Development of Yarrowia lipolytica for enhanced production of heterologous proteins

[摘要] English: Yarrowia lipolytica is a non-conventional yeast which is considered to be suitable forproduction of industrially important proteins at commercial scale. The yeast is nonpathogenic and is generally regarded as safe and as such can be used to producebiotechnological products for human consumption and applications. The genetic toolsfor the manipulation of the yeast have been developed over the years. This includeschemical and genetic based mutagenesis techniques to develop and isolate Y.lipolytica strains with enhanced properties in the production of endogenous andheterologous proteins. Protein expression systems comprised of a variety ofinducible and constitutive promoters are available; secretion signals for extracellularproduction of proteins and a choice of replicative or integrative expression systemshave also been developed. Auxotrophic and antibiotic based selectable markers areavailable some of which result in single or multiple copies of the integrativeexpression cassettes containing the target gene. The Y. lipolytica yeast can utilise awide variety of carbon sources and its fermentation processes in bioreactors are wellestablished.The property of Y. lipolytica as a prolific producer of the endogenous extracellularLip2p was exploited for the development of an expression system for production oftherapeutic peptides. To this end, the 38 amino acid long RANTES I peptide withtherapeutic applications in the treatment of HIV/AIDS was selected as a modelpeptide for co-expression with Lip2p. The sequence encoding the peptide was cloneddownstream of the complete LIP2 gene. The therapeutic peptide was separated fromthe LIP2 gene by the sequences encoding for 6X His and the DDDDK sequencerecognized by the enterokinase proteolytic enzyme. The expression cassette wasunder the hp4d quasi constitutive promoter with the rDNA as a target for multi copyintegration using the defective Ura3d4 integration cassette. The amount of RANTES Iproduced by the expression system was estimated to be 0.274 μg.L-1 as determinedby the CCL5/RANTES immunoassay method. The RANTES I peptide produced inthis study was reactive to polyclonal antibodies raised against human RANTES andfunctionality assays showed that the peptide inhibited the binding of the pseudoviralparticles to the TZM-bl cells through the CD4/CCR5 receptors.Another section of the study sought to identify genetic elements responsible forenhanced secretion of the extracellular Lip2p in Y. lipoyltica. Lipase hyperproducingmutants were generated using the zeta based mutagenesis system which integratesrandomly within the Y. lipolytica genome. One mutant denoted Y. lipolytica Y12 whichshowed lipase hyperproducing phenotype was found to be disrupted in the functionof the GPI7 gene, a protein encoding a polypeptide of 830 amino acids. The GPI7protein is 33% identical to the S. cerevisiae GPI7 known to play a role in themaintenance of the yeast cell wall integrity. Consistent with the disruption of cell wallintegrity through deletion of GPI7, the Y. lipolytica strain was more sensitive to cellwall hydrolysing enzymes, had defects in cell separation and a daughter cell-specificgrowth defect at the non-permissive temperature and exhibited hypersensitivity toCalcofluor white and Congo red which are known to interfere with yeast cell wallcomposition. While the Y. lipolytica Yl12 and the wild-type Po1d strains grewsimilarly in shake flask cultures, the Y. lipolytica Yl12 strain produced 7 times moreextracellular lipase activity in liquid shake flask cultures than the control Y. lipolyticaPo1d strain. To investigate the mode of GPI7 action in the enhancement of proteinproduction, a Y. lipolytica YlHmA25 strain expressing intracellular epoxide hydrolaseunder control of the hp4d promoter was deleted for the GPI7 gene to create a Y.lipolytica YlHmA25ΔGPI7 strain. The YlHmA25ΔGPI7 strain contained epoxidehydrolase activity in the extracellular in contrast to the Y. lipolytica YlHmA25. Inaddition, the Y. lipolytica YlHmA25ΔGPI7 accumulated more total extracellularprotein than Y. lipolytica YlHmA25 an indication that the strain accumulates moreproteins extracellulary in comparison with the Y. lipolytica YlHmA25 strain. Takentogether, the results suggested that the disruption of GPI7 affects the integrity of theyeast cell wall which in turn results in leakage or 'enhanced secretion of proteins tothe extracellular.The challenge in developing a host system with broad applications in heterologousproduction of proteins including therapeutics lies in the elimination of the endogenoushyper-mannosylated yeast glycans. The OCH1 gene encoding a protein with α-1,6-mannosyltransferase activity was deleted from the GPI7 deleted mutant of Y.lipolytica. The OCH1 and GPI7 double deletion mutant of Y. lipolytica showed similargrowth patterns to the GPI7 null mutant. Glycan analyses revealed that the mostabundant glycans are the Man8GlcNAc2 and Man9GlcNAc2 which are acceptablesubstrates of the Trichoderma reesei α-1,2-mannosidase I the enzyme widely used inthe engineering of yeast glycosylation pathway to produce Man5GlcNAc2, aprerequisite intermediate for the synthesis of complex glycans in human cells. TheOCH1 and GPI7 double deletion mutant of Y. lipolytica is a good candidate for furtherglycoengineering to enable production of extracellular therapeutic proteins withhuman-like glycans.

[发布机构] University of the Free State