Process for recovering water-immiscible isoprenoid compounds from microbial biomass
EP3665287B1 (en) Pisum sativum kaurene oxidase for high efficiency production of rebaudiosides
EP3574105B1 (en) Co-production of a sesquiterpene and a carotenoid
EP4627098A1 (en) Compositions and methods for using previously cultured cells
CN115697077A (en) Rebaudioside M sweetener compositions
CA3114953A1 (en) Stevia rebaudiana kaurenoic acid hydroxylase variants for high efficiency production of rebaudiosides
WO2024042486A1 (en) Compositions and methods for the production of polyurethanes
EP4646426A1 (en) Host cells capable of producing sequiterpenoids and methods of use thereof
EP4649142A1 (en) Production of canthaxanthin
HK40017319B (en) Co-production of a sesquiterpene and a carotenoid
HK40017319A (en) Co-production of a sesquiterpene and a carotenoid
HK40007834B (en) Udp-dependent glycosyltransferase for high efficiency production of rebaudiosides

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20250701

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC ME MK MT NL NO PL PT RO RS SE SI SK SM TR

19U Interruption of proceedings before grant

Effective date: 20250702

19W Proceedings resumed before grant after interruption of proceedings

Effective date: 20260302

RAP3 Party data changed (applicant data changed or rights of an application transferred)

Owner name: BIORBIS, LDA

Owner name: UNIVERSIDADE CATOLICA PORTUGUESA