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Synthetic biology encompasses the creation and assembly of new biological components, mechanisms, and structures, as well as the reconfiguration of natural biological systems for practical applications. Similar to other engineering disciplines, synthetic biologists adhere to the iterative DBTL cycle (design, build, test, learn) to effectively design innovative biological systems.

We utilized a newly discovered fungus, M. aphidis XM01, from mangroves to regulate its metabolic pathways for producing a large quantity of MEL. Additionally, we expressed corresponding enzymes to catalyze the synthesis of medium-chain α-olefins and 10-hydroxydecanoic acid. To address the issue of waste oil disposal, we designed a light-controlled degradation circuit to remove benzo[a]pyrene from the waste oil. Finally, we designed a NeuAc riboswitch to prevent gene leakage and enhance engineering security.

Design:

The marine yeast, Moesziomyces aphidis XM01, produces a substantial amount of intracellular lipids during MEL production. By identifying and knocking out key genes involved in intracellular lipid synthesis in the XM01 strain, the synthesis of MEL can be enhanced. Previous studies have shown that fungal intracellular lipid synthesis is closely related to genes such as DGA1, LRO1, and ARE1. Complete knockout of LRO1 results in the inability to generate intracellular lipids. With the help of our PI and advisor, Zhang Chao, we have identified the sequences of DGA1 and ARE1 genes in Moesziomyces aphidis XM01. Therefore, we have decided to knock out the DGA1 and ARE1 genes to observe whether it can reduce the formation of intracellular lipid droplets.



Figure 1 XM01 Fatty Acid Metabolism Diagram

Build:

We chose 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 Gene double knockout diagram

Test:

First, we verified the successful double gene knockout through DNA gel electrophoresis. We compared the growth curves of the XM01 strain with the ΔDGA1ΔARE1 strain using OD600 measurements and found that gene knockout had little impact on the growth of the XM01 strain. To qualitatively measure the changes in intracellular lipid droplets and MEL production, we used gas-liquid chromatography to determine the MEL production ability and intracellular lipid content of the original XM01 strain and the ΔDGA1ΔARE1 strain. Additionally, to visually observe the reduction of intracellular lipid droplets, we stained the intracellular lipids with Nile Red fluorescence staining and observed them under a fluorescence microscope. It was observed that there was a significant reduction in intracellular lipid droplets after the double gene knockout.

Learn:

The ΔDGA1ΔARE1 strain showed no significant change in growth rate compared to the wild-type strain, and there was a significant reduction in intracellular lipid droplet production. We have successfully achieved our goal.

Design:

To synthesize medium-chain α-olefins, we employed the OleTJE P450 enzyme from Jeotgalicoccus sp. ATCC 8456 to catalyze the single-step decarboxylation of fatty acids, which is a relatively simple approach in the biosynthesis of α-olefins. It directly utilizes free fatty acids as substrates to produce α-olefins in a straightforward and controllable manner. Additionally, to obtain a large amount of protein, we chose Escherichia coli BL21 as the host organism.

Build:

The gene fragment of OleTJE P450 was directly synthesized by a gene synthesis company and then ligated to the PET28a vector using restriction enzyme sites NcoI and XhoI. To facilitate protein purification, a 6×His tag was added at the C-terminus of the protein



Test:

After culturing the cells successfully transformed with the plasmid for a certain period of time, the cells were lysed, and the cell lysate was purified using Ni-NTA resin to collect the protein. Gel electrophoresis was performed to verify the presence of OleTJE P450 enzyme. Since the medium-chain fatty acid substrate produced by MEL has a chain length of C8-12, we first determined whether the purified P450 fatty acid decarboxylase OleTJE had the ability to catalyze medium-chain fatty acid substrates by testing different substrates including C8:0, C9:0, C10:0, C11:0, C12:0, C12:1, and C14:0. Finally, using the medium-chain fatty acids obtained in our experiments as substrates, the actual conversion rate of α-olefins was determined by GC-MS analysis. The amount of each fatty acid in the fatty acid composition was used as the denominator, corresponding to the measured amount of α-olefins obtained. The actual conversion rate of α-olefins for each medium-chain fatty acid was calculated.

Learn:

The results showed that fatty acid chain lengths between 10 and 12 were more easily catalyzed by the P450 fatty acid decarboxylase. The conversion rates for C8, C10, and C12 were 73.58%, 57.86%, and 75.67%, respectively. The higher substrate conversion rates demonstrate that using medium-chain fatty acids as substrates for the synthesis of medium-chain α-olefins is a promising and sustainable biosynthetic strategy. However, in the future, protein mutations can be explored to achieve higher specificity and conversion rates for C8-C12 substrates.

