Creation of an Industrial Bacillus thuringiensis Strain With High Melanin Production and UV Tolerance by Gene Editing

Abstract

Bacillus thuringiensis produces insecticidal crystal proteins (ICPs) which exhibit strong insecticidal toxicity. But when used in the field, ICPs would be destroyed by ultraviolet (UV) radiation in sunlight, thus decreasing the insecticidal activity and shortening the persistence. To improve the duration of B. thuringiensis preparations, we endowed a highly toxic industrial B. thuringiensis HD-1 with UV tolerance by making it produce melanin, a pigment that absorbs UV radiation. In B. thuringiensis, melanin is derived from homogentisate (HGA), an intermediate in the tyrosine pathway. And the absence of homogentisate-1,2-dioxygenase (HmgA) will lead to the formation of melanin. In this study, we used the CRISPR/Cas9 system to knock out the hmgA gene and obtained a melanin-producing mutant HD-1-ΔhmgA from strain HD-1. The melanin yield by mutant HD-1-ΔhmgA reached 3.60 mg/mL. And the anti-UV test showed that melanin serves as a protection to both the organism and the ICPs. After UV irradiation for 3 h, mutant HD-1-ΔhmgA still had an 80% insecticidal activity against the cotton bollworm, Helicoverpa armigera, while the control line only had about 20%. This study creates a light-stable biopesticide prototype based on a classic industrial strain that can be applied directly and takes the melanin-producing strain as a concept to improve the preparation validity.

Whole Genome Resequencing Revealed the Effect of Helicase yqhH Gene on Regulating Bacillus thuringiensis LLP29 against Ultraviolet Radiation Stress

“…UV was found to be able to significantly reduce the survival rate of Bt strains, but there were variations between different Bt strains ( Figure 6 B). Consistent with previous results, UV could affect the growth of microorganisms [ 17 , 36 ], which indicated that yqhH helicase could play a role in the repair of cells after damage. However, the knockout mutant was more sensitive to UV and grew more slowly than the other Bt strains harboring yqhH ( Figure 6 B).…”

“…In Bacillus , melanin can attenuate the effect of UV exposure on Bt insecticide products and has been used as a protective agent [ 9 ]. Currently, several strains have been screened for the production of high levels of melanin, which can effectively increase the UV resistance of the strain [ 15 , 16 , 17 ]. In addition, algae and cyanobacteria have evolved various avoidance and repair mechanisms to protect themselves against the damaging effects of UV radiation to acclimate to enhanced UV-B radiation.…”

Urea Cycle of Bacillus thuringiensis Affects Its Survival under UV Stress

“…22 At present, there are many studies focusing on improving the UV resistance of Bt, such as the photoprotection of melanin. 8,9 However, there are few studies on the anti-UV mechanism of Bt. Therefore, understanding the mechanism of Bt resistance to UV will be conducive to the long persistence of Bt in the field.…”

“…Thus, there are also a considerable number of research studies focused on improving the UV resistance of Bt. Most of these studies are based on the photoprotection of melanin, mainly by increasing the production of melanin synthesis to increase Bt resistance to UV stress, , while there are few studies on the anti-UV mechanism of Bt. In our previous study, we realized that the changes in several metabolic pathways might affect the resistance of Bt to UV stress .…”

Characterization of Bacillus thuringiensis bacteriophages: morphogenesis, lytic potentials & inter-simple sequence repeat (ISSR) analysis

“…There is a great number of B. thuringiensis subspecies (about 83%) that contain lysogenic phages, which can dramatically diminish the crop yield or result in a total loss (Yuan et al 2018). Using B. thuringiensis as a bio-pesticide is a successful protocol, however still experiences some di culties that sometimes shorten their application, for examples soil parameters, environmental temperature and pH, rain full, and ultraviolet (UV) uctuations can cause degeneration of the ICPs of at least shorten their validity (Turabik et al 2014;Zhu et al 2022) Different strains of B. thuringiensis showing different ICPs spectra and ability, and of course, the strains that have the highest insecticidal ability selected for application such as B. thuringiensis serovar kurstaki HD-1used to produce biopesticides known as Dipel, highly toxic against lepidopteran pests (Kati et al 2007;Sanahuja et al 2011).…”