Modular fatty acid chain length regulation
For synthesizing a broad range of fatty acids, several thioesterases of various organisms were heterologously expressed in two different microbes - Escherichia coli and Synechocystis sp. PCC 68031, as indicated in table 1.
| sample ID | organism | chain lenght | reference | Genkbank ID |
|---|---|---|---|---|
| ChFatB2 | Cuphea hookeriana | C8:0 and C10:0 | Dehesh, Katayoon, et al.2 | U39834 |
| TeMF | Marvinbryantia formatexigens | C4:0, C6:0 and C8:0 | Jawed, Kamran, et al.1 | EET61113.1 |
| TeBT | Bacteroides thetaiotaomicron | C4:0 | Liu, Xiping, et al.3 | AAO77182.1 |
| TeHP | Haematococcus pluvialis | C16:0 and C18:0 | Lei, Anping, et al.4 | HM560034 |
| ´TesA | Escherichia coli | C16:0 and C18:0 | Cho, Hyeseon, and John E. Cronan5 | 94512715 |
Therefore, we used the pSHDY or the derivate pSNDY, which enables the plasmid transfer via conjugation from E. coli to a cyanobacteria or other organisms (Fig. 1).
Results
To analyze the change in fatty acid yield of both organisms, we used the gas chromatography-mass spectrometry (GC-MS). E. coli cultures were grown overnight at 37°C and shaken at 250 rpm. On the following day, the cells were harvested at 4 ODU, i.e. an equivalent of 4 ml cells at OD600 = 1. They were centrifuged at 4500 rpm for 15 minutes and the pellet was used for the extraction and derivatization of fatty acids. Synechocystis cultures were grown under specific light conditions with 0.5% CO2 at 30°C and shaken at 150 rpm. These cells were also harvested at an ODU of 4 and centrifuged for further extraction and derivatization. The leaderless thioesterase of E. coli, ‘TesA (BBa_K2924051), was additionally induced with 0.5 mM rhamnose 24 h before harvesting.
In Fig. 2, changes can be seen in the yield of some fatty acids, for example C16:1 and C18:1 fatty acids. In E. coli transformants with TeHP, there is clearly a higher yield in C20:1 fatty acids. In contrast, E. coli transformants with ‘TesA show a clearly higher yield in C16:1, C18:1 and C20:1. This suggests the activities of the thioesterases TeHP and ‘TesA.
As figure 3 indicates, a change in the fatty acid profile and fold change in Synechocystis can be seen as well, for example in C18:0, C18:1 and 20:2. The Synechocystis conjugants with the thioesterase ChFatB2 are showing a high fold change for C18:1 fatty acids. For the Synechocystis conjugants with the thioesterase TeHP, there is a higher yield in C18:0 and C18:1 detected, in contrast, a decrease can be seen in C18:3 and C20:1 fatty acids. In the Synechocystis conjugants with the thioesterase ‘TesA, there was an increase in C18:0 and C20:2 detected, whereas a decrease of C16:0, C16:1 and C18:3 fatty acids was detected. This suggests the activity of the thioesterases.
Surprisingly, the different thioesterase works differently for E. coli and Synechocystis.
By integrating the leaderless thioesterase ‘TesA from E. coli into Synechocystis the organism changes its fatty acid profile to a more likely fatty acid profile of E. coli (Fig. 4).
To summarize, the fatty acid yield for one or a few more fatty acids increased and this may be due to the overexpression or regulated heterologous expression of different thioesterases.
The thioesterases TeBT and TeMF could not be analysed via GC-MS, because of there are not precisely enough established methods for detecting short-chain fatty acids.
Use of CRISPRi/dCas9-system
Modeling of the fatty acid metabolism of Synechocystis showed promising gene candidates for down-regulation. Since it was not clear how huge the impact of deletion of these genes will be and if it might disrupt cell viability, we chose to try an alternative method - an inducible downregulation at the transcriptional level via the previously published CRISPRi/dCas9-system6, to increase the yield of free fatty acids.
The CRISPRi/dCas9-system was kindly provided to us by kindly provided by Yao et al. (2015)14. Both dCas9 and the gene-specific sgRNA are inducible with anhydrotetracycline (aTc). The used dCas9 is catalytically inactive and therefore does not have an endonuclease activity like the ordinary Cas9. Therefore, this method is perfect for down-regulating enzymes essential for the organism.
