Overview

For the proof of concept, our main goal is to prove that lycopene and patchoulol with different yield can be achieved by adjusting the strength of the promoter of PTS in the recombinant Pichia pastoris co-expression system. Therefore, most of our related work focuses on the regulation of promoter strength, which mainly included promoter screening and characterization of Blue-Light-Induced Activation During Fermentation. In addition, as a project starting from silk dyeing, we dyed silk and tracked its dyeing effect, which showed that our project has application prospects and value.

Promoter screening

In this part, we quantify the promoter strength through a variety of instruments, such as microplate Reader, gas chromatography, HPLC. In previous experiments, we have constructed many biobricks composed of promoters and their downstream reporter genes, which include EGFP(Enhanced Green Fluorescent Protein) and PTS. After several rounds of fermentation, we quantified the green fluorescence intensity and the production of patchoulol and lycopene in recombinant Pichia pastoris. In the comparison of data, we characterized the strength of the promoter.

First, we selected EGFP as the reporter gene and analyzed the fluorescence of yeast corresponding to different promoters (excitation wavelength 488 nm, emission wavelength 520 nm) (figure 1). According to the results of fluorescence per OD, we preliminarily ranked the intensity of promoters. Overall, among the promoters we selected, the strength of methanol inducible promoters is generally greater than that of constitutive promoters, among which P_DAS2 and P_AOX1 are inducible promoters, and the others are constitutive promoters. To our delight, Pichia pastoris strain with P_DAS2 has stronger transcription intensity.

Fig 1. Fluorescence per OD of different promoters within 120 hours

Next, we used HPLC(High Performance Liquid Chromatography) and GC(Gas Chromatography) to analyze the engineered yeast which was capable of synthesizing lycopene and patchoulol, and measured the production of lycopene and patchoulol of the yeast corresponding to different promoters (figure 2). In this process, we found that the production of the two terpenoids of the yeast strains corresponding to the promoters P₀₅₄₇ and P_DAS2 were inversely proportional (figure 3&4), which provided the possibility for our subsequent experiments to carry out visualized PTS mutants screening.

Fig 2. The production of two terpenoids

Fig 3. Lycopene and patchoulol titer of yeast transformants corresponding to P_PGK1. The lycopene production of P_PGK1-1, P_PGK1-2 and P_PGK1-3 was 177 mg/L (15.63mg/g DCW), 194.75 mg/L (17.70mg/g DCW) and 199.95 mg/L (18.18 mg/g DCW), and the patchoulol production of P_PGK1-1, P_PGK1-2 and P_PGK1-3 was 4.77mg/L (0.525mg/g DCW), 3.95 mg/L (0.448mg/g DCW) and 3.02 mg/L (0.343 mg/g DCW).

Fig 4.Lycopene and patchoulol titer of yeast transformants corresponding to P₀₅₄₇. The lycopene production of P₀₅₄₇-2, P₀₅₄₇-3 and P₀₅₄₇-4 was 158.69 mg/L (14.43mg/g DCW), 208.58 mg/L (19.17mg/g DCW) and 204.3 mg/L (18.07 mg/g DCW), and the patchoulol production of P₀₅₄₇-2, P₀₅₄₇-3 and P₀₅₄₇-4 was 5.10mg/L (0.580mg/g DCW), 3.76 mg/L (0.437mg/g DCW) and 4.02 mg/L (0.427 mg/g DCW).

In order to further prove that the changes in lycopene production caused by these two promoters are visible to the naked eye, we carried out color complementary analysis experiments. 2OZPP yeast was used as the control group, and six engineered 2OZPP yeast containing PTS synthesis pathway and different upstream promoters were used as the experimental group. In the color complementation experiment, we found that the difference in yield between patchoulol and lycopene in the experimental group can be distinguished by observing the color of colonies with naked eyes, which proves that the color complementation experiment is feasible for PTS mutation experiments (figure 5).

Fig 5. Identification of two terpenoids in color complementation experiments. Blue represents the production of patchoulol, and red represents the production of lycopene.

Characterization of Blue-Light-Induced Activation During Fermentation

After constructing a double expression plasmid with PTS as the reporter gene, we obtained a strain of P.pastoris 2OZPP containing an optogenetic system by electric transformation (figure 6). To study the temporal behavior of our system, investigate whether pPIcza can be used to express patchoulol synthase and prove that the optogenetic system plays a role in regulating the yield of lycopene and patchoulol, we designed an experiment to characterize the blue-light-induced activation during fermentation. Total two strains were constructed and named 2OZPP-pPIcza-P_AOX1-PTS(2OZPP+PTS), and 2OZPP-pPIcza-(C120)₅-P_minCYC-PTS-P_GAP-TF.

