A method of producing a substance includes synthesizing a molecule at least by mixing substrates, a synthase, adenosine triphosphate (ATP), a polyphosphate kinase 2, and a polyphosphoric acid mixture. The polyphosphoric acid mixture includes 50% or more of polyphosphoric acid with a degree of polymerization of not less than 15. Adenosine diphosphate (ADP) is generated from the ATP during the synthesis. The synthesis is coupled with an ATP regeneration reaction in which the ATP is regenerated by the polyphosphate kinase 2 from the ADP and the polyphosphoric acid.

The present invention is an inducible coexpression system, capable of controlled induction of expression of each gene product.

A yeast extract containing 0.2% or more of -Glu-Abu based on dry weight of the yeast extract is produced by culturing a yeast, such as Saccharomyces cervisiae or Candida utilis, in a medium containing a compound selected from Abu (L-2-aminobutyric acid) and -Glu-Abu (L--glutamyl-L-2-aminobutyric acid), and preparing a yeast extract from the obtained cells.

One or more embodiments of the present invention provide a microbial strain with improved productivity of ?-glutamylcysteine, bis-?-glutamylcystine, ?-glutamylcystine, reduced glutathione, and/or oxidized glutathione. Such microbial strain has disruption of [1] a gene encoding ?-glutamyltransferase and [2] a gene encoding phosphoglycerate mutase and enhanced expression of [3] a gene encoding glutamate-cysteine ligase and/or a gene encoding glutathione synthetase or [4] a gene encoding bifunctional glutathione synthetase. This invention also discloses a method for producing the substances mentioned above via culture of the microbial strain.

The present invention is an inducible coexpression system, capable of controlled induction of expression of each gene product.

The present invention relates to a method of producing glutathione, wherein photosynthetic membrane vesicles and enzymes catalyzing glutathione synthesis are combined and glutamate, cysteine and glycine are used as reaction substrates. As enzymes catalyzing glutathione synthesis, -glutamylcysteine synthetase and glutathione synthetase may be used together, or bifunctional glutathione synthetase may be used alone. According to the conventional methods, there is a problem in that expensive adenosine triphosphate should be continuously supplied when glutathione is produced. However, according to the present invention, since photosynthetic membrane vesicles are used as a source to regenerate adenosine triphosphate, it is possible to continuously produce glutathione without additionally adding adenosine triphosphate, thereby reducing production costs of glutathione.

Aggregation is a major cause of the misbehavior of proteins. A system for modifying a protein to create a more stable variant is provided. The method involves identifying non-conserved hydrophobic amino acid residues on the surface of a protein, suitable for mutating to more hydrophilic residues (e.g., charged amino acids). Any number of residues on the surface may be changed to create a variant that is more soluble, resistant to aggregation, has a greater ability to re-fold, and/or is more stable under a variety of conditions. The invention also provides GFP, streptavidin, and GST variants with an increased theoretical net charge created by the inventive technology. Kits are also provided for carrying out such modifications on any protein of interest.

A mutant glutathione synthetase (GSHB) suitable for generating -Glu-Val-Gly, and a method for producing -Glu-Val-Gly using the same are provided. -Glu-Val-Gly is produced by using a mutant GSHB having a mutation at such a position as V7, N13, I14, N15, K17, F22, F95, M165, N199, Y200, P202, I274, T285, and P287.

A polypeptide includes a mutant sequence of the amino acid sequence of SEQ ID NO: The mutant sequence includes one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1 at positions 13, 17, 20, 23, 39, 70, 78, 101, 113, 125, 126, 136, 138, 149, 152, 154, 155, 197, 200, 215, 226, 227, 230, 239, 241, 246, 249, 254, 260, 262, 263, 270, 278, 299, 305, 307, and 310. The mutant sequence may further include one or more amino acid substitutions, additions, insertions, or deletions. The mutant sequence, excluding the substituted residue(s), may have a sequence identity of 80% or more with the amino acid sequence of SEQ ID NO: 1.

A microorganism useful as an expression host for -Glu-Val synthetase and a method for producing -Glu-Val-Gly using -Glu-Val synthetase expressed in the microorganism are provided. By using -Glu-Val synthetase expressed in a bacterium, such as Escherichia bacteria, modified so that the activity of a protein encoded by a ybdK gene (YBDK) is reduced as an expression host, -Glu-Val-Gly is produced from Glu, Val, and Gly as raw materials.