Abstract:

The codon usage of Thermomyces lanuginosus lipase (tll) and Vitreoscilla hemoglobin (vhb) gene were optimized
by online software according to P. pastoris preferred codon usage. The G + C contents of the codon-optimized genes tll-opt
and vhb-opt were increased from 46% to 49% and 42% to 51%, respectively. The nucleotides A, T, C and G dispersed evenly
in the optimized genes, thereby eliminating AT- or GC-rich motifs. The tll and tll-opt genes were ligated into pPICZαA and
transformed into P. pastoris X33, respectively. The recombinant strains containing tll and tll-opt were cultivated in shaking
flask and 50 L bioreactor, respectively. In shake-flask level, the maximum lipase activities of X33/tll and X33/tll-opt were
7 and 16 U/mL, respectively. In 50 L bioreactor, the maximum lipase activities produced by X33/tll and X33/tll-opt were
201 U/mL and 430 U/mL. To further increase the expression of tll-opt, the vhb-opt gene was transformed into the
recombinant strain containing tll-opt to form recombinant strain VHb+. In shake-flask level, the maximum lipase activity
of VHb+ was 23 U/mL, which was 139% and 328% higher than that from the tll-opt gene and the wild-type gene (tll),
respectively. The maximum lipase activity of VHb+ in 50 L bioreactor was 610 U/mL, which was 141% and 303% higher
than that from the tll-opt gene and the wild-type gene (tll).

Key words: codon optimization, lipase, Vitreoscilla hemoglobin, high cell density fermentation