Evolutionary design of gene expression systems
Daisuke UMENO
Graduate School of Engineering, Chiba University

Research aims
Various types of transcriptional controlling mechanisms have been investigatedin the search for useful gene induction systems suitable for the microbial
production of valuable chemicals. Although some of them, including arabinose-,lactose-, and tetracycline-inducible systems, perform well in the laboratory, they
are not used in the large-scale commercial production of chemicals, simply due
to high costs of inducer molecules. We aimed to develop a highly stringent, yethigh-yield induction system using a safe and cheap induction system truly
useable in real-world applications.
Methods
1. Improvement of the sensitivity of choline-inducing systems
Choline is a cheap and safe edible/nutrient compound found in egg yolk. We
have previously conducted directed evolution of the BetI/BtO system, which isthe choline induction system in Escherichia coli, into high-stringent and highpower transcriptional induction systems (ver.1) 1) 2). These systems have been

demonstrated as highly useful both in metabolic engineering and as tools forconstructing genetic circuits. However, there remains one technical issue: itrequires as much as 10 mM of choline to completely induce the genes.
We first conducted directed evolution of BetI in search for high-sensitive
variants, but all our attempts were unsuccessful. Based on this failure, weconcluded that choline import might be the bottle-neck. Therefore, we placed thecholine transporter BetT under the control of Bet promoter (ver.2). The
switching behavior of the resultant induction system was analyzed bytransforming the E. coli strain (BW25113) harboring resultant systems wherethe fluorescent protein (sfGFP) is placed under PBet. The same experiment wasconducted for a strain lacking betA (gene coding choline-metabolizing enzyme)

harboring the same plasmids.
2. Improving the switching property of Ptrp
Amino acids constitute other candidate molecules that can be safely used asinducers. Among these, tryptophan-responsive promoter systems (Ptrp) havebeen widely used, including in the industrial production of insulin. For thissystem, the endogenous regulator TrpR is strong enough to act on the Trp
promoter multi-copy plasmid, but non-negligible leakiness exists. Using aplasmid expressing TrpR, we could have drastically improved the stringency ofthe system, but drastic reduction of induction level was observed. We therefore
tried to optimize the operator/promoter sequences to obtain higher inductionlevels while maintaining the stringency.
To this end, we scrambled the -10 sequence of the natural Trp promoter,

which was significantly off from the consensus sequence of E. coli promoters. We
also randomized one of the six TrpR-binding sequences (TrpOs), because TrpO
located at the most upstream of TrpP was significantly off from the consensus
sequence (GNACT).
The resultant TrpP variants library (containing five randomized nucleotides;
1,024 possible sequences) was inserted upstream of a plasmid encoding GFP, tosearch for those with elevated induction level and high-stringency.
Results
1. Reduction of choline inducers required by 3,500-fold increased sensitivity
When BetT was additionally expressed by placing the gene encoding betT under
the control of Bet promoter, a drastic shift of transfer function of the BetI/BetO
system was observed (Fig. 1). This new system (ver.2) only required less than 0.1
mM of choline to induce genes, which is 200 times less than that for the originalsystem. Given the low price (ca. 5 USD/kg) of choline, this system requires 2
cents per liter of culture for inducer molecules. When installed into theBetA-deleted strains (ver.3), we observed another 20× reduction in the amountof choline required for induction, reaching down to ~5 µM. Thus, the inducer
cost of the choline-inducible system becomes virtually negligible.
2. Ten times improvement in the Tryptophan induction system
Although the sequence diversity of the promoter library we designed wasminimal (1,024), we could have isolated a variety of TrpP variants. Some of theobtained TrpP variants were found to show more than 10 times improvement in
induction levels, while preserving stringency in un-induced conditions (Fig. 2).

Conclusion
Nature harbors countless genetic switches and all of them could be potentially
useful as inducer systems for induction of enzymes/ pathways. However, it israre that the specification of these switches is suitable for biotechnological uses
without modification. To establish reliable induction systems without eliciting aphysiological response in host organisms, the specifications of each naturalgenetic switch should be altered. This work demonstrated the surprising
plasticity of switching properties of genetic switches. Using the directed
evolution platform we have been developing3) 4), we should be able to rapidly
provide a variety of genetic switches in each and every biotechnological contexts.
References
1) Ike K., et al.: Evolutionary design of choline- inducible and –repressible T7-based
induction systems. ACS Synthetic Biology, 4, 1352-1360 (2015).
2) Saeki, K, et al.: Rapid diversification of BetI-based transcriptional switches for thecontrol of biosynthetic pathways and genetic circuits. ACS Synthetic Biology, 5,1201-1210 (2016).
3) Tominaga M, et al.: Rapid and liquid-based selection of genetic switches using
nucleoside kinase fused with aminoglycoside phosphotransferase. PLOS One, 10,e0120243, 1-11 (2015).
4) K. Ike and Umeno D: Nucleotide kinase-based selection system for genetic switches:
Methods in Mol Biol, 1111, 141-152 (2014).

Figure.１ Choline-inducible systems. (A) Plasmid expression system based on
the Bet system. BetI repressed the transcription of T5 promoter fused with BetO,

and choline induces this promoter by binding and altering the structure of BetI,
thereby departing from BetO. In Ver.2, the choline importer (BetT) is expressed
under the control of T5P/BetO promoter, increasing the effective cellularconcentration of choline. Choline concentration is also elevated by blocking its
consumption (via deletion of BetAB). (B) Transfer function of Bet-systems. Ver.1（◼），ver.2（▲），ver.3（●）.

Figure. 2 A series of tryptophan induction systems. (A) Library design: threebases at -10 sequence and two bases at the first TrpO sequence were randomized,yielding 1,024 unique PTrp variants. (B) Switching properties of TrpP variants.