Team:Duesseldorf/Plant

Our project aims to synthesize the milk proteins and a broad range of fatty acids for creating most of the components of the cow’s milk, while reducing the amount of CO₂ in the atmosphere. The other components are minerals and vitamins, which are already synthesized without using the cow. The most abundant ingredient in cow’s milk is water. With our goal in mind, we decided to use the photosynthetic cyanobacterium Synechocystis. It has the ability to perform oxygenic photosynthesis and therefore use CO₂ in the atmosphere and produce O₂. We specifically used Synechocystis sp. PCC 6803 because it is a well established model organism, which can be easily genetically modified, for example to heterologously express milk proteins. Furthermore, not only the whole fatty acid metabolism is well known, but a genome-scale metabolic model is well-established for this organism, which is really helpful in modifying the organism to synthesize our fatty acids of interest. We were able to successfully model our project toward increased fatty acid production, as well as engineer various strains using synthetic biology by either modifying the genome directly or using a plasmid-based approach. First, our aim was to heterologously express a wide range of different, specific thioesterases from various other organisms ^{1, 2, 3}. Each thioesterase is able to specifically determine the chain-length of fatty acids by hydrolyzing the fatty acyl-ACP or -CoA bond. Therefore, we cloned each of the thioesterase genes via Gibson Cloning into the pSHDY and the pSNDY backbones.

**Table 1**: Overview of used thioesterases.
Sample ID	Organism	Chain length	Reference	Genbank ID
ChFatB2	Cuphea hookeriana (cigar plant)	C8:0 and C10:0	Dehesh, Katayoon, et al. ¹	U39834
TeMF	Marvinbryantia formatexigens	C4:0, C6:0 and C8:0	Jawed, Kamran, et al. ²	EET61113.1
TeBT	Bacteroides thetaiotaomicron	C4:0	Liu, Xiping, et al. ³	AAO77182.1
TeHP	Haematococcus pluvialis	C16:0 and 18:0	Lei, Anping, et al. ⁴	HM560034
tes’A	Escherichia coli	C16:0 and C18:0	Cho, Hyeseon, and John E. Cronan. ⁵	945127⁸

The pSHDY or the pSNDY backbone has a broad host range, which enables the conjugative transfer from E. coli to cyanobacteria and other microorganisms. For the uptake of the plasmid, the triparental mating method was used.

**Fig. 1**: Conjugation plasmids. Example of pSHDY/pSNDY plasmid with a thioesterase gene inserted with the P_J23119 (BBa_J23119), RBS* (BBa_K2924009) and double terminator (BBa_B0015). It contains the restriction sites EcoRI (E), XbaI (X), SpeI (S) and PstI (P) at the BioBricks. For ’tesA a rhamnose-inducible promoter was used ⁶.

After testing via colony PCR whether the colonies are positive, the conjugants were inoculated and grown in BG11 medium. The cells were harvested at 4 OD units, i.e., an equivalent of 4 ml cells at OD₆₀₀ = 1 and used for extraction and derivatization of fatty acids for further use by gas chromatography-mass spectrometry (GC-MS).

**Fig. 2**: GC-MS analysis of Synechocystis conjugants with the thioesterases ChFatB2, TeHP and ‘TesA

As seen in Fig. 2, respective changes in the fatty acid yield compared to the Synechocystis control are shown. The conjugants with the thioesterase ChFatB2 (BBa_K2924010) from the cigar plant showed an increase in C18:1 fatty acid, but a decrease in C20:2 fatty acids. In contrast, the conjugants with the thioesterase TeHP (BBa_K2924011) showed a higher fatty acid yield in C18:0 and C18:1 fatty acids and a decrease of fatty acids in C18:3 and C20:1. Further, the cells containing the leaderless thioesterase ‘TesA (BBa_K2924051) from E. coli showed a higher yield in C18:0 and C20:2 fatty acids, but a clear decrease in C16:0, C16:1 and C18:3 fatty acids.

To summarize, it can be said that the fatty acid yield for one or more chain lengths increased due to the overexpression or heterologous expression of the different thioesterases, like ’tesA. Unfortunately, the other thioesterases, which primarily produce short chain fatty acids such as C4:0 and C6:0 could not be tested, because the method for them is different and has not yet been established for our GC-MS facility.

Another promising method is the inducible down-regulation of enzymes involving the fatty acid metabolism for example in fatty acid degradation. Therefore, we used the previously published CRSIPRi/dCas9-system⁷, which was kindly provided to us by Dr. Elton P. Hudson from the KTH Royal Institute for Technology in Sweden. Since the system was already established for Synechocystis sp. PCC 6803, we used the background strain from Yao et al. encoding dCas9 and integrated a gene-specific small guide RNA (sgRNA) for each target, which is activated by anhydrotetracycline (aTc) and is responsible for finding the targeted gene and recruiting dCas9, into the genome of Synechocystis sp. PCC 6803 to enable the complex formation of dCas9 with the sgRNA and inhibit the transcription temporarily.

**Fig. 3**: Scheme of construct. The inserts, which were obtained by overlap extension PCR, containing the aTc-inducible promoter P_L22, the specific sgRNA (orange), dCas9 binding site, a terminator and a kanamycin resistance, were cloned *via* Gibson Cloning into the NS4 backbone, which enables the integration into the genome of Synechocystis sp. PCC 6803 *via* homologous recombination.

