Methods and strains for producing bioproducts in Aureobasidium pullulans
Inventors
Skory, Christopher D. • Leathers, Timothy D. • Price, Neil P.
Assignees
US Department of Agriculture USDA
Publication Number
US-10612063-B2
Publication Date
2020-04-07
Expiration Date
2036-07-18
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Abstract
The present disclosure provides methods for producing bioproducts from novel genetically altered strains of Aureobasidium pullulans. Methods and materials for the construction of these strains, examination of the bioproducts and analysis and isolation of the bioproducts from genetically altered strains is provided. Genetically altered A. pullulans strains in which one or more genes encoding biosynthetic enzymes are knocked out is detailed and the benefits of using such strains described.
Core Innovation
The invention relates to genetically modified strains of Aureobasidium pullulans capable of producing desirable bioproducts. By genetically altering these strains through targeted disruption of one or more genes encoding biosynthetic enzymes, strains have been created that no longer produce bio-contaminants such as melanin and melanin-related pigments. These pigments add significant cost to bioproduct production due to the need for their removal prior to further processing.
The background section identifies a problem wherein most strains of A. pullulans produce dark melanin-associated pigments contaminating the final desired bioproduct. Removing these pigments requires costly and complex purification steps which also result in loss of desired products. The invention addresses this problem by providing methods and strains of A. pullulans genetically modified to lack production of melanin or other undesired products, thereby producing bioproducts such as liamocins, pullulan, and massoia lactone directly in a substantially melanin-free form.
The disclosure details construction of genetically altered A. pullulans strains deficient in key biosynthetic genes such as mannitol-1-phosphate dehydrogenase (MPD1), mannitol dehydrogenase (MDH2), and polyketide synthase (PKS4). These deletions shift metabolic pathways favoring production of desired compounds like arabitol-liamocins and massoia lactone while eliminating melanin synthesis. The invention also provides methods of cultivating these strains under specific conditions to optimize production and purification of the bioproducts.
Claims Coverage
The patent includes two independent method claims focusing on the production of arabitol-liamocins and other bioproducts by genetically modified Aureobasidium pullulans strains.
Method of producing arabitol-liamocins using a strain lacking functional MPD1 gene
Growing a culture of Aureobasidium pullulans that lacks a functional mannitol-1-phosphate-dehydrogenase (MPD1) gene under conditions sufficient to support production of arabitol-liamocins, notably in media substantially free of arabitol, and collecting the arabitol-liamocins produced.
Method of producing one or more bioproducts using strains lacking functional MDH2 and MPD1 genes
Growing a culture of Aureobasidium pullulans lacking both a functional mannitol-dehydrogenase (MDH2) gene and a functional MPD1 gene under conditions sufficient to support production of bioproducts such as liamocins and exophilins, and collecting said bioproducts. The growth medium may contain glucose or fructose as the sole carbon source, and the targeted bioproducts include various liamocins with specific carbohydrate head groups and exophilins.
The independent claims cover methods using genetically altered A. pullulans strains deficient in MPD1 alone or both MPD1 and MDH2 genes to selectively produce valuable bioproducts like arabitol-liamocins, liamocins, and exophilins under specific culture conditions, thereby shaping the biosynthetic output by genetic modification and cultivation parameters.
Stated Advantages
Melanin-free bioproducts that are easier and cheaper to purify, reducing production costs.
Production of desired bioproducts on cheaper substrates, improving economic feasibility.
More ecologically responsible production methods for various industries including food, pharmaceutical, industrial, and biofuel.
Genetically modified strains produce target bioproducts preferentially and reduce bio-contaminant synthesis.
Documented Applications
Production of bioproducts such as liamocins with specific polyol head groups (mannitol, arabitol, fructose, and others).
Production of melanin-free pullulan for food, pharmaceutical, and cosmetic applications.
Production of massoia lactone as a natural, renewable alternative to extraction from Massoia tree bark.
Production of poly(β-malic acid), β-glucan, aureobasidin, intracellular fatty acids, triacylglycerols, and exophilins.
Potential applications in food preservatives, veterinary treatments, antifouling agents, phytopathogen control, biofuels, and industrial enzymes.
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