Since the system was already established for Synechocystis sp. PCC 6803, we used the background strain from Yao et al. (2015)14 encoding dCas9 and integrated a gene-specific small guide RNA (sgRNA) for each target gene. The sgRNA 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 leading to temporary inhibition of the transcription.
Results
Similar to the heterologous expression of thioesterases, the GC-MS method was also used for the detection of the fatty acid composition in Synechocystis when target genes were knocked down. Synechocystis was grown under specific light and CO2 conditions at 30°C and 150 rpm. As proof of concept of the CRISPRi/dCas9 methodology, a sgRNA for mVenus was designed and coexpressed in a strain constitutively expressing mVenus (BBa_K2924036). The knockdown was tested by measuring the fluorescence at λex/em = 511 / 552 nm. First, the temporal response was checked. Maximum reduction of fluorescence could be observed approximately 24 h after induction with aTc, as shown in Fig. 6, which correlates with the maximal activity of the dCas9.
Afterwards the fluorescence of mVenus under the constitutive promoter (BBa_K2924000) was compared to the fluorescence of the induced knockdown 24 h after induction with aTc (Fig. 7).
As seen in Fig. 7, the overall fluorescence of the induced Synechocystis knockdown strain decreased compared to the uninduced control strain. Nevertheless, the induced Synechocystis knockdown strain still showed some fluorescence compared to the empty vector control (EVC).
This proves our basic concept of knocking down enzymes by the CRISPRi/dCas9-system7 without causing lethality by abolishing the function completely.
The strains were further characterized by confocal microscopy. For this purpose, Synechocystis sp. PCC 6803 with sgRNA_mVenus + pSHDY_Pcpc560_mVenus and Synechocystis sp. PCC 6803 with pSHDY_Pcpc560_mVenus cultures were diluted to an OD750 = 0.2 and induced with 500 nM aTc. As shown in Fig. 6, the most activity was detected after approximately 24 h. At this time point 1 ml of each induced and uninduced culture was sampled. These samples were further diluted 1:10 and used for fluorescence confocal microscopy (Fig. 8).
When the culture is induced with 500 nM aTc, there is an overall lower fluorescence compared to the control culture (compare: Fig. 8 A and B), which was already shown by the plate reader measurements. 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. Also 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. 8.
After testing and proving the method of CRISPRi/dCas9 by Dr. Elton P. Hudson with the fluorescent protein mVenus on our project, the mentioned sgRNAs for enzymes involved in the fatty acid metabolism were created and cloned into the NS4 backbone (Fig. 5).
Therefore, Synechocystis sp. PCC 6803 were transformed with NS4 plasmids containing the specific sgRNA. These knockdown (KD) strains were tested for their transcriptional activity via qPCR and the change in fatty acid yield via GC-MS.
First, the transcriptional level was tested of the KD strains. As seen in Fig. 9, the targets of choice were the enzymes acetyl coenzyme A acetyltransferase (thiolase), beta ketoacyl-acyl carrier protein synthase, enoyl-[acyl-carrier-protein] reductase and long-chain-fatty-acid CoA ligase. Therefore, the KD strains were cultivated in BG11 with an appropriate amount of antibiotics. After adding 500 µM anhydrotetracycline (aTc), the cultures were grown for another 24 h and 1.5 ml harvested. Furthermore, the RNA were isolated and for qPCR prepared.
Fig. 10 shows the transcriptional activity of the four different KD strains. The long-chain-fatty-acid CoA ligase (A) seems the most promising candidate for a successfully downregulation. In contrast, enoyl-[acyl-carrier-protein] reductase (B) seems to have no change in the transcriptional activity. The KD strains with beta ketoacyl-acyl carrier protein synthase (C) and acetyl coenzyme A acetyltransferase (D) as targets shows mixed results. Clone 1 of both KD strains seemed to be working correctly. Several biological and technical replicates of Synechocystis sp. PCC 6803 cultures, encoding the specific sgRNA for long-chain-fatty-acid CoA ligase, acetyl coenzyme A acetyltransferase or beta ketoacyl-acyl carrier protein synthase, were cultivated at 30°C and shaken at 150 rpm. The cultures were diluted to an OD750 of 0,2 and induced with 500µM aTc. After this the cells were harvested at an ODU of 4, i.e. an equivalent of 4 ml cells at OD600 = 1, and used for extraction and derivatization of fatty acids.
