Abstract
Bacillus licheniformis is widely used to produce multiple enzymes and chemicals in industrial fermentation. It is also an organism that is hard to genetically manipulate, which is mainly attributed to its extremely low transformation efficiency. The lack of genetic modification technology severely limits its further application. In this study, an all-in-one conditional clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 plasmid was developed for B. licheniformis with the cas9 gene under the control of a xylose-inducible promoter. By means of this design, the expression of the cas9 gene could be repressed without xylose, which significantly improved the transformation ratio from less than 0.1 cfu/μg to 2.42 cfu/μg DNA. Compared with this conditional system, a constitutive overexpression system led to significant growth retardation in bacterial cells. Both the biomass and specific growth rate decreased greatly. After transformation, successful genome editing could be triggered by 0.5% xylose. When the α-amylase gene amyL was used as a genomic target, the efficiencies of its disruption using three different protospacer-adjacent motif (PAM) sequences were 64.3%, 70.9%, and 47.1%, respectively. Moreover, temperature plays a pivotal role in the function of the constructed CRISPR system. The maximum success rate reached 97% at 20 °C, while higher temperatures negatively impacted the function of the system. These results suggested that the design with a cas9 gene under the strict control of a xylose-inducible promoter significantly improved the success rate of genome editing in this host. This work contributes to the development of genetic manipulation and furthers the use of B. licheniformis as an efficient industrial workhorse.
Highlights
Bacillus licheniformis, which is a gram-positive bacterium of the phylum Firmicutes, plays a versatile role in the fermentation industry
The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) proteins, in addition to CRISPR RNA and transactivating CRISPR RNA, can recognize and introduce double-strand breaks (DSBs) in foreign nucleic acids with the aid of a protospacer-adjacent motif (PAM) sequence, which is the spacer region in the single guide RNA that is highly specific for the targeted gene [14,15]
When the cas9 gene was expressed during the process, with either a constitutive pattern (P43) or xylose induction, the transformation rate was clearly much lower than the control’s transformation rate and achieved with a silent cas9 design (Figure 2A) despite the plasmids being similar in size
Summary
Bacillus licheniformis, which is a gram-positive bacterium of the phylum Firmicutes, plays a versatile role in the fermentation industry. The CRISPR-associated (Cas) proteins, in addition to CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA), can recognize and introduce double-strand breaks (DSBs) in foreign nucleic acids with the aid of a protospacer-adjacent motif (PAM) sequence, which is the spacer region in the single guide RNA (sgRNA) that is highly specific for the targeted gene [14,15] This system is functional in both prokaryotes and eukaryotes with the difference being that a donor fragment is mandatory when performing genomic mutations in prokaryotes because most prokaryotic species lack the nonhomologous DNA end-joining (NHEJ) recombination system that is, in contrast, universal in eukaryotes [16]. The cas gene, encoding a DNA endonuclease, was under the strict control of a B. licheniformis xylose-inducible promoter, which significantly improved the success rate of genome editing in this host
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