Design:

In order to achieve a high yield of 10-hydroxydecanoic acid and generate a high concentration of alkane hydroxylase, we chose the BL21 strain as our expression host. For the ω-hydroxylase system, we selected the alkBGT alkane hydroxylase system, which consists of three enzymes, AlkB, AlkG, and AlkT, derived from the alk operon of Pseudomonas oleovorans GPo1. In this system, AlkB is a membrane-bound monooxygenase, which is a key component of the alkane hydroxylase system. AlkG and AlkT are electron transfer proteins involved in the oxidation of NADH and electron transfer in the system. Studies have suggested that intracellular NADPH in Escherichia coli may not be sufficient for sustained biosynthesis of 10-hydroxydecanoic acid. Therefore, an efficient coenzyme regeneration system can be designed to improve biosynthetic efficiency. We chose glucose dehydrogenase (GDH) from Bacillus subtilis as the driving enzyme for the NADPH cofactor recycling cycle. To further enhance the catalytic capacity of the system, we used the SpyCatcher-SpyTag system to design an intracellular self-assembling multienzyme complex, assembling the cofactor GDH and AlkT to enable more efficient electron transfer. This allows for the formation of an ordered cofactor transfer channel between the two enzymes, thereby achieving efficient biosynthesis.

Build:

Due to the large number of proteins in the gene circuit, we chose to use two plasmids, PET28a and PCDFDuet-1, with different replication origins for plasmid co-transformation. They both contain the T7 promoter, which is a strong promoter that can effectively express the enzymes we desire. Therefore, we cloned the target DNA into the PET28a and PCDFDuet plasmids and constructed two vectors according to our plan. The first vector, PET28a+alkBGT+SpyCatcher, was designed to carry alkBGT and SpyCatcher, while the second vector, PCDFDuet-1+GDH+SpyTag, was designed to carry GDH and SpyTag.



Test:

First, the expression of proteins was detected by SDS-PAGE, and the gas-liquid chromatograms of the generation of 10-hydroxydecanoic acid after catalysis by three strains, alkBGT, alkBGT+GDH, and alkBGT+GDH+SpyCatcher+SpyTag, were compared to determine whether the yield of 10-hydroxydecanoic acid was successfully increased.

Learn:

It was found that under the catalysis of 0.2mM IPTG, the expression levels of the proteins were low, and there were significant differences in expression levels among the proteins, with the fusion protein alkT+SpyCatcher having the highest expression level. No peak of the target product was observed after gas extraction and GC analysis, suggesting that methylation was not performed and the boiling point of the product was high, resulting in unsuccessful results.

Improve:

Different IPTG concentrations (0.2mM, 0.5mM, 1mM) were set for induction to observe the results of SDS-PAGE and determine the optimal induction concentration for protein expression. At the same time, the samples were methylated before HPLC analysis. The parameters were also modified: the initial injection temperature was changed from 250°C, holding at 50°C for 1 min, and then increasing at a rate of 15°C/min to 250°C, holding for 10 min to an injector temperature of 250°C and a detector temperature of 300°C. The initial temperature was set at 80°C for 2 min and then increased at a rate of 5°C/min to 250°C, holding for 10 min.

Learn:

It was found that the protein induction was most effective under the 0.5mM condition. However, the target product still did not appear. In the future, we will try cell disruption for HPLC analysis, as it is speculated that the low product yield combined with failure to transport it out of the cells may have resulted in the inability to detect the product. We also consider redesigning the RBS to achieve similar expression levels of the proteins, as this is also a factor affecting catalysis.

Design:

In order to prevent leakage, a suicide switch was designed based on the NeuAc riboswitch. The sequence reported by Schyung Cho, obtained through in vitro screening, was used. The switch consists of an RNA aptamer and a hammerhead ribozyme. The principle of its action is that NeuAc binding to the RNA aptamer causes a conformational change in the entire switch, leading to self-cleavage of the hammerhead ribozyme, creating a gap. Then, with the action of the RecJ nuclease in Escherichia coli cells, the downstream genes are cleaved, reducing the abundance of mRNA expression of the downstream genes. We chose ΦX174 as the lysing protein downstream to achieve the suicide of Escherichia coli.

Build:

We chose to have the company directly synthesize the fragment and then connect it to the plasmid PET28a.