As seen in Fig. 11, it seems that long-chain-fatty-acid CoA ligase KD strain might enables the increase of long-chain fatty acids.
Fig. 12 shows the relative fatty acid yields of acetyl coenzyme A acetyltransferase (A) and beta ketoacyl-acyl carrier protein synthase (B) KD strains compared to the control. Both of the KD strains shows no change or a slight decrease in long-chain fatty acids, which suggests that the yield increased in short-chain fatty acids, but this could not be detected and proved.
These results depends on several effects. One effect that could influence the result is the efficiency of the used sgRNA, which depends on other factors, too. The efficiency of the sgRNA can only experimentally determined3. Another aspect could be the light-sensible degradation of aTc, which results in a lower signal that can be received. And therefore can influence the knockdown of the enzyme.
Also, there could be several execution mistakes be the reason for a slight difference in the fatty acid yield to see, like the timing of isolating the cells and execution of the extraction and derivatization of the fatty acids in the cell pellet.
Results Biosensor
Biosensors are an efficient in vivo tool, especially useful for screening a large number of strains on their metabolic abilities, for example 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. 13)8.
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 PAR and the reporter gene for RFP9.
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. 13.
The different PAR 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 culturing the cells. An empty vector control (EVC) treated in the same manner was included in each measurement.
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 empty vector control (EVC) should remain at a basal level. Fig. 14 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. 15).
Fig. 16 shows the same dose-response experiments for the new part, PAR: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 PAR: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. 17).
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 PaldA10. The promoter aldA was published as an oleic acid (C18:1) sensitive promoter enabling for the measurement of free fatty acids present in microorganisms. In Escherichia 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 metabolism9. 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)8. PaldA was isolated from the Escherichia coli wild type genome.
The measurement showed that the PaldA produced a high amount of eYFP when oleic acid (C18:1) was added to the culture. In addition, PaldA 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.
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)11. This promoter was isolated from the Escherichia coli wild type genome. In the wild type the short chain fatty acids have an impact on the flagellar expression. The PfliC 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 leucine11.
The promoter was tested for the sensitivity to butyric acid in the culture medium by combining the promoter to an eYFP (BBa_E0030)9 as a reporter gene. The concentrations of butyric acid were from 0.5 mM to 20 mM.
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 PfliC 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. 23 shows the biosensor data compared to GC-MS data, 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. 23 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. 23 shows that the biosensor experiments with PaldA:eYFP + ´TesA have a higher fold change than the GC C18:0 + ´TesA experiment.
Results ACC
Considering the aim of SynMylk-Project, to create a synthetic alternative to native cow’s milk, many directions of interest had been pointed out. One of the major research objects is the application of enzymes which are involved in the biosynthesis of fatty acids. To generate one of the major milk components, the lipids - these are important for the texture and taste of the native cow’s milk, generating necessary fatty acids represents the main challenge. In this frame, the rate-limiting steps were figured out by biological modelling. The Acetyl-CoA carboxylase complex (ACC), involved in the fatty acid biosynthesis represents one of the major protein complexes in fatty acid biosynthesis12.
Cloning of Acc genes in suitable expression systems
The single acc genes as well as an operon-like composite of the genes coding for the whole ACC protein complex were cloned into an inducible pET21a (Novagen) vector carrying an ampicillin resistance and containing strong T7 promoter and C-terminal histidine tag. The theoretical cloning was conducted using SnapGene 4.3.1.1. The parts were created using BioBrick restriction sites. In this frame, initially faced challenging cloning steps contain the amplification of the single acc genes from E. coli strain K12 gDNA. While accA and accB amplification via PCR worked, it was not straightforward applicable for the genes accC and accD. Thus, the composite construct containing the accABCD was commercially synthesized.
Expression of AccABCD protein complex
The heterological overexpression of ACC protein subunits were conducted using Escherichia coli BL21 (DE3) as an expression host. The cells were grown at 37°C until reaching OD0.6 and induced with 0.5 mM IPTG. After 3 hours the cells were harvested by centrifugation. The cell pellets were lysed by using lysis buffer (BPER). Further purification of overexpressed proteins were performed by immobilized metal ion affinity chromatography (IMAC) using NiNTA beads. The results are shown in Fig. 25.
The heterologous overexpression of single AccA and AccB worked out. Fig. 24 indicates the SDS-PAGE of the IMAC purified proteins AccA and AccB. There are bands of approximately proper molecular weight. Further, the Acc protein complex was overexpressed concerning the same conditions.
