Background

Overview of Random Mutagenesis

In terms of reducing fermentation costs, it is easier to select a strain that can synthesize a higher proportion of products using the same amount of raw materials than to improve the fermenter design engineering. By altering the genetic sequence (gene recombination and mutation), strains can be affected to produce key biosynthetases, improving microbial strains, resulting in the production of excess metabolites to the level required for industrial use, reducing production costs. It is important to note that in addition to modification, the successful development of strains depends on effective screening and identification methods. Random mutagenesis is a common method for strain improvement. After inducing the mutation, the strains were randomly selected and tested for their ability to produce the target metabolite. Screening a large number of mutants usually identifies improved mutants. In addition, compared to genetic engineering, it can reap benefits in the shortest time and maintain this improved effect for several years.

However, a disadvantage of the random selection method is that it is not targeted and non-specific for mutation types, so many strains need to be screened to isolate improved mutants in a mixed population. The process of selecting improved strains has been successfully used in many industrial processes. It involves repeated applications of three basic primordials: (1) mutagenesis of populations to induce genetic variation, (2) random selection and screening from viable populations to find improved strains through small-scale model fermentation, and (3) determination of product fermentation solution/AGAR and evaluation of improved strains. The improved strain was obtained by each mutation, and then the parent strain was used for a new round of mutation, fermentation (liquid or solid) screening and determination. Repeat the above steps until the target strain appears.

Fig. 1. Random mutagenesis combined with adaptive evolution to improve the yeasts stress tolerance. (Wan, et al., 2024)

Creative BioMart Microbe strives to serve clients by providing random mutagenesis service that meet the industry and production expectations. Please feel free to contact us for more information.

Service Procedure

Platforms

Services Details

Atmospheric Room Temperature Plasma (ARTP) Mutagenesis

If you don't want to mutate with chemicals, or you never get the strains you want, Creative BioMart Microbe also offers a more efficient form of mutagenesis: ARTP mutagenesis. ARTP is a new RF discharge technology that uses inert gas discharge to generate a large number of high-energy active particles at atmospheric pressure and room temperature. Active particles can be effectively applied to the genetic material of cells and cause structural damage to DNA, and then use the repair mechanism of cells with high fault tolerance rate to generate a large number of mutation sites, and finally obtain a large capacity of gene mutation library. It has the characteristics of abundant mutation, safety and high efficiency.

Ultraviolet (UV) Mutagenesis

UV mutagenesis is formed by photochemical reactions, the efficiency of which depends on the wavelength, and Creative BioMart Microbe has extensive experience in mastering the optimal mutagenesis conditions for optimal mutant strains. UV mutagenesis is formed after the DNA bases directly absorb UV energy, which can produce only single nucleotide changes: base substitutions from cytosine (C) to thymine (T) at the dipyrimidine site, and tandem base substitutions from CC to TT. The mechanism of formation of these mutations involves translesion DNA synthesis (TLS) on UV-induced DNA base damage.

Ethyl Methane Sulfonate (EMS) Mutagenesis

EMS is a commonly used mutagen capable of generating genome-wide single nucleotide polymorphisms, resulting in DNA breakage, deletion or repair. EMS primarily induces GC to AT transitions, but it can also cause other types of mutations, such as small deletions or insertions, although at a lower frequency. After EMS mutagenesis, Creative BioMart Microbe ensures that mutagenesis strains are validated by multiple means to obtain more specific mutant strains. The mutation spectrum is relatively simple and predictable, making it a useful tool for genetic analysis.

In addition, other chemicals besides EMS can also induce mutations. Chemicals induced mutation: nucleic acid analogs such as bromodeoxyuridine (BrdU), hydroxylamine, alkylating agents, agents that form DNA adducts, DNA intercalating agents, DNA crosslinkers, etc.

