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The solution for addressing the issues related to pollution from medium-chain fatty acid production and the inefficient utilization of kitchen waste oil can be divided into three parts: optimization of medium-chain fatty acid synthesis, synthesis of derivatives of medium-chain fatty acids, and benzo[a]pyrene degradation. We conducted experiments in a sequential manner to validate these components. Additionally, we demonstrated the feasibility of a suicide switch and identified current shortcomings in suicide switch technology. We proposed a more suitable, controllable, and simplified suicide switch that aligns better with the requirements.

Part1:

MEL synthesis system

Due to the coexistence of different fatty acid pathways within the XM01 strain, the formation of MEL leads to the accumulation of intracellular lipids. Whether it is the synthesis of intracellular lipids or MEL, energy, fatty acid precursors, and acetyl-CoA are essential for these metabolic processes. The synthesis of intracellular lipids will inevitably hinder the extensive production of MEL to a certain extent.



Figure 1 Schematic diagram of double gene knockout plasmid construction.

We chosed to construct double knockout plasmids for the DGA1 and ARE1 genes. Using the genomic DNA of the XM01 strain as a template, we amplified the DGA1-5’arm and DGA1-3’arm, as well as the ARE1-5’arm and ARE1-3’arm fragments of the XM01 strain. The correctly sequenced and digested fragments were then ligated to the FL4A-HTP-loxp vector provided by the PI using T4 DNA ligase. Finally, using the FL4A-NAT-loxp-ARE1(DGA1) plasmid as a template, we performed PCR amplification with corresponding primers to obtain linearized knockout fragments. Since the probability of simultaneous double gene knockout is relatively low, we chose to perform the ARE1 gene knockout on the basis of DGA1 knockout.



Figure 2 PCR verification of double gene knockout.

After inoculating the ΔDGA1ΔARE1 strain and the original XM01 strain into 5 mL of seed liquid culture medium and incubating them at 28°C with shaking at 180 rpm for 48 hours, they were transferred to MEL fermentation medium for 7 days of oscillatory cultivation. Changes in MEL production capacity and intracellular lipid content of both the original XM01 strain and the ΔDGA1ΔARE1 strain were measured. The results are shown in the graph below.

Under the same culture conditions, the ΔDGA1ΔARE1 strain exhibited a significantly increased MEL production capacity compared to the original XM01 strain. The production of MEL increased from 62.8 g/L to 90.6 g/L, demonstrating that the double knockout of ARE1 and DGA1 greatly enhanced the strain's ability to produce MEL. At the same time, the intracellular lipid content decreased from 68% to 12.5%.



Figure 3 MEL production, biomass, and intracellular oil weight per gram of biomass in the ΔDGA1ΔARE1 strain

In addition, we conducted measurements to determine whether the double knockout of the DGA1 and ARE1 genes would have an impact on the cell's growth rate. As shown in the graph below, the results indicate that, compared to the original XM01 strain, the growth rate of the ΔDGA1ΔARE1 strain was not affected by the knockout of the DGA1 and ARE1 genes.



Figure 4 Growth rate changes in the ΔDGA1ΔARE1 strain

The cells cultured for seven days from both the ΔDGA1ΔARE1 strain and the original XM01 strain were subjected to staining of intracellular lipids using the Nile Red fluorescent staining method. Subsequently, they were observed under a fluorescence microscope. In comparison to the original strain, which exhibited a long chain of golden-yellow lipid droplets inside the cells, the knockout of ARE1 and DGA1 genes clearly had a significant impact on intracellular lipid synthesis.



Figure 5 Staining images of intracellular lipids in the original XM01 strain and the ΔDGA1ΔARE1 strain.

To investigate the relationship between the composition of carbon sources (natural plant oils) and the resulting products, specifically the production of MEL (mannosylerythritol lipids), you used soybean oil, peanut oil, rapeseed oil, corn oil, sunflower oil, and safflower oil as carbon sources for fermentation in shake flasks. We collected samples and performed methylation on both the carbon sources (natural plant oils) and the corresponding generated MEL. Subsequently, we conducted gas chromatography/mass spectrometry (GC/MS) analysis to analyze the fatty acid composition of the carbon sources and the fatty acid composition of the produced MEL. The fatty acid compositions of the carbon sources and the corresponding MEL are shown in the figure below.



