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US-20260125718-A1 - GENETICALLY MODIFIED STRAIN OF AUREOBASIDIUM PULLULANS AND USE THEREOF FOR PRODUCING BIOPRODUCT IN HIGH YIELD

US20260125718A1US 20260125718 A1US20260125718 A1US 20260125718A1US-20260125718-A1

Abstract

Provided are genetically modified strains of Aureobasidium pullulans that can produce a bioproduct with increased productivity and a method of producing a bioproduct using the same, wherein the genetically modified strain of Aureobasidium pullulans comprises an overexpression of a functional VHb protein and an overexpression of a functional PacC protein and is grown under conditions required to support the production of the bioproduct to collect the bioproduct in a high yield.

Inventors

  • Lauren Ann Riley DRUMM
  • Andrew John Borchert
  • Julia Rae Friedman

Assignees

  • SAFION RENEWABLES INC.

Dates

Publication Date
20260507
Application Date
20250828

Claims (20)

  1. 1 . A method of producing a bioproduct comprising: growing a genetically modified strain of Aureobasidium pullulans under conditions required to support the production of the bioproduct, wherein the genetically modified strain of Aureobasidium pullulans comprises an overexpression of a functional VHb protein having at least 90% sequence homology with the sequence set forth in SEQ ID NO. 1, and an overexpression of a functional PacC protein; and collecting the bioproduct from the genetically modified strain of Aureobasidium pullulans.
  2. 2 . The method of claim 1 , wherein the genetically modified strain of Aureobasidium pullulans further comprises an overexpression of pullulanase.
  3. 3 . The method of claim 1 , wherein the genetically modified strain of Aureobasidium pullulans is derived from Aureobasidium pullulans NRRL 50384 Δpks::ura3.
  4. 4 . The method of claim 1 , wherein the genetically modified strain of Aureobasidium pullulans is modified by a serine phage recombinase enzyme ΦC31.
  5. 5 . The method of claim 1 , wherein the genetically modified strain of Aureobasidium pullulans lacks a cellular byproduct comprising poly-malic acid (PMA), pullulan, or a combination thereof.
  6. 6 . The method of claim 1 , wherein the genetically modified strain of Aureobasidium pullulans lacks function of a UDPG-pyrophosphorylase enzyme.
  7. 7 . The method of claim 1 , wherein the bioproduct is a biosurfactant comprising glycolipids, liamocins, aglycone oligo-dihydroxydecanoic acids (DDA), rhamnolipin, or a combination thereof.
  8. 8 . The method of claim 7 , wherein the bioproduct comprises of 3,5-dihydroxydecanoyl and/or 5-hydroxy-2-decenoyl esters of arabitol and mannitol.
  9. 9 . The method of claim 7 , wherein the genetically modified strain of Aureobasidium pullulans is grown in a lipid production medium comprising glucose, lignocellulosic hydrolysate, corn steep liquor, fructose, high fructose corn syrup, waste sugars, sucrose, xylose, arabinose, syrup from corn grind ethanol processes, or a combination thereof.
  10. 10 . The method of claim 1 , wherein the genetically modified strain of Aureobasidium pullulans is grown by agitation at 180 RPM for seven days at a temperature of 30° C.
  11. 11 . The method of claim 7 , wherein the bioproduct comprises a single head group comprising mannitol, arabitol, xylitol, threitol, sorbitol, galactitol, or glycerol.
  12. 12 . The method of claim 7 , wherein the bioproduct is collected by mechanical cell disruption, centrifugation, solvent extraction, supercritical fluid extraction, or a combination thereof.
  13. 13 . The method of claim 12 , wherein the bioproduct is collected by the solvent extraction, and wherein the solvent comprises an organic chemical solvent selected from the group consisting of chloroform, methanol, butanol, isopropanol, and a combination thereof, or a non-polar solvent selected from the group consisting of hexane, toluene, petroleum ether, methyl ethyl ketone (MEK), acetonitrile, ethyl acetate, and a combination thereof, or a supercritical solvent.
  14. 14 . The method of claim 1 , wherein the genetically modified strain of Aureobasidium pullulans is grown by batch fermentation, fed-batch fermentation, continuous fermentation, repeated fed-batch fermentation, or a combination thereof.
  15. 15 . The method of claim 14 , wherein the genetically modified strain of Aureobasidium pullulans is grown in a bioprocessing system, a stirred tank bioreactor, a jet loop reactor, an air lift reactor, or a combination thereof.
  16. 16 . The method of claim 1 , wherein the genetically modified strain of Aureobasidium pullulans is grown by fermentation in an acidic growth medium.
  17. 17 . A composition comprising a strain of Aureobasidium pullulans , wherein the strain comprises: an overabundance of a functional VHb protein having at least 90% sequence homology with the sequence set forth in SEQ ID NO: 1; and an overabundance of a functional PacC protein.
  18. 18 . The composition of claim 17 , wherein the strain of Aureobasidium pullulans is a genetically modified strain of Aureobasidium pullulans NRRL 50384 Δpks::ura3.
