Experience of Pichia pastoris fermentation
Pichia Pasteuris is a kind of methanotrophic yeast which can use methanol as the only carbon source and energy source.
Like other yeast, it mainly exists in the form of haploid during the asexual growth period, and when the environmental nutrition is limited, it often induces two zygotic haploid cells of different physiological types to mate and fuse into diploid.
Cell proliferation and reproduction;
The transition stage of batch addition (glycerol or glucose);
Induction expression stage (methanol);
The carbon source in each stage is a restricted matrix, and the dynamic model of its complementary acceleration rate is the basis of efficient expression.
There is no natural plasmid in Pichia pastoris, so the expression vector needs to be homologous recombination with the host chromosome, and the foreign gene expression framework is integrated into the chromosome to achieve the expression of foreign genes. Including promoter, foreign gene clone site, termination sequence, screening markers and so on.
The expression vectors were all shuttle plasmids, which were first replicated and expanded in Escherichia coli, and then introduced into host yeast cells. In order to secrete the product extracellular, the expression vector also needs to carry the signal peptide sequence.
1. Advantages
(1) It has a promoter of the alcohol oxidase AOX1 gene, which is one of the strongest promoters with the strictest regulatory mechanism.
(2) The expression efficiency is high, and the foreign protein expressed by the protein can account for more than 90% of the total expressed protein, which is conducive to the separation and purification of the target protein.
(3) High density culture can be achieved in simple synthetic medium.
(4) The expression plasmid can integrate stably in the form of single or multiple copies at specific sites of the genome.
(5) Because the yeast can use methanol as the only carbon source and energy, while most microorganisms cannot use methanol as a carbon source, pollution can be reduced.
2. Insufficient
(1) Long fermentation cycle;
(2) Methanol is flammable, explosive and toxic, and has certain dangers.
(3) The drugs required for screening high-yield strains are expensive.
(4) The medium and culture condition are not mature.
Influencing factors of Pichia pastoris fermentation.Generally speaking, the more important influencing factors are glucose concentration, temperature, pH, dissolved oxygen, nitrogen sources, nutrients and the concentration of the bacteria itself.
1. Temperature
Except for some special strains, the vast majority of yeasts are usually suitable for growth at 20-25°C. In addition, the higher the temperature, the more beneficial it is to avoid bacterial contamination.
The optimum fermentation temperature of Pichia Pastoris is 30°C and the growth temperature is 25~30°C. Industrial fermentation is generally controlled within the range of 30 to 35°C, in which the yeast normally grows and multiplies. However, once the temperature range is exceeded, the enzyme is quickly deactivated as the temperature rises, and the yeast cells are prone to senescence, shortening the fermentation cycle and thus affecting the expression of the target protein.
2. pH
Pichia Pastoris cells can adapt to a relatively broad pH environment, and can grow in an environment of pH2.0~7.5. For different protein types, the pH range of adaptation is different, and different proteins have different tolerance to different proteases. Choosing an appropriate pH can promote cell growth while avoiding the decrease of protease activity. Prevent degradation of the target protein.
In industrial production, the pH is generally controlled between 4.5 and 5.5. In the fermentation process, the method of online adding ammonia water is used to adjust the pH in the fermentation liquid. At the same time, ammonia water can also be used as a nitrogen source in the fermentation process to make the bacteria grow rapidly and finally allow the target product to be fully expressed.
3. Glucose concentration
Experiments showed that when the initial glucose concentration was above 25g/L, the yield of the bacteria would gradually decrease, indicating that higher glucose concentration had a greater inhibitory effect on the growth of the bacteria, and the growth rate of Pichia Pastoris was higher when the initial glucose concentration was around 20g/L.
4. Dissolved oxygen
Pichia Pastoris is an aerobic microorganism, and its growth and metabolism require the participation of oxygen, and the dissolved oxygen is one of the most important indicators in the growth of yeast cells.
If DO is kept at a low level, it indicates that the bacteria are consuming the carbon source in the fermentation liquid, and the rise of DO indicates that the carbon source is exhausted, and the bacteria are hungry, and the carbon source needs to be replenished in time. When the oxygen is insufficient, the growth of the bacteria will be limited, when the DO is too high, the high concentration of oxygen free radicals in the fermentation liquid will cause the bacteria to die. The dissolved oxygen is generally controlled at 30% during the fermentation process, while it is generally maintained at 20% during the induction stage.
5. Bacterial concentration
It was found that the fermentation performance of Pichia Pastoris was significantly affected by the concentration of initial yeast cells. When the initial cell concentration was lower, the cell growth rate was faster and the protein expression was less. However, when the initial cell concentration is higher, the substrate utilization rate is slower and the protein concentration and production rate are higher, because the yeast cells use the substrate to express the protein rather than for cell growth.
In the methanol induction stage, before induction, the bacteria should reach a large concentration to adapt to the methanol environment and reduce the poisoning situation. At present, methanol induction is generally stopped when the wet weight reaches 180~220g/L.
6. Nitrogen source
In industrial production, organic nitrogen sources such as peptone are still used as nitrogen sources in seed media. During the fermentation process, a large amount of ammonia is added when the pH value is adjusted, which also provides nitrogen sources for yeast cell growth.
7. Inorganic salt
The essential nutrient salts of Pichia Pastoris include CaCl2, MgSO4, K2SO4, KH2PO4 and K2HPO4. Trace elements such as zinc, cobalt, manganese, molybdenum, copper, iron and other elements are also indispensable for yeast cells, they are co-factors of enzymes, some enzyme activators. The low concentration of these trace elements can promote the growth of yeast and the synthesis of products, while the high concentration will cause yeast poisoning and show inhibitory effect.
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