Test:

We integrated the plasmid successfully and validated its expression. Subsequently, varying concentrations of N-acetylneuraminic acid (NeuAc) ranging from 0g/L to 8g/L were introduced into the culture medium. The optical density (OD) values of E. coli BL21 were monitored over time to ascertain the optimal NeuAc concentration necessary for the bacteria's normal viability.

Learn:

The results showed that when the culture medium contained 10g/L NeuAc, Escherichia coli could maintain normal growth status. However, once removed from the environment, it would quickly die, which took about 1-2 hours.

Design:

In order to achieve controllability of degradation, we designed a red light switch to control the expression of benzo[a]pyrene-degrading enzyme. Among them, phyA is a functional photosensitive pigment receptor, composed of a lipoprotein and a photosensitive pigment-binding protein (chromophore). Since the chromophore cannot be synthesized in yeast, a similar compound called phycocyanobilin (PCB), purified from cyanobacteria, was added to the culture medium. PCB is easily absorbed by yeast cells and binds to the photosensitive pigment lipoprotein, forming a photosensitive pigment photoreceptor. Under red light (λmax = 660 nm) or far-red light (λmax = 730 nm), PhyA can reversibly change its conformation, allowing it to bind to FHY1 and through a linker, bind PhyA to BD (DNA-binding domain) and PHY1 to AD (transcription activation domain). By utilizing the principle of yeast double hybrid, the target gene can be expressed under the induction of red light.

Build:

We chose to directly synthesize the two fusion proteins of the red light system and then connect them to the existing yeast eukaryotic expression plasmid with the PGK promoter and terminator (provided by PI).



Test:

Due to time constraints, we only separately validated the function of the benzo[a]pyrene-degrading enzyme UPO1, and did not integrate it with the red light system to achieve overall control. However, we have used modeling to explore the conditions for future experiments. We plotted the relationship between the time required for the cP concentration to reach its maximum and the time required for the cP concentration growth rate to reach its maximum with the red light intensity RL. This helps in controlling the separation time in semi-continuous fermentation and selecting the light intensity in wet experiments, narrowing down the range of light intensity selection. The results showed that the expression level and expression rate of the target protein have a strong marginal effect on the time required to reach the maximum as the light intensity increases. When the light intensity reaches 50 umol/m^2/nm, it has almost reached the optimal state. Therefore, we chose a light intensity range of 50-60 umol/m^2/nm for subsequent testing.



Learn:

Upo1 has good benzo[a]pyrene degradation ability, and we have selected a light intensity range of 50-60 umol/m^2/nm for subsequent testing of the red light system.

● Sensing system

Design:

In order to control the fermentation, we designed a receptor for initiating fermentation in engineered bacteria, and initially we intended to use a benzo(a)pyrene receptor to perform this function, so we modeled the benzo(a)pyrene receptor to test whether it could detect benzo(a)pyrene in kitchen waste oil and activate downstream gene expression.

Build:

We modeled the benzo(a)pyrene receptor to simulate its opening at a certain benzo(a)pyrene concentration, and since the actual concentration of benzo(a)pyrene in kitchen waste oil is very low, we used this to set the initial conditions to verify whether it can function or not.

Test:

We explored the relationship between the binding rate at specific benzo(a)pyrene concentrations as a function of its KA value, n value.



Learn:

It is concluded that if we want the binding rate P to reach more than 0.5, we need to mutate the binding site of benzo(a)pyrene to make its KA close to the order of magnitude of 10^-7, and this is almost impossible. We therefore decided to replace the switch.

Improve:

So we considered using a red light system to control the activation of the XM01 strain at specific times.



Learn:

The results show that the changes of each substance under different red light intensities are as expected, indicating that the model itself can well describe the kinetic process of red light switching.

Improve:

During the actual experiment, we found that the yeast culture would attenuate the red light. Therefore, we took this factor into consideration and redesigned the model to obtain the optimal red light intensity for application in the final experiment.



Learn:

The fermentation tank we used is a cylindrical vessel with a radius of approximately 10cm and a height of approximately 40cm. Considering the actual fermentation process, most of the fermentation culture is located in the upper layer, while the lower layer is mainly occupied by the product MEL. After continuous adjustment of the number and positioning of the lights, we finally determined that wrapping 8 light bulbs with an intensity of 180 umol/m^2/nm at distances of 4cm, 12cm, and 20cm from the top of the tank would ensure that the upper 2/3 region within the fermentation tank reaches a light intensity of 50 umol/m^2/nm.

