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). The glk 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 . The E. coli BW25113 and SG104 were chosen as starting strains to construct host strains that slow carbon flux of glycolysis to supply precursor G-6-P. The key genes involving pgi, pfkA and pykF were deleted, respectively (Fig. 1). The pgi directly enhance precursor G-6-P accumulation by blocking glycolysis. The pfkA redirected carbon flux in glycolysis pathway, showing most of 6-phosphofructokinase activity . Previous study has showed the pykF with major pyruvate kinase activity was a regulatory factor of glycolysis . Our previous study indicated TbIPS from Trypanosoma brucei and EcIMP from E. coli have high specific activity . As such, the plasmid p01 expressing TbIPS and p02 expressing EcIMP were co-transformed in E. coli BW25113, SG104, R1, R2, R3, R4, R5 and R6 respectively to construct recombinant strains for inositol production.
The expressions of IPS and IMP in different host strains were shown as Fig. 2a. The whole-cell bioconversions with different strains were performed to select an appropriate host strain for inositol production. After 10 h of bioconversion, 29.6 mM inositol (highest concentration) was achieved by using R04, a stoichiometric yield of 0.6 mol inositol/mol glucose was reached without glucose residue (Fig. 2b). Compared with host strains R01 that deletes pgi to block glycolysis, R04 integrating SG104 character further increased inositol production. The results showed that the pgi was an effective target to accumulate precursor G-6-P. The strains R05 and R06 strains showed faster glucose consumption compared with R02 and R03, yet 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 biosynthetic pathways and usually give a high yield of a target product . To investigate the effect of expression orders of TbIPS and EcIMP within a single plasmid (Fig. 3a), the plasmids pR01 and pR02 were constructed and transformed into host strain R04 to produce inositol. The result showed that the plasmid pR01 contributed to the production of inositol, 0.62 mol inositol/mol glucose was produced with a titer of 31.1 mM (Fig. 3b). The expressions of key enzymes (TbIPS and EcIMP) were shown in Fig S1 a.
The overexpression of the key enzymes (TbIPS and EcIMP) contributed to the biosynthesis of inositol, and 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-biosynthetic pathway. The plasmids pR03 and p03 were constructed, and the plasmid combinations of pR01+ p03, and pR03 were separately transformed into host strain R04 to produce inositol. The results showed that 0.71 mol inositol/mol glucose was achieved with a titer of 35.5 mM by the double plasmids combination of pR01+p03 (Fig. 3c). The expressions of key enzymes (TbIPS, ScIPS, and EcIMP) were shown in Fig S1 b.
Further strains optimization by regulating zwf and deleting pgm
To further enhance the stoichiometric yield of inositol-biosynthetic pathway, the gene zwf strength in PPP was adjusted by replacement of promoter or RBS. The combined strain R04 was chosen as platform strain to construct seven host strains (R7 to R13). The strains R07 and R13 were used as controls to evaluate the effects of blocking and enhancing PPP, respectively. The gene zwf was weakened in the strains R08 to R12 by using different RBS strength (RBSL1 to RBSL 5).
The resulting host strains were then transformed with the plasmid combination pR01+p03. The expressions of key enzymes (TbIPS, ScIPS, and EcIMP) in host strains (R04, R07 to R15) were shown in Fig 4a. The recombinant strain R12 exhibited increased inositol production to 44.7 mM after 10 h of bioconversion, corresponding to stoichiometric yield of 0.9 mol inositol/mol glucose (Fig. 4b). The gene pgm encoding phosphorglucomutase acting on 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 recombinant strain R15, corresponding to concentration of 48 mM with consuming 50 mM glucose (Fig. 4b).
High-density fermentation through synergetic utilization of glucose and glycerol
To evaluate fermentation capacity of recombinant strains, the four host strains R04, R12, R14 and R15 with plasmids pR01+p03 were chosen for high-density fermentation by using inorganic salt medium. The stoichiometric yields of four recombinant strains in shake flasks level were all above 0.7 mol inositol/mol glucose (Fig. 4b). E. coli follows the glycolysis pathway as the central metabolism system to perform cell growth. However, glycolysis was blocked due of deleting pgi, thus glycerol was selected as a carbon source to supply cell growth.
The four recombinant strains were cultivated in inorganic salt medium by using glycerol and glucose as mixed carbon source. The recombinant strain R04 reached a density of OD600 = 135 after 48 h, yet other three strains could not grow or grew slowly in our optimized inorganic salt medium (Fig. 5a). The expressions of key enzymes (TbIPS, ScIPS, and EcIMP) in high-density fermentation level with recombinant strain R04 were shown in Fig. S2. The bioconversion of recombinant strain R04 in shake flask level and high-density fermentation showed the approximately equivalent stoichiometric yields (> 0.7 mol inositol/mol glucose) were achieved without glucose residue (Fig. 5b). The results indicated that the high-density fermentation of recombinant strain R04 could be improved through synergetic utilization of glucose and glycerol.
Scale-up production of inositol using recombinant strain R04
Recombinant strain R04 reached a high-density fermentation level through synergetic utilization of glucose and glycerol. To comprehensively evaluate the overall production performance of inositol, scale-up bioconversion using high-density fermentation strain R04 was carried out in a 1-L fermenter by fed-batch. After 25 h of bioconversion ex situ, 375 mM inositol was obtained, while 580 mM glucose was consumed. The stoichiometric yield reached 0.65 mol inositol/mol glucose (Fig. 6a). Surprisingly,, 0.82 mol inositol/mol glucose was achieved 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).