The LacI-Type Transcriptional Activator NCgl2689 Directly Regulates an Operon Involved in <scp>l-</scp>Cysteine Synthesis by the Reverse Transsulfuration Pathway in <i>Corynebacterium glutamicum</i>

l-Cysteine is widely used in food, pharmaceutical, animal feed, and cosmetic industries. Microbial fermentation is an economically effective method for producing l-cysteine. However, research on the synthesis and regulation mechanism of l-cysteine through the reverse transsulfuration pathway in important amino acid producing cell factory <i>Corynebacterium glutamicum</i> is still limited. In this study, we demonstrated that NCgl2688 had cystathionine γ-lyase (CGL) activity (2.98 × 10² M^-1 s^-1), which played an important role in the conversion of cystathionine to l-cysteine by the reverse transsulfuration pathway. In the Δ<i>NCgl2689</i> strain, the mRNAs of <i>NCgl2689</i> and the l-cysteine biosynthetic gene <i>NCgl2688</i> were significantly reduced and the activity of <i>NCgl2688</i> was relatively low. Moreover, the expression of the <i>NCgl2688</i> or <i>NCgl2689</i> gene was significantly upregulated in response to H₂O₂ or l-methionine. The ability of NCgl2689 to bind to the promoter region of the <i>NCgl2689</i> and <i>NCgl2688</i> genes was confirmed through EMSA. Furthermore, the growth of the Δ<i>NCgl2689</i> strain was significantly inhibited by antibiotics, alkylating agents, or oxidants. This work reveals that NCgl2689 is the first regulator of CGL, converting cystathionine into l-cysteine in <i>Corynebacteriales</i>, and provides new insights into the regulatory mechanisms involved in the stress resistance and l-cysteine biosynthesis through the reverse transsulfuration pathway.