Project

Communication

Highschool Teams HP Experience Exchange

We were invited by SYSU-CHINA to visit their laboratory and communicate about our projects. They showed us their advanced and practical scientific research, and provided us with experience and advice on the use of the equipment, which made great contributions to the direction and operation of our team’s experiment. At the same time, we learned each other’s speech skills, which provided us with valuable experience in speaking and writing manuscripts.

Lab Visit

Online Conferences

Later, we also had a video conference with SYSU-CHINA team. We presented and shared our story of patchoulol, questionary experiences, and failures to SYSU-CHINA team members. They gave us some valuable suggestions. SYSU-CHINA team also presented their research content about salidroside. The communication with SYSU-CHINA team gave us extraordinary inspiration for our project.

Online Metting

Propaganda Speech

In order to bring our project closer to the masses, our team held public talks to introduce ourselves and our results. Adhering to the principle of making synthetic biology enjoyable, we selected three speakers from our team to describe our team’s struggles and research results along the way. After returning to campus in September, we also held another club presentation to show new students our latest progress and basic information. At the club festival of our school, we publicized our project in the biology club, so that all the high school freshmen and part of the high school seniors who participated in the activity knew about our project and activity, and learned something from it. In all the lectures we held, we received a lot of compliments and suggestions, so we would adjust our presentation each time and do it better next time to give everyone a better understanding of the project.

Public Speaking

Interview with Experts

Considering that the light-induced promoter is used in our project, and the professor’s research field is about the regulation of photosynthesis, we asked the professor to give us some suggestions for improving the knowledge of photobiology used in our project.

Initially, the professor says, you need to learn to screen when doing experiments. Photobiology is a very cutting-edge branch of life science. There have been previous achievements in this field. When we make use of it, we should screen the suitable conditions for our experiments according to the actual situation. For example, the light-induced promoter of HCF173 used in this experiment should be screened before use to screen out the light-induced promoter with the best-carrying effect with our target gene to ensure that its activity matches each other with the gene.

In addition, promoter optimization should be carried out in combination with the actual situation. For example, the light we are using to activate now in our experiments, white light, means that daily light can activate the promoter. However, in the actual production, because white light is too easy to appear, it is impossible to achieve a real light environment. To solve this problem, the professor suggested that the light we used to induce could be selected from specific bands of light, such as those rare in nature, so as to avoid the interference of light in nature and make the experiment more accurate.

What is more, the promoter’s light requirements need to be feasible. If we have a light promoter that has very high light intensity requirements, and you have to meet these ultra-high requirements to start, then most of the time you can’t produce. And if very strong light is required, it will greatly increase the cost of our production.

Considering the problems that arise when moving from the laboratory to mass production, we also discussed the whole synthetic biology industry with the professor. The professor pointed out that the laboratory environment is very different from the actual production environment. For example, in this project, we have a light-induced promoter, and in the lab, the volume that we synthesize is actually very small, maybe a thin layer in yeast. However, in mass production, it is still very difficult to ensure that each yeast light promoter can feel the light in the face of the large volume of the factory. For these problems, the professor pointed out a clear way for us: we should first form a theoretical basis in the laboratory, and when we find problems in large-scale production, we should timely feedback, optimize and solve them in the laboratory, and then put them into large-scale production for trial. And so on and so on until the desired effect is achieved.

Finally, we asked professors how they could connect the projects they were working on to society and make a real contribution. First of all, the professor affirmed our research project on patchouli and patchoulol, which are very distinctive ingredients derived from Lingnan’s traditional Chinese medicine. It is a contribution to society to bring characteristic plants into play. As for the role of patchoulol, the professor pointed out the following points: one is that patchoulol can be directly used as a component in medicine, and the other is that patchoulol can be used as a chassis for other products, providing a prerequisite for other drugs.

According to the suggestions made by the professor during the interview, we plan to make the following improvements in the future: First, for the optical promoter itself, reduce its requirements on light and increase the practicability of the product. Second, if in the future will be put into large-scale production, the problems encountered in the production should be timely improved in the laboratory.

Interview with doctor

Considering the particularity of the research objects of our project (patchouli and patchouli alcohol), the team decided to interview some practitioners of traditional Chinese medicine working on the frontline in the community clinic, and further discuss the current situation of the use of patchouli and patchouli alcohol and the social value of our project.

