Medium: The Foundation of Fermentation

The organic combination of genetic engineering technology and large-scale culture techniques enables the mass production of natural proteins with low content. By introducing the nucleic acid sequences encoding target proteins into host cells through genetic engineering,

These cells can be induced to express the target proteins in large quantities. High-density cultivation of recombinant E. coli cells is an important strategy for obtaining high yields of exogenous proteins. This technology not only reduces the culture volume, simplifies downstream separation and extraction processes, but also shortens production cycles, decreases equipment investment, reduces production costs, and improves efficiency.

As demand for rapid protein production has increased in the post-genomic era, people are paying more attention to the production process of proteins. Using multi-dimensional strategies based on systems biology, the traditional processes are being reshaped and optimized to establish more comprehensive and efficient high-density fermentation processes for recombinant proteins.

The fermentation medium, which serves as the foundational raw material for E. coli growth and product synthesis, which is crucial for the growth of the bacteria and the expression of recombinant proteins. It plays an essential role in fermentation engineering by providing the necessary nutrients for protein expression and creating a suitable environment for bacterial growth. Whether a good fermentation medium can be designed is one of the primary factors determining whether industrialization of fermentation products can be successful by improving yield and lowering production costs.

Composition of E. coli Fermentation Medium

Carbon Sources

Carbon sources provide the framework for cell material and metabolic products and supply the energy needed for cellular physiological activities.

1.Common carbon sources: 

include glucose, glycerol, lactose, and mannose. Among them, glucose is the most commonly used carbon source in coli fermentation. However, when choosing glucose, attention should be paid to the following:

• Appropriate glucose concentration: 

High concentrations of glucose can cause the “glucose effect,” leading to the production of metabolic by-products like acetic acid, which can negatively impact bacterial growth, recombinant protein expression, and bioactivity.

• Carbon-to-nitrogen ratio (C/N ratio): 

This ratio refers to the balance between carbon and nitrogen concentrations in the medium. A lower C/N ratio may cause excessive bacterial growth, leading to cell aging and autolysis. A higher C/N ratio may inhibit bacterial growth. The effect of the C/N ratio varies depending on the stage of fermentation, and it can also influence changes in the pH during fermentation. A higher C/N ratio may lower pH, while a lower C/N ratio may raise pH.

2.Strategies for optimizing carbon sources: 

Optimization can be achieved by fine-tuning feeding strategies and adjusting culture conditions to maintain the ideal concentration and C/N ratio. Additionally, alternative carbon sources such as glycerol, lactose, and mannose, which produce fewer acidby-products, can be explored. Glycerol, for instance, enters the tricarboxylic acid (TCA) cycle through a different pathway than glucose, effectively reducing the production of acetic acid and preventing excessive bacterial growth. In some cases, glycerol may be more suitable for high-density fermentation of recombinant proteins in coli.

A case study from HZYMES BIOTECH shows that replacing glucose with an equivalent amount of glycerol during media optimization resulted in reduced bacterial growth but significantly increased the yield of exogenous proteins.

Nitrogen Sources

Nitrogen sources are used in the synthesis of cellular materials like amino acids, proteins, nucleic acids, and nitrogen-containing metabolites.

1.Nitrogen sources

include both organic and inorganic forms. The choice of nitrogen source type, concentration, C/N ratio, and supply strategy all need to be carefully optimized to significantly increase bacterial density and product yield.

A case study from HZYMES BIOTECH showed the effect of organic nitrogen sources on high-density fermentation of E. coli. The fermentation was initially carried out in M9 medium by flow addition of glucose and ammonia. The enzyme activity of the fermentation broth reached 152.5 U/mL (before optimization). After systematic optimization of carbon and nitrogen sources, with glycerol, peptone, and yeast extract as the main carbon and nitrogen sources, enzyme activity increased to 452 U/mL, and protein-specific activity reached 82 U/mL. Keeping the culture medium constant, adding glycerol at 150 g/L, peptone at 120 g/L, and yeast extract at 200 g/L further increased the enzyme activity of the fermentation broth to 532 U/mL. However, under these conditions, the specific activity of the protein decreased to 71 U/mL. Although the expression level of the protein increased, the proportion of active protein decreased (Optimization 2).

2.Organic nitrogen sources

can vary greatly between different manufacturers and batches, leading to significant differences in fermentation outcomes. For example, in shake-flask fermentation, different organic nitrogen sources and their specific impact on protein expression need to be tested.

In addition, adding amino acids or nitrogen-rich compounds can compete with host cell proteases, thereby reducing the degradation of target proteins.

 During the expression of exogenous proteins, degradation by host proteases can easily occur. Adding protein hydrolysates or peptone, which are rich in amino acids and peptides, serves as a nitrogen source for Escherichia coli growth and metabolism. Additionally, these components can act as substrates for proteases, competitively binding to cellular proteases and thereby reducing the degradation of the target protein.

