Improving and Streamlining Gene Editing in Yarrowia lipolytica via Integration of Engineered Cas9 Protein.

The oleaginous yeast Yarrowia lipolytica is a prominent subject of biorefinery research due to its exceptional performance in oil production, exogenous protein secretion, and utilization of various inexpensive carbon sources. Many CRISPR/Cas9 genome-editing systems have been developed for Y. lipolytica to meet the high demand for metabolic engineering studies. However, these systems often necessitate an additional outgrowth step to achieve high gene editing efficiency. In this study, we introduced the eSpCas9 protein, derived from the Streptococcus pyogenes Cas9(SpCas9) protein, into the Y. lipolytica genome to enhance gene editing efficiency and fidelity, and subsequently explored the optimal expression level of eSpCas9 gene by utilizing different promoters and selecting various growth periods for yeast transformation. The results demonstrated that the integrated eSpCas9 gene editing system significantly enhanced gene editing efficiency, increasing from 16.61% to 86.09% on TRP1 and from 33.61% to 95.19% on LIP2, all without the need for a time-consuming outgrowth step. Furthermore, growth curves and dilution assays indicated that the consistent expression of eSpCas9 protein slightly suppressed the growth of Y. lipolytica, revealing that strong inducible promoters may be a potential avenue for future research. This work simplifies the gene editing process in Y. lipolytica, thus advancing its potential as a natural product synthesis chassis and providing valuable insights for other comparable microorganisms.

Cell-free supernatant of Bacillus velezensis suppresses mycelial growth and reduces virulence of Botrytis cinerea by inducing oxidative stress.

As a notorious pathogenic fungus, Botrytis cinerea has been reported to infect more than 1400 species of plants and cause postharvest gray mold of numerous economic fruits, leading to substantial economic losses. Traditional chemical fungicides in pathogen control have potential issues regarding environmental pollution, disease resistance and human health. More safety and efficacious prevention technique of postharvest gray mold are in urgent demand. This study aims to investigate the potential function and mechanism of Bacillus velezensis to control gray mold for harvested fruits. The results showed that the cell-free supernatant (CFS) generated from B. velezensis strain A4 was able to inhibit spore germination, germ tube elongation and hyphal growth of B. cinerea in vitro, and impair the pathogenicity of B. cinerea on the four tested fruits. Further analysis demonstrated that CFS significantly reduced the expression of genes associated with growth and pathogenicity and weakened the ability of B. cinerea spores to penetrate plant cell walls in a dose-dependent manner. Moreover, the CFS destroyed the membrane of hyphae, resulting in exosmosis of cell contents and caused hyphal cells to accumulate excessive reactive oxygen species (ROS), leading to hyphal oxidative damage. Our findings indicate that B. velezensis CFS can damage B. cinerea mycelial cells by promoting excessive accumulation of ROS to realize its biological control function.