Biotin (vitamin H) is one of the most fascinating cofactors involved in central pathways in pro- and eukaryotic cell metabolism. Since its original discovery in 1901, research has led to the discovery of the complete biotin biosynthesis pathways in many different microbes and much work has been done on the highly intriguing and complex biochemistry of biotin biosynthesis. While humans and animals require several hundred micrograms of biotin per day, most microbes, plants and fungi appear to be able to synthesize the cofactor themselves. Biotin is added to many food, feed and cosmetic products, creating a world market of 10–30 t/year. However, the majority of the biotin sold is synthesized in a chemical process. Since the chemical synthesis is linked with a high environmental burden, much effort has been put into the development of biotin-overproducing microbes. A summary of biotin biosynthesis and its biological role is presented; and current strategies for the improvement of microbial biotin production using modern biotechnological techniques are discussed.

Biotin in microbes, the genes involved in its biosynthesis, its biochemical role and perspectives for biotechnological production

The present invention provides systems for producing engineered oleaginous yeast or fungi that express carotenoids.

Production of carotenoids in oleaginous yeast and fungi

The invention provides a non-naturally occurring microbial biocatalyst including a microbial organism having a 4-hydroxybutanoic acid (4-HB) biosynthetic pathway having at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, or α-ketoglutarate decarboxylase, wherein the exogenous nucleic acid is expressed in sufficient amounts to produce monomeric 4-hydroxybutanoic acid (4-HB). Also provided is a non-naturally occurring microbial biocatalyst including a microbial organism having 4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (BDO) biosynthetic pathways, the pathways include at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA transferase, 4-butyrate kinase, phosphotransbutyrylase, α-ketoglutarate decarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase or an aldehyde/alcohol dehydrogenase, wherein the exogenous nucleic acid is expressed in sufficient amounts to produce 1,4-butanediol (BDO). Additionally provided are methods for the production of 4-HB and BDO.

Compositions and methods for the biosynthesis of 1,4-butanediol and its precursors

Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to express recombinant genes encoding UDP-glycosyltransferases (UGTs). Such microorganisms, plants, or plant cells can produce steviol glycosides, e.g., Rebaudioside A and/or Rebaudioside D, which can be used as natural sweeteners in food products and dietary supplements.

Recombinant production of steviol glycosides

The invention provides a non-naturally occurring microorganism comprising one or more gene disruptions, the one or more gene disruptions occurring in genes encoding an enzyme obligatory to coupling 1,4-butanediol production to growth of the microorganism when the gene disruption reduces an activity of the enzyme, whereby the one or more gene disruptions confers stable growth-coupled production of 1,4-butanediol onto the non-naturally occurring microorganism. The microorganism can further comprise a gene encoding an enzyme in a 1,4-butanediol (BDO) biosynthetic pathway. The invention additionally relates to methods of using microorganisms to produce BDO.

Methods and organisms for the growth-coupled production of 1,4-butanediol

The present invention is directed to a recombinant enzymes having alcohol and aldehyde dehydrogenase activity which comprises one or mom recombinant polypeptides selected from the group consisting of polypeptides which are identified by SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 and chimeric recombinant polypeptides that are a chimeric combination of at least two of the following amino acid sequences identified by SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 and functional derivatives of the polypeptides identified above which contain addition, insertion, deletion and/or substitution of one or more amino acid residues, wherein said enzymatic polypeptides have said alcohol and aldehyde dehydrogenase activity. DNA molecules encoding the recombinant polypeptides, vectors comprising such DNA molecules, host cells transformed by such vectors and processes for the production of such recombinant enzymes are provided. Furthermore the recombinant enzymes having alcohol and aldehyde dehydrogenase activity are used for obtaining aldehydes, ketones or carboxylic acids, and specifically, 2-keto-L-gulonic acid an intermediate for the production of L-ascorbic acid (vitamin C).

Alcohol-aldehyde dehydrogenase

The present invention relates to a process for producing 2-keto-L-gulonic acid by fermentative conversion of D-sorbitol in high yield. 2-Keto-L-gulonic acid is an important intermediate for the production of L-ascorbic acid into which it can be converted.

Process for producing 2-keto-L-gulonic acid

The present invention is directed to a recombinant enzyme preparation having an alcohol and/or aldehyde dehydrogenase activity which comprises one or more enzymatic polypeptide(s) selected from the group consisting of polypeptides which are identified by SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8 and chimeric recombinant enzymes between the polypeptides identified by SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8 and functional derivatives of the polypeptides identified above which contain addition, insertion, deletion and/or substitution of one or more amino acid residue(s), wherein said enzymatic polypeptides have said alcohol and/or aldehyde dehydrogenase activity, DNA molecules encoding such polypeptides, vectors comprising such DNA molecules, host cells transformed by such vectors and processes for the production of such recombinant enzyme preparations, aldehyds, ketones or carboxylic acids by using such enzyme preparations and specifically 2-keto-L-gulonic acid and more specifically L-ascorbic acid (vitamin C).

Alcohol-aldehyd-dehydrogenases

A homogeneous alcohol/aldehyde dehydrogenase isolated from Gluconobacter oxydans DSM 4025 FERM BP-3812 is disclosed. The enzyme is capable in the presence of an electron acceptor, of catalyzing the conversion of L-sorbose to 2-keto-L-gulonic acid via L-sorbosone. The enzyme has a molecular weight of 130,000-140,000 daltons as determined by gel filtration column chromatography, a pyrroloquinoline quinone prosthetic group, an optimum pH for enzyme activity of 7.0 to 9.0, an optimum temperature for enzyme activity from about 20° C. to 40° C. and an isoelectric point of about 4.4. Also disclosed is a process for producing the enzyme and a process for producing aldehydes, carboxylic acids and ketones, especially 2-keto-L-gulonic acid utilizing the enzyme.

Alcohol/aldehyde dehydrogenase from Gluconobacter oxydans DSM 4025 FERM BP-3812

A process for producting vitamin B6 comprises cultivating a microorganism belonging to the genus Rhizobium and being capable of producing vitamin B6 in a culture medium under aerobic conditions, and separating the resulting vitamin B6 from the fermentation broth. As well as containing assimilable carbon sources, digestible nitrogen sources, inorganic salts and other nutrients necessary for the growth of the microorganism, the culture medium preferably additionally contains pyruvate, D-glyceraldehyde, glycolaldehyde, glycine, 1-deoxy-D-threo-pentulose, 4-hydroxy-L-threonine or a mixture thereof. This process affords high yields of vitamin B6, a vitamin essential for the nutrition of animals, plants and microorganisms and useful as a medicine and in foodstuffs.

Hoshino Tatsuo

Papers

Alcohol-aldehyde dehydrogenase

Process for producing 2-keto-L-gulonic acid

Alcohol-aldehyd-dehydrogenases

Alcohol/aldehyde dehydrogenase from Gluconobacter oxydans DSM 4025 FERM BP-3812

Fermentative production of vitamin B6