High-Level Production of a Thermostable Mutant of Yarrowia lipolytica Lipase 2 in Pichia pastoris

Abstract

As a promising biocatalyst, Yarrowia lipolytica lipase 2 (YlLip2) is limited in its industrial applications due to its low thermostability. In this study, a thermostable YlLip2 mutant was overexpressed in Pichia pastoris and its half-life time was over 30 min at 80 °C. To obtain a higher protein secretion level, the gene dosage of the mutated lip2 gene was optimized and the lipase activity was improved by about 89%. Then, the YlLip2 activity of the obtained strain further increased from 482 to 1465 U/mL via optimizing the shaking flask culture conditions. Subsequently, Hac1p and Vitreoscilla hemoglobin (VHb) were coexpressed with the YlLip2 mutant to reduce the endoplasmic reticulum stress and enhance the oxygen uptake efficiency in the recombinant strains, respectively. Furthermore, high-density fermentations were performed in a 3 L bioreactor and the production of the YlLip2 mutant reached 9080 U/mL. The results demonstrated that the expression level of the thermostable YlLip2 mutant was predominantly enhanced via the combination of these strategies in P. pastoris, which forms a consolidated basis for its large-scale production and future industrial applications.

Recent Advances in the Physicochemical Properties and Biotechnological Application of Vitreoscilla Hemoglobin

“…Furthermore, many processes of ethanol produc-Figure 2. The potential applications of VHb in the field of metabolic engineering for bacteria [55,56], fungi [48,49], plants [57] and animals [58]. ROS: reactive oxygen species; RNS: reactive nitrogen species.…”

“…[ 46 ], constitutive ermE promoter in Streptomyces sp. [ 47 ], and AOX1 promoter in Pichia pastoris [ 48 , 49 ]. In addition, the CaMV35S promoter has been chosen in higher plant systems [ 9 , 50 ].…”

“…For antibiotics, the synthesis of pyocyanin and rifamycin B can be increased 3-fold in E. coli [ 64 ] and 2.2-fold in Amycolatopsis mediterranei [ 65 ], respectively. For enzymes, the expression of lipase 2, coenzyme Q10 and xylanase can be enhanced by 87.84% in P. pastoris [ 48 ], 71% in Rhodobacter sphaeroides [ 66 ] and 31% in P. pastoris [ 49 ], respectively. For organic acids, the VHb expression has a greater effect on arachidonic acid (8-fold) in Mortierella alpina [ 67 ], docosahexaenoic acid (2.74-fold) in Aurantiochytrium sp.…”

Development of synthetic biology tools to engineer Pichia pastoris as a chassis for the production of natural products

“…The exploration of the genetic background of P. pastoris has laid a solid foundation for customized modification of P. pastoris . Currently, besides therapeutic proteins and enzymes [ 12 ], P. pastoris has been engineered to produce various chemicals and value-added compounds, such as d -lactic acid [ 13 ], 2,3-butanediol (BDO) [ 14 ], 2-phenylethanol [ 15 ], isobutanol and isobutyl acetate [ 16 ], carotenoids [ 17 ], lovastatin [ 18 ], and nootkatone [ 19 ] ( Fig. 1 ).…”

Amylose starch with no detectable branching developed through DNA-free CRISPR-Cas9 mediated mutagenesis of two starch branching enzymes in potato

“…Similar results were obtained on sweet potato mutated in Sbe2 , with an increase in amylose content from ca. 27 to 40% 18 .…”