1、 Introduction to Pichia pastoris expression system

The Pichia pastoris expression system, as an efficient eukaryotic expression system, has a wide range of applications and significant advantages. Compared with other expression systems, the Pichia pastoris expression system has significant advantages. It has one of the currently strong and tightly regulated promoters - the AOX1 promoter of the alcohol oxidase gene, which makes the expression regulation of exogenous genes more precise and efficient. Its expression level is high, which can achieve both intracellular expression and secretory expression, greatly meeting the needs of different research and production. The fermentation process is mature and easy to scale up, and has the ability for large-scale industrial high-density production, providing convenient conditions for industrial development. The cultivation cost is low, the products are easy to separate, and the fermentation medium used is inexpensive and protein free, which is beneficial for the separation and purification of downstream products and reduces production costs. Exogenous protein genes are genetically stable, and after integration onto the chromosomes of Pichia pastoris, they can exist stably with chromosome replication and are not easily lost. In addition, as an eukaryotic expression system, Pichia pastoris also has a subcellular structure of eukaryotes, which can perform post-translational modifications such as glycosylation, fatty acylation, and protein phosphorylation on expressed proteins, making the expressed proteins structurally and functionally closer to natural proteins and having higher biological activity.

2、 Composition of Pichia pastoris expression system

1. Host bacteria

At present, the host bacteria of Pichia pastoris used for exogenous protein expression include Mut+methanol utilizing strains GS115, X-33, SMD1165, SMD1163, and SMD1168; MutS methanol utilization slow type KM71; Mut methanol non utilization type MC100-3. Please refer to the table below for a detailed list of different host bacteria and expression characteristics of Pichia pastoris

Table 1 Common Host Bacteria and Characteristics of Pichia pastoris Expression

strain	feature
GS115	Host bacteria with high expression levels, high cell density, and high soluble protein production, currently widely used
KM71	Methanol utilization slow type, suitable for expressing proteins that are difficult to dissolve
X33	Can increase expression level and activity, suitable for expressing recombinant proteins
SMD1168H	Better expression efficiency and higher cell density, suitable for expressing large molecular proteins
SMD116（his4 pep4）	Belonging to the protease deficient strain, it can reduce protein degradation and solve the problem of uneven distribution of expression products caused by protein degradation. However, this strain grows relatively slowly, which may result in lower production of exogenous proteins expressed.
Y-11430	Efficient expression ability and controllable expression

2. Carrier

The expression vectors of Pichia pastoris are mainly integrated vectors, and common vectors can be divided into intracellular expression vectors and secretory expression vectors.

Table 2 Common expression hosts and characteristics of Pichia pastoris

3、 Fermentation process of Pichia pastoris

1. Fermentation stage of Pichia pastoris

The fermentation of Pichia pastoris is generally divided into two stages: 1. Cell growth stage: Firstly, the growth stage is carried out in a medium with glycerol or glucose as the carbon source, accumulating sufficient cell density and biomass. When the bacterial concentration reaches a certain stage, when glycerol or glucose is exhausted, the dissolved oxygen value suddenly rises to around 100%. Then, glycerol or glucose solution is added and the ventilation and stirring speed are maintained to maintain the dissolved oxygen level at around 30%, further increasing biomass (bacterial concentration). 2. Induction stage: When the biomass reaches the required level, stop supplementing the carbon source, deplete the carbon source, and after the transition period, switch the carbon source to methanol for the fermentation stage of inducing the expression of the target protein. Usually, 0.3% methanol is added intermittently twice for induction, and then methanol is introduced to induce protein expression. This method can increase the expression level of exogenous proteins and enable Pichia pastoris to accumulate high concentrations of target proteins in a short period of time.

