S were checked by flask fermentation, and for each transformant three replicates were conducted. The transformant with the highest lipase activity in flask was selected for the high density fermentation in a 5-L Biostat fermentor (B.Braun Biotech International, Melsungen, Germany). A fed-batch fermentation process was performed according to the model protocol described by the Invitrogen (http://toolszh. invitrogen.com/content/sfs/manuals/ pich_man.pdf). The fermentation basal salts (BSM) (H2PO4 26.7 mL, CaSO4 0.93 g, K2SO4 18.2 g, MgSO4N7H2O 14.9 g, KOH 4.13 g, glycerol 40.0 g, per liter) were used for yeast cell culture, and the parameters were monitored and controlled throughout the whole fermentation process. Briefly, the fermentation parameters were maintained as follows: temperature (27.0uC), dissolved oxygen (DO,.30 ), pH (6.0), agitation (rpm, 550?50) and aeration (0.1?.0 vvm). For the inducible expression of lipase, methanol was added into the broth at a final concentration of 0.5 . The time point for methanol induction was 30 h, and the methanol wasHigh-level Expression of CALB by de novo DesigningFigure 1. Sequence comparison between the native and purchase DprE1-IN-2 codon-optimized genes. (A). a-factor; (B). CALB gene. Dots represent the same nucleotides between the native and codon-optimized genes. Solid line box and dash line box indicate the signal peptide and pre-sequence of CALB, respectively, and * indicates the possible glycosylation site. indicate the catalytic triad Ser130 sp210 is249 and the conserved penta-peptide motif TWS130QG. Bold solid line box indicate the link sequence of F1 and F2 fragments for OE-PCR. doi:10.1371/journal.pone.0053939.gNHigh-level Expression of CALB by de novo Designingfed every 12 h with 0.5 mL/min speed. The whole fermentation time was 140 h and the methanol-induction time was 110 h. Samples were collected at intervals, and the fresh cell weight, lipase activity and protein content in broth were analyzed. Cell growth was monitored at various time points by determining the fresh cell weight (g/L). Purification of the lipase was conducted according to the description of Yang et al. [26], and the protein content was determined by the Bradford method [27].Lipase Activity and Protein Content AssaysTo qualitatively analyze the lipase activity, the yeast transformants were inoculated onto the GMMY agar plate (containing 0.5 tributyrin), and the halo diameter around the colonies was measured. Lipase activity was determined at pH 15755315 7.5 by free butyric acid titration using 50 mM NaOH. after incubated in a thermostated vessel for 10 min. The assay mixture consisted of 5 mL Tris-HCl buffer (50 mM), 50 mM NaCl, 4 mL emulsified tributyrin and 1 mL diluted enzyme solution. One unit (U) of the activity was defined as 
the amount of enzyme liberating 1 micromole of butyric acid per min at 45uC.55 , the second high-frequency codon for Phe (TTC, 18.9) and the third high-frequency codon for Leu (CTG, 15.5) were selected and the nucleotide sequencs of these blocks becoming 59TTCATGCTGAAC-39 and 59-TACCTGTTCAAC-39, respectively (Fig. 1). 5) Since the expression level of glycosylation-site-free CALB is equal to that with the glycosylation site [11], therefore, the glycosylation site (74Asn) of CALB was retained (Fig. 1). Comprehensively, about 170 rarely used codons were optimized (Fig. 1B). The GC content of gene was decreased from 61.89 to 53.99 . Moreover, we also optimied the codon of a-factor by simply replacing nine rarely u.