Amino acids are the basic raw materials for protein synthesis. During the synthesis of the target protein, when amino acid supply is insufficient, E. coli preferentially synthesizes essential amino acids for cell growth. Supplementing specific amino acids can enhance the expression of exogenous proteins. When considering the specific addition of amino acids or amino acid-rich organic nitrogen sources, one can selectively supplement the amino acids that are in higher demand based on the amino acid composition of the target protein. Furthermore, dynamic monitoring of the consumption of various amino acids in the culture medium during fermentation can be performed using specific detection and analysis methods. This monitoring can be correlated with key indicators such as cell yield, recombinant protein expression, and protein activity to identify crucial amino acids. By adding these key amino acids to the culture medium or performing a comprehensive analysis of amino acids in complex nitrogen sources, suitable nitrogen sources can be selected to achieve the necessary supplementation of specific amino acids

Inorganic Salts and Trace Elements

Inorganic salts help maintain pH, osmotic pressure, and are involved in the synthesis of cellular membranes and nucleic acids. Trace elements, though required in very small amounts, are crucial for microbial metabolism and product synthesis.

Inorganic salts
Element	Function
P	High-energy molecules such as nucleic acids, nucleoproteins, phospholipids, coenzymes, and ATP, as well as pH buffering systems
S	Composition of sulfur-containing amino acids and vitamins
Mg	Related to the composition of enzyme active centers, ribosomes, cell membranes, nucleic acids, etc.
Ca	Cofactors for enzymes, maintaining enzyme stability
K	Cofactors for enzymes, maintaining cell osmotic pressure
Na	Maintain cell osmotic pressure, involved in cell transport, and the stability of certain enzymes
Trace elements
Element	Function
Co	Cofactors for some enzymes, component of vitamin B12
Fe	Components of bacterial cytochromes, cytochrome oxidase, and peroxidase
Mn	Cofactors or activators of some enzymes, influencing cell membrane stability and certain metabolic pathways
Zn	Components of some enzymes, especially those involved in protein synthesis and DNA replication
Cu	Participates in various oxidation-reduction reactions, components of certain metalloenzymes
Mo	Components of enzymes such as flavoproteins, nitrogenase, nitrate reductase
Ni	Participates in the activity of certain enzymes, particularly in amino acid metabolism

Both too low and too high concentrations of inorganic salts or trace elements are detrimental to cell growth and protein expression. When designing culture media, the optimal ratios and concentrations of various inorganic salts or trace elements must be considered comprehensively. Supplementation during cultivation can also be used to avoid the inhibition caused by high initial concentrations of nutrients while preventing the deficiency of limiting nutrients.

A case study from HZYMES BIOTECH demonstrated that by supplementing the fermentation medium with specific concentrations of cobalt (Co²⁺) and manganese (Mn²⁺), protein-specific activity increased by 330%, and the total activity of the cells increased by 500%. This indicates that two metal ions not only promote cell growth to a certain extent but also significantly increase the protein expression level per unit of biomass and the specific activity of the protein.

Cofactors

Some cofactors are vital for the growth of E. coli and for the expression of recombinant proteins.

1.Vitamins can act as growth-promoting factors, enhancing bacterial growth and reducing cell death.

2.Certain cofactors are essential for the biological activity of recombinant enzymes. By analyzing the protein structure and optimizing the addition of key cofactors, improvements in the quality and yield of recombinant enzymes can be achieved.

3.The addition of glycerol, sorbitol, arabinitol, myo-inositol, trehalose, amino acids, and amino acid derivatives can act as auxiliary factors to regulate osmotic pressure, improve cell membrane permeability, promote the soluble expression of recombinant proteins, and increase protein yield.

For instance, in one of HZYMES BIOTECH’s projects, the addition of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) as cofactors in the fermentation medium significantly enhanced protein activity and fermentation performance.

2.Optimization Strategies for E. coli Fermentation Medium

Designing and optimizing fermentation medium is a complex process involving multiple factors that often interact with one another. It requires careful selection of materials, adjustment of process parameters, and evaluation of final product performance.

Optimization Process of Medium:

• Literature research and medium classification to gather different medium combinations.
• Screening and selecting the most promising media combinations.
• Using statistical methods to design multi-factor experiments and analysingresults to find the optimal medium.
• Verification of the optimal formula.

In one of HZYMES BIOTECH ‘s projects, by optimizing the medium and fermentation conditions for a recombinant enzyme expressed in E. coli, enzyme activity increased from an initial 26.46 U/mL in shake-flask experiments to over 4200 U/mL in a high-density fermentation tank.

3.HZYMES BIOTECH’s Recombinant Protein Fermentation CMO Services

As a leader in the specialty enzyme industry, HZYMES BIOTECH has extensive experience in the development and production of recombinant proteins. We excel in both E. coli and yeast expression systems and have developed a series of highly adaptable fermentation processes tailored to different expression systems. With experience across 5L-60T production lines, we can meet production needs at all stages, from small-scale to industrial-scale production.