de novo design and synthesis of Candida antarctica lipase B gene and α-factor leads to high-level expression in Pichia pastoris

Abstract

Candida antarctica lipase B (CALB) is one of the most widely used and studied enzymes in the world. In order to achieve the high-level expression of CALB in Pichia, we optimized the codons of CALB gene and α-factor by using a de novo design and synthesis strategy. Through comparative analysis of a series of recombinants with different expression components, we found that the methanol-inducible expression recombinant carrying the codon-optimized α-factor and mature CALB gene (pPIC9KαM-CalBM) has the highest lipase production capacity. After fermentation parameters optimization, the lipase activity and protein content of the recombinant pPIC9KαM-CalBM reached 6,100 U/mL and 3.0 g/L, respectively, in a 5-L fermentor. We believe this strategy could be of special interest due to its capacity to improve the expression level of target gene, and the Pichia transformants carrying the codon-optimized gene had great potential for the industrial-scale production of CALB lipase.

Figure 1. Sequence comparison between the native and codon-optimized genes.

(A). α-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–Asp₂₁₀–His₂₄₉ and the conserved penta-peptide motif TWS₁₃₀QG. Bold solid line box indicate the link sequence of F1 and F2 fragments for OE-PCR.

Figure 2. in vitro synthesis of α-factor, native CALB and codon-optimized CALB genes.

A single-step strategy (A-PCR) was conducted to synthesize the codon-optimized α-factor (A and B), and a two-step strategy combining A-PCR and OE-PCR (C) was conducted to synthesize the native CALB (D) and codon-optimized CALB (E) genes. In order to synthesize the native CALB, the oligonucleotides were firstly assembled into F1 (541 bp) and F2 (510 bp), and then they were assembled into the genes with native signal peptide (CalBSP), native pre-sequence (CalBP) and mature CALB (CalB) with different primer pairs at OE-PCR step (D). In order to synthesize the codon-optimized CALB, the oligonucleotides were firstly assembled into F1M (510 bp) and F2M (553 bp), and then they were assembled into genes with signal peptide (CalBSPM), pre-sequence (CalBPM) and mature CALB (CalBM) with different primer pairs at OE-PCR step (E).

Figure 3. Lipase activity of the recombinants.

(A). The phenotypes of the recombinants on the tributyrin-MS plates; (B). The expression products of the recombinants. In Fig. 3A, A: pGAPZα-CalBM, B: pPIC9KαM-CalB; C: pPIC9K-CalBM, D: pPIC3.5K-CalBSP, E: pPIC9KαM-CalBM, F: pPIC9K-CalBP, G: pPIC9K-CalB, H: pGAPZα-CalB. In Fig. 3B, a total of 30 µL fermentation broth of pPIC3.5K-CalBSP and pPIC9KαM-CalBM were added into the wells, respectively. The purified CALB was deglycosylation by Endo H and then directly loaded into the well.

Figure 4. Lipase production capacity of the recombinants checked by flask fermentation.

Figure 5. Lipase production of recombinant pPIC9KαM-CalBM in 5-L fermentor.

(A). Fermentation condition; (B). Lipase production capacity. The fermentation parameters were maintained as follows: temperature (27.0°C), the pH (6.0) was sustained by ammonia titration, and the dissolved oxygen (DO, >30%) was lined with agitation (rpm, 550–650). For the inducible expression of lipase, methanol was added into the broth at a final concentration of 0.5%. Short dash line indicated the time point for methanol induction.