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Biosensors are an efficient in vivo tool, especially useful for screening a large number of strains on their metabolic abilities, e.g. over-producing fatty acids. The production of long chain fatty acids (LCFA) is regulated by the transcription factor FadR. This transcription factor can bind to Acyl-CoA which leads to the release of FadR from its cognate operator sequence, thereby increasing gene expression (as shown in Fig. 1)¹.

Fig. 1: Regulation of the fatty acid metabolism by the transcriptional factor FadR. FadR recognizes its cognate binding site (white), thereby repressing transcription. Upon binding of an Acyl-CoA to FadR, the promoter region is freed, enabling gene expression..

In frame of SynMylk, a synthetic fatty acid biosensor already present in the iGEM registry was tested, comprising a double terminator, the fatty acid inducible promoter P_AR and the reporter gene for RFP². Since RFP has a relatively slow folding activity, it was chosen to improve this part by adding more reporter genes, namely, sfGFP (BBa_I746916), which has improved folding characteristics, and amilCP (BBa_K592009), which can be quantified by measuring absorption. The regulatory function of the biosensor is shown in Fig. 1.

The fatty acid stocks for the experiments were prepared. All of the fatty acids were dissolved in ethanol with the exception of butyric acid, which was dissolved in water. The stock solutions of the fatty acids are shown in Table 1.

Table 1: Stock solutions for different fatty acids.

Fatty acid	Chain length	MW [g/mol]	Solvent	Solubility	Stock concentration
Butyric acid	C4:0	88,11	water	60 g/L	200 mM
Capric acid	C10:0	172,26	ethanol	30 g/L	100 mM
Lauric acid	C12:0	200,32	ethanol	20 g/L	100 mM
Myristic acid	C14:0	228,37	ethanol	15 g/L	75 mM
Palmitic acid	C16:0	256,42	ethanol	20 g/L	75 mM
Stearic acid	C18:0	284,48	ethanol	20 g/L	50 mM
Oleic acid	C18:1	282,46	ethanol	100 g/L	200 mM

Table 2: List of all part improvement experiments. Concentrations are listed as final concentrations used for induction of the cultures.

Biosensor	fatty acid	Final concentrations
PAR:RFP	lauric acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM
PAR:RFP	myristic acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM
PAR:RFP	palmitic acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM
PAR:RFP	stearic acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM
PAR:sfGFP	lauric acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM
PAR:sfGFP	myristic acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM
PAR:sfGFP	palmitic acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM
PAR:sfGFP	stearic acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM
PAR:amilCP	lauric acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM
PAR:amilCP	myristic acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM
PAR:amilCP	palmitic acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM
PAR:amilCP	stearic acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM
EVC	lauric acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM
EVC	myristic acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM
EVC	palmitic acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM
EVC	stearic acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM

The different P_AR biosensor variants were tested in a dose-dependent manner by adding different concentrations of the fatty acids with a chain length from C14:0 to C18:0 to the culture medium prior to culturing the cells. An empty vector control (EVC) treated in the same manner was included in each measurement.

Fig. 2: Response of P_AR:RFP (red) to different chain lengths of fatty acids compared to an empty vector control (gray). Plot A presents the response for lauric acid, plot B for myristic acid, plot C for palmitic acid and plot D for stearic acid.

By adding fatty acids to the culture medium, a dose dependent increase of fluorescence, consistent with the results shown by UPF_CRG_Barcelona iGEM team 2018 (BBa_K2581012), was expected, while the EVC should remain at a basal level. Fig. 2 shows that the addition of fatty acids to the culture medium results in an increase in fluorescence in a dose-dependent fashion compared to the EVC. The most clear result could be shown with the fatty acids palmitic acid (C16:0) and stearic acid (C18:0). Adding myristic acid (C14:0) to the culture medium, fluorescence of the reporter gene RFP was increased, but the fluorescence of the EVC increased slightly as well. These results indicate that the promoter is more specific to the chain lengths C16:0 and C18:0 than to the chain lengths C12:0 and C14:0. The Escherichia coli cells with the RFP were also monitored with confocal fluorescence microscopy to visualize the intracellular localization of RFP in the cells. The RFP appears to be located in the cytosol instead of being bound to a membrane (Fig. 3).

Fig. 3: Overview (A) and a detailed (B) image from RFP in a E. coli strain after being induced with 1 mM palmitic acid. The RFP can be detected in the whole cytosol.

Fig. 4: Response of P_AR+sfGFP (green) to different chain lengths of fatty acids compared to an empty vector control (gray). Plot A presents the response for lauric acid, plot B for myristic acid, plot C for palmitic acid and plot D for stearic acid.

