Application of a Light-Inducible Metabolic Switch for Enhancing
Bio-Production in Microorganisms
Yoshihiro TOYA
Graduate school of Information Science and Technology, Osaka University
Research objective
The production of useful compounds using microorganisms is important for achieving asustainable society. Thus far, much effort has been dedicated to changing metabolic flux
distributions to enhance target production through static pathway modifications. To further
improve productivity, it is highly desirable to fine-tune the fluxes to achieve an ideal state.
Although chemically inducible systems are widely used for metabolic regulation, thesechemicals cannot be removed from the broth, it is difficult to perform flux fine-tuning.
Furthermore, many useful compounds compete for the formation of biomass, and these areproduced with repressing cell growth. However, cellular activity gradually decreases during
the stationary phase. Once metabolism switches to the production mode by adding inducers,
it becomes difficult to return to the growth mode. To overcome these issues, we developed ametabolic switch to alter flux distribution by light irradiation using an optogenetic tool(CcaS/R). Because light can irradiate cells from outside the vessels, it allows for the finetuning of flux with on-off regulation. The optogenetic switch may also contribute to enhanced
production by repeating the production and growth modes.
In the present study, a light-inducible switch was applied to 1,3-propandiol (1,3-PDO)
production in Escherichia coli. We developed an engineered strain that could change themetabolic state between growth and 1,3-PDO production modes using green and red lights,
respectively (Fig. 1). The effects of flux control on 1,3-PDO production were also evaluated.
Furthermore, because the metabolic network has many branches, orthogonal switches arenecessary to widely alter the flux distributions. We developed another optogenetic switch
using a blue-light-inducible transcription factor and regulated the flux ratios at the twobranches.
Methods
During 1,3-PDO production, triosephosphate isomerase (TPI) expression is regulated by
CcaS/R. After deleting tpiA from the MG1655(DE3) strain, we introduced genes encoding
CcaS/R. tpiA is expressed under the control of the cpcG2 promoter which is regulated by
CcaS/R. To implement the 1,3-PDO synthesis pathway, dhaB and gdrAB derived fromKlebsiella pneumoniae were introduced. The cells were inoculated into 20 mL of synthetic

medium containing glycerol, at an initial OD600 of 0.05, and incubated at 37oC and 150 rpm
with LED light irradiation. 1,3-PDO was measured using high-performance liquid
chromatography.
In multidimensional flux control, the flux ratio between the Embden-Meyerhof-Parnas(EMP), pentose phosphate (PP), and Entner-Doudoroff (ED) pathways is regulated by CcaS/Rand EL222. Because a switch for controlling the flux ratio between the EMP and PP pathways
has already been developed using CcaS/R, we constructed another switch to control the flux
ratio between the PP and ED pathways using EL222. The MG1655(DE3)∆pgi∆gntR strain
was used as the parental strain. The EL222 binding sequence was inserted into the appropriate
regions of the target gene promoters for up and down regulations. The cells were inoculated
into 1 mL of synthetic medium containing [1-13C] glucose at an initial OD600 of 0.1 in a 24well plate. The plates were then incubated at 37oC and 500 rpm under each light condition.
The flux ratio was determined based on the 13C-enrichment of proteinogenic amino acids.
Results
1. Production of 1,3-PDO using a light-inducible metabolic switch
TPI expression was induced and repressed under green and red light, respectively. Thus, theconstructed strain is expected to grow well under green light and produce 1,3-PDO under red
light (Fig. 1B). To verify whether the TPI switch functioned properly, we first cultured thecells under continuous light conditions (Fig. 1C). As expected, growth was faster under green
light than under red light. No differences in 1,3-PDO production were observed under green
and red light.
Greenlight(A)
CcaS

(B)

RedlightPPCcaR

PP

TPI

1,3-PDO

201,3-PDO (mM)

4Glycerol

TPI

PcpcG2
tpiA

(C)
3

OD600

152

1010→ OFFRed light

048
96Time (h)

144

50048
96
Time (h)Production
mode

GrowthmodeGreen light → ON

DHAP

TPI

GAP

Glycerol

Cell 1,3-PDO

DHAP

TPI

GAP
Cell

Fig. 1 Mechanism of light-inducible metabolic switch andapplication for 1,3-PDO production(A) Gene expression regulation by the CcaS/R system, (B)Metabolic shift between growth and production modes by
TPI regulation, (C) Growth and 1,3-PDO production undercontinuous green or red light. The line colors corresponds to144 the color of the irradiated light.

