Specific bioproduction using thermophilic cells that produce
mesophilic enzymes under temperate conditions
Hirokazu SUZUKI
Graduate School of Engineering, Tottori University
Report: Abstract for the international conference BACELL2020 (conference canceled)Research objectives
Microorganisms can serve as hosts for the efficient production of beneficial metabolites
where the productivity is often enhanced by metabolic engineering by enabling hostorganisms to activate specific pathways leading to the metabolites. The engineering
generally involves disruption and/or forcible expression of genes relevant to the metabolicpathway. The process is challenging, requiring substantial effort and time, and occasionallyresults in involuntary phenotypes such as inefficient growth of host cells. Here, we designed
an engineering approach for more facile bioproduction of specific metabolites. In thisapproach, mesophilic enzymes are overproduced in thermophiles at moderate temperatures.
Since thermophilic enzymes in thermophiles are less active at moderate temperatures, themesophilic enzymes could dominate in thermophiles to catalyze specific reactions under theconditions. In order to evaluate this approach, the present study was conducted to produce
mesophilic enzymes for inositol conversion1) in a thermophile (Geobacillus kaustophilus
HTA426) and to analyze whether the cells can specifically convert inositol at moderatetemperatures.
Methods
The cspX promoter (PcspX) was previously identified to express at moderatetemperatures in G. kaustophilus. This promoter was cloned in the pGKE119 vector2) toconstruct pGKE120, which was then used to clone iolG–iolX and iolG–iolW genes fromBacillus subtilis at the PcspX downstream. Geobacillus kaustophilus was transformed with theresultant plasmids and cultured at 60℃ in LB medium until the optical density at 600 nm
was equal to 1. The cells were additionally incubated at 40℃ for 24 h and thenpermeabilized by the addition of surfactants and lysozyme to the culture. The culture wasincubated at 30℃ for 6 h in the presence of myo-inositol (MI; 10 g/L) and NAD+ (1 mM)
and subjected to high performance liquid chromatography for the detection of scyllo-inositol(SI), that was converted from MI.

ResultsIolG catalyzes NAD+-dependent MI oxidation to produce scyllo-inosose, whereas IolX
and IolW reduce scyllo-inosose using NADH and NADPH, respectively, as electron donors,
to produce SI (Fig. 1A). Geobacillus kaustophilus that produced IolG and IolX converted
30% of MI (10 g/L) into SI, following incubation at 30℃ for 6 h. A prolonged incubation
did not increase the conversion rates potentially because of the chemical equilibrium
between MI and SI. The cells converted 25% of MI even when incubated at 10℃ for 6 h.
The cells also converted a higher concentration (60 g/L) of MI at 30℃ for 24 h at a rate of27%, which indicates that this system can produce 16 g/L SI. The reaction proceeded in thecell culture without washing cells. Cell permeabilization with a higher concentration oflysozyme decreased the conversion rates. A lower concentration (0.1 mM) of NAD+
decreased the conversion rates but a higher concentration (2 mM) had no effect. Geobacillus
kaustophilus that produced IolG and IolW also produced SI; however, the reaction required
NADPH and the rate was lower compared to the cells that produced IolG and IolX.
Unfortunately, the cells could not produce SI without permeabilization even when iolT wassimultaneously expressed along with iolG and iolX. This was attributable to inefficient MIuptake at moderate temperatures.

Fig. 1 Evaluation of an engineering approach that produces mesophilic enzymes inthermophiles at moderate temperatures. (A) Reactions catalyzed by Iol enzymes fromBacillus subtilis. IolG and IolX (or IolW) convert myo-inositol (MI) into scyllo-inositol (SI).
IolT is potentially involved in MI uptake. Geobacillus kaustophilus can intrinsicallycatabolize MI. (B) MI degradation by G. kaustophilus is repressed at moderate temperatures.
MI was incubated with the permeabilized cells for 24 h at respective temperatures.
To see whether MI is involuntarily degraded by intrinsic enzymes in G. kaustophilus,

cells carrying pGKE120 were cultured and incubated with MI following permeabilization.
MI was notably decreased at 60℃ but not at 30℃ (Fig. 1B). This observation suggested that
Iol enzymes catalytically dominated in thermophiles at moderate temperatures, supporting
our concept. We also analyzed the Geobacillus spp. in which PcspX functions. The geneencoding for a yellow fluorescence protein (Venus) was cloned in pGKE120 and introduced
into the Geobacillus spp. The cells were cultured at 60℃ in LB medium until the opticaldensity at 600 nm was equal to 1 and then at 40℃ for 24 h. Fluorescence analysis showedthat Venus was produced more efficiently in G. stearothermophilus 10 than in G.
kaustophilus, suggesting that G. stearothermophilus 10 is a promising host for our approach.
In contrast, Venus was negligibly produced in G. subterraneus DSM 13552, G.
thermoglucosidasius DSM 2542, and G. thermoleovorans DSM 5366; therefore, Geobacillus
spp. in which PcspX functions may be in minority.
Conclusion
Geobacillus kaustophilus that produced IolG and IolX were readily permeabilized andefficiently produced SI from MI. Since involuntary MI degradation was repressed atmoderate temperatures, our approach could be effective for specific SI production. There are
two similar approaches to prepare specific enzyme cocktails. One approach produces
thermophilic enzymes in mesophiles and inactivates mesophilic enzymes by heat treatment.
This approach requires reactions at elevated temperatures that cause vitamin degradation andcannot use mesophilic enzymes. The other produces mesophilic enzymes in psychrophiles
and inactivates psychrophilic enzymes by incubation at moderate temperatures. Although
this approach allows reactions at moderate temperatures, psychrophiles generally require acooling system for cultivation and grow slowly. In contrast to these approaches, ourapproach does not require heat treatment and the reaction proceeds at moderate temperatures.
The cells rapidly grow and can efficiently produce enzymes. Importantly, our approach
could use viable cells for bioproduction when the substrates are incorporated into cells. Thisapproach has the potential to serve as a new strategy for the facile and efficient production ofdiverse metabolites.
References
1) Tanaka, K., Natsume, A., Ishikawa, S., Takenaka, S., and Yoshida, K. (2017) A
new-generation of Bacillus subtilis cell factory for further elevated scyllo-inositolproduction. Microb. Cell Fact. 16: 67.
2) Kurashiki, R., Mizuno, T., Murata, K., Ohshiro, T., and Suzuki, H. (2020) A plasmid
vector that directs hyperproduction of recombinant proteins in the thermophiles
Geobacillus species. Extremophiles 24: 147.