High cell density fermentation strategies for the production of bovine lactoferrin in Pichia pastoris

Abstract

Bovine lactoferrin (bLf) has been well characterized as a multifunctional glycoprotein belonging to the transferrin family with antibacterial, antioxidant, anti-tumor, and antiviral activities. The main aim of this study was to determine the feeding strategies in fed-batch fermentation to obtain high-cell density in Pichia pastoris KM71H-3 for production of bLf. During the growth phase, two feeding strategies were performed: (i) Feeding glycerol solution; and (ii) feeding glycerol and nitrogen source solution with a carbon-to-nitrogen ratio of 3:1. In addition, two feeding induction strategies were applied: One-time feeding and continuous feeding with 0.5% of methanol every 24 h. The results showed that the highest cell density at OD 600 was 362.67 ± 2.04 when the feeding phase used a mix-feed solution of glycerol and nitrogen sources with a carbon to nitrogen ratio of 3:1 in the 2-L scale. Up-scaled production of lactoferrin from this strain was successfully employed in the 10-L and 100-L bioreactor with the highest OD 600 reaching 338.20 ± 3.38 and 375.50 ± 2.98, respectively. For optimal lactoferrin expression, methanol was fed continuously, corresponding to an induced methanol concentration of 0.5% per 24 h. The appropriate induction time was 48 h. This research provides information on cell growth and fed-batch strategies for enhanced bLf production using P. pastoris as a host, which may be applicable to the expression of other proteins from P. pastoris strains.

Characterization of Recombinant Human Lactoferrin Expressed inKomagataella Phaffii

“…Unlike most recombinant systems, K. phaffii can be modified to produce rhLF with glycan structures similar to hmLF. Also distinct from other host expression systems, K. phaffii can carry out high-density fermentation, which is a cost-effective way to generate high yields of recombinant proteins [33]. K. phaffii has been used as a production organism for numerous food ingredients that have received "no questions" letters from the U.S. Food and Drug Administration in the generally recognized as safe (GRAS) notification program [36][37][38].…”

“…Both hmLF and rhLF from transgenic cows contain sialylated and fucosylated glycans, whereas oligomannose glycans were reported with high abundance in the rhLF from transgenic cows and low abundance in hmLF [11,15]. K. phaffii is a eukaryotic, methylotrophic yeast commonly used to express recombinant proteins efficiently, including animal-derived and human-derived recombinant LF [32][33][34][35]. Unlike most recombinant systems, K. phaffii can be modified to produce rhLF with glycan structures similar to hmLF.…”