The induction expression stage is a crucial step in the fermentation process of Pichia pastoris, during which methanol is used as a specific inducer to replace the previous carbon source. The alcohol oxidase gene (such as AOX1) in Pichia pastoris is activated in the presence of methanol, driving efficient expression of exogenous genes. The condition control at this stage has a significant impact on the yield and quality of exogenous proteins. It is necessary to strictly control the amount and rate of methanol addition, as well as parameters such as temperature, pH, and dissolved oxygen in the fermentation environment, to ensure the sustained and efficient expression of exogenous genes, while avoiding the toxicity and other adverse effects of methanol on cells.

2. Types and formulas of fermentation media

There are various types of fermentation media for Pichia pastoris, and different media formulations are suitable for different fermentation stages and purposes.

BSM (Basic Salt) medium is one of the commonly used basic culture media for Pichia pastoris fermentation. Its typical formula is: 85% phosphoric acid 26.7ml/l, calcium sulfate dihydrate 0.93g/l, potassium sulfate 18.2g/l, magnesium sulfate heptahydrate 14.9g/l, potassium hydroxide 4.13g/l, glycerol 40.0g/l, PTM1 4.35ml/l. This culture medium can provide basic nutrients for the growth of Pichia pastoris, including inorganic salts such as nitrogen, phosphorus, potassium, magnesium, and glycerol as a carbon source. PTM1 supplements the trace elements and vitamins required for yeast growth, helping to maintain the normal growth and metabolism of yeast cells.

F22 medium is also a common fermentation medium for Pichia pastoris. Its formula may be adjusted in different research and production applications, but it generally contains appropriate amounts of carbon sources (such as glucose or glycerol), nitrogen sources (such as yeast extract, peptone, etc.), inorganic salts (such as magnesium sulfate, calcium chloride, potassium dihydrogen phosphate, etc.), and other additives (such as biotin, trace element solutions, etc.). Compared with BSM medium, F22 medium may be optimized in terms of nutrient composition and proportion to meet specific fermentation needs, such as in some cases, it may be more conducive to increasing the expression level of exogenous proteins or improving protein quality.

In addition to the two common culture media mentioned above, there are many other Pichia pastoris fermentation media, and the formulations of these media are often optimized and adjusted according to specific experimental purposes, strain characteristics, and fermentation process requirements. For example, in some studies, in order to improve the utilization efficiency of specific substrates or enhance the tolerance of Pichia pastoris to environmental pressure, special components such as amino acids, organic acids, polysaccharides, etc. are added to the culture medium; Alternatively, by adjusting the ratio of carbon sources, nitrogen sources, and inorganic salts in the culture medium, the growth and metabolic pathways of yeast cells can be optimized to achieve higher fermentation efficiency and product yield.

3. Fermentation process parameters

In the fermentation process of Pichia pastoris, precise control of process parameters plays a crucial role in the success of fermentation, including pH, temperature, dissolved oxygen, etc. These parameters are interrelated and affect each other, jointly determining the growth status, metabolic activity, and expression level of exogenous proteins of Pichia pastoris.

PH value is one of the key factors affecting the fermentation of Pichia pastoris. Pichia pastoris has different pH requirements during different growth stages and metabolic processes. In the stage of cell growth, the generally suitable pH range is 5.0-6.0. Under this pH condition, yeast cells can maintain a good growth state, and enzyme activity can also be well exerted, which is conducive to cell proliferation and accumulation of metabolites. The pH value during the fermentation process of Pichia pastoris not only affects the growth status of the bacterial cells, but also has a significant impact on the expression of exogenous proteins. The impact of pH on Pichia pastoris mainly includes the following aspects: (1) affecting the activity of intracellular enzymes, which in turn affects cell metabolism, thereby affecting the expression of exogenous proteins; (2) Affects the permeability of the cell membrane and has a certain impact on the absorption of nutrients and excretion of metabolites by cells; (3) The pH value can affect the dissociation of components and metabolites in the culture medium, thereby affecting the absorption of nutrients by cells; (4) The pH value may also affect the metabolic processes of cells, causing changes in the content of cellular metabolites. In addition, during high-density fermentation, in order to reduce the degradation of exogenous proteins by proteases, the optimal pH value for protease action should be avoided, and the pH value should be set at a deviation from the isoelectric point of exogenous proteins, which can also increase the yield of exogenous proteins.