It is indicated in Fig. 25 that the overexpression of AccB and AccD worked out, while it remains unclear whether the other subunits had been seriously overexpressed. For AccA and AccC there are only tiny bands on the SDS-PAGE observable. This might be due to the approached cloning strategy. The control of T7 promoter may not be strong enough to guide the transcription process of all the subsequently added acc genes.
The rise in absolute fatty acid amount of ACC and tesA modified E. coli strains in comparison to wild type and HP (thioesterase of Haematococcus pluvialis) is not significant.
Protein Production in Escherichia coli BL21 DE3
For this composite part (BBa_K2924032) the strong T7 expression system was used, the PT7 promoter (BBa_K2406020) expresses α-s1-casein (BBa_K2924026) with the T7 terminator (BBa_B0012).
Fig. 29 shows a band of almost 25 kDa in the cell pellet sample of E. coli (BL21) with α-s1-casein, there is no band visible in comparison to the empty vector control of E. coli. This band is very likely the α-s1-casein protein, which ran slightly lower than expected. The band is faint compared to most other protein bands on the gel, suggesting that the yield is low for this expression. Additionally there is a new band visible around 55 kDa, which is not present in the empty vector, which might be a dimer of α-s1-casein, since it has roughly twice the mass of a monomer. The results indicate that E. coli could be suitable for the production of milk proteins, however, further experiments need to be carried out to observe the secretion efficiency for the protein and to determine how much of the protein can be produced. Additionally other milk proteins should be produced as well.
Protein Production in Bacillus subtilis DB430
Bacillus subtilis PHpaII + RBS + SPNprE+ α-s1-casein + 6xHis-tag + fd Terminator
This composite part (Fig. 30) contains the constitutive promoter PHpall (BBa_K2924043), expressing α-s1-casein (BBa_K2924026) with the secretion signal SPNprE (BBa_K2924047) and the fd terminator (BBa_K2924044) for expression of the protein in Bacillus subtilis.
Neither B. subtilis native nor heterologous expressed α-s1-casein secreted proteins were detectable on a coomassie stained SDS-PAGE at all (Fig. 31.1). This might be caused since the 30 ml culture might be not concentrated enough to enable detection of the proteins. To detect smaller amounts of protein Immunoblots on different B. subtilis fractions were executed. α-s1-casein has a molecular weight of 25.4 kDa without the secretion signal and 28.5 kDa with the secretion peptide. No bands could be detected in the SDS-PAGE gel for WT and pBSMUl1-SPNprE+α-s1-casein (Fig. 31.1). The expressed protein athands a 6xHis tag. For detection, a anti-his antibody is used and further treated on Immunoblot. An Immunoblot of the lysed cell pellet shows no strong bands for the WT, while showing two intense bands for pBSMUl1-SPNprE+α-s1-casein at 15 kDa and ~25 kDa respectively (Fig. 31.2). The band at 25 kDa corresponds to the expected size of α-s1-casein, while the band at 15 kDa might be degraded protein (Fig. 31.2). To prove, that our protein of interest is secreted into the growth medium another Immunoblot was executed with the concentrated fraction which should contain proteins over 30 kDa (Fig. 31.3). This fraction and not the fraction below 30 kDA was used, since the cutoff is close to the molecular size of the protein of interest and it therefore should be retained in this fraction. The immunoblot of the concentrated protein fraction containing proteins >30 kDa show no difference between the wildtype control and the strain expressing SPNpre+α-s1-casein indicating, that the protein is retained in the cells and not secreted into the medium (Fig. 31.3). In summary, The gene α-s1-casein was successfully cloned and expressed in Bacillus subtilis. With the focus on optimizing the expression of the protein of interest, five signal peptides (sslipA ,SPNprE, SPYurl, SPPel, and SPEpr) (BBa_K2924046-BBa_K2924050) were tested out. Immunoblot analysis of lysed pellet proteins illustrate the protein of interest inside the cells, indicating that the signal peptide SPNprE was suitable for α-s1-casein production (Fig. 31.2), but not for secretion, since no protein is shown in the growth media (Fig. 31.1) or in the concentrated media fraction containing proteins >30 kDa (Fig. 31.3). The band at 25 kDa resembles our protein of interest, while the band at 15 kDa might be degraded protein. After 12 hours the cells should have reached the stationary phase and do not produce new protein. The produced protein could be degraded after that time point. In further experiments the induction time should be decreased to see if the unspecific band at 15 kDa is decreased and the specific protein band is increased.