First, we asked doctors what kind of concoctions they typically prescribe to patients, such as sliced or ground medicine. Doctors say there are only one or two medicines that usually contain patchouli or patchoulol in hospitals, such as Huoxiang Zhengqi capsule and Huoxiang Zhengqi liquid.

Then, we briefly introduced our project and discussed the impact of the increased production of patchoulol on doctors’ consultations and even society as a whole. Doctors say that if the production of patchoulol can be increased and more patchoulol becomes more available, the price of patchoulol will fall accordingly. Among them, pharmacists in the pharmacy told us that the price of Chinese patent medicine has been soaring. If the price of patchoulol can be reduced, doctors will have fewer concerns when prescribing medicine to patients, and it will also be able to ensure that some poor people have access to medicine, which is of great significance to society.

Interviewing Doctors

Contribution

1.Characterization of previous iGEM parts

In this project, basic parts of previous projects are characterized, including pPGK1 promoter (BBa_K122000), pTEF1 promoter (BBa_K2765041), TADH1 terminator (BBa_K122004), and TCYC1 terminator (BBa_K122003). Patchoulol synthase (PTS) gene and farnesyl diphosphate synthase (ERG20, also named FPS) gene are expressed by this promoters and terminators. The sequences were assembled into pSP-GM2 plasmids and constructed 4 recombinant vectors, namely pSP-GM2-FPS, pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS and pSP-GM2-PTS-FPS (Figure 1-4). Then these vectors are all transformed to Saccharomyces cerevisiae BY4741. The colony PCR were conducted for further verification of this recombinant vectors (Figure 5).

Figure 1. The construction of pSP-GM2-FPS plasmid with pPGK1 as promoter and TADH1 as terminator.

Figure 2. The construction of pSP-GM2-PTS plasmid with pPGK1 as promoter and TADH1 as terminator.

Figure 3. The construction of pSP-GM2-PTS-linker-FPS plasmid with pPGK1 as promoter and TADH1 as terminator.

Figure 4. The construction of pSP-GM2-PTS-FPS plasmid with pPGK1 and pTEF1 as promoter, TADH1 and TCYC1 as terminators, respectively.

Figure 5 A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR results of pSP-GM2-PTS-FPS.

2.Add New Documentations To An Existing Part

We will artificially cloned 2 new genes including HCF173 promoter and PTS. The patchoulol synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces more than 20 valuable sesquiterpenes including patchoulol by conversion of the natural substrate FPP.[1-3] HCF173 is the promoter of HIGH CHLOROPHYLL FLUORESCENCE173 (HCF173), which is required for Arabidopsis thaliana PSII biogenesis. HCF173 transcript levels and expression were induced by light. HCF173 promoter contains ACE motif and G-box element.[4] In our project, the CDS sequence of PTS was optimized according to yeast codon preference. PCR was conducted first for cloning PTS, FPS (ERG20) and HCF173 (Figure 6). Later a gene fusion of PTS and FPS namely PTS-linker-FPS are acquired by removing the stop codon of PTS and replacing it by a short peptide (Gly-Ser-Gly) to introduce a linker between the FPS and PTS ORFs.

Figure 6. A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR results of pSP-GM2-PTS-FPS.

Later, we assembled HCF173 promoters, PTS gene and ERG20 (FPS) and constructed 4 recombinant vectors, namely pSP-GM2-HCF173-FPS, pSP-GM2-HCF173-PTS, pSP-GM2-HCF173-PTS-linker-FPS and pSP-GM2-HCF173-PTS-FPS (Figure 7-10). The colony PCR were conducted for further verification of this recombinant vectors (Figure 11). .. image:: https://static.igem.wiki/teams/4270/wiki/contributions7.png

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Figure 7. The construction of pSP-GM2-HCF173-FPS plasmid with HCF173 as promoter, TADH1 as terminators.

Figure 8. The construction of pSP-GM2-HCF173-PTS plasmid with HCF173 as promoter, TADH1 as terminators.

Figure 9. The construction of pSP-GM2-HCF173-PTS-linker-FPS plasmid with HCF173 as promoter, TADH1 as terminators.

Figure 10. The construction of pSP-GM2-HCF173-PTS-FPS plasmid with HCF173 as promoter, TADH1 as terminators.

