Microorganisms with improved 1,3-propanediol and butyric acid production
A technology of propylene glycol and butyric acid, which is used in the production of 1,3-propanediol and butyric acid, Clostridium species consortium, and polytrimethylene terephthalate field, which can solve the problem of high cost, discontinuity, and unstable production strains, etc. question
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Embodiment 1
[0102] Example 1: Identification of mutations present in one clone that was an outlier from the 174P population
[0103] clone isolation
[0104] Clonal isolation on agar plates was initiated from continuous cultures of a population of Clostridium acetobutylicum strain DG1 pSPD5 type 174P that overexpressed the 1.3-propanediol operon from Clostridium butyricum and was adapted to be obtained from industrial plants with high concentrations. Glycerol (as described in patent application WO 2012 / 062832A1 ) medium that grows and produces PDO populations. Synthetic medium for isolation on agar plates contains per liter of deionized water: 25 g agar; 30 g commercial glycerol; 0.5 g KH 2 PO 4 ;0.5g K 2 HPO 4 ;0.2g MgSO 4 ,7H 2 O; 0.01g CoCl 2 6H 2 O; 99.8% acetic acid, 2.2ml; 1.65g NH 4 Cl; 23.03g MOPS; 0.16mg biotin; 32mg p-aminobenzoic acid; 0.028g FeSO 4 ,7H 2 O; 1 mg resazurin and 0.5 g cysteine. use NH 4 OH 6N to adjust the pH of the medium to 6.5.
[0105] After...
Embodiment 2
[0121] Example 2: Characterization of mutations identified in the genes glpK and hydA
[0122] a. Characterization of glycerol kinase wild-type (GlpK) and mutant (GlpK*) proteins
[0123] Three different point mutations were identified in the glpK gene in clone PD0557Vc05, resulting in the mutein GlpK* with H22Y, A347T and V466M (Table 2 above). To better characterize the effects of the mutations, we tested them individually or in combination. Therefore, the allele encoding the mutant glycerol kinase was cloned into the expression plasmid pPAL7 , and the obtained plasmid was transformed into Escherichia coli (E. coli) strain BL21(DE3) star to purify each mutant protein and perform biochemical experiments.
[0124] Table 3: Bacteria constructed and used to determine kinetic parameters of glycerol kinase mutants from PD0557Vc05 strain
[0125]
[0126] protein overproduction
[0127] Cultivation of seven strains 1 to 7 for protein overproduction was achieved in 2...
Embodiment 3
[0155] Example 3: Effects of GlpK* and HydA truncation on two production strains (Clostridium acetobutylicum DG1pSPD5 and The role of PD0001VE05c08) in producing PDO and BA
[0156] To confirm the effect of the mutations identified in clone PD0557Vc05 on the production of PDO / BA (see Example 2), we introduced the glpK* and hydA* alleles, alone or in combination, into two production strains that had no modifications at these genes .
[0157] The two recipient strains chosen for genetic modification are:
[0158] - DG1 pSPD5: Clostridium acetobutylicum strain DG1 pSPD5, described in the publication Gonzalez-Pajuelo et al., 2006
[0159] - PD0001VE05c08: Clostridium acetobutylicum strain DG1 pSPD5 isolated clone and adapted to high concentration of glycerol as described in patent application WO2012 / 062832
[0160] Mutations in the genes glpK* (triple mutant) and hydA* identified in the above strain PD0557Vc05 were introduced into the DG1 pSPD5 strain to generate strains 8, ...
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