Microbial approach for the production of long chain compounds
Inventors
Westpheling, Janet • Guss, Adam M. • Riley, Lauren A.
Assignees
University of Georgia Research Foundation Inc UGARF • UT Battelle LLC • University of Tennessee Research Foundation
Publication Number
US-12325859-B2
Publication Date
2025-06-10
Expiration Date
2042-08-09
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Abstract
This disclosure describes recombinant Megasphaera microbes designed to include increased consumption of acetate, increased carbon flux to butyryl-CoA and/or hexanoyl-CoA, increased production of butyrate and/or hexanoate, or a combination thereof, than a comparable control. This disclosure also describes methods that generally include growing such recombinant microbes under conditions effective for the recombinant microbes to consume greater amounts of acetate, produce increased amounts of butyryl-CoA and/or hexanoyl-CoA, produce increased amounts of butyrate and/or hexanoate, or a combination thereof.
Core Innovation
The invention is directed to recombinant Megasphaera microbes that have been genetically engineered to increase the consumption of acetate, enhance carbon flux to butyryl-CoA and/or hexanoyl-CoA, and elevate the production of butyrate and/or hexanoate compared to a comparable control microbe. These modifications can involve mutations, such as deletions, in coding regions for specific enzymes including CoA-transferases, glyoxalases, or lyases, which effectively redirect the metabolic pathways within the microbe for greater yield of desired organic acids.
This invention addresses several limitations in the current biofuel industry, particularly the need to produce longer-chain alcohols such as butanol and hexanol more efficiently and with a lower carbon footprint. Conventional methods rely heavily on organisms like E. coli, which have proven inadequate for industrial scale production due to limited flux in heterologous pathways. In contrast, Megasphaera species, especially M. elsdenii, possess a native capacity for high-flux fatty acid biosynthesis but have historically been genetically intractable.
Advanced genetic tools and metabolic engineering strategies described in this disclosure enable targeted and efficient manipulation of Megasphaera's genome, overcoming previous barriers to genetic modification. Methods include the use of counter-selectable markers such as upp and pyrF for flexible genome editing. The engineered microbes and associated methods thus open new possibilities for the sustainable biosynthesis of industrially relevant long-chain organic acids and potentially their conversion to alcohols.
Claims Coverage
The independent claims of this patent describe three main inventive features relating to recombinant Megasphaera microbes and associated genetic and metabolic engineering methods.
Recombinant Megasphaera microbe with specific metabolic pathway mutations
The invention encompasses a recombinant Megasphaera microbe genetically modified to: - Consume a greater amount of acetate than a comparable control microbe, or - Produce a greater amount of butyrate, hexanoate, or a combination thereof than a comparable control microbe, or - Increase carbon flux to butyryl-CoA and/or hexanoyl-CoA than a comparable control microbe, or a combination thereof. This inventive feature is defined by the presence of a mutation in a CoA-transferase coding region, glyoxalase coding region, or lyase coding region in the microbe, compared against a control microbe differing only by that mutation.
Recombinant Megasphaera microbe with undetectable pyrF expression
This feature covers a recombinant Megasphaera microbe having a mutation of a pyrF coding region to result in undetectable expression of the pyrF coding region (SEQ ID NO: 29). The mutation may be a deletion and can be present in any Megasphaera, specifically including M. elsdenii and also especially modified ATCC 25940 strains.
Method for engineering and selection in Megasphaera using pyrF and a mutagenic cassette
A method is claimed for genetically engineering a Megasphaera microbe comprising: 1. Providing a recombinant Megasphaera with a mutated pyrF gene, 2. Transforming this microbe with a plasmid comprising a pyrF coding region and a mutagenic cassette, where the cassette contains a marker flanked by DNA sequences for homologous recombination, 3. Incubating transformed microbes under conditions for positive selection and counter selection to select those that have lost the pyrF coding region, 4. Identifying transformed microbes carrying the desired mutation in the targeted coding region. This method optionally includes attachment sites in the cassette for recombinase-mediated excision of the marker, and extends to deletion-type mutations.
In summary, the claims provide broad coverage for metabolically engineered Megasphaera strains with defined mutations that drive increased acetate consumption and long-chain organic acid production, as well as specific genetic tools and methods to facilitate precise engineering and selection of desired phenotypes.
Stated Advantages
The engineered Megasphaera microbes have increased carbon flux toward butyryl-CoA and/or hexanoyl-CoA, resulting in greater production of butyrate and/or hexanoate.
The disclosed genetic tools, such as counter-selectable markers upp and pyrF, enable efficient construction and selection of targeted mutations in Megasphaera species.
The invention enables production of longer chain-length organic acids and alcohols from alternative carbon sources, providing a potential platform for drop-in biofuels with reduced carbon emissions.
A defined medium for Megasphaera allows for controlled manipulation and selection, facilitating strain construction and metabolic analysis.
Documented Applications
Use of recombinant Megasphaera to produce butyrate and/or hexanoate from lactate under fermentation conditions.
Further processing of butyryl-CoA and/or hexanoyl-CoA produced by engineered microbes to butanol and hexanol for biofuel applications.
Application of genetic tools (upp, pyrF) for strain construction, marker selection, and counter-selection in Megasphaera engineering.
Use of defined medium for mass balance analysis, study, and manipulation of carbon and nitrogen sources to enhance organic acid production and select for uracil prototrophy or auxotrophy.
Potential use of engineered Megasphaera as a platform for the conversion of lignocellulosic biomass sugars and organic acids into longer chain alcohols such as hexanol and other valuable chemicals.
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