Strain Development Service
BOC Sciences offers strain development and strain improvement services to enhance their metabolic capacities. With over 20 years of experience in traditional strain technology, we are capable of providing our customers with ideal strains whose advantages include rapid growth, genetic stability, and reduction of cultivation costs. Our strain development and improvement services cover various microbial systems, including Escherichia coli, streptomyces, actinomyces, mycobacterium and Paracoccus, and the eukaryotic systems such as yeast (Pichia pastoris, saccharomyces cerevisiae), aspergillus, penicillium, acremonium, filamentous fungi and excavate.
BOC Sciences has successfully developed and improved various strains for our customers and helped them transform primitive strains into industrialized strains effectively.
Introduction
In nature, microorganisms provide various valuable bioactive products, yet they produce a minimum amount of metabolites for survival. These metabolites are not overproduced naturally. However, overproduction of these metabolites revealed significant enhancement in the chemical, pharmaceutical, food industries. Therefore, it is essential to improve these microbial strains for industrial products.
The science and technology of manipulating and improving microbial strains by enhancing their metabolic capacities are known as strain development or strain improvement. Strain improvement is meant for obtaining microbial isolates possessing certain desirable characteristics for solving a specific problem. Traditionally, strain improvement has been achieved by mutation, selection, cloning and genetic recombination. Overproduction of primary or secondary metabolites is a complex process, and successful development of improved strains requires the combination of knowledge in physiology, molecular biology, genetic engineering, and the design of creative screening procedures.
Application of Strain Development
Microorganisms produce various essential compounds for the industrial, such as small molecules essential for vegetative growth (primary metabolites), and inessential (secondary metabolites), large molecules such as proteins, nucleic acids, carbohydrate polymers and more. Regulatory mechanisms have evolved in microorganisms that enable a strain to avoid an excessive production of metabolites; thus strain improvement programs are required for commercial application. The strain improvement strategy greatly increases fermentation productivity and significantly reduces costs. These genetic programs are also used for other goals, such as eliminating unwanted products or analogs, discovering novel structural compounds, and deciphering biosynthetic pathways.
Methods for strain development
Strain development in fermentation is a complicated process that combines traditional microbiological approaches with current genetic engineering and bioprocess optimization. The procedure involves various processes and approaches for improving the genetic and phenotypic characteristics of microbial strains.
Traditional methods are used for strain development
Traditionally, strain development is performed by random mutation and screening or selection, known as the classical technique. This empirical approach has been used for over 50 years and has a proven track record of effectiveness. The best-known example is the titer improvement for penicillin. Given its lengthy history, the classical technique remains the principal strain improvement strategy for each newly founded strain development program. The classical technique remains popular since it does not require prior understanding of the metabolite metabolic process, regulation, or transport. Another explanation is that advances in the accuracy and sensitivity of analytical equipment throughout time have significantly increased detection reliability and sensitivity. Furthermore, automation and downsizing of screening methods have greatly decreased system variability while increasing screening throughput.
(1) The first stage entails the thorough selection of a parent strain, which can be either a wild-type strain, chosen for its naturally occurring desirable qualities, or a laboratory strain, recommended for its genetic stability and detailed characterization within scientific literature.
(2) Genetic manipulation techniques: To produce a vast range of genetic variety, mutations can be introduced deliberately or spontaneously. To produce this genetic variety, chemical mutagenesis, UV radiation, and transposon mutagenesis are frequently used techniques. Under selection pressures, microorganisms are cultivated to generate advantageous features, such improved resilience under process conditions or greater product production.
(3) Random screening: After being separated, mutation survivors are grown on either liquid or solid agar substrate to create separate colonies. In small-scale fermentation tanks, single colonies are selected at random and fermented. It is more probable to uncover mutants with a slight productivity gain rather than a high-producing, blockbuster mutation while looking for mutants with higher productivity. To detect mutants with a tiny titer increase, nevertheless, the screening method has to be sensitive and consistent enough to lessen the possibility of discovering fake mutants.
The process for selecting improved strains. (Parekh S., et al., 2000)
Genetic engineering for strain development
Genetic engineering is currently a common method of deriving better microbial strains. This strategy has a variety of benefits, such as the ability to specifically regulate both desired and helpful genes, the reduction of the number of mutants tested, and the creation of several beneficial mutations. It is necessary to have prior understanding of the metabolic process in order to apply this strategy, though. Furthermore, the availability of genetic tools is necessary for the manipulation of the generating organism.
