Corynebacterium glutamicum is widely used in the production of amino acids. C. glutamicum possesses seven sigma factors, among which SigD is responsible for the transcription of genes involved in the synthesis of mycolic acid (MA) and its derivatives, the unique cell envelope of C. glutamicum. To understand the influence of MA synthesis on amino acid production and membrane phenotype of C. glutamicum, the expression of sigD gene and some mycolyltransferase genes, i.e., cmt1, cop1 and cmt2, were regulated by several growth-regulated promoters in this study. Except for 2 mutant strains of Pcg3096-sigD and Pcg1633-cop1, the growth and 4-hydroxyisoleucine (4-HIL) titer of most modified strains did not change significantly. But the 4-HIL titer of PodhI-sigD strain increased by 20.73

To explore an l-isoleucine (Ile)-induced biosensor for down-regulation of Ile synthesis pathway and enhancement of 4-hydroxyisoleucine (4-HIL) production in Corynebacterium glutamicum SN01. Four Ile-induced riboswitches (IleRSN) with different strength were screened from mutation library based on TPP riboswitch. Firstly, IleRSN were integrated into the chromosome of strain SN01 immediately upstream of ilvA gene. The 4-HIL titer of strains carrying PtacM-driven IleRS1 or IleRS3 (14.09 ± 1.07, 15.20 ± 0.93 g 4-HIL L−1) were similar with control strain S-D5I (15.73 ± 2.66 g 4-HIL L−1). Then, another copy of IleRS3-ilvA was integrated downstream of the chromosomal cg0963 gene in SN01-derived strain D-RS with down-regulated l-lysine (Lys) biosynthesis. The Ile supply and 4-HIL titer increased in ilvA two-copy strains KIRSA-3-D5I and KIRSA-3-9I, and Ile concentration was maintained less than 35 mmol L−1 under the control of IleRS3 during fermentation. The resulting strain KIRSA-3-9I produced 22.46 ± 0.96 g 4-HIL L−1. The screened IleRS was effective in the dynamic down-regulation of Ile synthesis pathway in C. glutamicum, and IleRSN with different strength can be applied in various conditions.

Corynebacterium glutamicum, an important industrial producer, is a model microorganism. However, the limited gene editing methods and their defects limit the efficient genome editing of C. glutamicum. To improve the screening efficiency of second-cross-over strains of traditional SacB editing system, a universal pCS plasmid which harbors CRISPR-Cpf1 system targeting kan gene of SacB system was designed and established to kill the false positive single-cross-over strains remained abundantly after the second-cross-over events. The lethality of pCS plasmid to C. glutamicum carrying kan gene on its genome was as high as 98.6

4-hydroxyisoleucine (4-HIL) is a potential drug for diabetes and weight control. 4-HIL was produced by expressing ido gene in L-isoleucine (Ile)-producing Corynebacterium glutamicum . But L-lysine (Lys) was also accumulated as the main by-product in this recombinant strain SN02. To attenuate Lys synthesis, two genes in Lys synthetic pathway, i.e., ddh encoding the diaminopimelic acid dehydrogenase and lysE encoding the specific Lys exporter were deleted in SN02. However, the deletion of ddh increased 4-HIL titer by 28.1%, but did not decrease Lys content; while the deletion of lysE significantly reduced Lys content by 66.7%, but 4-HIL titer also decreased by 19.3%. Therefore, we carried out transcriptome analysis to reveal the global variation in these mutants. Deletion of ddh and lysE (especially lysE ) enhanced the transcription of key enzymes in succinylase branch of Lys synthesis pathway (DapD and DapC) and several enzymes involved in succinyl-CoA accessibility (SucC, SucD and OdhI), suggesting the compensatory synthesis of Lys via succinylase branch. In addition, the transcription of ilvBN in Ile synthesis pathway was improved, while the transcription of some genes in the 2-methylcitrate cycle and inositol metabolism pathway was weakened in these mutants. Mere deletion of ddh enhanced the transcription of aceA , ppc and pck , thus promoting oxaloacetate supply and 4-HIL synthesis. Deletion of lysE affected the transcription of some stress-related genes and transporter genes, suggesting that this mutant would be under stress, thus attenuating its 4-HIL synthesis. These findings will be helpful for systematic microbiology and bio-manufacturing of C. glutamicum .