Improved extracellular endo-1,4-β-mannosidase activity of recombinant Pichia pastoris by optimizing signal peptide

Abstract

In order to improve the extracellular endo-1,4-β-mannosidase (MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides (W1, MF4I, INU1A, αpre, HFBI) were chosen to be analyzed by SignalP 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector pGAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4I, INU1A, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12. 95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/mL, while those mediated by MF4I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/mL, respectively. The maximum MAN activity reached 347.5 U/mL with 4 gene copies mediated by MF4I. These results indicate that replacing the signal peptide α-factor with MF4I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.

Improving the Secretion Yield of the β-Galactosidase Bgal1-3 in Pichia pastoris for Use as a Potential Catalyst in the Production of Prebiotic-Enriched Milk

“…Therefore, signal-peptide optimization for Bgal1-3 was performed. Five signal peptides were chosen, including a modified α-factor, MF4I; the preregion of α-factor, apre; a signal peptide from Trichoderma reesei, HFBI; a signal peptide from Kluyveromyces marxious, INU1A; and a synthetic signal peptide, W1 . The coding sequences of these signal peptides were optimized using the Optigene platform (Generay Biotechnology), which is shown in Table .…”

“…In addition, modification of the proregion can also enhance the secretion of some proteins. For example, the insertion of two spacer peptides (AIP and EEAEAEAEPK) into the α-factor signal peptide (MF4I) significantly enhances the secretion of insulin, phytases, , and β-mannosidase by several times.…”

“…The most efficient signal peptide for Bgal1-3 secretion was W1, a synthetic signal peptide based on proposed signal design rules. , It contains a positively charged N-terminus of three R followed by a three-residue spacer from the AOX1 promoter (AIP), a hydrophobic core of 11 repeated L, and a C-terminus comprising the preregion of α-factor (SSALA). In a previous study on the secretion of an acidic phytase, introduction of the spacer AIP into the α-factor signal peptide significantly improved its secretion yield from 15 to 40 mg/L.…”

“…Quantitative real-time PCR (qPCR) was performed by using the SYBR green PCR Master Mix (Thermo Fisher Scientific) in a StepOnePlus PCR System (ABI, Waltham, MA) with P29/P30 and P31/P32 as the primers (Table S1). The GAP (glyceraldehyde-3-phosphate dehydrogenase) fragment was used as the reference sequence . This fragment and the bgal1-3 gene were amplified using P25/P26 and P27/P28, respectively, and inserted into pUC119, generating plasmids pUC19-GAP and pUC19-Bgal1-3 (Table ).…”

“…The signal peptide sequences of apre, HFBI, W1, INU1A, and MF4I were codon optimized and synthesized by Generay Biotechnology (Shanghai, China, Table 2). 24 They were amplified using the primer pairs P9/P10, P11/P12, P13/P14, P15/P16, and P17/P18, respectively, and digested by Af lII and BamHI. Then, they were ligated to vector-2, producing plasmids pPICZ-S apre Bgal, pPICZ-S HFBI Bgal, pPICZ-S W1 Bgal, pPICZ-S INUIA Bgal, and pPICZ-S MF4I Bgal.…”