An improved Bacillus subtilis cell factory for producing scyllo-inositol, a promising therapeutic agent for Alzheimer’s disease

Abstract

BackgroundBacillus subtilis 168 possesses an efficient pathway to metabolize some of the stereoisomers of inositol, including myo-inositol (MI) and scyllo-inositol (SI). Previously we reported a prototype of a B. subtilis cell factory with modified inositol metabolism that converts MI into SI in the culture medium. However, it wasted half of initial 1.0% (w/v) MI, and the conversion was limited to produce only 0.4% (w/v) SI. To achieve a more efficient SI production, we attempted additional modifications.ResultsAll “useless” genes involved in MI and SI metabolism were deleted. Although no elevation in SI production was observed in the deletion strain, it did result in no wastage of MI anymore. Thus additionally, overexpression of the key enzymes, IolG and IolW, was appended to demonstrate that simultaneous overexpression of them enabled complete conversion of all MI into SI.ConclusionsThe B. subtilis cell factory was improved to yield an SI production rate of 10 g/L/48 h at least. The improved conversion was achieved only in the presence of enriched nutrition in the form of 2% (w/v) Bacto soytone in the medium, which may be due to the increasing demand for regeneration of cofactors.

A second-generationBacilluscell factory for rare inositol production

“…To solve this problem, iolABCDEFHIJ, iolX, and iolR were deleted from the chromosome using a marker-free deletion technique 6 to yield strain MYI04. 7 As expected, MI was not wasted in MYI04, but the conversion rate of MI to SI was not improved. We accordingly attempted the overexpression of iolG and iolW, which encode the key enzymes for the conversion, under the control of strong and constitutively active promoters, even during stationary growth; the promoters were chosen from global B. subtilis transcriptome analysis data.…”

“…11 In the previous study, we showed that the reduction of scylloinosose catalyzed by IolW requiring NADPH could be the most important step in SI conversion and also that the reduced amounts of the major nutrient Bacto soytone drastically reduced SI conversion. 7 The results suggested that higher concentrations of Bacto soytone might stimulate NADPH regeneration. In addition, our preliminary observation suggested that inactivation of some of NADPH regenerating enzymes could result in decreased SI conversion even in the presence of higher concentrations of Bacto soytone.…”

A new-generation of Bacillus subtilis cell factory for further elevated scyllo-inositol production

“…As reported in a previous study, when 2% (w/v) Bacto soytone contained in the conversion medium was reduced, SI production was impaired significantly in strain KU106, although no severe effect on cell growth was observed [15]. Therefore, we tested the conversion in the medium containing a higher concentration of Bacto soytone, such as 4% (w/v), using the strains mentioned above.…”

Permeabilized Escherichia coli Whole Cells Containing Co‐Expressed Two Thermophilic Enzymes Facilitate the Synthesis of scyllo‐Inositol from myo‐Inositol

“…Whole‐cell catalytic reactions for SI production were performed at 5 mL scale at 55 °C for 6 h in 20 m m phosphate buffer (pH 7.0) containing 20 g L −1 MI and whole cells harboring pETDuet‐ idh ‐ sidh or pETDuet‐ sidh ‐ idh with OD 600 of 20. Then a same volume of acetonitrile was added to terminate the reaction and the concentration of SI was quantified by the method described previously with slight modification (see Supporting Information).…”

“…Compared to chemical methods, biological strategies are preferable for the synthesis of SI, even on an industrial scale, since they are environmentally benign and reaction conditions are generally mild. Bacillus subtilis cell factories were recently developed for the production of SI by modifications of inositol metabolism and culture conditions including knockout of several genes related toinositol metabolism, overexpression of key enzymes, and addition of nutrients (bacto soytone) in the medium . However, the conversion efficiency of these cell factories was limited by some factors such as metabolic factors and the efflux system of SI.…”