Fig 6. Single-plasmid design of the pPIcza. (C120)₅ is located upstream of the shortened constitutive promoter (P_minCYC). Patchoulol synthase gene (PTS) is the reporter of our single-plasmid pPIcza. Meanwhile, the expression EL222-containing TF(SV40-VP16-EL222) is driven by the constitutive promoter GAP(P_GAP). PpHIS4 will be recognized as a homologous integration fragment and integrated into the genome of 2OZPP, and then provide histidine synthesis function for 2OZPP.

In the proof of concept experiment of optogenetic system, we took Pichia pastoris strain 2OZPP which did not have patchoulol synthesis pathway as the control group-1 (Control-1), 2OZPP-pPIcza-P_AOX1-PTS(2OZPP+PTS) as the control group-2 (Control-2), and 2OZPP-pPIcza-(C120)₅-P_minCYC-PTS-P_GAP-TF as the experimental group. In order to compare the effect of light induced promoter on the expression of patchoulol synthase, we divided the experimental group into short blue light duration group (Shorter group) and long blue light duration group (Longer group).

For control group-1, control group-2 and long blue light duration group, the culture was grown under blue light for 48 h. For shorter blue light duration group, the culture was grown in the absence of the blue light for 16 h, thus exhibiting activation, and then, was grown under the blue light for the rest 32 h.

After 48 h of shaking table fermentation, we analyzed the titer of patchoulol and lycopene (figure 7), and the titer of patchoulol in long blue light duration group was significantly higher than that in short blue light duration group, indicating that the blue light absence for 16 hours led to the inactivation of blue light induction. At the same time, due to the continuous expression of PTS gene in the long blue light duration group, the precursor FPP entering the lycopene synthesis pathway was subject to competition, resulting in low lycopene production, which also proved our concept of promoter screening. In addition, compared with Control-2, the titer of patchoulol in long blue light duration group was 2.5 times that of Control-2. We thought that this proved that the light induced promoter had better induction activity than the methanol-induced promoter.

Due to lack of time, we did not detect the leakage of the optogenetic system, and did not carry out experiments on its spatial activation. However, the existing experimental results have proved our concepts that the induction intensity of the optogenetic system is positively correlated with the dose and periodicity of blue illumination, and has good time control characteristics, which can be used to regulate the production of lycopene and patchoulol in the fermentation process.

Fig 7. Titer of lycopene and patchouli alcohol by shaker flask cultivation in 48 hours in different groups.

Table 1. Different conditions of 48 h of shaker flask fermentation

Silk Dyeing

In this part, what we want to prove is the feasibility of lycopene and patchoulol dyeing on silk.

After fermentation, the recombinant Pichia pastoris was crushed, centrifuged, extracted and concentrated to produce red dyes containing lycopene. We soaked the silk in the dye for a period of time, and then took it out to dry. By repeated soaking and drying, we strengthened the attachment of the dye molecules on the silk, and succeeded in getting the red silk. By changing the concentration of lycopene in the dye, we succeeded in producing silk with different shades of red. Although the color of silk is slightly uneven due to our lack of exquisite dyeing technology, we believe that this problem can be solved in the future industrial production optimization.

Fig 8. The red silk we dyed

In the industrial production, we hope to use the solvent that can dissolve the patchoulol as the extractant agent, so that the patchoulol can be extracted during the fermentation process and can be concentrated for dyeing. However, in the laboratory, we used n-dodecane as the extraction agent in the fermentation process, which is not easy to concentrate. Therefore, in the feasibility verification experiment of dyeing, we used store-bought patchoulol for dyeing experiment.

We added patchoulol and lycopene to ethyl acetate to get our dye. After repeated soaking and drying, the silk shows obvious red color and the fragrance of patchoulol.

Fig 9. The fragrant red silk we dyed

At the same time, we carried out the verification experiment of silk color and odor durability. We exposed the dyed silk to air for a period of time to record changes in color and smell. After two weeks of experiment, the color of the silk was slightly lighter, but there was no obvious fading, and the aromatic smell of patchoulol still remained after two weeks. Although the time is limited, the existing experimental results can prove the feasibility of this project in dyeing.

In addition, we also changed the dyeing time, temperature, solvent and other different conditions, hoping to find the most suitable dyeing conditions.