  19. 19 . The composition of claim 17 , wherein the strain of Aureobasidium pullulans produces a heavy oil with titers exceeding 30 g/L.
  20. 20 . The composition of claim 17 , wherein the strain of Aureobasidium pullulans lacks function of a UDPG-pyrophosphorylase enzyme.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims the benefit of U.S. Provisional Patent Application No. 63/689,130 filed Aug. 30, 2024, which is incorporated herein by reference in its entirety. REFERENCE TO A SEQUENCE LISTING XML The instant application contains a Sequence Listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML file, created on Aug. 27, 2025, is named 2025-08-28_Sequence-Listing_19995.0001USU1.xml and is 47,700 bytes in size. FIELD OF THE DISCLOSURE The present disclosure generally relates to methods and compositions of Aureobasidium pullulans strains for producing industrial-levels of renewable bioproducts. Particularly, the present disclosure relates to a genetically modified strains of Aureobasidium pullulans that can produce a bioproduct with increased productivity, yields, and titers, and a method of producing the bioproduct using the same. BACKGROUND OF THE DISCLOSURE Sustainable biotechnology has seen an increase in investment and development as natural resources continue to be depleted and environmentally harmful chemical processes are unsustainable long-term. Microorganisms are often utilized as biocatalysts for manufacturing sustainable products for applications in a variety of industries such as pharmaceuticals, energy, raw materials, pollution prevention, and so forth. Species of a black, yeast-like fungi (belonging to the Aureobasidium genus), have shown promise as scalable, genetically tractable microorganisms with versatile metabolic networks that can be easily modified for producing a plethora of natural products including pullulan, β-glycan, polymalate, melanin, polyol lipids, and aglycone oligo-dihydroxydecanoic acids, which have broad commercial applications within cosmetics, healthcare, chemicals, and fuels industries. Bioproducts produced by Aureobasidium pullulans strains include mixtures of extracellular polyol lipids (amphiphilic glycolipids comprising a sugar alcohol head group and ester-linked fatty acid tails) and aglycone oligo-dihydroxydecanoic acids (lacking a sugar alcohol head group), or water-insoluble “heavy oils”, that possess renewable applications as antimicrobials, biosurfactants, coconut oil substitutes, and as substrates for low carbon intensity biofuels, cosmetics, flavoring chemicals, and healthcare products. The polyol lipids within heavy oils typically are composed of a mannitol head group bound via ester linkages to between three to five decanoic acid groups. The mannitol headgroup is synthesized from the central metabolite, fructose-6-phosphate via the combined action of mannitol-1-phosphatase and mannitol-1-phosphate dehydrogenase enzymes. The decanoic acid group is created by a highly reduced polyketide synthase (HR-PKS) combining the metabolites acetyl-CoA and malonyl-CoA. Three to five dihydroxydecanoic acid tails can undergo esterification to form the aglycone oligo-dihydroxydecanoic acids. To produce the polyol lipid fraction of heavy oil, a mannitol headgroup and dihydroxydecanoic acid tail are assembled by an esterase enzyme. The heavy oil is secreted from the cell and is capable of spontaneously phase separating from the aqueous growth media. But complex eukaryotes such as Aureobasidium pullulans are not solely dedicated to producing a single bioproduct, as heavy oil must compete for carbon and energy resources with other biological processes, lowering the potential titers, rates, and yields of heavy oils during fermentation. Previous methods for optimizing the production of heavy oils have included alterations of growth medium, fermentation conditions, and the introduction of an induction medium. One earlier work related to heavy oil production is described in U.S. Pat. No. 11,352,633, which describes an Aureobasidium pullulans recombinant strain with high-yield heavy oil that was obtained by knocking out a pullulan synthetase gene while overexpressing an ATP-citrate lyase gene. Another work of genetically modifying Aureobasidium pullulans removes a functional polyketide-synthase (PKS4) enzyme for omitting the byproduct melanin from produced bioproducts. This genetic alteration reduces the cost of processing the produced bioproducts such as heavy oils and pullulan, but the production of heavy oils is limited to no more than 22 g/L at maximum titer. SUMMARY OF THE DISCLOSURE Various details of the present disclosure are hereinafter summarized to provide a basic understanding. This summary is not an exhaustive overview of the disclosure and is neither intended to identify certain elements of the disclosure, nor to delineate the scope thereof. Rather, the primary purpose of this summary is to present some concepts of the disclosure in a simplified form prior to the more detailed description that is presented hereinafter. To improve the heavy oil production on a commercial level, the present disclosure provides a genetically modified strain of A