Figure 6 Fatty acid composition of natural plant oil and MEL

The results of the correlation analysis have demonstrated that the proportion of fatty acid components in the substrate determines the changes in the composition of medium-chain fatty acids in MEL synthesis. The higher the content of a particular component in the substrate, the more it will be reflected in the corresponding component in the produced MEL. Understanding this pattern allows us to produce a wide range of medium-chain fatty acids in large quantities, enabling customized production of medium-chain fatty acids.

Conclusion:
In summary, by knocking out the ARE1 and DGA1 genes, we significantly reduced the formation of intracellular lipid droplets during MEL synthesis, which facilitated both the synthesis and secretion of MEL. Under the experimental conditions of shake flask fermentation, we achieved a MEL yield of 90.6g/L, with the intracellular lipid content reduced to 12.5%. This successful transformation of the original XM01 strain demonstrates the effectiveness of our approach. Additionally, we have uncovered the potential for customized production of medium-chain fatty acids.

Part2:

Medium chain fatty acid derivatives

Synthesis of α-olefins

The recombinant expression vector, provided by the Institute of Bioenergy and Process Research, Chinese Academy of Sciences, was introduced into Escherichia coli BL21 cells for seed culture in LB liquid medium. Subsequently, these cells were inoculated into LB liquid medium supplemented with glycerol and a rare salt solution for further cultivation. When the culture reached a certain optical density (OD), isopropyl-β-D-thiogalactoside (IPTG) and δ-aminolevulinic acid were added to induce the expression of the P450 enzyme. After a specific period of cell cultivation, the cells were lysed, and the cell lysate was purified using Ni-NTA resin. The protein was collected, and gel electrophoresis was performed to verify the presence of the OleTJE P450 enzyme.

It was observed that the protein secreted by Escherichia coli BL21 cells, after purification and processing, exhibited a single and clear band on the gel electrophoresis, with a size ranging from 45 kDa to 60 kDa. The OleTJE P450 enzyme used in this study has a size of 50.53 kDa, which aligns well with the position of the band shown on the protein gel.



Figure 7 (A) Gene circuit (B) SDS-PAGE result image

Due to the production of medium-chain fatty acids by MEL, with substrate chain lengths ranging from C8-12, we initially determined the in vitro activity of the P450 fatty acid decarboxylase OleTJE through a recombinant in vitro reaction. We conducted experiments using various fatty acids, including C8:0, C9:0, C10:0, C11:0, C12:0, C12:1, and C14:0, as substrates, with the addition of 0.2 μM of the P450 fatty acid decarboxylase OleTJE. The reaction was carried out for two hours, and by measuring the changes in substrate content, we determined whether the purified P450 fatty acid decarboxylase OleTJE possesses the ability to catalyze medium-chain fatty acid substrates in vitro. As a control group, we used C14:0 palmitic acid substrate to confirm the protein activity of P450 fatty acid decarboxylase OleTJE.



Figure 8 Conversion rate of fatty acid substrates catalyzed by P450 enzyme.

Using the MEL obtained from the experiments described in this study, we prepared medium-chain fatty acids as substrates for the reaction. The reaction system included 0.5 μM of the enzyme, 200 μM of medium-chain fatty acids, and 500 μM of H2O2. The reaction was carried out at 28°C for 2 hours. The products obtained from the reaction were analyzed using GC-MS. Based on the GC-MS analysis results, the actual conversion rates of α-olefins were calculated. For each type of fatty acid in the fatty acid composition, the corresponding content of α-olefins obtained from the measurements was used as the numerator, with the content of each fatty acid type as the denominator, to calculate the actual conversion rate of α-olefins for each medium-chain fatty acid.



Figure 9 Conversion yield of α-olefins.

