A cofactor self-sufficient Escherichia coli and its construction method and application in the synthesis of L-glufosinate are provided. The present invention expresses a NADH kinase and key enzymes of the cofactor synthesis pathway in E. coli, and knocks out the genes of enzymes that catabolizes cofactor, and with the addition of co-metabolic intermediates during cell incubation, the intracellular NADP(H) concentration is increased by at least 50% and the catalytic activity of glutamate dehydrogenase by 2-fold, resulting in a significant increase in the spatiotemporal yield of the-glufosinate synthesis reaction.

A process comprising (i) providing a gaseous stream including greater than 1% by volume carbon dioxide; (ii) providing water; (iii) converting the carbon dioxide and the water to an organic intermediate and oxygen gas in the presence of light; (iv) separating the oxygen gas from the organic intermediate; and (v) converting the organic intermediate to ethylene and carbon dioxide after said step of separating the oxygen gas from the organic intermediate.

The present invention concerns a method for the production of derivatives of 4-hydroxy-2-ketobutyrate chosen among 1,3-propanediol or 2,4-dihydroxybutyrate by culturing a genetically modified microorganism for the production of the desired derivative of 4-hydroxy-2-ketobutyrate, the microorganism further comprising a gene coding for a mutant glutamate dehydrogenase converting by deamination L-homoserine into 4-hydroxy-2-ketobutyrate. The invention also concerns said genetically modified microorganism.

The present disclosure concerns recombinant yeast host cells having a first genetic modification for downregulating a first metabolic pathway that converts NADP.sup.+ to NADPH, as well as a second genetic modification for upregulating a second metabolic pathway that converts NADP.sup.+ to NADPH. The second genetic modification allows the expression of a glyceraldehyde-3-phosphate dehydrogenase lacking phosphorylating activity, which can, in some embodiments, be from enzyme commission 1.2.1.9 or 1.2.1.90. The second pathway is distinct from the first metabolic pathway. The present disclosure also concerns a process for making and improving the yield of a fermented product, such as ethanol, using the recombinant yeast host cell.

Disclosed are a gene mining method combining functional sequence and structure simulation, an NADH-preferring phosphinothricin dehydrogenase mutant and an application thereof. The gene mining method comprises the following steps: (1) analyzing a characteristic sequence which an NADH-type glutamate dehydrogenase should have; (2) searching a gene library based on the characteristic sequence; (3) performing clustering analysis and protein structure simulation on genes obtained by the searching; (4) selecting genes that feature high gene aggregation and a protein structure similar to that of the known phosphinothricin dehydrogenase as candidate genes. A wild-type phosphinothricin dehydrogenase with an amino acid sequence as set forth in SEQ ID No.2 derived from Lysinibacillus composti is obtained through the gene mining, and then mutated, and an NADH-preferring phosphinothricin dehydrogenase mutant is screened out, which has a mutation site selected from one of the following: (1) A144G-V375F-M91A; (2) A144G-V345A-M91A; (3) A144G. This mutant enzyme can be used for catalytic reaction with an inexpensive coenzyme NAD.

The present invention provides for a mechanism to reduce glycerol production and increase nitrogen utilization and ethanol production of recombinant microorganisms. One aspect of this invention relates to strains of S. cerevisiae with reduced glycerol productivity that get a kinetic benefit from higher nitrogen concentration without sacrificing ethanol yield. A second aspect of the invention relates to metabolic modifications resulting in altered transport and/or intracellular metabolism of nitrogen sources present in corn mash.

The invention discloses a glutamate dehydrogenase mutant and an application thereof. The mutant is one of the following: a mutant of the amino acid sequence of SEQ ID NO. 1 which has a mutation at lysine at position 402 to phenylalanine or aspartic acid; a mutant which has a mutation at isoleucine at position 406 to phenylalanine or threonine; a mutant which has a mutation at threonine at position 121 and leucine at position 123; a mutant which has a mutation at alanine at position 379 and leucine at position 383. In the invention, the catalytic activity of glutamate dehydrogenase derived from Pseudomonas putida to 2-carbonyl-4-(hydroxymethylphosphonoyl)butanoic acid (PPO) is significantly improved by a molecular transformation method combining directed evolution and a semi-rational design; and the issue of low glutamate dehydrogenase activity in the process of preparing L-glufosinate by reductive amination is solved.

A method for producing an active-form mutant enzyme, by specifying a protein of which a native form exhibits an enzyme activity but which has 10% or less enzyme activity of the native form when a gene of the protein is expressed to provide an inactive-form enzyme; determining a sequence conservation of amino acid residues in an amino acid sequence of the inactive-form enzyme and specifying amino acid residue(s) for which sequence conservation in the inactive-form enzyme is lower than sequence conservation of other amino acid(s) of the same residue; preparing a gene having a base sequence that codes for the amino acid sequence of the inactive-form enzyme in which at least one said specified amino acid residue is substituted by another amino acid with a higher sequence conservation; and expressing the gene to obtain an enzyme that exhibits an enzyme activity of the native form protein.

Disclosed are a machine learning gene mining method and a phosphinothricin dehydrogenase mutant for amino translocation. The phosphinothricin dehydrogenase mutant for amino translocation is obtained by mutation of a wild-type phosphinothricin dehydrogenase with an amino acid sequence as shown in SEQ ID No.2 at one of the following sites: (1) E263D-K134R-H96A-R290V; (2) E263D-K134R-H96A; (3) E263D-K134R; (4) E263D; (5) E263N; (6) E263C; and (7) E263G. The present invention utilizes the site-saturation mutagenesis technology to mutate a phosphinothricin dehydrogenase gene as shown in SEQ ID No. 1, finds that the 263rd, 134th, 290th and 290th positions are the key sites affecting enzyme activity and stereoselectivity, and obtains a mutant with enzyme activity and ee value much higher than those of the parent phosphinothricin dehydrogenase.

A method of the bio-production of methionine and/or of its derivatives thereof from a reduced source of sulfur, such as MeSH or MeSNa including genetically modified yeasts, having an increased ability to produce methionine and/or its derivatives thereof, as compared to the parent yeasts.