Bacillus subtilis Hpall + RBS + SPNprE + alpha-s2-casein + 6xHis-tag + fd terminator
This composite part (Fig. 32) contains the constitutive promoter PHpall (BBa_K2924043), expressing α-s2-casein (BBa_K2924027) with the secretion signal SPNprE (BBa_K2924047) and the fd terminator (BBa_K2924044) for expression and secretion of the protein into the Bacillus subtilis medium.
Neither B. subtilis native nor heterologous expressed α-s2-casein secreted proteins were detectable on a Coomassie stained gel at all (Fig. 40.1). This might be caused since the 30 ml culture might be not concentrated high enough to see the proteins. To detect smaller amounts of protein Immunoblot on different B. subtilis fractions were executed. α-s1-casein has a molecular weight of ~27 kDa without the secretion signal and ~30 kDa with the secretion peptide. No bands could be detected in the SDS-PAGE gel for WT and pBSMUl1-SPNprE+α-s1-casein (Fig. 33.1). The expressed protein athands a 6xHis tag. For detection, a anti-his antibody is used and further treated on Immunoblot . α-s2-casein has a molecular weight of ~27 kDa without and ~30 kDa with the signal peptide. The Immunoblot of the lysed pellet shows no strong band for the wildtype, while showing a faint band for pBSMUl1-SPNprE+α-s2-casein at 15 kDa (Fig. 40), however there is no strong band at the expected sizes at ~27 or 30 kDa as there is for pBSMUl1-SPNprE+α-s1-casein. Possible reasons for that might be that the protein was digested or cleaved. Furthermore, another protein bearing the same or similar epitope might be detected differently by the antibody.
Another, more favorable, explanation could be, that the secretion tag works more efficient for α-s2-casein then for α-s1-casein and therefore the protein is secreted more efficient to the medium. To prove, that our protein of interest is secreted into the growth medium another Immunoblot was executed with the concentrated fraction which should contain proteins over 30 kDa (Fig. 33.3). This fraction and not the fraction below 30 kDA was used, since the cutoff is close to the molecular size of the protein of interest and it therefore should be retained in this fraction. The Immunoblot of the concentrated protein fraction containing proteins >30 kDa for SpNprE+α-s2-casein shows a band at ~25 kDa, while the wildtype shows no band, indicating that the protein is secreted into the cell medium and is contained in the concentrated fraction (Fig. 33.3).
With our protein of interest, being ~27 kDa without and ~30 kDa with the signal peptide, it is highly possible that during the filter step proteins with this particular size did not flow through the column, since the cutoff was not chosen properly. For further experiments it should be chosen as: (Protein size / 2). Also it can be because the spinning time was too short for the proteins of interest to flow through the column.
In summary, α-s2-casein was successfully cloned and expressed in B. subtilis. With the focus on optimizing the expression of the protein of interest, five signal peptides (sslipA ,SPNprE, SPYurl, SPPel, and SPEpr) (BBa_K2924046, BBa_K2924047, BBa_K2924048, BBa_K2924049, BBa_K2924050) were tested out. No proteins were detected in the Coomassie stained growth media (Fig.29.1), indicating that further test need to be carried out on concentrated fractions to get clearer results. Immunoblot analysis of lysed pellet proteins illustrate no protein of interest in the pellet, but an unspecified signal that might be degraded or cleaved protein (Fig. 33.2). Immunoblot analysis of concentrated media fraction containing proteins >30 kDa illustrates the protein of interest indicating that the α-s2-casein protein fused to the SPNprE signal peptide is secreted into the growth media (Fig. 33.3).
In comparison to pBSMUl1-SPNprE+α-s1-casein (BBa_K2924033) pBSMUl1-SPNprE+α-s2-casein (BBa_K2924052) were it seems, that the protein of interest is retained in the cells alpha S2 casein seems to be secreted by the same secretion tag. Showing, that an optimal tag needs to be found for each individual protein. Many known B. subtilis secretion signals are available for that purpose.
With SPNprE a suitable secretion tag for α-s2-casein is now already identified.
The band at 25 kDa resembles our protein of interest, while the band at 15 kDa might be degraded protein. After 12 hours the cells should have reached the stationary phase and do not produce new protein. The produced protein could be degraded after that time point. In further experiments the induction time should be decreased to see if the unspecific band at 15 kDa is decreased and the specific protein band is increased.