Figure 11. A. The colony PCR results of pSP-GM2-HCF173-FPS. B. The colony PCR results of pSP-GM2-HCF173-PTS. C. The colony PCR results of pSP-GM2-HCF173-PTS-linker-FPS. D. The colony PCR results of pSP-GM2-HCF173-PTS-FPS.

3.Add New Parts

In our 2022 project, we provide 10 new parts:

part name	part number	website
HCF173 promoter	BBa_K4270001	http://parts.igem.org/Part:BBa_K4270001
PTS (patchoulol synthase)	BBa_K4270004	http://parts.igem.org/Part:BBa_K4270004
erg20 K197P	BBa_K4270007	http://parts.igem.org/Part:BBa_K4270007
erg20 K197H	BBa_K4270008	http://parts.igem.org/Part:BBa_K4270008
FPS-linker-PTS	BBa_K4270010	http://parts.igem.org/Part:BBa_K4270010
pPGK1+PTS+TADH1	BBa_K4270011	http://parts.igem.org/Part:BBa_K4270011
pPGK1+ERG20+TADH1	BBa_K4270012	http://parts.igem.org/Part:BBa_K4270012
pTEF1+ERG20+TCYC1	BBa_K4270016	http://parts.igem.org/Part:BBa_K4270016
HCF173+ ERG20+TADH1	BBa_K4270013	http://parts.igem.org/Part:BBa_K4270013

4.Improvement of Existing Parts

4.1 Optimized of farnesyl diphosphate synthase (ERG20, FPS) (BBa_K517004)

The ERG20 is a basic part comprised from a synthesized farnesyl diphosphate synthase coding sequence. The basic part BBa_K4270007 (erg20 K197P) and BBa_K4270008 (erg20 K197H) are optimized versions of ERG20 (BBa_K517004) that can be constructed into pSP-GM2 plasmid. The resulted vectors are named pSP-GM2-FPS, pSP-GM2-FPS K197P and pSP-GM2-FPS K197H respectively. Then this vectors were transformed into Saccharomyces cerevisiae BY4741 to produce patchoulol.

4.2 Plasmid Construction

PCR was conducted for cloning FPS (ERG20), erg20 K197P and erg20 K197H. The purified PCR products were assembled into pSP-GM2 plasmids and constructed 3 recombinant vectors, namely pSP-GM2-FPS, pSP-GM2-FPS K197P and pSP-GM2-FPS K197H (Figure 12-13). Then these vectors are all transformed to Saccharomyces cerevisiae BY4741. The colony PCR were conducted for further verification of this recombinant vectors (Figure 14).

Figure 12. The construction of pSP-GM2-FPS K197P plasmid with pPGK1 as promoter, TADH1 as terminators.

Figure 13. The construction of pSP-GM2-FPS K197H plasmid with pPGK1 as promoter, TADH1 as terminators.

Figure 14. The colony PCR results of pSP-GM2-FPS K197P (left side) and pSP-GM2-FPS K197H (right side).

4.3 Results

Gas chromatography (GC) method to observe the patchoulol in Yeast fermentation broth. Unfortunately, we did not detect patchoulol in the supernatant but detected patchoulol in the standard substance containing patchoulol (Figure 15). The reason we did not detect patchoulol in the supernatant may due to the low yield of patchoulol and not enough fermentation.

Figure 15. The detection of patchoulol in the supernatant. A-C. The detection of patchoulol in Yeast fermentation broth from Yeast transformed with pSP-GM2-FPS, pSP-GM2- FPS K197P, pSP-GM2- FPS K197H plasmids, respectively. D. The detection of patchoulol in the standard substance containing patchoulol.

Reference

[1] Albertsen L, Chen Y,Bach L. S, Rattleff S, et al. Diversion of flux toward sesquiterpene production in Saccharomyces cerevisiae by fusion of host and heterologous enzymes. Appl. Environ. Microbiol. 2011, 77, 1033−1040.

[2] Shuiqin W, Michel S, Anthony C, et al. Redirection of cytosolic or plastidic isoprenoid precursors elevates terpene production in plants. Nature Biotechnology, 2006, 24: 1441-47.

[3] Ma B, Liu M, Li Z H, et al. Significantly enhanced production of patchoulol in metabolically engineered Saccharomyces cerevisiae [J]. J Agric Food Chem, 2019, 67(31): 8590-8.

