Which microbial host is best for your process or project?

An Overview of Alternative Microbial Hosts for Recombinant Bioactive Production: Benefits and Industrial Use Cases

Selecting the right microbial host is a major step for any biotechnology project, whether you are producing food-grade enzymes, pharmaceutical ingredients or industrial bioproducts. Your choice of production host can affect your project’s yield, product quality, scalability and regulatory approval with administrations such as the FDA. In this article, we share how to navigate these choices while analysing the advantages and use cases of our four IsoChassis^TM microbial hosts. These strains include Escherichia coli, Bacillus subtilis, Pichia pastoris (Komagataella phaffii) and Saccharomyces cerevisiae. 

Bacteria for Industrial Production

Using E. coli

Escherichia coli has been the most widely utilised organism for recombinant protein production since 1977^[1]. This platform is a rapid, cost-effective and scalable choice for simple systems. Notably, industries extensively use BL21(DE3) for the production of bioproducts such as biofuels^[2].

Industrial advantages of choosing E. coli as your microbial host:

1. Toxic and low yielding proteins: Our E. coli strains are optimized for the production of historically low titre proteins. Additionally, E. coli is highly resilient against toxic product formation. This resistance is useful when synthesising antibiotics, biopesticides and solvents, which can be harmful to other strains. 
2. Quality: We offer protease deficient strains, with optional co-expressed chaperones, disulfide bond formation and rare codon expression. Our modified strains ensure recombinant protein and biologic quality.
3. Low temperatures: Particularly useful for overcoming misfolding and solubility issues, we offer E.coli  strains which operate efficiently at temperatures of ~54^oF. 
4. Constitutive and inducible promoters: We can tightly regulate gene expression using our tuneable IPTG induction ΔlacY strain. Alternatively, production can be "always on" with a constitutive promoter depending on partner requirements. 
5. Metabolic and chromosomal engineering: We metabolically and chromosomally engineer all our E. coli strains for high-yield production, minimal byproducts, and improved purity. 
6. Isolation: Our range of tags allow for periplasmic and extracellular secretion, as well as isolation of target heterologous proteins. Combined these additions simplify the downstream purification process. 
7. Yield: We achieve greater production per strain using a variety of replicative high copy number vectors. 

Using B. subtilis ^(GRAS)

Similar to E. coli, B. subtilis has been extensively researched and characterised. Industrial interest in B. subtilis emerged due to its natural ability to secrete large amounts of proteins directly into the culture medium. Today, B. subtilis is used in the manufacture of proteases, amylases, and other industrial enzymes, as well as in probiotic and agricultural applications^[3]. Our B. subtilis expression hosts are designed for robust, high-yield extracellular protein production and are particularly well-suited for applications requiring rapid growth with endotoxin-free secretion.

Industrial advantages of choosing B. subtilis as your microbial host: 

1. Purification and yield: As opposed to Gram-negative bacteria, external secretion is much simpler in Bacillus. Therefore, alongside our expression machinery, B. subtilis has an impressive ability to secrete up to 25 g/L of protein into the culture medium, which is extremely efficient for purification purposes. 
2. Plasmid and genomic integration: We offer a selection of high copy number and genomic vectors depending on partner requirements. 
3. Adaptive metabolism: Forming spores allows B. subtilis to survive in extreme conditions (e.g. atypical temperatures, low moisture and toxic environments)^[4]. 
4. Quality: We have strains derived from B. subtilis Marburg which are protease deficient, ensuring consistently high end product quality. 
5. Controlled expression: Both constitutive and inducible promoters allow for finetuned expression.
6. Short fermentation cycle: B. subtilis is known for its ability to grow rapidly, with a fermentation cycle time as low as 24 hours^[5].

Yeast for Industrial Production

Using Pichia pastoris (Komagataella phaffi) ^(GRAS)

Researchers in France first isolated Pichia in 1920^[6]. Discoveries between 1950 and the mid 1990s uncovered the variety of species we know of today. Of these, Pichia pastoris is known for its effectiveness at recombinant expression^[7]. Our Pichia pastoris expression platform specialises in high titre enzyme production, which includes extracellular secretion and complex eukaryotic post-translational modifications. As such, many sectors, particularly food and ingredients, have employed our system.