Bacillus subtilis Hpall + RBS + SPNprE + alpha-s2-casein + 6xHis-tag + fd terminator
This composite part (Fig. 34) contains the constitutive promoter PHpall (BBa_K2924043), expressing α-s2-casein (BBa_K2924027) with the secretion signal SPYurl (BBa_K2924050) and the fd terminator (BBa_K2924044) for expression and secretion of the protein into the Bacillus subtilis medium.
Due to time issues only the concentrated protein fraction containing proteins around the size and higher than 30 kDa was analysed by Immunoblot (Fig. 34).
The Immunoblot of the concentrated media fraction containing proteins over and around 30 kDa indicates, that some of our target protein is secreted out of the B. subtilis cells, however not as strong as with another signal peptide (SPNprE)
In summary, α-s2-casein was successfully cloned and expressed in B. subtilis. With the focus on optimizing the expression of the protein of interest, five signal peptides (sslipA ,SPNprE, SPYurl, SPPel, and SPEpr) (BBa_K2924046, BBa_K2924047, BBa_K2924048, BBa_K2924049, BBa_K2924050) were tested out. Immunoblot analysis of concentrated media fraction containing proteins >30 kDa illustrates the protein of interest indicating that the α-s2-casein protein fused to the SPYurl signal peptide is secreted in low quantities into the growth media (Fig. 34).
In comparison to pBSMUl1-SPNprE+α-s1-casein (BBa_K2924033) the secretion is increased, but in comparison to pBSMUl1-SPNprE+α-s2-casein (BBa_K2924052) the protein secretion is decreased. Many known B. subtilis secretion signals are available for that purpose. With SpYurl we found a signal peptide, that is less suitable than SPNprE for α-s2-casein secretion in B. subtilis.
The band at 25 kDa resembles our protein of interest. After 12 hours the cells should have reached the stationary growth phase and do not overproduce new protein. The produced protein could be degraded after that time point. In further experiments the induction time should be decreased to see if the specific protein band is increased.
Protein Production in Synechocystis sp. PCC 6803
The protein production in Synechocystis sp. PCC 6803 is based on the strong expression of proteins in cyanobacteria enabled by the Pcpc560 promoter.The strong constitutive cyanobacterial promoter PPcpc560 (BBa_K2924000) was used for the expression of mVenus (BBa_K2924035) with the T1/T7 double terminator (BBa_B0015). The promoter was cloned into the pSHDY plasmid. The pSHDY plasmid [Fig. 36] is an RSF1010-based, low-copy self-replicating vector derived from pVZ321 and has a broad host range, which can ensure the conjugation from Escherichia coli to cyanobacteria and other microorganisms12.
To test the strength of the Pcpc560 promoter (BBa_K2924000), it was cloned in front of mVenus (BBa_K2924035), a fluorescent protein originally isolated from Aequorea victoria with improved brightness. The sequence was provided codon-optimized for Synechocystis sp. PCC 6803. All experiments were carried out in Synechocystis sp. PCC 6803, into which the plasmid was conjugated by triparental mating with a transformed E. coli strain.
Based on the growth of the different Synechocystis strains expressing an empty vector control, mVenus or ɑ-s1-casein it is possible to say that heterologous protein are strongly expressed leading to a metabolic burden, which is shown in decreased growth (Fig. 37).
The fluorescence per OD750 decreased over time, likely due to limitations in light and nutrients, which force the cells to put more energy into photosynthetic pigments.
After 2 days’ growth, 10 ml samples were taken and used for protein measurements and SDS PAGE. The Synechocystis cells were disrupted using glass beads to shred the cells in a Precellys® 24 homogeniser. The cell extract was centrifuged to obtain a pellet of insoluble protein and a supernatant of soluble protein, which were separated. The protein content of the fractions was quantified by a Bradford protein assay.
The total amount of protein produced by the cells with Pcpc560 expressing mVenus did not differ significantly from the empty vector control. The fraction of insoluble protein was larger in those cultures however, suggesting that not all mVenus is present as a soluble protein, as the protein folding apparatus might be overloaded by the strong expression.
The gel showed no difference between the EVC an the mVenus cultures in the insoluble protein. Between 25 and 35 kDa in the soluble fraction, a band is visible for mVenus, which is not visible in the EVC. mVenus has a molecular weight of 26.9 kDa, so the band is in the correct area.