Fig. 4 shows the same dose-response experiments for the new part, P_AR:sfGFP. The graphs show a significant increase in fluorescence compared to the original part of Barcelona with RFP. Fluorescence is strongest in the experiment with stearic acid (C18:0), but also in the other experiments a large increase in fluorescence can be seen. For example, addition of the fatty acid palmitic acid (C16:0), doubled fluorescence from 0.01 mM to 1 mM. EVC also shows a better result. The fluorescence of the samples also increased only minimally and is significantly lower compared to sfGFP, indicating some kind of excitation resulting directly from some of the fatty acids in the case of RFP-exciting wavelengths, instead of from the reporter protein. This is not the case for excitation wavelengths relevant for sfGFP, further supporting the improvement of our part. For induction with lauric acid (C12:0), the results appear more erratic at higher concentrations , which is probably due to a decreasing specificity of the promoter AR to the fatty acids - this result is also consistent with the data measured for P_AR:RFP. The E. coli cells expressing sfGFP were also monitored with confocal fluorescence microscopy to visualize the localization of sfGFP in the cells. As for RFP, sfGFP is also not bound to the membrane, but is localized in the cytosol (Fig. 5).

Fig. 5: Overview (A) and a detailed (B) image from sfGFP in a E. coli strain after being induced with 1 mM palmitic acid. The sfGFP can be detected in the whole cytosol.

Fig. 6: Response of P_AR+amilCP (blue) to different chain lengths of fatty acids compared to an empty vector control (gray). Plot A presents the response for lauric acid, plot B for myristic acid, plot C for palmitic acid and plot D for stearic acid.

The same experiments with the blue chromoprotein amilCP (BBa_K592009) showed that the production of the chromoprotein is increased by a higher concentration of fatty acids in the medium. The best result was achieved with stearic acid. The biosensor also worked for the chain lengths from C14:0 and C16:0. Here, the EVC is lower than the samples with amilCP. By adding lauric acid (C12:0) to the culture medium, the production of amilCP increased, but the EVC also increased in a similar manner close to amilCP, so it is not certain if this result can be used for distinct conclusion.

Another promising biosensor candidate obtained from the preexisting literature is P_aldA³. P_aldA was published as an oleic acid (C18:1) sensitive promoter enabling for the measurement of free fatty acids present in microorganisms. In E. coli the growth on fatty acids requires many different proteins which are repressed by the transcriptional factor FadR¹. The long chain Acyl-CoA ester is an effector on the transcriptional factor FadR for the regulation of fatty acid metabolism². After adding oleic acid (C18:1) many proteins showed an altered expression level and new proteins like AldA were synthesised. The promoter for aldA was also used for the production of green fluorescent protein (GFP)¹. P_aldA was isolated from the E. coli wild type genome.

Fig. 7: P_aldA is bound to the reporter gene eYFP and cloned into a pBb backbone. The restriction enzymes EcoRI and XbaI were used for the cloning. The pBb backbone has a kanamycin resistance and a medium copy ori p15A.

Table 3: List of all experiments with the P_aldA. Concentrations are listed as final concentrations used for induction of the cultures.

Biosensor	fatty acid	Final concentrations
PaldA:eYFP	palmitic acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM
PaldA:eYFP	stearic acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM
PaldA:eYFP	oleic acid	0 mM; 0,5 mM; 1 mM; 5 mM; 10 mM; 20 mM
EVC	palmitic acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM
EVC	stearic acid	0 mM; 0,01 mM; 0,1 mM; 0,2 mM; 0,4 mM; 0,6 mM; 0,8 mM; 1 mM
EVC	oleic acid	0 mM; 0,5 mM; 1 mM; 5 mM; 10 mM; 20 mM

Fig. 8: Response of P_aldA+eYFP (yellow) to different chain lengths of fatty acids compared to an empty vector control (gray). Plot A presents the response for palmitic acid, plot B for stearic acid and plot C for oleic acid. The fluorescence was measured at an excitation wavelength from 497 nm and an emission wavelength from 540 nm.

The measurement showed that the P_aldA produced a high amount of eYFP when oleic acid (C18:1) was added to the culture. In addition, P_aldA can also detect stearic acid (C18:0), the saturated form of oleic acid, as well as palmitic acid (C16:0), although with a weaker absolute fluorescence value compared to oleic acid (C18:1).

In vivo detection of fatty acids in our production strains
After testing the dose-dependent response of the created biosensor constructs to fatty acids added extracellularly, we were interested in exploring the biosensor response to the intracellular increase of fatty acids via coexpression with heterologous genes in vivo. Production strains, where we expected a higher production of long chain fatty acids, were co-transformed with biosensor constructs for a fast measurement method to show which cultures can produce more fatty acids.

Table 4: List of all in vivo biosensor experiments with the P_aldA.

