title: The trade-off of availability and growth inhibition through copper for the production of copper-dependent enzymes by Pichia pastoris.
abstract: BACKGROUND
  Copper 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.
  
  
  RESULTS
  At 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 (13)C-metabolic flux analysis.
  
  
  CONCLUSIONS
  Copper 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.
authors.affiliation: Centre for Biotechnology, Anna University, Sardar Patel Road, Guindy, Chennai, 600025, India. genbalu86@gmail.com.
authors.forename: Palanisamy Athiyaman
authors.initials: PA
authors.lastname: Balakumaran
authors.affiliation: iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany. jan.foerster@rwth-aachen.de.
authors.forename: Jan
authors.initials: J
authors.lastname: Förster
authors.affiliation: iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany. martin.zimmermann@rwth-aachen.de.
authors.forename: Martin
authors.initials: M
authors.lastname: Zimmermann
authors.affiliation: Centre for Biotechnology, Anna University, Sardar Patel Road, Guindy, Chennai, 600025, India. mathij28@gmail.com.
authors.forename: Jayachandran
authors.initials: J
authors.lastname: Charumathi
authors.affiliation: iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany. Andreas.Schmitz@rwth-aachen.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schmitz
authors.affiliation: iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany. eik.czarnotta@rwth-aachen.de.
authors.forename: Eik
authors.initials: E
authors.lastname: Czarnotta
authors.affiliation: iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany. mathias.lehnen@rwth-aachen.de.
authors.forename: Mathias
authors.initials: M
authors.lastname: Lehnen
authors.affiliation: iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany. suresh.sudarsan@rwth-aachen.de.
authors.forename: Suresh
authors.initials: S
authors.lastname: Sudarsan
authors.affiliation: iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany. birgitta.ebert@rwth-aachen.de.
authors.forename: Birgitta E
authors.initials: BE
authors.lastname: Ebert
authors.affiliation: iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany. lars.blank@rwth-aachen.de.
authors.forename: Lars Mathias
authors.initials: LM
authors.lastname: Blank
authors.affiliation: Centre for Biotechnology, Anna University, Sardar Patel Road, Guindy, Chennai, 600025, India. meenakshi@annauniv.edu.
authors.forename: Sankaranarayanan
authors.initials: S
authors.lastname: Meenakshisundaram
mesh_terms: D003300:Copper / Q000378:metabolism* / Q000494:pharmacology*; D005656:Fungal Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D042845:Laccase / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D064688:Metabolic Flux Analysis; D010843:Pichia / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D003300:Copper; D042845:Laccase