Leuconostoc citreum has been used as a starter culture for industrial kimchi fermentation in Korea. The lactic acid bacterium produces dextan using sucrose in vegetables. However, dextran synthesis often causes unpleasant mouse feeling to the consumers. The aim of this study was to develop no dextran-producing L. citreum by employing DNA-free genome editing tools of CRISPR-Cas. For this, preassembled Cas9 protein and sgRNA targeting dextransucrase gene were transformed into competent cells of L. citreum by electroporation. As result, mutants were successfully obtained on sucrose agar medium which do not produce dextransucrase or dextran. Sequencing analysis showed that one nucleotide in the mutant DNA was deleted and it resulted in gene knock-out by a frame shift. When the mutant strain was inoculated as a starter in kimchi (dongchimi) fermentation, the fermentation characteristics such as viable cell counts, pH, and metabolites were not significantly different compared to the wild type strain. Whereas, dextran content in dongchimi inoculated with mutant strain was significantly decreased after fermentation. This study highlights that DNA-free genome editing tools using preassembled Cas9 and sgRNA can be used to successfully knock-out target gene in L. citreum.

Development of DNA-free genome editing technology for no dextran producing Leuconostoc citreum using CRISPR-Cas9 system

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ACS Fall 2022

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