The trade-off of availability and growth inhibition through copper for the production of copper-dependent enzymes by Pichia pastoris

Abstract

BackgroundCopper is an essential chemical element for life as it is a part of prosthetic groups of enzymes including super oxide dismutase and cytochrome c oxidase; however, it is also toxic at high concentrations. Here, we present the trade-off of copper availability and growth inhibition of a common host used for copper-dependent protein production, Pichia pastoris.ResultsAt copper concentrations ranging from 0.1 mM (6.35 mg/L) to 2 mM (127 mg/L), growth rates of 0.25 h−1 to 0.16 h−1 were observed with copper uptake of as high as 20 mgcopper/gCDW. The intracellular copper content was estimated by subtracting the copper adsorbed on the cell wall from the total copper concentration in the biomass. Higher copper concentrations led to stronger cell growth retardation and, at 10 mM (635 mg/L) and above, to growth inhibition. To test the determined copper concentration range for optimal recombinant protein production, a laccase gene from Aspergillus clavatus [EMBL: EAW07265.1] was cloned under the control of the constitutive glyceraldehyde-3-phosphate (GAP) dehydrogenase promoter for expression in P. pastoris. Notably, in the presence of copper, laccase expression improved the specific growth rate of P. pastoris. Although copper concentrations of 0.1 mM and 0.2 mM augmented laccase expression 4 times up to 3 U/mL compared to the control (0.75 U/mL), while higher copper concentrations resulted in reduced laccase production. An intracellular copper content between 1 and 2 mgcopper/gCDW was sufficient for increased laccase activity. The physiology of the yeast could be excluded as a reason for the stop of laccase production at moderate copper concentrations as no flux redistribution could be observed by 13C-metabolic flux analysis.ConclusionCopper and its pivotal role to sustain cellular functions is noteworthy. However, knowledge on its cellular accumulation, availability and distribution for recombinant protein production is limited. This study attempts to address one such challenge, which revealed the fact that intracellular copper accumulation influenced laccase production and should be considered for high protein expression of copper-dependent enzymes when using P. pastoris. The results are discussed in the context of P. pastoris as a general host for copper -dependent enzyme production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0251-3) contains supplementary material, which is available to authorized users.

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“…Hence, lower concentrations should be appraised. Based on reports in the literature with promissory laccase activity results (Balakumaran et al, 2016), copper sulfate -1 concentration was reduced by 50% in the PBED to be established as a fixed factor (0.05mM) in the BBED.…”

Evaluation of Metal Tolerance of Fungal Strains Isolated from Contaminated Mining Soil of Nanjing, China

“…Cytochrome-b5 reductase helps Pichia to survive in high heavy metal concentrations [ 38 ]. P. pastoris also has the ability to accumulate copper [ 39 ].…”