Synergistic codon optimization and bioreactor cultivation toward enhanced secretion of fungal lignin peroxidase in Pichia pastoris: Enzymatic valorization of technical (industrial) lignins

B.M.Majeke^a, M.García-Aparicio^a,b, O.DBiko^b, M.Viljoen-Bloom^b, W.H.van Zyl^b, J.F.Görgens^a

Department of Process Engineering, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa.

Abstract

Lignin peroxidase (LiP) is a well-recognized enzyme for its ability to oxidize lignins, but its commercial availability is limited, which hinders the biotechnological application of LiP-based bioprocesses in lignocellulose biorefineries. This study evaluated a combination strategy to improve the expression of LiP to promote its practical use. The strategy included optimization of the lipH8 gene of Phanerochaete chrysosporium according to the codon usage of Pichia pastoris, followed by fed-batch fermentation using a 14 L bioreactor (10 L working volume). The combination strategy achieved a maximum volumetric LiPH8 activity of 4480 U L^-1, protein concentration of 417 mg L^-1 and a specific activity of 10.7 U mg^-1, which was higher than previous reports. Biochemical characterization showed that the recombinant LiPH8 (rLiPH8) was optimum at pH 3.0, 25℃ and 0.4 mM H₂O₂. Using the optimized conditions, rLiPH8 was used to treat isolated technical lignins namely soda-anthraquinone (SAQ) lignin and steam explosion (S-E) lignin. High-performance gel permeation chromatography (HP-GPC) analysis showed that the molecular weight (Mw) of SAQ and S-E lignins were increased by 1.43-and 1.14-fold, respectively, after the enzymatic treatment. Thermogravimetric analysis (TGA) also showed that the thermal stability of the lignins was improved, indicating that the enzyme treatment of lignins with rLiPH8 resulted in lignin re-polymerization. As the first report on rLiPH8 production using P. pastoris, this study has shed light on the possible route for the enhancement of rLiPH8 production and its potential application for upgrading technical lignins.

Keywords: Codon optimization, Fed-batch fermentation, Lignin peroxidase, Pichia pastoris, Lignin polymerization, Technical lignin.