In order to enhance the yield of medium-chain fatty acids in XM01 bacteria, we delete the ARE1 and DGA1 genes in XM01, diverting a greater portion of carbon towards the synthesis of MEL rather than intracellular lipids. Following this, we introduce multiple genes into Escherichia coli for fermentation, resulting in the production of 10-hydroxydecanoic acid and alpha-olefin.Finally, a kill switch is designed to prevent gene leakage.

We choose to knock out ARE1 and DGA1 to increase medium-chain fatty acid production.First, we construct the XM01 strain ARE1 gene knockout vector (Figure 1) and the DGA1 gene knockout vector (Figure 2) separately.

Figure 1 DGA1 gene knockout vector

Figure 2 ARE1 gene knockout vector

After the double-gene knockout, the genomic DNA is extracted for PCR amplification, and the knockout results were validated by gel electrophoresis (Figure 3).

Figure 3 M represents the D2000 plus DNA Marker, and the bands from top to bottom correspond to 5000, 3000, 2000, 1000, 750, 500, 250, 100bp. "XM01" represents the genomic 5F-3R PCR results of the XM01 strain. "ΔARE1" represents the genomic 5F-3R PCR results of the double knockout strain with ARE1 and DGA1 genes.

We inoculated the ΔDGA1ΔARE1 strain and the XM01 wild-type strain separately into seed culture media and then transferred them to MEL fermentation media for oscillation cultivation. We measured the MEL production, biomass, and intracellular oil weight per gram of biomass for both strains (Figure 4). The results showed that the ΔDGA1ΔARE1 strain had increased MEL production and decreased intracellular oil content.

Figure 4 MEL production, biomass, and intracellular oil weight per gram of biomass for both strains. "ΔDGA1ΔARE1" represents the double knockout strain with ARE1 and DGA1 genes.

We also measured the impact of the double-gene knockout on cell growth rate. The results showed 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 5).

Figure 5 Growth rate of strain ΔDGA1ΔARE1

In order to enhance the yield of medium-chain fatty acids in XM01 bacteria, we delete the ARE1 and DGA1 genes in XM01, diverting a greater portion of carbon towards the synthesis of MEL rather than intracellular lipids. Following this, we introduce multiple genes into Escherichia coli for fermentation, resulting in the production of 10-hydroxydecanoic acid and alpha-olefin.Finally, a kill switch is designed to prevent gene leakage.

Figure 6 OleTJE P450 enzyme protein electrophoresis results

Afterwards, the in vitro activity of the P450 fatty acid decarboxylase OleTJE is determined through a recombinant in vitro reaction. C8:0, C9:0, C10:0, C11:0, C12:0, C12:1, and C14:0 fatty acids are used as substrates, and 0.2μM of the P450 fatty acid decarboxylase OleTJE is added to the reaction for a duration of two hours. The change in substrate content is then measured to determine the proportion of conversion.

Figure 7 Conversion rate of P450 enzymes catalyzing fatty acid substrates

Subsequently, the substrates are replaced with medium-chain fatty acids derived from our produced MEL (Microbial oil-derived fatty acids). The in vitro decarboxylation reaction is performed again, and the conversion rates of α-olefins with different fatty acid chain lengths are determined. The results indicate that higher conversion rates offer promising prospects for the production of α-olefins using the OleTJE P450 enzyme.

Figure 8 Conversion yield of α-olefins

Firstly, we constructed two plasmids for the expression of ALKB, ALBG, ALKT, GDH, and SpyCather-SpyTag dual enzyme complex in Escherichia coli BL21.

Figure 9 Plasmid profile of the dual enzyme complex comprising ALKB, ALBG, ALKT, GDH, and SpyCather-SpyTag.

Verification of the sequence after plasmid transformation using colony PCR (Figure 10).

Figure 10 Colony PCR. M represents the D5000plus DNA Marker, 1 is the test strain, and the plasmid size is approximately 5000 bp.

After confirming successful plasmid transformation, we induced protein expression using IPTG and detected the proteins using SDS-PAGE.

Figure 11 Protein electrophoresis gel. M represents the Protein Marker, 1 is the control group without IPTG, and 2 to 4 are the IPTG-induced groups.The protein bands from top to bottom are alkT+GDH (87KDa), alkT (56KDa), alkB (45KDa), GDH (31KDa), and alkG (18KDa).

It is unfortunate to find that we did not detect the expected 10-hydroxydecanoic acid during the catalytic fermentation process. It is speculated that the significant differences in the expression levels of the catalytic proteins may have affected the final fermentation results. In the future, we will redesign the ribosome binding site (RBS) and explore catalytic conditions to achieve the production of 10-hydroxydecanoic acid.

We first constructed a plasmid to introduce the kill switch into Escherichia coli BL21.

Figure 12 Plasmid profile of kill switch

After plasmid transformation, colony PCR was performed to validate the cloned sequences.

Figure 13 Colony PCR , with M representing DNA Marker, and 1 to 6 representing the tested bacterial strains. The expected size of the target gene is approximately 750 bp.

After inducing protein expression with IPTG, protein detection was performed using SDS-PAGE.Due to the release of a large amount of hydrolytic enzymes from cell death, proteins are degraded. It can be observed that compared to the control group, the induced group has almost no protein bands, indicating a significant amount of cell death.

Figure 14 Protein electrophoresis gel, M represents the Protein Marker, and 1 represents the tested bacterial strain.

We added six concentrations of NeuAc to the culture medium and incubated the engineered bacteria for 0.5 hours. We measured the absorbance at OD 600nm at 0.5-hour intervals. The results showed that the suicide switch could promptly kill the engineered bacteria when they escaped from the culture medium.

Figure 15 Growth curve of the engineered bacteria.Control: Culture medium without IPTG and NeuAc.0g/L: Culture medium containing only IPTG. 0.5g/L: Culture medium containing IPTG and 0.5g/L of NeuAc. 1g/L: Culture medium containing IPTG and 1g/L of NeuAc. 2g/L: Culture medium containing IPTG and 2g/L of NeuAc. 4g/L: Culture medium containing IPTG and 4g/L of NeuAc. 8g/L: Culture medium containing IPTG and 8g/L of NeuAc.

The experimental data is fitted using a mathematical model to obtain a curve graph depicting the relationship between N-acetylneuraminic acid concentration and the suicide effect. It is found that when the N-acetylneuraminic acid concentration reaches 10g/L, it can ensure the normal growth of Escherichia coli in the culture medium as much as possible. However, once it is removed from the culture medium environment, it will die within 1-2 hours.

Figure 16 The effect of inducer concentration on bacterial suicide.

In order to achieve better stirring and mixing during the production of MEL, we have successfully designed a propeller blade through iterative design that greatly enhances the stirring effect. We conducted an oil-water mixing experiment to explore the impact of different propeller arrangements on mixing efficiency, and obtained the following data graph.

The final conclusion is that the combination of clockwise helical for the upper impeller and counterclockwise helical for the lower impeller showed higher initial and complete mixing efficiency. Additionally, it demonstrated excellent layering efficiency after agitation cessation. This combination is deemed the most suitable impeller configuration for our fermentation project. Based on this, we will implement this combination in the actual fermentation experiment to study its fermentation performance.