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A Myo-Inositol-Inducible Expression System for Corynebacterium glutamicum and Its Application.

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.

SUBMITTER: Lu N

PROVIDER: S-EPMC8634428 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Inositols (cyclohexanehexols) comprise nine isomeric cyclic sugar alcohols, several of which occur in all domains of life with various functions. Many bacteria can utilize inositols as carbon and energy sources via a specific pathway involving inositol dehydrogenases (IDHs) as the first step of catabolism. The microbial cell factory Corynebacterium glutamicum can grow with myo-inositol as a sole carbon source. Interestingly, this species encodes seven potential IDHs, raising the question of the reason for this multiplicity. We therefore investigated the seven IDHs to determine their function, activity, and selectivity toward the biologically most important isomers myo-, scyllo-, and d-chiro-inositol. We created an ΔIDH strain lacking all seven IDH genes, which could not grow on the three inositols. scyllo- and d-chiro-inositol were identified as novel growth substrates of C. glutamicum. Complementation experiments showed that only four of the seven IDHs (IolG, OxiB, OxiD, and OxiE) enabled growth of the ΔIDH strain on two of the three inositols. The kinetics of the four purified enzymes agreed with the complementation results. IolG and OxiD are NAD+-dependent IDHs accepting myo- and d-chiro-inositol but not scyllo-inositol. OxiB is an NAD+-dependent myo-IDH with a weak activity also for scyllo-inositol but not for d-chiro-inositol. OxiE on the other hand is an NAD+-dependent scyllo-IDH showing also good activity for myo-inositol and a very weak activity for d-chiro-inositol. Structural models, molecular docking experiments, and sequence alignments enabled the identification of the substrate binding sites of the active IDHs and of residues allowing predictions on the substrate specificity. IMPORTANCE myo-, scyllo-, and d-chiro-inositol are C6 cyclic sugar alcohols with various biological functions, which also serve as carbon sources for microbes. Inositol catabolism starts with an oxidation to keto-inositols catalyzed by inositol dehydrogenases (IDHs). The soil bacterium C. glutamicum encodes seven potential IDHs. Using a combination of microbiological, biochemical, and modeling approaches, we analyzed the function of these enzymes and identified four IDHs involved in the catabolism of inositols. They possess distinct substrate preferences for the three isomers, and modeling and sequence alignments allowed the identification of residues important for substrate specificity. Our results expand the knowledge of bacterial inositol metabolism and provide an important basis for the rational development of producer strains for these valuable inositols, which show pharmacological activities against, e.g., Alzheimer's disease, polycystic ovarian syndrome, or type II diabetes.

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A Myo-Inositol-Inducible Expression System for Corynebacterium glutamicum and Its Application.

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