Effect of cultivation conditions on the dimorphism of and heterologous protein production by Arxula adeninivorans

[摘要] Arxula adeninivorans strain LS3, a dimorphic yeast with potential for biotechnological applications, has in recent years has been studied for the production of heterologous proteins. The growth kinetics of this species and the effect of environmental conditions on its morphology under controlled cultivation conditions have not been well documented. Because genetic transformation might affect the yeast physiology, a comparative investigation of the growth characteristics of strains LS3, G1211 (LEU2+) (the auxotroph derived from strain LS3) and LS3/pXynA, derived from strain LS3 by transformation with the Thermomyces lanuginosus xylanase gene under the control of the TEF1 promoter, was conducted, focusing on the effect of temperature and dissolved oxygen tension (DOT) on the morphology and growth parameters of the strains. This xylanase gene (XynA) was integrated in the 25S rDNA locus and expressed under control of the strong constitutive Arxula-derived TEF1 promoter. The plasmid copy number integrated into the rDNA locus was unknown. Little to no activity was found with the A. adeninivorans LS3 transformants, namely only 5.86 nkat ml-1-1 (0.35 U ml) compared to the 4 418 nkat ml-1-1 (265 U ml) obtained with the T. lanuginosus strain SSBP positive control. The protein itself might have been defective and since it accumulated intracellularly, this could also have resulted in the diminished activity observed. Cultivation in a temperature gradient incubator over a wide temperature range revealed significant differences in the maximum specific growth rates of the strains LS3 and G1211(LEU2+), namely 0.48 and 0.52 h-1, respectively. The minimum and maximum temperatures were 18 and 46°C, respectively, with the optimum temperature in the range of 37 to 39°C. In accordance with the literature, in shake flask cultures the morphology of LS3 changed with an increase in temperature from predominantly budding cells at 30°C to pseudohyphae at 42°C and resembled a mycelial culture at 45°C. By contrast, the morphology of strain G1211 (LEU2+) remained pseudohyphal at 45°C. During batch cultivations no true hyphae were observed, but a change in morphology, from budding cells to pseudohyphae, was observed during cultivation at different dissolved oxygen concentrations at different temperatures for LS3 and LS3/pXynA. G1211 (LEU2+) remained a pseudohyphal culture. The critical dissolved oxygen concentration (Ccrit) value, determined from the intersection of tangent lines of a plot of specific rate of oxygen uptake as function of dissolved oxygen concentration, of strains LS3 and G1211 (LEU2+) was 0.016 mmol l-1-1 (9% of saturation) and 0.013 mmol l (7% of saturation), respectively. This revealed that A. adeninivorans strain G1211 (LEU2+) had a slight but significantly lower Ccrit than strain LS3. This study showed that insertion of plasmid pAL-ALEU2m has a significant effect on the specific growth rate and on the morphology of Arxula adeninivorans LS3. Insertion of plasmid pXynA had only a slight effect on the morphology but no effect on the specific growth rate.

[发布机构] University of the Free State