Industrial advantages of choosing Pichia as your microbial host:

• Antibiotic free: Antibiotic use is often banned in the food and agritech sectors due to strict regulations. We instead select for our auxotrophic mutants by including their sole carbon source on a vector containing your target sequence. We have developed this system over numerous years as an effective alternative to antibiotic resistance markers.
• Methanol-free: Pichia usually uses methanol as its carbon source, however methanol's combustion risk poses significant challenges during scaled chemical manufacturing. We have therefore created multiple systems which instead rely on alternative feedstocks. An example is in biodiesel manufacturing where waste-products can be utilised by the production strain in a cyclical fashion.
• Quality and yield: Pichia integrates DNA into its genome, avoiding inconsistencies seen with plasmid-based systems. This ensures consistent quality of protein expression and yields at industrial scale. 
• Scalability: Pichia can be grown to very high cell densities in simple media, enabling cost-effective, large-scale protein production.  
• Purification: Pichia can secrete recombinant proteins into the culture medium, simplifying purification^[8,9]. 
• Minimal endotoxin production: Pichia does not produce endotoxins, which are harmful contaminants when synthesising therapeutic proteins. This makes it a more attractive system for producing biopharmaceuticals, where endotoxin contamination is a major concern
• Correct folding and Quality: Our proprietary protease deficient strains reduce protein degradation. Additionally, as a eukaryote, Pichia can perform key post-translational modifications such as glycosylation, disulfide bond formation, and proteolytic processing. This is particularly useful in biopharmaceuticals as it reduces immunogenicity. 

Using S. cerevisiae - Baker's yeast ^(GRAS)

Saccharomyces cerevisiae has been used for sugar fermentation from as early as 7000 BC in China^[10]. Since then, humans have routinely used S. cerevisiae for the fermentation food and beverages, making it the most studied eukaryote on Earth. 

Industrial advantages of choosing S. cerevisiae as your microbial host: 

1. Acidic conditions: Tolerance to pH as low as 3 makes S. cerevisiae excellent in situations where an acidic environment is required for fermentation and preservation. Some examples of this are in the production of many foods and beverages, waste treatment, bioremediation and certain pharmaceuticals^[11].
2. Ethanol production: S. cerevisiae is extremely efficient at producing ethanol, making it the key organism in the brewing of alcoholic beverages. In biomanufacturing, this ability also makes it an excellent choice for the synthesis of bioethanol. 
3. Quality and yield: Like Pichia, S. cerevisiae does not need to rely on plasmids for heterologous expression as it can easily integrate genetic cassettes into its genome, ensuring consistency of yield.
4. Simplified purification: Secretion of synthesised proteins into the medium makes downstream processing easier. 

Matching Platform to Project: Considerations When Choosing a Microbial Host:

• End use: Food-grade enzymes and nutritional proteins require GRAS hosts like B. subtilis, Pichia, and S. cerevisiae, with non-GMO and antibiotic-free options often favoured for international markets. For API production, you need hosts that are free from endotoxins and capable of handling the folding or post-translational modifications your molecule requires. 
• Technical demands. Some products require advanced folding, glycosylation, or secretion for purification. Yeast systems are often the first choice for these complex proteins, while E. coli is preferred for fastest and cost-effective production of simple peptides or proteins. 
• Regulatory requirements: GRAS status is non-negotiable for food and beverage applications. Pharmaceuticals require full documentation, traceability and safety data (including non-GMO status, if required) to streamline approval.
• Scale and economics: For high-volume, cost-sensitive projects, B. subtilis and S. cerevisiae provide proven, reliable and scalable production. Smaller-scale, high-value products like APIs, yeast or engineered E. coli often offer the best combination of yield and compliance. 

IsoChassis^TM within the IsoZym platform

Our IsoChassis^TM hosts can be seen as a modular part of our IsoZym production platform, meaning packages can either be carried out as standalone or integrated workflows. Learn more about IsoZym as an end-to-end solution and how IsoChassis^TM fits into its "Design Phase" here.

Selecting the correct industrial microbial host can save considerable costs down the line. Whether your project involves developing a food enzyme using Saccharomyces cerevisiae, boosting therapeutic protein yields with Pichia pastoris or producing industrial enzymes at scale with Bacillus subtilis, our team provides practical support at every step. If you would like to discuss E. coli, B. subtilis Pichia or S. cerevisiae as your microbial host contact us or enquire below.