The invention discloses a method for improving L-malic acid synthesis through fermentation of starch by using Aspergillus oryzae, and belongs to the field of gene engineering. According to the present invention, on the basis of genetic engineering bacteria A. Oryzae PN5, glucosidase coding gene agdA and amylase coding gene amyB are simultaneously subjected to overexpression, and the obtained recombinant strain A. Oryzae GAA is subjected to shake flask fermentation for 96 h, such that 100 g/L of starch can be converted into 72 g/L of L-malic acid, 6.4 g/L of succinic acid and 0.18 g/L of fumaric acid, wherein the conversion rate (g/g) of the malic acid relative to the starch is 0.72, and the production intensity is 0.75. With the method of the present invention, the foundation is established for the further metabolic transformation of Aspergillus oryzae in the starch fermentation production of L-malic acid.

Method for improving L-malic acid synthesis through fermentation of starch by using Aspergillus oryzae

The invention discloses aspergillus oryzae capable of reducing accumulation of a by-product in a synthesis process of aspergillus oryzae and an application of the aspergillus oryzae, and belongs to the field of genetic engineering. According to the invention, on the basis of A.oryzae GAA, over-expression of a fumarase coding gene derived from saccharomyces cerevisiae, a pyruvate carboxylase codinggene derived from rhizopus oryzae, a succinic acid-fumaric acid mitochondria transport protein coding gene derived from saccharomyces cerevisiae is implemented, so that conversion of fumaric acid andpyruvic acid in cytoplasm and mitochondria towards the malic acid and material exchange of cytoplasm and mitochondria are further enhanced. As for the finally obtained recombinant bacterium A.oryzaeCMPFS, a yield of L-malic acid undergoing shake-flask fermentation for 102h reaches 105.3g/L, concentration of succinic acid is 3.8g/L and concentration of fumaric acid is 0.21g/L; therefore, the concentration of the by-product is obviously reduced and product yield is improved; and a foundation is laid for production of the L-malic acid which is higher in purity through further metabolic transformation.

Aspergillus oryzae capable of reducing accumulation of by-product in synthesis process of malic acid and application of aspergillus oryzae

The invention discloses a promoter for expressing Aspergillus oryzae glucoamylase, and an application thereof, and belongs to the field of gene engineering. The novel glucoamylase promoter PN5 is constructed through serially connecting the core regions of a glaA promoter. Overexpression of glaA gene is carried out in Aspergillus oryzae under the mediation of an original promoter and a new promoter, and the novel glucoamylase promoter PN5 can be used in starch fermentation to produce L-malic acid. A result shows that the average expression activity and the malic acid output of a recombinant bacterium of the PN5-glaA gene are obviously higher than those of PglaA-glaA gene. A method for reconstructing and applying the promoter is simple and highly-efficient, provides theoretic basis for homologous or heterogenous expression of the target gene in the Aspergillus oryzae, and is of practical production significane to produce the target product through fermenting starch by the Aspergillus oryzae.

Promoter for expressing Aspergillus oryzae glucoamylase, and application thereof

The invention discloses a method for effectively improving L-malic acid production intensity of Aspergillus oryzae pp25, belonging to the field of genetic engineering According to the invention, Aspergillus oryzae pp25 is used as a starting strain, a C4-dicarboxylate transport protein gene C4T318 of the Aspergillus oryzae pp25 and a malic acid transport protein gene maeI of Schizosaccharomyces pombe are expressed to obviously reduce the intracellular accumulation of malic acid, and finally, the shake flask yield of the L-malic acid is up to 9317+/-210g/L and is increased by 474%; the concentration of succinic acid is 517+/-049g/L and is reduced by 418%; and the conversion rate of the malic acid relative to glucose is 085g/g and is increased by 491% Meanwhile, the fermentation period is shortened from 90 hours to 80 hours, and the production intensity is up to 116g/Lh and is increased by 657%

Method for effectively improving L-malic acid production intensity of Aspergillus oryzae pp25

The invention discloses a method for increasing the carbon source utilization rate in the aspergillus oryzae L-malic acid synthesizing process and belongs to the field of genetic engineering According to the method, Aspergillus oryzae NRRL 3488 serves as an original strain, metabolic flux for synthesizing malic acid from oxaloacetic acid is enhanced by expressing pyruvic carboxylase and malic dehydrogenase in aspergillus oryzae cytoplasm, and the shake flask yield of L-malic acid is increased to 505+/-106 g/L and is increased by 384%; then, the synthesizing efficiency of oxaloacetic acid from phosphoenolpyruvic acid in aspergillus oryzae cytoplasm is improved by expressing pyruvic carboxylase and pyruvate carboxykinase of Escherichia coli in the converter Aspergillus oryzae p16, finally, the yield of L-malic acid reaches 632+/-132 g/L, the concentration of succinic acid is 888+/-045 g/L, the production intensity is also improved to 07 g/(Lh) from original 024 g/(Lh) of Aspergillus oryzae NRRL 3488, and a foundation is laid for further metabolic engineering reform of Aspergillus oryzae for producing L-malic acid

Method for increasing carbon source utilization rate in aspergillus oryzae L-malic acid synthesizing process

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