Our Advantages

• Capable of effectively improving various types of strains covering the bacterial and eukaryotic system which are over 20,000 strains in microbial library.
• Our improved strains have the characteristics of rapid growth, genetic stability, reduction of cultivation cost, etc.
• Advanced strain culture technology.
• Optimized medium selection, biomass control and product induction.
• Cost-effective and high-quality products.

Case Study

Case Study 1: Successive rounds of mutagenesis triggered by ARTP, subjected to various selective forces, aimed at enhancing the yield of coenzyme Q10 in Rhodobacter sphaeroides.

In an investigation of enhancing coenzyme Q10 (CoQ10) biosynthesis in R. sphaeroides, the application of ARTP for iterative mutagenesis was explored. This was the inaugural use of a combination of selective agents, namely vitamin K3 (VK3), sodium sulfide (Na2S), and benzoic acid (BA), to exert multiple selection pressures. Following two cycles of mutagenesis coupled with screening processes, a derived mutant strain, designated R.S 17, was isolated. This strain exhibited an 80.37% increase in CoQ10 production. In fed-batch fermentation, the mutant achieved a CoQ10 concentration of 236.7 mg/L and a cell density of 57.09 g/L, marking a 22.1% increase in CoQ10 content compared to its progenitor strain.

Fig.
Fig. 2. Glucose consumption, CoQ10 production and biomass accumulation in fed-batch fermentations using R.S 17 and the parent strain. (Wang, et al., 2022)

Case Study 2: The wild-type Beauveria bassiana was induced by ultraviolet light to obtain hypervirulent EPF isolates.

Two mutants (named 6M and 8M) were obtained by exposing wild-type Beauveria bassiana ARSEF2860 to ultraviolet light to induce mutagenesis. The mutants showed higher tolerance to osmosis, oxidation and UV stress. The activities of protease, chitinase, cellulose and chitinase of the mutants were higher than those of the wild type. Both mutants and WT are compatible with matrine, spinetoram and chlorantranilprole. The results of insect bioassays showed that the two mutants were more virulent against S. frugiperda and the greater wax moth Galleria mellonella.

Fig. 3. Protease, lipase, cellulose of wild-type (WT) and mutants. (Sun, et al., 2023)

Case Study 3: UV and EMS induce the Fusarium incarnatum strain LD-3 to produce the optimal laccase-producing strain.

A total of forty-seven mutant strains were developed from the progenitor Fusarium incarnatum strain LD-3 following UV irradiation, and an additional seventeen mutants arose after treatment with EMS. Prominent among them was the mutant strain UC-14, which demonstrated the highest laccase yield, tripling that of the original LD-3 strain. When employing solid substrate tray fermentation with wheat straw and rice bran, the productivity of laccase by mutant UC-14 increased twofold, while the total organic matter (TOM) decreased fivefold in comparison to the wild strain LD-3. Scanning electron microscopy indicated that the slower conidiation process in the UC-14 mutant might be the key factor contributing to its enhanced laccase output.

Fig. 4. Comparison of control isolate LD-3 and mutant isolate UC-14 under solid substrate tray fermentation using rice bran as a solid substrate. (Chhaya, et al., 2019)

FAQs

References:

1. Wan Z.; et al. Engineering industrial yeast for improved tolerance and robustness. Crit Rev Biotechnol. Published online March 19, 2024.
2. Wang Y.; et al. Iterative mutagenesis induced by atmospheric and room temperature plasma treatment under multiple selection pressures for the improvement of coenzyme Q10 production by Rhodobacter sphaeroides. FEMS Microbiol Lett. 2022;368(21-24):fnab154.
3. Sun YX.; et al. UV-induced mutagenesis of Beauveria bassiana (Hypocreales: Clavicipitaceae) yields two hypervirulent isolates with different transcriptomic profiles. Pest Manag Sci. 2023;79(8):2762-2779.
4. Chhaya U, Gupte A. Studies on a better laccase-producing mutant of Fusarium incarnatum LD-3 under solid substrate tray fermentation. 3 Biotech. 2019;9(3):100.