Abstract
Corynebacterium glutamicum is one of the important industrial microorganisms for production of amino acids and other value-added compounds. Most expression vectors used in C. glutamicum are based on inducible promoter (P tac or P trc ) activated by isopropyl-β-D-thiogalactopyranoside (IPTG). However, these vectors seem unsuitable for large-scale industrial production due to the high cost and toxicity of IPTG. Myo-inositol is an ideal inducer because of its non-toxicity and lower price. In this study, a myo-inositol-inducible expression vector pMI-4, derived from the expression vector pXMJ19, was constructed. Besides the original chloramphenicol resistance gene cat, multiple cloning sites, and rrnB terminator, the pMI-4 (6,643 bp) contains the iolR q cassette and the myo-inositol-inducible promoter P iolT1 . The pMI-4 could stably replicate in the C. glutamicum host. Meanwhile, the non-myo-inositol degradation host strain C. glutamicumΔiolGΔoxiCΔoxiDΔoxiE for maintaining the pMI-4 was developed. Overexpression of hemA M and hemL using pMI-4 resulted in a significant accumulation of 5-aminolevulinic acid, indicating its potential application in metabolic engineering and industrial fermentation.
Highlights
As a non-pathogenic Gram-positive bacterium, Corynebacterium glutamicum has been widely used in industrial biotechnology for the production of several million tons of amino acids annually, especially L-glutamate, L-lysine, and L-valine (Hasegawa et al, 2012; Eggeling and Bott, 2015; Wendisch, 2020)
To verify the non-toxicity of myo-inositol to cell growth, C. glutamicum ATCC 13032 cells were cultivated in a medium containing different concentrations of myo-inositol (0.1–100 mM)
PiolT1 is a myo-inositolinducing promoter, which is repressed by the repressor IolR in the absence of myo-inositol, but was active in the presence of myo-inositol (Figure 1)
Summary
As a non-pathogenic Gram-positive bacterium, Corynebacterium glutamicum has been widely used in industrial biotechnology for the production of several million tons of amino acids annually, especially L-glutamate, L-lysine, and L-valine (Hasegawa et al, 2012; Eggeling and Bott, 2015; Wendisch, 2020). Benefiting from the genome annotation of C. glutamicum and the availability of molecular biology techniques and tools, significant progress has been made in designing production strains by metabolic engineering via rational approaches (Ikeda and Nakagawa, 2003; Nesvera and Patek, 2011; Suzuki and Inui, 2013; Wang et al, 2021). Several vectors for gene overexpression in C. glutamicum have been developed, such as pXMJ19, pEC-XK-99E, and pDXW8. Most of these expression vectors are based on inducible promoters (Ptac or Ptrc) regulated by isopropyl-β-D-thiogalactopyranoside (IPTG)
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