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

Abstract

Background: The biosynthesis of high value-added compounds using metabolically engineered strains has received wide attention in recent years. Myo-inositol (inositol), an important compound in the pharmaceutics, cosmetics and food industries, is usually produced from phytate via a harsh set of chemical reactions. Recombinant Escherichia coli strains have been constructed by metabolic engineering strategies to produce inositol, but with a low yield. The proper distribution of carbon flux between cell growth and inositol production is a major challenge for constructing an efficient inositol-synthesis pathway in bacteria. Construction of metabolically engineered E. coli strains with high stoichiometric yield of inositol is desirable. Results: In the present study, we designed an inositol-synthesis pathway from glucose with a theoretical stoichiometric yield of 1 mol inositol/mol glucose. Recombinant E. coli strains with high stoichiometric yield (> 0.7 mol inositol/mol glucose) were obtained. 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 was constructed by deleting the key genes pgi, pfkA and pykF. Plasmid-based expression systems for IPS and IMP were optimized, and expression of the gene zwf was regulated to enhance the stoichiometric yield of inositol. The highest stoichiometric yield (0.96 mol inositol/mol glucose) was achieved from recombinant strain R15 (SG104, Δpgi, Δpgm, and RBSL5-zwf). Strain R04 (SG104 and Δpgi) reached high-density in a 1-L fermenter when using glucose and glycerol as a mixed carbon source. In scaled-up fed-batch bioconversion in situ using strain R04, 0.82 mol inositol/mol glucose was produced within 23 h, corresponding to a titer of 106.3 g/L (590.5 mM) inositol. Conclusions: The biosynthesis 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 provides an essential reference to obtain a suitable distribution of carbon flux between glycolysis and inositol synthesis.

Synergetic Fermentation of Glucose and Glycerol for High-Yield N-Acetylglucosamine Production in Escherichia coli

“…However, when the competing pathways are related to the central metabolism, especially the EMP and PPP, application of this strategy becomes challenging due to their important effects on cell growth. Carbon cofeeding has been successfully used to balance growth and production metabolism, demonstrating the effectiveness of this strategy [ 18 , 19 ]. In this study, the carbon cofeeding strategy was successfully adopted for high-yield GlcNAc production.…”

“…Based on this strategy, production enhancements have been widely reported. For example, in myo-inositol fermentation, a creative strategy has been exploited for efficient inositol production (reaching as high as 106.3 g/L) by synergetic utilization of glucose and glycerol as carbon sources [ 18 ]. Additionally, the productivity of lycopene was significantly improved in the fed-batch culture of glycerol supplemented with glucose and arabinose, which was 11.7-fold higher than that without auxiliary carbon sources [ 19 ].…”

Industrially Relevant Enzyme Cascades for Drug Synthesis and Their Ecological Assessment

“…After its extraction, phytate is chemically dephosphorylated, which suffers from various drawbacks such as phosphorous waste, high production costs and difficult myo -inositol separation [ 21 ]. An alternative route is a cell-based production by metabolically engineered microorganisms such as Escherichia coli with a titer of 106.3 g L −1 (590.5 mM) and a yield of 0.82 mol mol −1 glucose in 23 h [ 51 ]. Notably, several in vitro synthetic routes were developed with sugar as starting materials such as starch [ 20 , 52 ], cellulose [ 53 ], xylose [ 54 ], sucrose [ 55 ] or glucose [ 56 ].…”

Myo‐Inositol in Fermented Sugar Matrix Improves Human Macrophage Function

“…Fermentation, optimized for high stoichiometry yield of myo-inositol, is known to be desirable in the pharmaceutical and food industry. [20,21] Interestingly, multiple sugar alcohols including inositols have been detected in FPP. [16] Inositols are natural polyols present in animal and plant cells either in its free form or as component of phospholipids or inositol phosphate derivatives.…”