Microorganism with knock-in at acetolactate decarboxylase gene locus
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Publication Number
US-12264348-B2
Publication Date
2025-04-01
Expiration Date
Abstract
Provided herein is a genetically engineered microorganism comprising knock-in of DNA at an acetolactate decarboxylase gene locus. Replacement of the acetolactate decarboxylase gene with DNA encoding one or more native or nonnative enzymes confers certain advantages, including fermentation stability and increased production of native and nonnative products from gaseous substrates.
Core Innovation
The invention concerns a genetically engineered C1-fixing microorganism that is modified by knock-in of DNA at an acetolactate decarboxylase gene locus. In this knock-in arrangement, the acetolactate decarboxylase (budA) coding region is replaced while the acetolactate decarboxylase promoter is not replaced, so that expression occurs under the budA promoter (PbudA).
The knock-in directs the microorganism’s carbon flux away from 2,3-butanediol and toward expression of one or more heterologous enzyme/pathway copies at the budA locus. The engineered locus expresses enzymes under the acetolactate decarboxylase promoter, and expression can be supported by an additional promoter.
The engineered microorganism comprises oscillations in metabolite production and gas consumption, and it is configured for production of heterologous products from gaseous C1 substrates such as CO, CO2, and H2, including syngas and industrial waste gas. Documented examples include an acetone pathway with thlA/ctfAB/adc, and an optionally adjusted route involving secAdh disruption for acetone versus a functional secAdh for isopropanol.
Claims Coverage
The independent claim covers a genetically engineered C1-fixing microorganism with a budA-locus knock-in and an oscillatory phenotype in metabolite production and gas consumption. The inventive features further specify which genomic regions are replaced or not replaced, promoter combinations controlling enzyme expression, particular enzyme sets, and gaseous C1-carbon-source definitions.
Knock-in of DNA at an acetolactate decarboxylase gene locus
The microorganism comprises knock-in of DNA at an acetolactate decarboxylase gene locus.
Oscillations in metabolite production and gas consumption
The microorganism comprises oscillations in metabolite production and gas consumption.
Replacing the acetolactate decarboxylase coding region
The microorganism comprises DNA that replaces the coding region of the acetolactate decarboxylase gene.
Not replacing the acetolactate decarboxylase promoter
The microorganism comprises DNA that does not replace the acetolactate decarboxylase promoter.
Regulation by an acetolactate decarboxylase promoter and an additional promoter
The microorganism comprises one or more enzymes regulated by an acetolactate decarboxylase promoter and at least one additional promoter.
Specified enzyme set for a pathway via defined decarboxylases
The one or more enzymes include a thiolase, a CoA transferase, and a decarboxylase selected from acetoacetate decarboxylase or alpha-ketoisovalerate decarboxylase.
C1-carbon source comprising CO, CO2, and/or H2
The gaseous substrate includes a C1-carbon source comprising CO, CO2, and/or H2.
Overall, the claim coverage centers on a budA-locus DNA knock-in that preserves the budA promoter context while replacing the budA coding region, combined with an oscillatory behavior in metabolite production and gas consumption, and further specifies promoter architecture, enzyme composition, and gaseous C1 feed definition.
Stated Advantages
Enables production of heterologous products from gaseous C1 substrates such as CO/CO2/H2, including syngas and industrial waste gas.
Directs carbon flux away from 2,3-butanediol.
Shows oscillations in metabolite production and gas consumption.
Contrasts improved stability of high acetone production with budA-locus knock-in versus instability when knock-in is placed at an adhE1+adhE2 locus.
Documented Applications
Cultivation/methods for producing heterologous products from gaseous C1 carbon sources, including syngas and industrial waste gas.
Production of acetone from gaseous C1 substrates.
Production of isopropanol from gaseous C1 substrates.
Delivery of additional listed target products via cultivation with gaseous C1 feeds.
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