Electrochemical control of bacterial metabolism and its application
to bioproduction
Atsushi KOUZUMA
School of Life Sciences, Tokyo University of Pharmacy and Life Science
Research objective
Currently, various useful chemicals are produced by fermentation using
microorganisms. However, there are many chemicals that cannot be produced byconventional fermentation processes at a practical level owing to their low production
efficiencies. One reason for the low production efficiencies is that organic substrates, such as
sugars, are consumed for the growth and maintenance of microbial cells. The fermentative
production of highly reduced chemicals at high yields is particularly difficult as it requires
more substrates to generate reducing equivalents (e.g., NADH) and ATP. This can be solved
through the use of external energy sources for the bioconversion of substrates; however, suchenergy sources must be less expensive than organic substrates to maintain the economic
feasibility of the entire process.
Electrochemically active bacteria (EAB) have recently gained attention in the fieldof environmental microbiology1. In the presence of high-potential electrodes, these bacteria
grow by releasing electrons generated by the oxidative metabolism of organic substrates tothe electrodes. However, in the presence of low-potential electrodes, EAB can take up
electrons from the electrodes and use them for reductive metabolism. Since electricity ischeaper than sugars (e.g., glucose), its use is expected to reduce the cost of fermentation
processes.
Therefore, this study aimed to establish the basis of “electrofermentation” (EF) as atechnology that can be widely used to improve the efficiency of the fermentation process. In
particular, the present study aimed to develop a process to improve the production yield of
1,4-butanediol (1,4-BDO) by supplying electricity to genetically modified Shewanella, anEAB, from low-potential electrodes.
Methods
Shewanella oneidensis MR-1 and its derivatives were cultured at 30 °C in LB or
minimal medium2 containing lactate or glucose as the sole carbon and energy source.
Transformation and gene disruption were carried out by filter mating and double-crossover
methods, respectively2. For transcriptome analysis using DNA microarrays,MR-1(pBBR-glk-galP) strains were precultured in glucose-minimal medium underfumarate-reducing conditions. The cells were then cultured under fermentation (in theabsence of electron acceptors) or fumarate-reducing conditions for 3 h, after which total
RNA was extracted. DNA microarray analysis was performed as described previously3.
Results
1. Evaluation of the glucose fermentation ability of genetically modified Shewanella
S. oneidensis MR-1 is a promising host for EF due to its ability to receive electrons
from low-potential electrodes and use them for intracellular reductive metabolism1. Althoughwild-type MR-1 cannot utilize sugars (glucose), a previous study has shown that anengineered strain of MR-1 (MR-1[pBBR-glk-galP] ) expressing the genes responsible forglucose uptake and phosphorylation (galP and glk, respectively) can utilize glucose underfumarate-reducing conditions2. However, it remains unclear whether this mutant strain could
grow on glucose under fermentation conditions. Therefore, we first inoculatedMR-1(pBBR-glk-galP) into glucose minimal medium and examined whether it was able togrow under fermentation conditions. The results revealed that this strain cannot grow on
glucose minimal medium under fermentation conditions but can grow in the presence ofelectron acceptors, such as oxygen and fumarate. The inability to grow under fermentation
conditions may be attributed to decreased activity of the TCA cycle and amino acid synthesis
because MR-1 is known to repress the TCA cycle activity under reductive conditions. Thishypothesis was supported by the results of the comparative transcriptome analysis of MR-1
grown under fermentative and fumarate-reducing conditions. We cultivatedMR-1(pBBR-glk-galP) in glucose minimal medium in the presence of 0.1% (w/v) tryptone
as the amino acid source and found that supplementation with tryptone enabled thefermentative growth of this strain on glucose. This finding suggests that theMR-1(pBBR-glk-galP) strain can be used as a host for EF using glucose as a substrate if asmall amount of an amino acid source is supplied.
2. Introduction of 1,4-BDO synthesis genes and disruption of byproduct synthesis genes
To confer the ability to synthesize 1,4-BDO on MR-1(pBBR-glk-galP), we
introduced the genes required for 1,4-BDO synthesis (Fig. 1)4 into this strain and disruptedthe pathways involved in the production of byproducts (i.e., lactate and acetate). In addition,
we introduced the pyruvate decarboxylase gene (pyc) derived from Bacillus subtilis intoMR-1(pBBR-glk-galP) to facilitate the conversion of glucose to succinate, a precursor of
1,4-BDO. Currently, we are confirming the expression of the genes shown in Fig. 1 and theactivity of each enzyme.

Fig. 1. Gene cassette for 1,4-BDO synthesis

Conclusion
Here, we showed that an engineered strain of MR-1 expressing the genes forglucose uptake and phosphorylation (galP and glk, respectively), MR-1(pBBR-glk-galP), iscapable of fermentatively metabolizing glucose and is suitable as a host for EF. In addition, agene cassette for the synthesis of 1,4-BDO was designed and introduced intoMR-1(pBBR-glk-galP). In the future, we plan to examine whether an
MR-1(pBBR-glk-galP)-based engineered strain can synthesize 1,4-BDO by EF in thepresence of a low-potential electrode (-0.6 V vs. Ag/AgCl).
References
1
Kouzuma, A. (2021) Molecular mechanisms regulating the catabolic andelectrochemical activities of Shewanella oneidensis MR-1. Biosci. Biotechnol.
2Biochem. 85: 1572-1581.
Nakagawa, G. et al. (2015) Metabolic Characteristics of a glucose-utilizing
Shewanella oneidensis strain grown under electrode-respiring conditions. PLOS ONE
10: e0138813.

3
4Hirose, A. et al. (2018) Electrochemically active bacteria sense electrode potentials
for regulating catabolic pathways. Nat. Commun. 9: 1083.
Yim, H. et al. (2011) Metabolic engineering of Escherichia coli for direct productionof 1,4-butanediol. Nat. Chem. Biol. 7: 445-452.