Transformation is the most common technique for introducing DNA into bacteria. In order for transformation to occur, recipients must absorb plasmid DNA during a physiological period of competence, which typically happens at a certain growth stage. On the other hand, naturally occurring competence-based DNA absorption is typically ineffective. Chemical treatments of bacterial cells that promote DNA absorption can produce competence. Plasmid DNA absorption in Escherichia is facilitated by pretreating cells with either calcium chloride or rubidium chloride. Plasmid DNA transformation into the species Streptomyces, which makes most antibiotics, is more intricate than that of Escherichia. Protoplasts are prepared by lysozyme-treating mycelia to remove the majority of the cell wall. To facilitate DNA absorption, plasmid DNA is combined with protoplasts in the presence of polyethylene glycol (PEG). When double-stranded plasmid DNA is employed, substantial restriction barriers in some Streptomyces species prevent effective protoplast transformation. Sometimes using single-stranded plasmid DNA can solve this issue.
Conjugation is an additional technique for plasmid DNA introduction into microorganisms. This technique uses a donor strain that has the desired gene(s), the origin of transfer (oriT) on a plasmid, and the chromosomal genes encoding transfer functions. DNA is transferred when the donor and receiver come into short touch. Following conjugation, recipient cells become resistant to the antibiotic used to destroy the donor cells. The plasmid's antibiotic resistance marker is used to identify recipient cells that will receive the transplanted plasmid.
An alternate technique to PEG-mediated DNA uptake is electroporation. When recipient cells are exposed to ultrasonic pulses in the presence of plasmid DNA, the cells become electrocompetent. The electroshock causes transient holes in the cell membrane, which open up for DNA absorption. By introducing new genes or altering existing ones, advanced molecular tools like plasmids, CRISPR-Cas9, and other gene-editing technologies are used to improve the strain's capabilities.
Industrial production model. (Liu J., et al., 2022)
Optimization of Fermentation Conditions for strain development
Media optimization: Carbon, nitrogen, and minerals are the three essential ingredients that bacteria need to develop. Extra nutrients like vitamins, nucleotides, amino acids, or even specialized chemicals could be needed, according to the organism and the conditions of the fermenter used for growth and metabolite formation. A variety of sources of nitrogen and carbon are available for selection. While some are found in relatively pure forms, others, like the byproducts of the food and agriculture sectors, are found in more complicated forms. While more costly, pure or premium raw materials offer greater consistency among batches and vendors. The quality of the agriculture and food sectors' outputs is frequently inconsistent. High-value products like therapeutic proteins are often made from pure or superior-quality carbon and nitrogen sources, while low-cost and high-volume commodity products like organic acids or bulk chemicals are made from less expensive raw materials. The development of fermentation medium requires careful consideration of lot uniformity, availability, and cost. Furthermore, fermentation medium that make downstream processing easier offer an extra benefit.
Fermentation process scale-up: Scaling up a fermentation process is the process of moving it from a laboratory to an industrial setting. Replicating the ideal circumstances under which the enhanced strains are grown in the lab in big production fermenters is the aim of the scale-up process. The process involves managing several environmental factors, such as power supply, capacity to mix, transfer of oxygen, shear stress, heat transmission, sterilization of medium, and preparation of seed cultures.
Our Advantages
Over 20 years of experience in traditional strain breeding.
Provision of ideal strains characterized:
- Rapid growth
- Stabilized Genetic
- Non-toxic to humans
- Cost-effective substrates
- Downstream processing interferences elimination
- Improved carbon and nitrogen usages
- Reduced cultivation cost
- Shorten fermentation period
Advanced strain culture technology.
Optimized medium selection, biomass control and product induction.
Improved feedstock and feed regime.
Complete strain expression systems cover bacterial systems and eukaryotic systems.
Our Methods
Production strain choice and optimization
Developing an appropriate medium for fermentation
- Chemicals induced mutation
- Radiation induced mutation
- Site-directed mutagenesis
Genetic recombination is the exchange of genetic material between different organisms. There are three recombination techniques: transduction, transformation and conjugation
High-throughput screening and analytical technologies
- Agar plate-based screening
- Fluorescence-activated cell sorting
- Droplet microfluidic screening
Project Workflow
- Customer advisory
- Project discussion
- Selection of microbial host
- Strain optimization: cloning, gene editing, mutation, screening, etc.
- Evaluation of the novel strain, transform primitive strains into industrialized strains
- Project delivery
References
- Liu J., et al., Industrial production of L-lysine in Corynebacterium glutamicum: Progress and prospects, Microbiological Research, 2022, 262: 127101.
- Han L., et al., Development of improved strains and optimization of fermentation processes, Microbial processes and products, 2005: 1-23.
- Mans R., et al., Under pressure: evolutionary engineering of yeast strains for improved performance in fuels and chemicals production, Current opinion in biotechnology, 2018, 50: 47-56.
- Parekh S., et al., Improvement of microbial strains and fermentation processes, Applied microbiology and biotechnology, 2000, 54: 287-301.