Next, we initially irradiated the cells with green light to facilitate growth and then changed
to red light to alter the metabolism from growth to production modes (Fig. 2). Compared withthe continuous green light condition, the cells grew to a similar OD600 and produced 1.6 times
the amount of 1,3-PDO under red light. Furthermore, the effect of periodic light (1 min green

and 9 min red) during the stationary phase on 1,3-PDO production was evaluated. 1,3-PDO
production was 2.3 times higher than that under the condition that initially facilitated growthwith green light and switched to red light (Fig. 2). However, because 1,3-PDO was mainly
produced during the early phase, recovery was not achieved. Growth reached a plateau at a
certain level, even under continuous green light conditions, suggesting a shortage of essential
nutrients. In the future, we will reconsider the components of the medium and aim to achieve
efficient 1,3-PDO production by optimizing the periodic light patterns.
41,3-PDO (mM)

20

3OD600152101004896
Time (h)

144

50048
96
Time (h)Fig. 2 Fermentation profiles with changing light during
the stationary phase
For the data shown with green and red lines, the growth wasinitially induced by green light. After the OD600 reached 1,
the green light was changed to red light to shift themetabolism to production mode. The line colors correspondto the color of the irradiated light. For the data shown with144
blue lines, after inducing the growth by green light similarly,
periodic light (1 min green and 9 min red) was irradiated.

2. Control of flux distribution with two branches by multicolor irradiation
To manipulate the flux distribution by light irradiation, we developed a metabolic switch
using another optogenetic tool that responds to light wavelengths different from those used by
the CcaS/R system. The flux ratios of the three glycolytic pathways were regulated using
green/red-light-responsive CcaS/R and blue-light-responsive EL222 (Fig. 3A). First, weaimed to control the flux ratio between the PP and ED pathways using blue light irradiation.
After blocking the EMP pathway, the gnd and edd promoters were engineered for up- anddownregulation, respectively, by EL222 under blue light. The flux ratios under dark and blue
light conditions are shown in Fig. 3B. The PP and ED fluxes increased under dark and blue
light conditions, respectively, suggesting that the blue-light-responsive metabolic switch
functioned correctly. A previously developed CcaS/R-based switch for controlling the flux
ratio between the EMP and PP pathways was implemented (Fig. 3C). The EMP flux increased
under green light and the sum of the PP and ED fluxes increased under red light. Under both
red and blue light, the PP flux decreased and the ED flux increased.

CcaS/R

On
Off
PGI

G6P

EMP

Gnd

PP

Pyruvate

On

Edd

6PG
ED

(C)

120
80
60
40
200120100100Flux ratio (%)

Glucose Off(B)EL222

Flux ratio (%)

(A)

Dark

Blue

80
60

EMP flux
PP flux
ED flux

40200GreenRed Red+Blue

Fig. 3 Control of glycolytic flux ratio using CcaS/R and EL222 by multicolor irradiation(A) Scheme of flux rewiring of glycolytic pathways by two optogenetic tools, (B) Flux ratios under dark and blue light
conditions in a PGI-deletion strain with repressed PP flux and inducing ED flux by EL222, (C) Flux ratios when EL222
and CcaS/R are combined to control three glycolytic pathways.

Conclusion
The metabolic state of 1,3-PDO-producing E. coli can be altered to growth and production
modes using a light-inducible metabolic switch. Furthermore, the flux ratios at the twobranches of the glycolytic pathways were successfully regulated by multicolor light irradiation.