Effective synthesis of high-content fructooligosaccharides in engineered Aspergillus niger

Abstract

Background: Aspergillus niger ATCC 20611 is an industrially important fructooligosaccharides (FOS) producer since it produces the β-fructofuranosidase with superior transglycosylation activity, which is responsible for the conversion of sucrose to FOS accompanied by the by-product (glucose) generation. This study aims to consume glucose to enhance the content of FOS by heterologously expressing glucose oxidase and peroxidase in engineered A. niger.

Results: Glucose oxidase was successfully expressed and co-localized with β-fructofuranosidase in mycelia. These mycelia were applied to synthesis of FOS, which possessed an increased purity of 60.63% from 52.07%. Furthermore, peroxidase was expressed in A. niger and reached 7.70 U/g, which could remove the potential inhibitor of glucose oxidase to facilitate the FOS synthesis. Finally, the glucose oxidase-expressing strain and the peroxidase-expressing strain were jointly used to synthesize FOS, which content achieved 71.00%.

Conclusions: This strategy allows for obtaining high-content FOS by the multiple enzymes expressed in the industrial fungus, avoiding additional purification processes used in the production of oligosaccharides. This study not only facilitated the high-purity FOS synthesis, but also demonstrated the potential of A. niger ATCC 20611 as an enzyme-producing cell factory.

Keywords: Aspergillus niger; Fructooligosaccharides; Glucose oxidase; Heterologous expression; Peroxidase; β-fructofuranosidase.

Fig. 1

Construction of the glucose oxidase expressing strains of A. niger. A Schematic diagram of the gox expression cassette containing the promoter of the fopA gene (PfopA), the gox gene, the trpC terminator (TtrpC) and the selection marker ptrA. The expression cassette was constructed as described in “Materials and methods” section. B Assay of glucose oxidase on the GOD-POD bienzymatic detection plate. The same concentrations of spores of the glucose oxidase-expressing transformants GOD-1, GOD-3 and GOD-39 were spotted on the CD plate. Then the amount of glucose released was detected by the GOD-POD bienzymatic system. Glucose oxidase activity was tested by a dark brown halo surrounding the colonies. A. niger ATCC 20611 was used as the control. C Relative expression levels of gox in the glucose oxidase expressing transformants. Actin was used as a reference gene and the 2-^ΔΔCt method was used for calculating relative expression levels. D Determination of the glucose oxidase activities in different cell fractions of transformants. Error bars represent the standard deviation of three independent experiments. “a/b/c” above the bars indicate significant differences at P < 0.01. n.d., not detected. 20611, A. niger ATCC20611

Fig. 2

Construction of the glucose oxidase-FopAC expressing strains of A. niger. A Schematic diagram of the gox-fopAC expression cassette containing the promoter of the fopA gene (PfopA), the gox gene, the encoding sequence for the C-terminal domain of the fopA (fopAC), the trpC terminator (TtrpC), and the selection marker ptrA. The expression cassette was constructed as described in “Materials and methods” section. B Assay of glucose oxidase activity on the GOD-POD bienzymatic detection plate. The same concentrations of spores of the glucose oxidase-expressing transformants GOF-1, GOF-2 and GOF-3 were spotted on the CD plate. Then the amount of glucose released was detected by the GOD-POD bienzymatic system. Glucose oxidase activity was tested by a dark brown halo surrounding the colonies. A.niger ATCC 20611 was used as the control. C Relative expression levels of gox in the glucose oxidase-FopAC expressing transformants. Actin was used as a reference gene and the 2-^ΔΔCt method was used for calculating relative expression levels. D Determination of fructofructofuranosidase activities of the glucose oxidase-FopAC transformants in different cell fractions. E Determination of the glucose oxidase activities in different cell fractions of transformants. Error bars represent the standard deviation of three independent experiments. “a/b/c” above the bars indicate significant differences at P < 0.01. n.d., not detected

Fig. 3

Construction of the peroxidase-FopA expressing strains of A. niger. A Schematic diagram of the pod-fopA expression cassette containing the promoter of the fopA gene (PfopA), the horseradish roots pod gene, the fopA sequence and the trpC terminator (TtrpC). The expression cassette was constructed as described in “Materials and methods” section. B Assay of peroxidase and FopA on the GOD-POD bienzymatic detection plate. The same concentrations of spores of the peroxidase-expressing transformants DPOF-1, DPOF-3 and DPOF-14 were spotted on the CD plate. Then the amount of glucose released was detected by the GOD-POD bienzymatic system. Peroxidase activity was tested by a dark brown halo surrounding the colonies. A.niger ATCC 20611 and A. niger ∆fopA were used as the control. C Relative expression of pod in the peroxidase-FopA expressing transformants. Actin was used as a reference gene and the 2-^ΔΔCt method was used for calculating relative expression levels. D Determination of the peroxidase activities in different cell fractions of transformants using glucose as the substrate. E Determination of fructofructofuranosidase activities of the peroxidase-FopA transformants in different cell fractions by ABTS method. Error bars represent the standard deviation of three independent experiments. “a/b/c” above the bars indicate significant differences at P < 0.01. n.d., not detected

Fig. 4

Construction of the peroxidase-FopAC expressing strains of A. niger. A Schematic diagram of the pod-fopAC expression cassette containing the promoter of the fopA gene (PfopA), the horseradish roots pod gene, the encoding sequence of the C-terminal domain of the fopA (fopAC), the trpC terminator (TtrpC), and the selection marker ptrA. The expression cassette was constructed as described in “Materials and methods” section. B Assay of peroxidase on the GOD-POD bienzymatic detection plate. The same concentrations of spores of the peroxidase-expressing transformants DPOC-1, DPOC-8, DPOC-9 and DPOC-11 were spotted on the CD plate. Then the amount of glucose released was detected by the GOD-POD bienzymatic system. Peroxidase activity was tested by a dark brown halo surrounding the colonies. A. niger ATCC 20611 was used as the control. C Relative expression of pod in the peroxidase-FopAC expressing transformants. Actin was used as a reference gene and the 2-^ΔΔCt method was used for calculating relative expression levels. D The peroxidase activities in the transformants. Error bars represent the standard deviation of three independent experiments. “a/b/c” above the bars indicate significant differences at P < 0.01. n.d., not detected

Fig. 5

The production of FOS by using the glucose oxidase-expressing strain A. niger GOF-3 and the peroxidase-expressing A. niger DPOC-11. A The glucose content in the product formed by A. niger GOF-3 and DPOC-11 during the transglycosylation reaction using 40% (w/w) sucrose as substrate. A. niger ATCC 20611, A. niger ATCC 20611 plus glucose oxidase (20611 + GOX) and A. niger GOF-3 were used as the controls. B The yield of FOS produced by A. niger ATCC 20611, A. niger ATCC 20611 plus glucose oxidase (20611 + GOX), A. niger GOF-3 and A. niger GOF-3 plus DPOC-11 (GOF-3 + DPOC-11) during the transglycosylation reaction using 40% (w/w) sucrose as substrate. The products were detected by HPLC. Error bars represent the standard deviation of three independent experiments. “a/b/c” above the bars indicate the significant differences at P < 0.01