Decreased Rhamnose Metabolic Flux Improved Production of Target Proteins and Cell Flocculation in Pichia pastoris

Abstract

Previously, several genes, including LRA1-LRA4 and LRAR, involved in rhamnose utilization pathway, were discovered in Pichia pastoris GS115; among them, LRA3 and LRA4 were considered as key rate-determining step enzymes. A P. pastoris expression platform based on the strong rhamnose-inducible promoter P _LRA3 did not meet the demands of industrial application due to poor production of recombinant proteins. To enhance recombinant protein production of this expression platform, a genetically engineered strain, P. pastoris GS115m, with decreased rhamnose metabolic flux was developed from P. pastoris GS115 by replacement of the rhamnose-inducible promoter P _LRA4 with another much weaker rhamnose-inducible promoter, P _LRA2 . Grown in MRH and YPR media using rhamnose as the main carbon source, the engineered strain showed decreased growth rate and maximal biomass compared with the parental strain. More importantly, grown in rhamnose-containing MRH and YPR media, the recombinant engineered strain harboring a β-galactosidase gene lacB, whose expression was regulated by rhamnose-inducible P _LRA3 , yielded substantial increases, of 2.5- and 1.5-fold, respectively, in target protein production over the parental strain. Additionally, grown in MRH and YPR media, the engineered strain had remarkable cell flocculation and rapid sedimentation with the increasing of cell density, providing an effective and convenient separation of the fermentation supernatant from strain cells. The engineered strain is a promising expression host for industrial production of target proteins due to its advantages over the parental strain as follows: (i) improved production of recombinant proteins, and (ii) remarkable cell flocculation and rapid sedimentation.

Keywords: Pichia pastoris; cell flocculation; promoter exchange; rhamnose metabolic; rhamnose-inducible promoter.

FIGURE 1

Growth profiles of different strains complemented with LRA4 under various P_LRA3 lengths. (A) Colonies formed by LRA4-disrupted strains transformed by different plasmids harboring various lengths of P_LRA3 controlling the expression of LRA4. 1, LRA4-disrupted strain; 2, 3, 4, and 5, colonies formed by LRA4-disrupted strains complemented with LRA4 under control of various lengths of P_LRA3: 100, 120, 140, and 210 bp, respectively. (B) Verification of positive transformants via PCR. An 890-bp DNA fragment of LRA4 was amplified from chromosomal DNA of different strains. Lanes 1–4, 5–8, and 9–12, strains complemented with LRA4 under control of various lengths of P_LRA3 (210, 140, and 120 bp, respectively); lane 13, LRA4-disrupted strain; lane 14, wild-type Pichia pastoris strain; lane M, DNA marker.

FIGURE 2

Relative transcription levels of rhamnose utilization related genes (LRA1, LRA2, LRAR, LRA3, and LRA4) in various strains grown in YPR. (A) The ratios of LRA4/LRA2 and LRA3/LRA2 in GS115, GS115/LacB, and GS115m/LacB are shown on the vertical axis. (B) Relative transcription levels of LRA1, LRA2, LRAR, LRA3, and LRA4 in GS115m/LacB, using these in GS115/LacB as controls. The GAPDH gene was used as a reference, and the relative expression value of LRA2 in each strain was designated as 1. Each test was performed in triplicate, and the results are presented as means ± SD of three replicates. GS115, P. pastoris GS115; GS115/LacB, P. pastoris GS115 harboring lacB; GS115m/LacB, P. pastoris GS115m harboring lacB.

FIGURE 3

Growth characteristics of strains when grown in media MRH (A), MDH (B), YPR (C), or YPD (D). WCW, wet cell weight. GS115, P. pastoris GS115; GS115m, P. pastoris GS115m; GS115/ΔLRA4, P. pastoris GS115 with LRA4 disruption. Each test was performed in triplicate, and the results are presented as means ± SD of three replicates.

FIGURE 4

Concentrations of residual rhamnose in cultures of strains grown in YPR at different duration. GS115, P. pastoris GS115; GS115/LacB, P. pastoris GS115/LacB; GS115m, P. pastoris GS115m; GS115m/LacB, P. pastoris GS115m/LacB. Each test was performed in triplicate, and the results are presented as means ± SD of three replicates.

FIGURE 5

β-Galactosidase production in culture supernatants of different strains grown in different media. β-Galactosidase activity in culture supernatants of strains grown in YPR (A) or MRH (B). β-Galactosidase production in culture supernatants of GS115m/LacB (C) and GS115/LacB (D) in YPR. GS115, P. pastoris GS115; GS115/LacB, P. pastoris GS115/LacB; GS115m/LacB, P. pastoris GS115m/LacB. Each test was performed in triplicate, and the results are presented as means ± SD of three replicates.

FIGURE 6

Green fluorescence in different strains grown in YPR. GS115/gfp, P. pastoris GS115 harboring gfp; GS115m/gfp, P. pastoris GS115 harboring gfp; GS115, P. pastoris GS115.

FIGURE 7

Cell flocculation profiles of GS115/LacB and GS115m/LacB grown in YPR at different cell density. (A) OD₆₀₀ ∼1.0; (B) OD₆₀₀ ∼3.0; (C) OD₆₀₀ ∼6.0. GS115/LacB, P. pastoris GS115/LacB; GS115m/LacB, P. pastoris GS115m/LacB.

FIGURE 8

Cell flocculation and sedimentation of different strains in different media. (A,E,I,M) 30 min; (B,F,J,N) 60 min; (C,G,K,O) 120 min; (D,H,L,P) 240 min. Cultures from left to right in (A–P) are P. pastoris GS115, P. pastoris GS115m, P. pastoris GS115/LacB, and P. pastoris GS115m/LacB, respectively.