Development of Yarrowia lipolytica for enhanced production of heterologous proteins

English: Yarrowia lipolytica is a non-conventional yeast which is considered to be suitable for production of industrially important proteins at commercial scale. The yeast is non pathogenic and is generally regarded as safe and as such can be used to produce biotechnological products for human consumption and applications. The genetic tools for the manipulation of the yeast have been developed over the years. This includes chemical and genetic based mutagenesis techniques to develop and isolate Y. lipolytica strains with enhanced properties in the production of endogenous and heterologous proteins. Protein expression systems comprised of a variety of inducible and constitutive promoters are available; secretion signals for extracellular production of proteins and a choice of replicative or integrative expression systems have also been developed. Auxotrophic and antibiotic based selectable markers are available some of which result in single or multiple copies of the integrative expression cassettes containing the target gene. The Y. lipolytica yeast can utilise a wide variety of carbon sources and its fermentation processes in bioreactors are well established. The property of Y. lipolytica as a prolific producer of the endogenous extracellular Lip2p was exploited for the development of an expression system for production of therapeutic peptides. To this end, the 38 amino acid long RANTES I peptide with therapeutic applications in the treatment of HIV/AIDS was selected as a model peptide for co-expression with Lip2p. The sequence encoding the peptide was cloned downstream of the complete LIP2 gene. The therapeutic peptide was separated from the LIP2 gene by the sequences encoding for 6X His and the DDDDK sequence recognized by the enterokinase proteolytic enzyme. The expression cassette was under the hp4d quasi constitutive promoter with the rDNA as a target for multi copy integration using the defective Ura3d4 integration cassette. The amount of RANTES I produced by the expression system was estimated to be 0.274 μg.L-1 as determined by the CCL5/RANTES immunoassay method. The RANTES I peptide produced in this study was reactive to polyclonal antibodies raised against human RANTES and functionality assays showed that the peptide inhibited the binding of the pseudoviral particles to the TZM-bl cells through the CD4/CCR5 receptors. Another section of the study sought to identify genetic elements responsible for enhanced secretion of the extracellular Lip2p in Y. lipoyltica. Lipase hyperproducing mutants were generated using the zeta based mutagenesis system which integrates randomly within the Y. lipolytica genome. One mutant denoted Y. lipolytica Y12 which showed lipase hyperproducing phenotype was found to be disrupted in the function of the GPI7 gene, a protein encoding a polypeptide of 830 amino acids. The GPI7 protein is 33% identical to the S. cerevisiae GPI7 known to play a role in the maintenance of the yeast cell wall integrity. Consistent with the disruption of cell wall integrity through deletion of GPI7, the Y. lipolytica strain was more sensitive to cell wall hydrolysing enzymes, had defects in cell separation and a daughter cell-specific growth defect at the non-permissive temperature and exhibited hypersensitivity to Calcofluor white and Congo red which are known to interfere with yeast cell wall composition. While the Y. lipolytica Yl12 and the ”wild-type” Po1d strains grew similarly in shake flask cultures, the Y. lipolytica Yl12 strain produced 7 times more extracellular lipase activity in liquid shake flask cultures than the control Y. lipolytica Po1d strain. To investigate the mode of GPI7 action in the enhancement of protein production, a Y. lipolytica YlHmA25 strain expressing intracellular epoxide hydrolase under control of the hp4d promoter was deleted for the GPI7 gene to create a Y. lipolytica YlHmA25ΔGPI7 strain. The YlHmA25ΔGPI7 strain contained epoxide hydrolase activity in the extracellular in contrast to the Y. lipolytica YlHmA25. In addition, the Y. lipolytica YlHmA25ΔGPI7 accumulated more total extracellular protein than Y. lipolytica YlHmA25 an indication that the strain accumulates more proteins extracellulary in comparison with the Y. lipolytica YlHmA25 strain. Taken together, the results suggested that the disruption of GPI7 affects the integrity of the yeast cell wall which in turn results in leakage or “enhanced” secretion of proteins to the extracellular. The challenge in developing a host system with broad applications in heterologous production of proteins including therapeutics lies in the elimination of the endogenous hyper-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 similar growth patterns to the GPI7 null mutant. Glycan analyses revealed that the most abundant glycans are the Man8GlcNAc2 and Man9GlcNAc2 which are acceptable substrates of the Trichoderma reesei α-1,2-mannosidase I the enzyme widely used in the engineering of yeast glycosylation pathway to produce Man5GlcNAc2, a prerequisite intermediate for the synthesis of complex glycans in human cells. The OCH1 and GPI7 double deletion mutant of Y. lipolytica is a good candidate for further glycoengineering to enable production of extracellular therapeutic proteins with human-like glycans.