Thermal adaptability of Kluyveromyces marxianus in recombinant protein production

Abstract

Background: Kluyveromyces marxianus combines the ease of genetic manipulation and fermentation with the ability to efficiently secrete high molecular weight proteins, performing eukaryotic post-translational modifications. It is able to grow efficiently in a wide range of temperatures. The secretion performances were analyzed in the host K. marxianus L3 in the range between 5°C and 40°C by means of 3 different reporter proteins, since temperature appears a key parameter for production and secretion of recombinant proteins.

Results: The recombinant strains were able to grow up to 40°C and, along the tested temperature interval (5-40°C), the specific growth rates (μ) were generally lower as compared to those of the untransformed strain. Biomass yields were slightly affected by temperature, with the highest values reached at 15°C and 30°C. The secretion of the endogenous β-fructofuranosidase, used as an internal control, was efficient in the range of the tested temperature, as evaluated by assaying the enzyme activity in the culture supernatants. The endogenous β-fructofuranosidase production was temperature dependent, with the highest yield at 30°C. The heterologous proteins HSA, GAA and Sod1p were all successfully produced and secreted between 5°C and 40°C, albeit each one presented a different optimal production temperature (15, 40, 5-30°C for HSA, GAA and Sod1p, respectively).

Conclusions: K. marxianus L3 has been identified as a promising and flexible cell factory. In a sole host, the optimization of growth temperatures for the efficient secretion of each individual protein can be carried out over a wide range of temperatures.

Figure 1

Effects of the growth temperature on secretion of the β-fructofuranosidase. β-fructofuranosidase has been measured as inulinase activity at 5 - 40°C, both at the growth and stationary phases. The values are means of three independent experiments ± standard deviations. Superscripts indicate statistically similar means (P > 0.05). Letters are used to compare samples collected at the same growth phase, grown at different temperatures. Symbols are used to compare samples grown at the same temperature, collected at diverse growth phases (exponential or stationary).

Figure 2

Effects of the growth temperature on HSA secretion. Culture samples, grown at 5 - 40°C, have been analyzed both at the growth and stationary phases. A) Western blot analysis of secreted rHSA; B) Western blot analysis of intracellular rHSA; C) Q-PCR analysis of rHSA mRNA. Data are reported as a function of growth phase and temperature. The values, means of three independent experiments ± standard deviations, have been normalized setting at 1 the highest value. Superscripts indicate statistically similar means (P > 0.05). Symbols are used to compare sample grown at the same temperature, collected at different growth phases (exponential or stationary). Letters are used to compare samples collected at the same growth phase, cultured at diverse temperatures.

Figure 3

Effects of the growth temperature on GAA secretion. Culture samples, grown at 5 - 40°C, have been analyzed both at the growth and stationary phases. A) extracellular GAA activity; B) northern blot analysis of GAA mRNA. Data are means of three independent experiments ± standard deviations. The values, means of three independent experiments ± standard deviations, have been normalized setting at 1 the highest value. Superscripts indicate statistically similar means (P > 0.05). Symbols are used to compare samples grown at the same temperature, collected at different phases (exponential or stationary). Letters are used to compare samples collected at the same growth phase, cultured at different temperatures.

Figure 4

Effects of the growth temperature on Sodp secretion. Culture samples, grown at 5 - 40°C, have been analyzed both at the growth and stationary phases. A) extracellular SOD activity measured by zymogram analysis; B) northern blot analysis of SOD1 mRNA. Data are means of three independent experiments ± standard deviations. The values, means of three independent experiments ± standard deviations, have been normalized setting at 1 the highest value. Superscripts indicate statistically similar means (P > 0.05). Symbols are used to compare sample grown at the same temperature, collected at different growth phases (exponential or stationary). Letters are used to compare samples collected in the same growth phase, cultured at different temperatures.