Biosensor	production construct
PaldA:eYFP
PaldA:eYFP	ACC
PaldA:eYFP	TeHP
PaldA:eYFP	´tesA

Fig. 9: Two thioesterases and an Acetyl-CoA carboxylase complex (ACC) were combined with the P_aldA and the reporter gene eYFP. The cultures were induced by 1 mM IPTG and 1 mM rhamnose (+) and compared to an uninduced strain (-) and the absolute fluorescence were measured. That was done at an excitation wavelength of 497 nm and an emission wavelength of 540 nm.

When the ACC and a biosensor are combined and induced, the engineered strains shows a slight increase of fatty acid production compared to the control. This increase was specific for the induced sample. When the thioesterase ´TesA and the biosensor were combined and induced in the cells, the fluorescence was much higher than the control (biosensor only). Similar to ACC, fluorescence was increased compared to the uninduced culture.

The promoter fliC was published as a sensitive promoter for short chain fatty acids, especially for butyrate (C4:0)⁴. This promoter was isolated from the E. coli wild type genome. In the wild type the short chain fatty acids have an impact on the flagellar expression. The P_fliC is repressed by leucine-responsive regulatory protein (Lrp). Butyrate can enhance the expression of the flagellar expression like leucine which is a ligand of Lrp. Difference between thus enhancers is that the promoter fliC is only sensitive for the butyrate and not for the leucine¹.
The promoter was tested for the sensitivity to butyric acid in the culture medium by combining the promoter to an eYFP (BBa_E0030)² as a reporter gene. The concentrations of butyric acid were from 0.5 mM to 20 mM.

Table 5: List of all experiments with the P_fliC. Concentrations are listed as final concentrations used for induction of the cultures.

Biosensor	fatty acid	Final concentrations
PfliC:eYFP	Butyric acid	0 mM; 0,5 mM; 1 mM; 5 mM; 10 mM; 20 mM
EVC	Butyric acid	0 mM; 0,5 mM; 1 mM; 5 mM; 10 mM; 20 mM

Fig. 10: Response of P_fliC+eYFP (red) to different chain lengths of fatty acids compared to an empty vector control (black).

The experiment showed that the fluorescence does not grow with higher concentrations of butyric acid. Surprisingly the fluorescence from the empty vector control rises with higher concentrations while the P_fliC shows a falling tendency. A big issue with this experiment was that the E. coli cultures were not able to grow in the high concentration of butyric acid and at lower concentrations the fluorescence stayed on the same level like the uninduced control.
Finally, the previously obtained GC-MS results to our measurements were compared with the biosensor results. Fig. 11 shows the biosensor data compared to GC-MS data, thus indicating a strong correlation between the two methods. In summary, the results show that the biosensor does not work as a fast and efficient read-out for intracellular fatty acids, although the data obtained from it corresponds to actual measured GC-MS data.

Fig. 11: Comparison of the in vivo biosensor (blue) to the GC (red) with stearic acid (18:0) about the fold change. The in vivo biosensor was diluted to an OD of 0.5 and induced by 1 mM IPTG and 1 mM rhamnose. For the GC, 4 ODU were used for the measurement.

Fig. 11 shows that the fold change from fatty acids by including ´TesA to the organism is clearly higher than the wild type (WT) in both experiments. The fold change from fatty acids by including ´TesA to the organism is also greater than the experiments where ACC or TeHP were included to the organism. In the experiment from ACC a slightly higher fold change could be detected compared to the WT. When both methods are compared to each other, Fig. 11 shows that the biosensor experiments with P_aldA:eYFP + ´TesA have a higher fold change than the GC C18:0 + ´TesA experiment.

1. D. P. Clark and J. E. Cronan Jr., "Two-carbon compounds and fatty acids as carbon sources," in Escherichia coli and Salmonella: Cellular and Molecular Biology, F. C. Neidhardt, R.Curtiss III, J. L. Ingraham, et al., Eds., pp. 343–357, ASM Press,Washington, DC, USA, 1996
2. Fuzhong Zhang, James M Carothers, Jay D Keasling. "Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids" Nature Biotechnology volume 30, pages 354–359 (2012)
3. Mee-Jung Han, Jeong Wook Lee, Sang Yup Lee, and Jong Shin Yoo. "Proteome-Level Responses of Escherichia coli to Long-Chain Fatty Acids and Use of Fatty Acid Inducible Promoter in Protein Production" Hindawi Publishing Corporation Journal of Biomedicine and Biotechnology Volume 2008, Article ID 735101,12 pages
4. Toru Tobe,* Noriko Nakanishi, and Nakaba Sugimoto "Activation of Motility by Sensing Short-Chain Fatty Acids via Two Steps in a Flagellar Gene Regulatory Cascade in Enterohemorrhagic Escherichia coli" INFECTION AND IMMUNITY, Mar. 2011, p. 1016–1024