● Fusion protein System

Design:

We first performed kinetic simulations of the reaction system and found that increasing the electron transfer efficiency can effectively increase the rate of product production in the early stages without changing the relevant enzyme activities. It can therefore be determined that the construction of fusion proteins can indeed increase the production of medium-chain fatty acids. In order to further provide the catalytic ability of the system, we intended to construct a fusion protein of AlkT and GDH to increase the efficiency of electron transfer.

Build:

Because alkT is a key enzyme in the production of electrons, its activity is critical to the success or failure of the entire experiment. To verify that alkT with the addition of the SpyCatcher-SpyTag system performs its original function, we molecularly docked alkT with its substrate, NADH, and performed the same test on the original alkT and various alkT-GDH connected with linkers to compare and analyze their substrate-binding abilities.

Test:

We first simulated the change in product expression over time as the electron transfer efficiency constant grew from 0.5 to 2 to observe the effect of electron transfer efficiency on product expression.



To validate the structure of the fusion proteins, we performed structure prediction on fusion proteins constructed by each of the linker and spytag-spyteacher systems.



To ensure that the monomer of the fusion protein is still active, we performed molecular docking using its substrate and judged the catalytic efficiency of the fusion protein from the docking data.




Learn:

Through structure prediction and screening, we finally decided to construct an intracellular self-assembling multi-enzyme complex designed to assemble cofactors GDH and AIKT using the SpyCatcher-SpyTag system.

Improve:

Since the simulated structures show that SpyTag and SpyCatcher will interact with the AlkT and GDH proteins causing them to move closer to them, this may cause the SpyTag-SpyCatcher system to not work properly or have an effect on the structure and activity of the protein itself. Considering that the linkage site of the SpyTag-SpyCatcher system is ASP on SpyTag and Lys on SpyCatcher, we decided to substitute glycine for some of the amino acids.



Learn:

Structural simulations were performed after replacing amino acids in the relevant sites, and SpyTag no longer interacted with the protein itself. This method can be used as a direction to optimize the performance of fusion proteins in the future, and the specific binding efficiency and enzyme activity changes need to be determined by subsequent experiments.

● imulation of MEL fermentation process

Design:

In order to better understand and improve the fermentation process of engineered bacteria, we intend to simulate the production of intracellular oil and MEL during fermentation of engineered bacteria.

Build:

We used MATLAB to model the fermentation part of the engineered bacteria, and established a fermentation model for the growth and production of MEL by the engineered bacteria. The relevant parameters were obtained experimentally.

Test:

We tested changes in the concentration of engineered bacteria, MEL concentration, and substrate concentration over time under a specific initial condition.



Learn:

The results give a good response to the growth of the engineered bacteria and the fermentation of MEL, but we intend to improve the model considering that intracellular lipids are also produced during the fermentation process and that we will be using a semi-continuous fermentation for the fermentation.

Improve:

We added to the model a relevant equation for measuring intracellular lipid production and made the fermentation process consistent with a semi-continuous fermentation: a product separation step was added.



Learn:

In the end, we obtained a model that can be applied under semi-continuous fermentation, which can respond to the growth of engineered bacteria, the production of MEL, and the production of intracellular oil, and the results initially met the expectations, which can help us to adjust the relevant fermentation parameters during the fermentation process in order to achieve optimization.

[1] Yu G, Wang X, Zhang C, et al. Efficient production of mannosylerythritol lipids by a marine yeast Moesziomyces aphidis XM01 and their application as self-assembly nanomicelles [J]. Marine Life Science & Technology, 2022, 4(3) : 373-383.

[2] Oelkers P, Cromley D, Padamsee M, et al. The DGA1 gene determines a second triglyceride synthetic pathway in yeast [J]. Journal of Biological Chemistry, 2002, 277(11) : 8877-8881.

[3] Cho, S, et al, In vitro selection of sialic acid specific RNA aptamer and itsapplication to the rapid sensing of sialic acid modified sugars. Biotechnol Bioeng,2013.110(3): p.905-13.

[4] Sorokina, O., Kapus, A., Terecskei, K. et al. A switchable light-input, light-output system modelled and constructed in yeast. J Biol Eng 3, 15 (2009).

[5] Hochrein L, Machens F, Messerschmidt K, Mueller-Roeber B. PhiReX: a programmable and red light-regulated protein expression switch for yeast. Nucleic Acids Res. 2017 Sep 6;45(15):9193-9205.

[6] Athenstaedt K. Yali0e32769g (dga1) and yali0e16797g (lro1) encode major triacylglycerol synthases of the oleaginous yeast yarrowia lipolytica [J]. BBA Molecular and Cell Biology of Lipids, 2011, 1811(10) : 587-596.