Efficient pathway-driven
scyllo
-inositol production from
myo
-inositol using thermophilic cells and mesophilic inositol dehydrogenases: a novel strategy for pathway control
Authors: Kurashiki Ryota , Takahashi Masahiro , Okumura Yuta , Ono Tatsuya , Endo Hirofumi , Makino Kohei , Fukui Kaho , Yokoyama Kyosuke , Ishikawa Shu , Yoshida Ken-ichi , Ohshiro Takashi , Suzuki Hirokazu 
Published: July 2024

Mesophilic enzymes, which are active at moderate temperatures, may dominate enzymatic reactions even in the presence of thermophilic crude enzymes. This study was conducted to investigate this hypothesis with mesophilic inositol dehydrogenases (IolG and IolX) produced in
Geobacillus kaustophilus
HTA426. To ensure the efficient production of mesophilic enzymes, we first screened for promoters induced at moderate temperatures using transcriptome analysis and identified four genes highly expressed at 30°C in the thermophile. We further characterized these promoters using fluorescent reporter assays to determine that the
mti3
promoter could direct efficient gene expression at 40°C. We cloned the promoter into an
Escherichia coli–Geobacillus
shuttle plasmid and confirmed that the resulting vector functioned in
G. kaustophilus
and other thermophiles. We then used this vector for the cooperative expression of the
iolG
and
iolX
genes from
Bacillus subtilis
168.
G. kaustophilus
cells carrying the expression vector were incubated at 60°C for cellular propagation and then at 40°C for the production of IolG and IolX. When the cells were permeabilized, IolG and IolX acted as catalysts to convert exogenous
myo
-inositol into
scyllo
-inositol at 30°C. In a scaled-up reaction, 10 g of
myo
-inositol was converted to 1.8 g of
scyllo
-inositol, which was further purified to yield 970 mg of pure powder. Notably,
myo
-inositol was degraded by intrinsic enzymes of
G. kaustophilus
at 60°C but not at 30°C, supporting our initial hypothesis. We indicate that this approach is useful for preparing enzyme cocktails without the need for purification.

IMPORTANCE

Enzyme cocktails are commonly employed for cell-free chemical synthesis; however, their preparation involves cumbersome processes. This study affirms that mesophilic enzymes in thermophilic crude extracts can function as specific catalysts at moderate temperatures, akin to enzyme cocktails. The catalyst was prepared by permeabilizing cells without the need for concentration, extraction, or purification processes; hence, its preparation was considerably simpler compared with conventional methods for enzyme cocktails. This approach was employed to produce pure
scyllo
-inositol from an economical substrate. Notably, this marks the first large-scale preparation of pure
scyllo
-inositol, holding potential pharmaceutical significance as
scyllo
-inositol serves as a promising agent for certain diseases but is currently expensive. Moreover, this approach holds promise for application in pathway engineering within living cells. The envisioned pathway is designed without chromosomal modification and is simply regulated by switching culture temperatures. Consequently, this study introduces a novel platform for both whole-cell and cell-free synthetic systems.