Expression of adenosine monophosphate deaminase in Kluyveromyces lactis

Abstract:

[Objective] We constructed a recombinant Kluyveromyces lactis GG799 strain to constitutively produce adenosine monophosphate (AMP) deaminase. [Methods] The codons of AMP deaminase gene derived from Streptomyces murinus were optimized and used as template. We designed primers and amplified the opt-AMPD gene. The opt-AMPD gene was cloned into the expression plasmid pKLAC1. The recombinant expression plasmid was linearized by Sac II and transformed into K. lactis GG799 by electrotransformation. We determined the AMP deaminase activity of positive transformants. The AMP deaminase was purified by His TrapTM HP and we preliminary optimized the fermentation medium of K. lactis GG799. [Results] The codons of AMPD gene were optimized and a recombinant K. lactis GG799/pKLAC1-opt-AMPD was constructed to constitutively produce AMP deaminase. The AMP deaminase activity reached 586±50 U/mL after optimizing codon. The purified AMP deaminase showed asingle band on SDS-PAGE and the molecular weight by SDS-PAGE was about 60 kD. The preliminary optimized medium contained 40 g/L glucose, 20 g/L peptone, 15 g/L yeast extract, 8 g/L NaCl, 10 g/L KCl, 2 g/L MgSO4. The activity of AMP deaminase reached 2 100±60 U/mL cultured in flask after 120 h at 30 °C with agitation 200 r/min. [Conclusion] These codons of AMPD gene were optimized and AMP deaminase was constitutively produced in the K. lactis GG799. It is a valuable exploration about high efficiency recombinant expression and production of AMP deaminase.

Key words:Kluyveromyces lactis, AMP deaminase, Recombinant expression, Fermentation optimization