This method was first tested with the fluorescent protein mVenus. Therefore, we created a sgRNA for mVenus and introduced it into the genome of Synechocystis sp. PCC 6803 via homologous recombination in combination with plasmid-based expression of P_cpc560:mVenus. The fluorescence of mVenus under the constitutive promoter (BBa_K2924000) was compared to the fluorescence of the induced knockdown of mVenus 24 h after induction with aTc.

**Fig. 4**: Fluorescence measurement of the mVenus knock-down (KD) strain in the plate reader 24 h after induction with 500 nM aTc (red) or 100 % EtOH (negative control, blue). 2 biological and 3 technical replicates were cultured in 6-well plates.

As seen in Fig. 4, the overall fluorescence of the induced Synechocystis knock-down strain decreased compared to the uninduced control strain. Nevertheless, the induced Synechocystis knock-down strain still showed some fluorescence compared to the empty vector control (EVC). We then chose to further characterize the strains microscopically.

**Fig. 5**: Overview (A and B) and detail image (C and D) of the mVenus knock-down strain which were either induced with aTc (500 nM) or EtOH, after 24 h of incubation. It shows clearly the chlorophyll autofluorescence (purple) and the mVenus fluorescent protein (green).

The localization of the chlorophyll in the membrane due to the autofluorescence, as well as the localization of the cytosolic mVenus fluorescent protein can be clearly seen in Fig. 5. As well as shown in Fig. 4, there is an overall lower fluorescence compared to the control culture, measured already in the plate reader. Interestingly, in contrast to our expectation that this may be due to a dose-dependent, gradual decrease of mRNA, resulting in a gradual decrease of protein, it is more comparable to an “ON/OFF”-switch. Most of the induced cells show no evidence of mVenus fluorescence at all, while a few remaining cells show the same fluorescence intensity compared to the control strain.

These results confirm the functionality and successful implementation of our basic concept of repressing specific genes by the CRISPRi/dCas9-system⁷.

This concept was then further implemented in our project by down-regulating promising candidates of the fatty acid metabolism previously evaluated and confirmed by modeling, such as the long-chain-fatty-acid CoA ligase, involved in β-oxidation, by inducing the strain with aTc and analysing the fatty acid profile via GC-MS.

**Fig. 6**: Relative fatty acid yield of *Synechocystis sp.* PCC 6803 transformants with the long-chain-fatty-acid CoA ligase-specific sgRNA compared to the control strains. Different fatty acids are listed and tested for two biological replicates. Measurements were carried out *via* GC-MS.

The results show that the long-chain-fatty-acid CoA ligase knockdown strain shows a slight difference in the fatty acid profile and yield as in the control strain (shown in Fig. 6). For the fatty acids C17:1, C18:0 and C18:1, there is a slight increase in fatty acid yield visible. This may be due to the long-chain-fatty-acid CoA ligase knockdown.

**Fig. 7**: Scheme of modified Synechocystis sp. PCC 6803, producing the proteins of interest.

It is likely that this method needs further optimization. At this point, the specific time point at which the reduction of the protein results in a change of the metabolic reaction is unclear. It is also highly likely that different sgRNA sequences have a different specificity toward their target.

With all of this, we were able to show that it is possible to synthesize a broad range of fatty acids with Synechocystis sp. PCC 6803. Next, our aim was to heterologously produce the milk proteins in Synechocystis sp. PCC 6803 as well, in addition to our other chassis.

Therefore, the plasmid containing ɑ-s1-casein under the control of the strong, constitutive cyanobacterial promoter P_cpc560 was transferred via mating triparental from E. coli to Synechocystis sp. PCC 6803. These conjugants were inoculated and ɑ-s1-casein production was investigated.

**Fig. 8**: *via* SDS-PAGE of cyanobacterial protein, separated into soluble and insoluble protein fractions. The gel was run at 220 V for 45 minutes and then stained with a Coomassie blue dye.

The gel in Fig. 8 shows a clear visible band at around 26 kDa, which is the expected size of our milk protein, whereas the empty vector control shows none. This clearly indicates successful production of the target protein in Synechocystis sp. PCC 6803.

**Fig. 9**: Growth analysis of Synechocystis protein production strains compared to the empty vector control (EVC) in black. Grey: P_cpc560:a-s1-casein. Yellow: P_cpc560:mVenus.

Fig. 9 shows the growth behavior of two strains heterologously producing different proteins in Synechocystis. In contrast to the empty vector control shown in black, both production strains show slower growth. As previously discussed in our metabolic model, targeted production directly interferes with growth and biomass production. A decrease in growth in our production strain therefore indicates redirection of cellular resources towards the target protein.

To summarize all of these results, we were able to genetically modify Synechocystis sp. PCC 6803 for our project-specific goals, thereby enabling synthesis of all building blocks needed to create synthetic cow’s milk only with this one organism. We present a collection of methods, synthetic biology strategies, and parts specifically tailored to future applications using photosynthetic bacteria in the hope that future iGEM teams may benefit from our work. Finally, by establishing this strain as a main organism for producing our products, we present an eco-friendly production alternative because of its phototrophic abilities to reduce CO₂ and increase O₂ in the atmosphere.

References

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