Temperature: It also has a significant impact on the growth and metabolism of Pichia pastoris. The growth temperature of Pichia pastoris is generally suitable between 28-30 ℃. Within this temperature range, yeast cells grow rapidly, have vigorous metabolic activity, and can efficiently utilize nutrients in the culture medium for growth and reproduction. However, if the temperature is too high, exceeding 32 ℃, it may lead to cell death because high temperature can damage the membrane structure of cells, the stability of biological macromolecules such as proteins and nucleic acids, thereby affecting the normal physiological functions of cells. On the contrary, at lower temperatures, although the growth rate of cells slows down, some studies have shown that appropriately reducing the culture temperature can improve the yield and quality of target proteins. This is because low temperature can reduce the stress of protein folding in cells, reduce the occurrence of misfolding, and also decrease the activity of proteases, reducing the degree of hydrolysis of exogenous proteins, thus facilitating the correct folding and accumulation of exogenous proteins.

Dissolved oxygen (DO): It is another key process parameter in the fermentation process of Pichia pastoris. Pichia pastoris requires the participation of oxygen for growth and metabolism using glycerol and methanol, so maintaining an appropriate level of dissolved oxygen is crucial to ensure smooth fermentation. During the entire fermentation process, it is usually necessary to maintain the dissolved oxygen concentration at a level greater than 20%. In the early stage of cell growth, due to the small number of bacterial cells, the demand for oxygen is relatively low. At this time, appropriate ventilation and stirring can meet the cell's dissolved oxygen demand. As cells grow and metabolic activity increases, the consumption of oxygen gradually increases. At this time, it is necessary to further adjust the ventilation rate, stirring speed, or use pure oxygen supplementation to increase the dissolved oxygen level, in order to ensure that cells can obtain sufficient oxygen for metabolism. If there is insufficient dissolved oxygen, it may lead to slow cell growth, reduced accumulation of metabolites, and even cell death, seriously affecting the efficiency of fermentation and the yield of products.

Cultivation time: It is one of the most critical factors in achieving the highest protein expression level in the Pichia pastoris expression system. In the Pichia pastoris expression system, the production time is relatively long (about 100 hours). The cultivation time is related to the number of yeast cells and the degree of degradation of target proteins. Studies have shown that the highest growth density of Pasteur Pichia pastoris cells is 96 hours of cultivation, while the highest protein expression occurs at 48 hours [11]. This suggests that longer cultivation time may lead to the hydrolysis and digestion of expressed proteins. Other studies have also shown that the optimal protein expression time occurs between 72 and 96 hours of cultivation [2]

The fermentation process of Pichia pastoris is a complex and sophisticated process that requires precise control and optimization of the fermentation stage, medium formulation, and process parameters. The fermentation conditions, such as pH, temperature, dissolved oxygen, and medium composition, can be systematically optimized through the Design of Experiments (DOE) method to fully leverage the advantages of Pichia pastoris and achieve efficient expression and production of exogenous proteins. By deeply understanding the fermentation characteristics and requirements of Pichia pastoris, combined with advanced fermentation technology and equipment, and continuously optimizing the fermentation process, it is expected to further improve the efficiency and product quality of Pichia pastoris fermentation.

4、 The tacit cooperation between Pichia pastoris and fermentation tanks

During the fermentation process of Pichia pastoris, the fermentation tank serves as the core equipment, providing critical environmental conditions for its growth and metabolism, ensuring its efficient expression of exogenous proteins.

The fermentation tank provides suitable growth temperature for Pichia pastoris through a precise temperature control system. As mentioned earlier, the temperature control system of the fermentation tank can accurately maintain the specific temperature requirements of Pichia pastoris during the growth stage of 28-30 ℃ and the induction expression stage, avoiding the adverse effects of high or low temperature on yeast cell growth and protein expression. In large-scale fermentation production, precise temperature control is particularly important as it ensures that each batch of Pichia pastoris can grow in a stable and suitable temperature environment, thereby improving production stability and repeatability.