Synthesis of 10- hydroxydecanoic acid

In order to enhance the efficiency of preparing 10-hydroxydecanoic acid, we introduced the cofactor GDH (Glucose Dehydrogenase) to provide more NADH. This was achieved by designing a SpyCatcher-SpyTag system to construct a dual-enzyme complex. Due to the relatively large quantities of both alkBGT and the cofactor GDH, expressing them in the same plasmid can often be challenging. Therefore, we considered the approach of dual-plasmid transformation and selected two plasmids, PET28a and pCDFuet-1, each with different origins of replication.

Firstly, the verification of the alkBGT system was conducted. Colony PCR results showed the appearance of the target band at around 4000 bp, indicating successful plasmid integration into Escherichia coli. SDS-PAGE analysis revealed the successful expression of the AlkB, AlkG, and AlkT proteins. Additionally, by setting a gradient of IPTG concentrations, it was observed that the protein expression was most efficient when induced with 0.5 mM IPTG, yielding the highest protein expression levels.In order to better visualize the expression intensity of proteins at different IPTG induction concentrations, we used software(GelAnalyzer) to analyze the results of SDS-PAGE and obtained a data graph of the relative protein expression intensity.



Figure 10 (A) Gene circuit (B) Colony PCR (C) SDS-PAGE M: Protein Marker 1: 0mM IPTG 2: 0.2mM IPTG 3: 0.5mM IPTG 4: 1mM IPTG. The protein bands from top to bottom are alkT (56KDa), alkB (45KDa), and alkG (18KDa).



Figure 11 Expression intensity of proteins at different induction concentrations

Next, the validation of the alkBGT+GDH+SpyTag+SpyCatcher system was conducted. Colony PCR results showed the appearance of target bands at approximately 4000 bp and 1000 bp, indicating successful plasmid integration into Escherichia coli. SDS-PAGE analysis revealed successful expression of the AlkB, AlkG, AlkT, and GDH proteins. By setting a gradient of IPTG concentrations, it was observed that the protein expression was most efficient when induced with 0.2 mM IPTG, yielding the highest protein expression levels.In order to better visualize the expression intensity of proteins at different IPTG induction concentrations, we used software to analyze the results of SDS-PAGE and obtained a data graph of the relative protein expression intensity.However, the obtained data graph is inconsistent with the gel electrophoresis results due to inconsistent light distribution during the image capturing process, leading to errors in the software analysis results. Therefore, when using relevant software(GelAnalyzer) to process gel electrophoresis results, it is important to pay attention to the distribution of light sources during image capturing.

Moreover, a distinct band was observed at around 90 KDa, indicating successful linkage of alkT and GDH through SpyTag and SpyCatcher. As a result, the band corresponding to GDH was lighter compared to the previous one due to partial migration to the 90 KDa position as a result of binding with GDH.



Figure 12 (A) Gene circuit (B) Colony PCR (C) SDS-PAGE M: Protein Marker 1: 0mM IPTG 2: 0.2mM IPTG 3: 0.5mM IPTG 4: 1mM IPTG.The protein bands from top to bottom are alkT+GDH (87KDa), alkT (56KDa), alkB (45KDa), GDH (31KDa), and alkG (18KDa).



Figure 13 Expression intensity of proteins at different induction concentrations

Additionally, we amplified the GDH fragment without the SpyTag from the previously constructed pCDFuet-1+GDH+SpyTag plasmid using PCR. The purpose was to verify that the catalytic efficiency of the engineered bacteria without the enzyme complex is lower than that of the engineered bacteria with the enzyme complex. However, to ensure that the primer’s Tm values did not differ too much, we adjusted the length of the PCR fragment and retained a portion of the SpyTag sequence, aiming to disrupt the formation of the enzyme complex. After sequencing the PCR fragment, we reconnected it to a new pCDFuet-1 vector to construct a new expression vector. The newly constructed expression vector was then introduced into the E.coli BL21 strain, which already contained the PET28a+AlkBGT+SpyCatcher plasmid. Colony PCR confirmed the successful introduction of the dual plasmids. SDS-PAGE results indicated the presence of bands corresponding to the enzyme complex between 75 kDa and 100 kDa. This suggests that we did not remove the amino acids responsible for binding with SpyCatcher. In the future, we will PCR amplify the GDH fragment without any SpyTag and repeat the validation process.



