Construction Of A Pichia Pastoris Strain Deficient In Glycerol Catabolite Repression And, In The Presence Of Glycerol, Expressing Heterologous Proteins Under Induction By Methanol

In 1983, Wegner, et al. in America had firstly developed the methylotrophic yeasts representative of the second generation of yeast expression system. Wherein, the expression system using Pichia pastoris as a host was developed rapidly and used extensively. Extensive application of Pichia pastoris expression system is due to the fact that, with the exception of all characteristics belonging to common yeasts, it has also the followings advantages: 1) it uses alcohol oxidase 1 (AOX1) promoter which is one of the strongest and most strict promoters at present; 2) the expression plasmids used are integrating vectors and can integrate in the form of single or multiple copies, overcoming genetic instability of episomal plasmid in Saccharomyces cerevisiae; 3) it grows rapidly, has a low requirement for nutrition with lost cost in medium, has a strong aerobic preference, and can tolerate a higher hydrodynamic pressure, which thus is able to be fermented in high density, be up to 120g/L for the dry weight of cells in fermentor, and suitable for large-scale industrialized production; 4) there is peroxisome in Pichia pastoris cell, in which the expressed proteins are stored, can avoid being degraded by protease, and reduces the toxicity to cell; 5) the core carbohydrate in secreted proteins is Man8GlcNAc2 or Man9GlcNAc2 with low probability of superglycosylation and average 8-14 mannose residues in sugar chain, which can avoid the superglycosylation of high mannose oligosaccharide chain in Saccharomyces cerevisiae, and there is branched sugar chain in Pichia pastoris but noα-1,3 mannan (it is not present naturally in human, so this structure may cause adverse immune reaction in human body), so its N-acetyl glycosylation modification is closer to that in high eukaryotes than that in Saccharomyces cerevisiae; 6) there is seldom endogenous proteins secreted to outside of Pichia pastoris cell and extracellular recombinant proteins have relative high purity and are easy to be purified, so it is believable that it is very suitable for fundamental study; 7) in addition, the expressed foreign proteins can be not only present intracellularly but also secreted extracellularly.So far, high performance expression using Pichia pastoris has been achieved for more than 700 heterologous proteins, e.g., HBsAg, HSA, TNF, EGF, the C fragment of tetanus toxin, genetic engineering antibodies, among which the expression level can be up to gram grade per liter, confirming that this system is an expression system for heterologous gene characterized by high performance, utility, convenience, increased expression, and retention of biological activity in product, and suitable extremely for industrial scale.Although the Pichia pastoris expression system is used, due to its some outstanding advantages, to both industrial production and fundamental study, it is still not a very complete expression system and, at present, cannot substitute for other expression system, which thus has some limitations: 1) there is longer fermentation period, tendency of being easy to be polluted, and detrimental effect on the expression of heterologous proteins during long-time fermentation; 2) some foreign proteins are degraded by some proteases during secretion from Pichia pastoris, and the degraded proteins are secreted along with intact proteins to cell outside, which not only reduces the yield of protein of interest but also cause adverse effect on subsequent purification because of the degraded proteins in fermentation supernatant; 3) there are more complete mechanisms of post-translational modification, processing and correct folding, although they are different somewhat from the counterparts in high eukaryotes, especially, for the glycosylation modification: the N-linked sugar chain in Pichia pastoris can be expressed to be Mann GlcNAc2, but the glycoproteins in high eukaryotes also contain some saccharide residues, e.g., sialic acid, galactose, fructose; therefore, due to the structure of oligosaccharide chain is often involved in protein immunogenicity, those glycoproteins derived from high eukaryotes may not have their own natural structure when expressed in Pichia pastoris expression system, thus influencing their biological activities; 4) so far, the expression level in Pichia pastoris are different for different recombinant