Strain improvement and statistical optimization as a combined strategy for improving fructosyltransferase production by Aureobasidium pullulans NAC8

Abstract

Strain improvement of a low fructosyltransferase-producing Aureobasidium pullulans NAC8 (Accession No. KX023301) was carried out using chemical mutagens such as ethidium bromide and ethyl methane sulfonate. The wild-type and mutant strain were distinguished using Random amplified polymorphic DNA PCR and DNA fingerprinting analysis. Plackett-Burman and Box Behnken design were statistical tools used to determine important media parameters and optimization, respectively. Phenotypically and genetically, the new improved strain was different from the wild-type. The most important media parameters from PDB influencing fructosyltransferase production were ammonium chloride, sucrose and yeast extract at p < 0.05. Some significant parameters obtained with the BBD exhibited quadratic effects on FTase. The F values (35.37 and 32.11), correlation coefficient (0.98 and 0.97) and the percent coefficient of variation (2.53% and 2.40%) were obtained for extracellular and intracellular FTase respectively. The validation of the model in the improved strain resulted in an overall 6.0 and 2.0-fold increase in extracellular and intracellular FTase respectively compared to the wild-type. A relatively low FTase-producing strain of Aureobasidium pullulans NAC8 was enhanced for optimum production using a two-pronged approach involving mutagenesis and statistical optimization. The improved mutant strain also had remarkable biotechnological properties that make it a suitable alternative than the wild-type.

Keywords: Box-Behnken design; DNA fingerprinting RAPD-PCR; Mutagenesis; Plackett-Burman design; RSM.

Fig. 1a

Mutant strain of A. pullulans NAC8.

Fig. 1b

Wild strain of A. pullulans NAC8.

Fig. 2

RAPD profile generated by all primers.

Fig. 3

Rectangular cladogram showing the Neighbor Joining (NJ) tree cluster analysis of agarose RAPD gel bands analyzed using GelQuest software (DNA fingerprint analysis software) to distinguish between parent and mutant strain of Aureobasidium pullulans NAC8.

Fig. 4

Plackett-Burman design based pareto chart for the effect of several media components on extracellular fructosyl transferase production by the improved mutant strain of A. pullulans.

Fig. 5

Plackett-Burman design based Pareto chart for the effect of several media components on intracellular fructosyl transferase production by the improved mutant strain of A. pullulans NAC8.

Fig. 6

The contour and three-dimensional surface plots for the combined effect of sucrose and NH₄Cl on extracellular fructosyltransferase production by the improved mutant strain of A. pullulans NAC8.

Fig. 7

The contour and three-dimensional surface plots for the combined effect of sucrose and yeast extract on extracellular fructosyltransferase production by the improved mutant strain of A. pullulans NAC8.

Fig. 8

The contour and three-dimensional surface plots for the combined effect of yeast extract and NH₄Cl on extracellular fructosyltransferase production by the improved mutant strain of A. pullulans NAC8.

Fig. 9

The contour and three-dimensional surface plots for the combined effect of sucrose and NH₄Cl on intracellular fructosyltransferase production by the improved mutant strain of A. pullulans NAC8.

Fig. 10

The contour and three-dimensional surface plots for the combined effect of yeast and NH₄Cl on intracellular fructosyltransferase production by the improved mutant strain of A. pullulans NAC8.

Fig. 11

The contour and three dimensional surface plots for the combined effect of sucrose and NH₄Cl on intracellular fructosyltransferase production by the improved mutant strain of A. pullulans NAC8.