| As a safe and multifunctional lipase,Rhizomucor miehei lipase(RML)is able to catalyze various reactions including hydrolysis,esterification and transesterification,and plays an important role in food,medicine,health care products and other strategic emerging industrys.Nonetheless,large-scale applications of RML are restricted by its low yield.Yarrowia lipolytica,a generally recognized as safe host species,has been used for high-efficiency expression of many proteins and other products.The existing genetic tools of Y.lipolytica are still difficult to rapidly realize targeted,repeated,and markerless gene integration,and there is lacking more strategies for efficient protein secretion in this host.Thus,the present study focused on overexpression of RML in Y.lipolytica,explored and optimized the related factors of protein production,developed a new gene integration tool and an effective protein expression strategy,accomplished high-level production of RML in Y.lipolytica,and keeped the safety of engineered strains.The main contents and results are summarized as follows:(1)The heterologous expression of RML was first and successfully realized in Y.lipolytica.The recombinant plasmid hp4d-orirml,carrying the original R.miehei lipase gene,was constructed and introduced into Y.lipolytica to produce the engineered strain Po1g/hp4d-orirml,which showed lipase activity as 19.5 U/m L in fermentation supernatant.There was not excessive N-glycosylation for recombinant RML,and its optimum reaction p H and temperature were respectively determinated as 8.0 and 50 °C.In addition,the result of mass spectrometry identification of recombinant RML was consistent with wild-type RML protein.(2)The RML expression level was improved by optimizing gene structure,gene expression cassette element and dosage,and strain culture condition.To obtain the optimal gene structure for RML expression,codon usage bias and the necessity of propeptide sequence were evaluated,enhancing the RML expression level to 26.9 U/m L.Subsequently,the effects of seventeen promoters,three signal peptides and two terminators on RML production were respectively analysed,indicating that hp12d-XPR2pre-rml-XPR2 t was the optimal expression cassette for RML,and the lipase activity of corresponding strain was 36.7 U/m L.After that,a series of multicopy strains harboring 1～4 copies of rml gene expression cassette were builded,among them the strain carrying 2 rml gene copies showed higher activity(56.8 U/m L).On this basis,the optimal culture factors for recombinant strain were determined: 5%(m/v)D-Sorbitol,inoculation density 2%(v/v),initial medium p H 7.0,medium volume 30 m L,and fermentation time84 h,rising RML activity to 157 U/m L.In brief,the RML expression level in Y.lipolytica was increased by 7.1 times under the combined action of the above conditions.(3)A novel genetic tool was designed to efficiently implement targeted,repeated,and markerless gene integration.In order to further explore the relevant factors(such as protein synthesis and secretion)that may affect RML expression level,and overcome the existing limitations of gene integration in Y.lipolytica,a new gene integration tool,comprising of three plasmids for initial strain construction and iterative gene integration,was designed based on the Cre/lox system.Using this tool,the positive strain proportions remained above 90% in seven rounds of rml gene integration in Y.lipolytica,and the obtained recombinant strains performed well in RML expression level texts.The genetic tool also has many advantages,such as only a single selection marker is adequate for repeated gene integration,excising all unnecessary sequences(yeast screening marker,cre gene expression cassette,bacterial DNA sequences and homologous integration site)in controllable integration process,and flexible plasmid elements.(4)Protein synthesis and secretion was improved via co-expressing helper proteins to further promote RML production.The influences of helper proteins Kar2,Sls1,Ire1,Hac1,Pdi,and VHb(Vitreoscilla hemoglobin)on RML secretion level in recombinant strains were investigated via the above gene integration tool,and Kar2,Sls1,Pdi and VHb respectively brought 54%,17%,99% and 12% improvement for RML activity.Then,the synergistic effects and optimal gene dosage of the above 4 proteins were successively analyzed and identified,generating the strain FY5-4rml-kspvp 4# with the highest lipase activity in this study.Its RML activity reached 430 U/m L in shaking flask fermentation and was 1.4-fold higher than that before co-expressing helper proteins.Moreover,the lipase activity of FY5-4rml-kspvp 4# could remain stable after serial passages and rose to 2,250 U/m L in 3 L fermentation,which was 115-fold of the original lipase activity in this work(19.5 U/m L).Therefore,this study confirmed the feasibility of efficiently expressing RML in Y.lipolytica via optimization of the related factors affecting the RML production level in the recombinant strains,indicated that improving the synthesis and secretion of the target protein could increase its final yield,overcomed the existing gene integration restrictions in this host,and provided a solid foundation for large-scale production of RML and further development of Y.lipolytica as protein expression host. |
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