For pH regulation, the fermentation tank is equipped with advanced pH detection and adjustment devices. At different stages of Pichia pastoris fermentation, the fermentation tank can automatically add acidic and alkaline solutions according to its changing pH requirements, accurately maintaining the pH value within the required range. Maintaining a pH of 5.0-6.0 during the cell growth stage and adjusting it to 3.0-5.0 during the induction expression stage helps to maintain the activity of enzymes in yeast cells, promote normal cell growth and metabolism, while reducing the degradation of exogenous proteins and improving protein yield and quality.

The control of dissolved oxygen levels is also one of the key functions of fermentation tanks. Pichia pastoris has a high demand for dissolved oxygen during the fermentation process. The fermentation tank ensures that the dissolved oxygen concentration is always maintained at a level greater than 20% through a reasonable ventilation system and stirring device. The ventilation system can evenly introduce air or oxygen into the fermentation broth, while the stirring device continuously stirs to fully disperse oxygen in the fermentation broth, improve the efficiency of oxygen transfer, meet the oxygen demand of Pichia pastoris at different growth stages, ensure its normal metabolic activity, and promote the efficient expression of exogenous proteins.

The synergistic effect of the stirring and ventilation system can also ensure the uniform distribution of nutrients in the fermentation broth, avoiding local nutrient deficiency or excess, allowing every Pichia pastoris cell to grow in a good nutritional environment. At the same time, the sealing performance of the fermentation tank is also very important. It can prevent contamination from external bacteria, ensure the purity and stability of the fermentation process of Pichia pastoris, and avoid problems such as fermentation failure or product quality decline caused by bacterial contamination.

Jinwei Sheng Gong's fermentation tank products are designed and manufactured based on these key requirements. They have advanced temperature control, pH adjustment, dissolved oxygen control, and efficient stirring and ventilation systems, which can provide optimized environmental conditions for the fermentation of Pichia pastoris, helping users achieve efficient and stable production of Pichia pastoris fermentation. Whether in the laboratory trial stage or industrial large-scale production, they can demonstrate their performance and meet the various needs of different users for Pichia pastoris fermentation, contributing to the vigorous development of the biotechnology industry. We will continue to pay attention to the research trends and application needs of Pichia pastoris, continuously optimize and innovate the design and manufacturing technology of fermentation tanks, improve the performance and stability of fermentation tanks, provide more advanced and efficient equipment support for the fermentation production of Pichia pastoris, and jointly promote the biotechnology industry towards a more brilliant future.

Introduction to Jinwei Shenggong Fermentation Tank
Jimway-be ® Lab Series - Desktop Reactor

Jimway-be ® PM series - Multi connected bioreactor

Applicable occasions: Cultivation, fermentation, and expression of prokaryotic/eukaryotic cells

Cultivation dish volume: 250ml/500ml/1L/3L/5L/10L

Unit quantity: 2-16 pairs

Cultivation method: Suspension&wall attachment

Blade types: Three inclined blades, six inclined blades, six flat blades, semi tubular, lifting blades and other combinations

Transmission mode: magnetic coupling drive/mechanical stirring

Control strategy: PID&stepwise control strategy (feeding, stirring speed, temperature, ventilation, etc.)

Feeding control: precise feeding control (error ± 1g)

Features:

Concise interface: Master the operation in a few minutes, achieve twice the result with half the effort

Unmanned: can be accessed and operated remotely, achieving unmanned operation

Formula function: One click distribution of formulas, reducing personnel operational errors and improving quality

Data analysis: enables comparison of multiple batches of data, and can also be integrated with Doe&PAT for in-depth multivariate analysis to optimize process data

Data sharing: Based on VPN for web publishing, it enables monitoring and historical curve viewing on mobile devices (Android, IOS, Windows) while ensuring data and device security