proteins, e.g., gram grade per liter for some proteins, only milligram even microgram grade per liter for others, especially the proteins with low molecular weight which are often lower in expression level; therefore, Pichia pastoris is still not a very extensive host for expression; 5) the Pichia pastoris strains with Mut+ phenotype are generally cultured by a three-step fermentation process during large-scale fermentation, i.e., glycerol batch phase, glycerol fed-batch phase, and methanol fed-batch induction phase, because of carbon catabolite repression to AOX1 promoter, but a large bulk of methanol, due to high-density cells achieved at glycerol batch phase and glycerol fed-batch phase, is required at methanol fed-batch induction phase to supply the carbon for maintaining cell growth and the inducer for high-performance induction of AOX1 promoter; however, high-concentration methanol volatilizes easily and is inflammable, and culture using methanol at high level as sole carbon has a certain risk which increases the design parameters of fermentor required for safety, thus increasing design cost of fermentor and finally the production cost; meanwhile, using the three-step fermentation process prolongs fermentation period, resulting in increased consumption during fermentation and thus increasing production cost.In these limitations, the problems caused due to the three-step fermentation process in the large-scale fermentation of Pichia pastoris are focuses in this study. The root cause leading to these problems are due to the fact that AOX1 promoter is regulated by carbon catabolite repression, here mainly showing that the induction and activation of AOX1 promoter by methanol are inhibited by some inhibitory carbon sources, e.g. glycerol. Therefore, to overcome these problem caused by the three-step fermentation process, first effort should be done for exploring cis-acting elements in AOX1 promoter and their trans-acting factors to make clear the mechanism of carbon catabolite repression occurring in AOX1 promoter.For Saccharomyces cerevisiae, Candida mycoderma, and Hansenula polymorpha, many genes relevant to carbon catabolite repression, e.g., GCR1, MIG1, are isolated using various kinds of mutation techniques. In addition, in the aspect of carbon catabolite repression in Pichia pastoris, some scientists in Japan had contructed a strain deficient in glucose catabolite repression using a chemical mutagenesis technique and obtained a relevant patent. Therefore, this study is to construct the Pichia pastoris strain deficient in glycerol catabolite repression in GS115 yps1? strain using LacZ as reporter by two mutation protocols: in the first one, random mutation is introduced into the genome of GS115 yps1? strain using REMI technique to inactivate the genes related to glycerol catabolite repression, and screening is then done for the strain deficient in glycerol catabolite repression; in the second one, error-prone PCR is used to amplify the AOX1 promoter, the product of which will contain multiple mutations and cloned into upstream of LacZ gene in pPIC9-LacZ plasmid to control its expression, and the resulting plasmid is used to transform GS115 yps1? cell followed by screening for the strain deficient in glycerol catabolite repression. A GS115-pPIC9-LacZ yps1? GR? able to expression LacZ in BMGMY medium was obtained by REMI technique and a gene related to glycerol catabolite repression, called GR1 gene, was also isolated with plasmid rescue and TAIL-PCR techniques from this strain. GS115 yps1? gr1? strain was obtained after disruption of GR1 gene using Cre-loxP system, and showed the deficiency in glycerol catabolite repression, that is, expression of heterologous proteins can be induced in GS115 yps1? gr1? strain by methanol in the presence of glycerol, e.g., LacZ, HSA-AX15(R13K). A random mutant library was constructed using Error-prone PCR and then introduced into GS115 yps1? cell by transformation. Finally, three strains that can express LacZ in BMGMY were obtained and refered to as GS115-pPIC9ZαA-LacZ yps1? ep1, 2 and 3, respectively. Based on the three strains, we initially determined those regions related to glycerol catabolite repression in AOX1 promoter, and cloned the relevant regions with mutations into pPIC9b vector. As a result, pPIC9AM vector was obtained and then introduced into GS115 yps1? cell by transformation. The resulting strain can express the heterologous proteins, e.g., LacZ, HSA-AX15(R13K), under induction by methanol in the presence of glycerol.