Efficient production of myo-inositol in Escherichia coli through metabolic engineering

Abstract

Background The biosynthesis of high value-added compounds through metabolically engineered strains has received widely attention in recent years. As an effective compound in pharmaceutical, cosmetic and food industry, myo-inositol (inositol) is mainly produced via a harsh set of chemical reactions from phytate. The proper distribution of carbon flux between cell growth and inositol production was a major challenge for constructing an efficient inositol-synthetic pathway. Recombinant E. coli strains have been constructed by metabolic engineering strategies to produce inositol, yet with a low yield. Therefore, construction of E. coli metabolically engineered strains with high stoichiometric yield will be attractive. Results In the present study, the recombinant E. coli strains with high stoichiometric yield (> 0.7 mol inositol/mol glucose) were obtained to efficiently synthesize inositol. Inositol was successfully biosynthesized after introducing two crucial enzymes, inositol-3-phosphate synthase (IPS) from Trypanosoma brucei , and inositol monophosphatase (IMP) from E. coli. Based on starting strains E. coli BW25113 (wild type) and SG104 ( ΔptsG::glk , ΔgalR::zglf , ΔpoxB::acs ), a series of engineered strains for inositol production were constructed by deleting the key genes pgi, pfkA or pykF . Furthermore, the plasmid expression systems of IPS and IMP were optimized, and the gene zwf was regulated to enhance stoichiometric yield. The highest stoichiometric yield (0.96 mol inositol/mol glucose) was achieved with the combined strain R15 of SG104, Δpgi , Δpgm , and RBSL5-zwf. Simultaneously, the engineered strain R04 reached high-density fermentation level in a 1-L fermenter by using glucose and glycerol as mixed carbon source. In the scale-up bioconversion in situ with R04, 0.82 mol inositol/mol glucose was produced by fed-batch within 23 h, corresponding to a titer of 106.3 g/L (590.5 mM). Conclusions The biosynthetic pathway of inositol from glucose in recombinant E. coli was optimized by metabolic engineering strategies. The metabolically engineered E. coli strains represent a promising method for future inositol production. This study provided an essential reference to obtain a suitable distribution of carbon flux between glycolysis pathway and product synthetic pathway.

Production of myo‐inositol: Recent advance and prospective

“…As G6P is the key node of central metabolism, only a small amount of G6P can be used as the precursor for inositol production. The difference is that the myo ‐inositol biosynthesis of S. cerevisiae is rigorously regulated and controlled, owing to the key enzymes are inhibited by myo ‐inositol and the synthesis of I1P is inhibited by at least three genes of OPI1, OPI2, OPI4, thus the titer of inositol produced by S. cerevisiae is only 9 g/L by OPI1 gene deletion and fermentation optimization, 53 while the titer of inositol produced by E. coli could reach to a high level of 106.3 g/L by synergetic utilization of glucose and glycerol as carbon sources 36 . The synthetic utilization of glucose and glycerol may be a hopeful method for high‐titer of myo‐ inositol production and high‐density fermentation.…”

“…It is presumed that the high yield of myo ‐inositol from glucose would not be available on metabolic engineering of strain. However, massive efforts have been devoted recently to improve myo ‐inositol production via the biosynthetic pathway of myo ‐inositol from different substrates 35,36 …”

“…Meanwhile, Tao and coworkers 36 had designed a myo ‐inositol biosynthesis pathway by synergetic utilization of glucose and glycerol in E. coli . Here, both improvements of G6P supply and optimization of high‐density fermentation were the key means to improve myo ‐inositol production.…”

“…However, massive efforts have been devoted recently to improve myo-inositol production via the biosynthetic pathway of myo-inositol from different substrates. 35,36 The great challenge for effective myo-inositol biosynthesis is the appropriate distribution of carbon flux. Two research teams have constructed similar microbial pathways for myo-inositol production (Figure 3).…”

Co-culturing experiments reveal the uptake of myo-inositol phosphate synthase (EC 5.5.1.4) in an inositol auxotroph of Saccharomyces cerevisiae

“…This knowledge enhances their commercial potential, especially as it pertains to inositol production. A recent research article has demonstrated the complexity involved in engineering inositol production in bacterial cells [ 50 ]. Given the importance of inositol in many industries, including those related to health, pharmaceuticals, and agriculture, information presented here will allow a more efficient production of inositol and an extracellular vesicle in which to transport it.…”