To confirm the identity of the protein, a western blot was performed with the soluble protein fraction. Three clones containing pSDHY Pcpc560 + mVenus were used for expression and protein extraction. The Western blot was carried out with an anti-gfp antibody.
The western blot shows no band at all for the EVC, while showing three distinct bands of the mVenus colonies. The middle band corresponds to the expected size of mVenus. The other bands might be misfolded or partly-degraded protein respectively.
Composite part BBa_K2924034:
After testing the strength of the promoter Pcpc560 (BBa_K2924000) with a fluorescent reporter (BBa_K2924036), it was used to express a protein from cow’s milk, a-s1-casein (BBa_K2924026) in Synechocystis sp. PCC 6803.
After 2 days of growth, samples corresponding to 20 OD units were taken and used for protein measurements and SDS PAGE. The Synechocystis cells were disrupted using glass beads to shred the cells in a Precellys® 24 homogeniser. The cell extract was centrifuged to obtain a pellet of insoluble protein and a supernatant of soluble protein, which were separated.
The samples were used for an SDS-PAGE. ɑ-s1-casein is a protein of approx. 25.4 kDa size and was therefore expected around that size.
In Synechocystis strains expressing ɑ-s1-casein a specific protein band was visible slightly under 25 kDa, which was not detected in the empty vector control (Fig. 42). This band is very likely the ɑ-s1-casein protein, which ran slightly lower than expected. It is visible to a smaller degree in the insoluble protein fraction as well.
The example result of ɑ-s1-casein indicates that bovine milk proteins can be successfully produced in Synechocystis. They are mostly located in the soluble fraction of the cells. However in the future they need to be produced and purified in large scales in order to examine if the organism is suitable for industrial production.
- Kamran Jawed, Anu Jose Mattam, Zia Fatma, Saima Wajid, Malik Z. Abdin, Syed Shams Yazdani "Engineered production of short chain fatty acid in Escherichia coli using fatty acid synthesis pathway." PloS one 11.7 (2016): e0160035.
- Katayoon Dehesh, Aubrey Jones, Deborah S. Knutzon, Toni A. Voelker "Production of high levels of 8: 0 and 10: 0 fatty acids in transgenic canola by overexpression of Ch FatB2, a thioesterase cDNA from Cuphea hookeriana." The Plant Journal 9.2
- Xiping Liu, Haiying Yu, Xu Jiang, Guomin Ai, Bo Yu, Kun Zhu "Biosynthesis of butenoic acid through fatty acid biosynthesis pathway in Escherichia coli." Applied microbiology and biotechnology 99.4 (2015): 1795-1804.
- Lei, Anping & Chen, Huan & Shen, Guoming & Hu, Zhangli & Chen, Lei & Wang, Jiangxin. (2012). Expression of fatty acid synthesis genes and fatty acid accumulation in Haematococcus pluvialis under different stressors. Biotechnology for biofuels. 5. 18. 10.1186/1754-6834-5-18.
- Cho, Hyeseon, and John E. Cronan. "Defective export of a periplasmic enzyme disrupts regulation of fatty acid synthesis." Journal of Biological Chemistry 270.9
- Lun Yao, Ivana Cengic, Josefine Anfelt, Elton P. Hudson "Multiple gene repression in cyanobacteria using CRISPRi." ACS synthetic biology 5.3 (2015): 207-212.
- Zhang, Jian-Hua, et al. "Optimization of genome editing through CRISPR-Cas9 engineering." Bioengineered 7.3 (2016): 166-174.
- 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
- 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)
- 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
- 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
- Faergeman, Nils Joakim, and Jens Knudsen. "Role of long-chain fatty acyl-CoA esters in the regulation of metabolism and in cell signalling." Biochemical Journal 323.Pt 1 (1997): 1.
- Behle, A., Saake P., Axmann, I. M. "Comparative analysis of inducible promoters in cyanobacteria." bioRxiv (2019): 757948.
- Lun Yao, Ivana Cengic, Josefine Anfelt, Elton P. Hudson "Multiple gene repression in cyanobacteria using CRISPRi." ACS synthetic biology 5.3 (2015): 207-212.
- Zheng, Yan-Ning, et al. "Optimization of fatty alcohol biosynthesis pathway for selectively enhanced production of C12/14 and C16/18 fatty alcohols in engineered Escherichia coli." Microbial cell factories 11.1 (2012): 65