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

Abstract

BackgroundA stereoisomer of inositol, scyllo-inositol (SI), has been regarded as a promising therapeutic agent for Alzheimer’s disease. However, this compound is relatively rare, whereas another stereoisomer of inositol, myo-inositol (MI) is abundant in nature. Bacillus subtilis 168 has the ability to metabolize inositol stereoisomers, including MI and SI. Previously, we reported a B. subtilis cell factory with modified inositol metabolism that converts MI into SI in the culture medium. The strain was constructed by deleting all genes related to inositol metabolism and overexpressing key enzymes, IolG and IolW. By using this strain, 10 g/l of MI initially included in the medium was completely converted into SI within 48 h of cultivation in a rich medium containing 2% (w/v) Bacto soytone.ResultsWhen the initial concentration of MI was increased to 50 g/l, conversion was limited to 15.1 g/l of SI. Therefore, overexpression systems of IolT and PntAB, the main transporter of MI in B. subtilis and the membrane-integral nicotinamide nucleotide transhydrogenase in Escherichia coli respectively, were additionally introduced into the B. subtilis cell factory, but the conversion efficiency hardly improved. We systematically determined the amount of Bacto soytone necessary for ultimate conversion, which was 4% (w/v). As a result, the conversion of SI reached to 27.6 g/l within 48 h of cultivation.ConclusionsThe B. subtilis cell factory was improved to yield a SI production rate of 27.6 g/l/48 h by simultaneous overexpression of IolT and PntAB, and by addition of 4% (w/v) Bacto soytone in the conversion medium. The concentration of SI was increased even in the stationary phase perhaps due to nutrients in the Bacto soytone that contribute to the conversion process. Thus, MI conversion to SI may be further optimized via identification and control of these unknown nutrients.

A bacterial cell factory converting glucose into scyllo-inositol, a therapeutic agent for Alzheimer’s disease

“…This could enable the conversion of glucose into myo-inositol, since its substrate G6P is naturally supplied from glucose and also because its product MI1P is dephosphorylated by the intrinsic and constitutive YktC to form myo-inositol 22 . Accordingly, in order to enable the production of scyllo-inositol from glucose, we next introduced a previously established artificial pathway to convert myo-inositol into scyllo-inositol involving two inositol dehydrogenases, IolG and IolW 15 . To achieve this, strain MC022 [amyE::(PrpsO-iolG-iolW-iolT kan) pbuE::(PrpsO-ino1Mt-His 6 cat) ΔiolABCDEF ΔiolHIJ ΔiolX ΔiolR] was newly constructed, with pbuE disrupted by the insertion of an ino1 gene cassette on the KU302 background to couple myo-inositol production from glucose with the conversion of myo-inositol into scyllo-inositol (Supplementary Table 1).…”

“…The bacterial cell factory created in this study gave a production efficiency about 2 g L −1 of scyllo-inositol from 20 g L −1 of glucose, and the produced scyllo-inositol would not be cheaper than USD 4.50-6.00 per kg considering the material cost. On the other hand, one of the most efficient cell factories previously created was capable of producing 27.6 g L −1 of scyllo-inositol from 50 g L −1 of myo-inositol 15 . In this case, the produced scyllo-inositol would be more expensive than USD 18.12-54.35 per kg.…”

“…In the last decade, B. subtilis has been genetically engineered to form a "cell factory" capable of performing the bioconversion of myo-inositol into rare inositol stereoisomers [15][16][17][18][19] . One of the cell factories recently created was capable of producing 27.6 g L −1 of scyllo-inositol from 50 g L −1 of myo-inositol within 48 h of culturing 15 . In this case, iolG, iolW, and iolT were simultaneously overexpressed in a strain that lacked all of the other iol genes.…”

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

“…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.…”

“…However, the conversion efficiency of these cell factories was limited by some factors such as metabolic factors and the efflux system of SI. In addition, the yield of SI also depended on some unknown nutrients in the medium . Thus, it is necessary to develop another new, simple, and efficient method for the production of SI.…”