Figure 14 (A) Gene circuit (B) Colony PCR (C) SDS-PAGE M: Protein Marker 1: 0mM IPTG 2: 0.5mM IPTG

After constructing the three engineered bacteria, we conducted catalytic experiments with decanoic acid. Despite varying IPTG concentrations, induction temperatures, and extraction conditions, we were unable to obtain the desired product, 10-hydroxydecanoic acid. We speculate that this is due to unequal expression levels of the proteins in the catalytic system, which is evident from the SDS-PAGE results. It is also possible that adding too many proteins to the same plasmid has overloaded the engineered bacteria, preventing efficient expression of each protein. In the future, we will continue by redesigning the RBS to achieve similar expression levels for each catalytic protein.

Part3:

Benzo[a]pyrene degradation system

Due to time constraints, we were unable to verify the red light switch. However, we independently verified the degradation effect of benzo[a]pyrene.According to the graph, it can be seen that after approximately 15 days, almost all of the benzopyrene has been degraded, which can demonstrate the strong benzopyrene degradation activity of the Uop1 enzyme.



Figure 15 The change in benzopyrene content

Part4:

Kill switch

To prevent the leakage of foreign genes into the environment, we constructed a suicide switch as shown in the diagram below. Successful integration of the plasmid into the bacterial strain was confirmed through colony PCR, as shown in the figure below, where a target band of approximately 750 bp was obtained. After inducing with IPTG for 5-6 hours, SDS-PAGE analysis revealed clear bands in the control group, while no distinct bands were observed in the induced group. This is because the induced group expressed a lysis protein, resulting in self-destruction and the release of a hydrolytic enzyme, which subsequently degraded all other proteins.



Figure 15 (A) Gene circuit (B) Colony PCR (C) SDS-PAGE M: Protein Marker 1: 0mM IPTG 2: 0.2mM IPTG

After successful plasmid integration and expression validation, different concentrations of NeuAc (N-acetylneuraminic acid) ranging from 0g/L to 8g/L were added to the culture medium. The OD (Optical Density) values of E. coli BL21 were measured over time to determine the optimal NeuAc concentration required to ensure the normal survival of the bacteria. In the graph, "0h" represents the time point at which IPTG induction was added.

It can be observed that the control group and E. coli BL21 without IPTG induction exhibited typical sigmoidal growth curves. In contrast, the OD values of E. coli BL21 in the experimental group decreased as the NeuAc concentration decreased. This phenomenon occurred because at lower NeuAc concentrations, fewer NeuAc molecules bound to the riboswitch. As a result, the ribozyme was unable to perform self-cleavage, and the downstream target gene remained hidden, avoiding degradation by the ReJ enzyme. Consequently, the lysis protein ΦX174 was highly expressed, leading to the death of E. coli.

This demonstrates the successful functionality of our NeuAc riboswitch. Through modeling and data analysis, it was determined that E. coli would undergo lysis and die when removed from the culture environment if NeuAc was added at a concentration of 10g/L in the culture medium.



Figure 17 The change in OD value at different NeuAc concentrations(0.2mM IPTG)



Figure 18 The change in OD value at different NeuAc concentrations(0.5mM TPTG)

It can be observed that in the end, the control group had lower OD values compared to the induced group. This is due to the fact that we used a 96-well plate for detection, and the smaller growth space in the wells allowed the inhibited growth of the induced bacterial strains to remain relatively stable for a longer period. Consequently, there would be a higher cell count in the induced group compared to the control group in the later stages. However, the actual effect should be assessed based on the OD values in the earlier part of the growth curve.

Additionally, we compared the differences in OD values under different IPTG concentrations and found that the suicide effect was less effective under 0.5 mM IPTG induction compared to 0.2 mM IPTG. This suggests that the reason behind this is likely due to excessive protein expression, which can lead to the formation of inclusion bodies, thereby reducing the activity of the proteins themselves. As a result, the lysis effect is compromised.

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