Multiple strategies for high-efficiency expression of Thermomyces lanuginosus lipase in Pichia pastoris and production of biodiesel in solvent-free system

With the development of enzymatic routes toward biodiesel production, the process can be concerned in a commercial viewpoint. The main bottleneck is the high cost of lipases. In this study, multiple strategies were used to improve the expression level of Thermomyces lanuginosus lipase (TLL) in Pichia pastoris. A recombinant P. pastoris with MF Delta signal peptide, 4 copies of tll gene and co-expression of Vitreoscilla hemoglobin was con-structed. Under the optimal culture condition, the TLL activity induced by methanol reached 7770 U/mL. In order to alleviate the inhibitory effect of methanol on cell growth, an additional constitutive promoter PGCW14 was introduced and a one-step methyl ester induction strategy based on the double promoter expression system was adopted. When using biodiesel as inducer, the TLL activity reached 8700 U/mL, which was 29-fold higher relative to the original strain. Moreover, the fermentation supernatant can be used in solvent-free system to produce biodiesel. The highest biodiesel yield of 96 % was achieved after 60 h at 35 degrees C with TLL concentration of 30 U/g oil, water content of 20 % (w/w) and step-wise addition of methanol (methanol to oil ratio of 4:1). The strains and procedures described here provided a solid foundation for sustainable and economical enzymatic production of biodiesel.