Production of inositol by slowing carbon flux in glycolysis pathway
The intake of glucose in E. coli involves two key enzymes: glucose kinase (glk) and protein-Npi-phosphohistidine-D-glucose phosphotransferase (ptsG). Glucose kinase converts glucose to G-6-P with high catalytic efficiency [26-28]. Our laboratory has developed an E. coli strain, SG104, by deleting ptsG and enhancing glk to increase glucose intake . E. coli BW25113 and SG104 were chosen as starting strains to construct host strains that slow carbon flux to glycolysis to enhance the supply of inositol precursor G-6-P. The key genes involved—pgi, pfkA and pykF—were respectively deleted (Fig. 1). Deletion of pgi directly enhances precursor G-6-P accumulation by blocking glycolysis. The pfkA redirected carbon flux in glycolysis pathway, and showed most of 6-phosphofructokinase activity . Previous study has shown that pykF, the major pyruvate kinase, is a regulatory factor of glycolysis . Our previous study indicated that TbIPS from Trypanosoma brucei and EcIMP from E. coli have high specific activity . As such, plasmid p01 expressing TbIPS and p02 expressing EcIMP were co-transformed into E. coli strains BW25113, SG104, R01, R02, R03, R04, R05 and R06 respectively to construct recombinant strains for inositol production.
The expression of IPS and IMP in different host strains was shown as Fig. 2a. Whole-cell bioconversions were performed using different strains to select an appropriate host strain for inositol production. After 10 h of bioconversion, 29.6 mM inositol was obtained using strain R04; a stoichiometric yield of 0.6 mol inositol/mol glucose was reached, and no residual glucose was observed (Fig. 2b). Compared with strain R01 in which pgi is deleted to block glycolysis, strain R04 derived from strain SG104 showed increased inositol production. The results showed that deletion of pgi was effective for accumulation of precursor G-6-P. Strains R05 and R06 showed faster glucose consumption than R02 and R03, but low inositol concentration was achieved (Fig. 2b).
Improvement of inositol production by optimizing plasmid expression systems
Plasmid expression systems are useful for the reconstruction of biosynthesis pathways and usually give a high yield of a target product . To investigate the effect of expression of TbIPS and EcIMP from a single plasmid, plasmids pR01 and pR02 were constructed (Fig. 3a) and respectively transformed into host strain R04 to produce inositol. The results showed that pR01 improved the production of inositol; 0.62 mol inositol/mol glucose was produced with a titer of 31.1 mM (Fig. 3b). The expression of key enzymes (TbIPS and EcIMP) is shown in Fig S1a.
The activity of IPS in the metabolic pathway is one of the most important factors . Therefore, ScIPS from Saccharomyces cerevisiae was introduced to enhance IPS activity in the inositol-biosynthesis pathway. Plasmids pR03 and p03 were constructed, and plasmid combinations pR01+p03, and pR03 were respectively transformed into host strain R04 to produce inositol. The cells transformed with pR01+p03 produced 0.71 mol inositol/mol glucose with a titer of 35.5 mM (Fig. 3c). The expression of TbIPS, ScIPS, and EcIMP is shown in Fig S1b.
Further strain optimization by regulating zwf and deleting pgm
To further enhance the stoichiometric yield of inositol, the strength of expression of gene zwf from the PPP was adjusted by replacement of its promoter or RBS. Strain R04 was chosen as the platform strain to construct seven host strains (R7 to R13), which were used as controls to evaluate the effects of blocking and enhancing the PPP, respectively. Expression of zwf was decreased in strains R08 to R12 by using different RBS strengths (RBSL1 to RBSL5).
The resulting host strains were then transformed with the plasmid combination pR01+p03. The expression of key enzymes (TbIPS, ScIPS, and EcIMP) in host strains (R04, and R07 to R15) is shown in Fig 4a. Strain R12 exhibited inositol production of 44.7 mM after 10 h of bioconversion, corresponding to a stoichiometric yield of 0.9 mol inositol/mol glucose (Fig. 4b). The gene pgm, encoding phosphoglucomutase, which acts in isomerization of G-6-P and glucose-1-phosphate, was deleted to construct host strains R14 and R15. The highest stoichiometric yield (0.96 mol inositol/mol glucose) was achieved in strain R15, corresponding to a concentration of 48 mM inositol and consumption of 50 mM glucose (Fig. 4b).
High-density fermentation through synergetic utilization of glucose and glycerol
To evaluate the fermentation capacity of recombinant strains, host strains R04, R12, R14 and R15 transformed with plasmids pR01+p03 were chosen for high-density fermentation using a modified inorganic salts medium containing glucose and glycerol. The stoichiometric yields produced by the four strains in shaken flasks were all >0.7 mol inositol/mol glucose (Fig. 4b). E. coli follows the glycolysis pathway as the central metabolic system for cell growth. However, glycolysis was blocked by deletion of pgi; thus glycerol was selected as a carbon source to enable cell growth.
The four recombinant strains were cultivated in modified inorganic salts medium with glycerol and glucose as a mixed carbon source. Strain R04 reached a density of OD600 = 135 after 48 h. However, the other three strains could not grow, or grew slowly, in our modified inorganic salts medium (Fig. 5a). The expression of key enzymes (TbIPS, ScIPS, and EcIMP) in strain R04 in the high-density fermentation is shown in Fig. S2. In both shaken flasks and high-density fermentation, strain R04 showed approximately equivalent stoichiometric yields (>0.7 mol inositol/mol glucose) without glucose residue (Fig. 5b). The other three strains (R12, R14 and R15) showed poor expression of key enzymes and low stoichiometric inositol yield (Fig. S3). The results indicated that the high-density fermentation of strain R04 could be improved through synergetic use of glucose and glycerol as carbon sources.
Scaled-up production of inositol using strain R04
Strain R04 reached high-density through synergetic use of glucose and glycerol. To comprehensively evaluate the overall production performance of inositol, scaled-up bioconversion using strain R04 was carried out in a 1-L fed-batch fermenter. After 25 h of bioconversion ex situ, 375 mM inositol was obtained, while 580 mM glucose was consumed. The stoichiometric yield was 0.65 mol inositol/mol glucose (Fig. 6a). 0.82 mol inositol/mol glucose was produced after 23 h of bioconversion in situ, while 720.5 mM glucose was consumed. The concentration of inositol reached 590.5 mM, corresponding to a titer of 106.3 g/L (Fig. 6b).