The present disclosure relates to a recombinant microorganism producing threonine, and a method for producing L-threonine using the same.

The present invention discloses a single-cell factory for efficiently synthesizing -aminobutyric acid and construction and application thereof, which belong to the technical field of microorganisms. The present invention expresses an L-threonine deaminase gene, an L-amino acid dehydrogenase gene and a dehydrogenase gene for providing cofactor NADH cycle in tandem to construct a recombinant Escherichia coli single-cell factory which is used for efficiently synthesizing -aminobutyric acid. The expression level of the L-threonine deaminase is optimized and controlled by an RBS sequence, so that the problem of transformation inhibition caused by the rapid accumulation of an intermediate product ketobutyric acid is solved, moreover, the expression level of the dehydrogenase for providing cofactor NADH cycle is optimized and controlled by a promoter and an RBS sequence, consequently, the NADH regeneration rate is increased, and ultimately, yield is increased. Utilizing the single-cell factory to carry out whole-cell transformation can reduce obstacles to substances getting in and out, increase the transformation rate and promote the intracellular cycle of cofactors without requiring exogenous addition, and the cost is low. Within 20 h, the yield of the recombinant Escherichia coli single-cell factory in a 5 L fermentation tank is 204 g.Math.L.sup.1, the space-time yield is 10.2 g.Math.L.sup.1.Math.h.sup.1, and a practical effective strategy is provided for industrialized production.

Provided herein are processes and microorganisms which utilize both protein hydrolysates and carbohydrates from biomass feedstocks to produce renewable hydrocarbon compositions. Advantages of the disclosed methods may be recognized in fuel blends comprising such hydrocarbon compositions.

A method for preparing glycine by using threonine relates to a fermentation process in which threonine is decomposed into glycine and acetaldehyde by aldolase. Glycine and acetyl coenzyme A can be produced in a fermentation process, in which acetaldehyde is reduced into acetyl coenzyme A or an acetyl coenzyme A derivative by acetylating acetaldehyde dehydrogenase; or threonine is dehydrogenated by threonine dehydrogenase to obtain 2-amino-3-ketobutyric acid, which is then ligated by 2-amino-3-ketobutyrate CoAligase to obtain acetyl coenzyme A. Coenzyme A can be converted into an acetyl coenzyme A derivative under different fermentation conditions.

Methods for evolving cells or strains towards improved methyltransferase activity, particularly SAM-dependent methyltransferase activity, as well as to cells and strains useful in such methods and methods of using the evolved cells in the production of methylated products.

The present invention discloses a single-cell factory for efficiently synthesizing -aminobutyric acid and construction and application thereof, which belong to the technical field of microorganisms. The present invention expresses an L-threonine deaminase gene, an L-amino acid dehydrogenase gene and a dehydrogenase gene for providing cofactor NADH cycle in tandem to construct a recombinant Escherichia coli single-cell factory which is used for efficiently synthesizing -aminobutyric acid. The expression level of the L-threonine deaminase is optimized and controlled by an RBS sequence, so that the problem of transformation inhibition caused by the rapid accumulation of an intermediate product ketobutyric acid is solved, moreover, the expression level of the dehydrogenase for providing cofactor NADH cycle is optimized and controlled by a promoter and an RBS sequence, consequently, the NADH regeneration rate is increased, and ultimately, yield is increased. Utilizing the single-cell factory to carry out whole-cell transformation can reduce obstacles to substances getting in and out, increase the transformation rate and promote the intracellular cycle of cofactors without requiring exogenous addition, and the cost is low. Within 20 h, the yield of the recombinant Escherichia coli single-cell factory in a 5 L fermentation tank is 204 g.Math.L.sup.1, the space-time yield is 10.2 g.Math.L.sup.1.Math.h.sup.1, and a practical effective strategy is provided for industrialized production

A method for producing L-isoleucine at a higher yield by fermentation includes the step of using a genetic engineering method to obtain a genetically engineered strain. The genetically engineered strain has a threonine deaminase gene substantially releasing the inhibition of L-isoleucine and/or an acetylated hydroxy acid synthetase III gene substantially releasing the inhibition of L-isoleucine; and performing fermentation culture on the genetically engineered strain, adding diketobutyric acid or a raw material capable of being converted into diketobutyric acid in a culture process, and separating L-isoleucine from a culture after the end of culturing. Further provided is a genetically engineered strain for realizing high yield of L-isoleucine.

Improved production of threonine from E. coli by fermentation is accomplished by attenuation but not elimination of the expression of either or both of the yajV gene encoding omega-amidase (a.k.a. 2-oxoglutaramate amidase) and the ilvA gene encoding threonine dehydratase (a.k.a threonine deaminase). In cases where there is attenuated expression of the ilvA gene, there is no need to express an exogenous cimA gene. In examples of both cases, attenuation is accomplished by engineering these genes to contain a weaker ribosome site. Further improvements in threonine production are made by expression of a heterologous pyruvate carboxylase gene exemplified by expression of the Corynebacterium glutamicum pyc gene under control of an E. coli promoter, to provide expression of pyruvate carboxylase that is not naturally expressed in E. coli.

This invention encompasses methods of making 1-propanol. In some embodiments the methods comprise providing a cultured bacterial biofilm; culturing the bacterial biofilm under conditions suitable for production of 1-propanol; and collecting 1-propanol produced by the biofilm culture. In some embodiments the methods comprise providing a bacterial culture comprising bacteria and culture media, wherein the culture media comprises a concentration of threonine higher than that present in LB; maintaining the bacterial culture under conditions suitable for production of 1-propanol; and collecting 1-propanol produced by the culture. This invention also encompasses bacterial culture systems. In some embodiments the bacterial culture systems comprise a bacterial biofilm comprising bacteria growing on an artificial solid substrate; culture media; 1-propanol in liquid and/or gas form; and a collection device configured to collect 1-propanol produced by the culture. In come embodiments the culture systems comprise bacteria; culture media, wherein the culture media comprises a concentration of threonine higher than that present in LB; 1-propanol in liquid and/or gas form; and a collection device configured to collect 1-propanol produced by the culture.

Multi-carbon compounds such as ethanol, n-butanol, sec-butanol, isobutanol, tert-butanol, fatty (or aliphatic long chain) alcohols, fatty acid methyl esters, 2,3-butanediol and the like, are important industrial commodity chemicals with a variety of applications. The present invention provides metabolically engineered host microorganisms which metabolize methane (CH.sub.4) as their sole carbon source to produce multi-carbon compounds for use in fuels (e.g., bio-fuel, bio-diesel) and bio-based chemicals. Furthermore, use of the metabolically engineered host microorganisms of the invention (which utilize methane as the sole carbon source) mitigate current industry practices and methods of producing multi-carbon compounds from petroleum or petroleum-derived feedstocks, and ameliorate much of the ongoing depletion of arable food source farmland currently being diverted to grow bio-fuel feedstocks, and as such, improve the environmental footprint of future bio-fuel, bio-diesel and bio-based chemical compositions.