[4] Xue L, Hong-Bin W, and Hong-Lei J. Light signaling-dependent regulation of PSII biogenesis and functional maintenance. Plant Physiology, 2020, 183, 1855-1868.

Description

Pogostemon cablin and patchouli oil

Pogostemon cablin is a kind of medicinal aromatic herb and one of the Top 10 drugs in Guangdong province. P. cablin is mainly grown in Indonesia, Singapore, Vietnam, Malaysia, China, and other areas.[1] Patchouli oil is made from the dried ground part of P. cablin by distillation and is mainly used in two aspects.[2] First, it is used as a drug because of its anti-tumor, anti-inflammatory, antibacterial, antioxidant, and other pharmacological effects.[3-5] On the other hand, its fragrance is unique and used as a spices and food additive.[6] Benefiting from its good efficacy, patchouli oil is widely used in the daily chemical, pharmaceutical, and food industries and has a broad market prospect. The price of high-quality patchouli oil can be as high as ¥1300 per kilogram. In terms of the retail price, it can rank 10th among the important essential oils in the world and has obvious economic benefits. [7, 8]

Pogostemon cablin

Patchoulol

Patchoulol, a sesquiterpene, is one of the most abundant components in patchouli oil. [6, 9] As an important index for evaluating the quality of P. cablin and patchouli oil in Chinese Pharmacopoeia, patchoulol is also considered the main component contributing to the pharmacological effects and aroma of patchouli oil. [10] Nowadays, there is a serious imbalance between the supply and demand of patchoulol and patchouli oil in the world. The global annual demand for patchoulol and patchouli oil can reach over 2 000 t, but the annual output is about 1300 t. Indonesia supplies 90 percent of all annual output, while China produces less than 10 percent of that, which has plenty of room for growth. [11, 12] Increasing the yield of patchoulol has become a key point in the study of p. cablin.

At present, patchoulol is obtained in three main ways, namely, plant extraction, chemical synthesis, and microbial fermentation.

Plant extraction

Typically, patchoulol is extracted from the leaves of P.cablin. However, the low content of patchoulol in P. cablin is the bottleneck of its large-scale application. [2]

Chemical synthesis

Although the chemical synthesis method can support patchoulol with high purity, it needs many steps, complex operation, high synthesis cost, and the yield are low, so it cannot be applied to large-scale industrial production. [13]

Microbial Fermentation

Compared with plant extraction and chemical synthesis, microbial fermentation synthesis exhibits several advantages in producing patchoulol, such as fast-growing, land-saving, and controllable culture conditions. With the development of metabolic engineering and synthetic biology, many valuable plant-derived terpenoids also have been produced in microbial cell factories, such as artemisinic acid [14], ginsenosides [15], and tanshinones [16]. Although naturally produced patchoulol can be obtained from plants, patchoulol production using biobased microbial platforms is an economical and sustainable alternative.

Patchoulol biosynthetic pathways

There are two biosynthetic pathways for patchoulol formation, namely the mevalonic acid (MVA) pathway located in the cytoplasm and the methylerythritol 4-phosphate (MEP) pathway located in plastids. [17, 18] In general, patchoulol is mainly synthesized through the MVA pathway. There are 3 key enzymes in the synthesis of patchoulol, namely 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), farnesyl diphosphate synthase (FPS), and patchoulol synthase (PTS). HMGR catalyzes the irreversible formation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) into MVA. Because it can affect the yield of IPP (isopentenyl diphosphate), the precursors of patchoulol, HMGR is the key to the metabolism of patchoulol. FPS catalyzes IPP to FPP (farnesyl diphosphate). PTS is a key rate-limiting enzyme in the formation of the patchoulol, which catalyzes the formation of patchoulol skeleton from the FPP precursor, and the skeleton is modified by secondary enzymes to form patchoulol.

Vision

Patchoulol production using biobased microbial platforms is an economical and sustainable alternative. Recently, based on the endogenous mevalonate (MVA) pathway or 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, heterologous production of patchoulol has been accomplished by introducing the patchoulol synthase gene in Saccharomyces cerevisiae [8] and Corynebacterium glutamicum [19], as well as in the moss Physcomitrella patens[20] and eukaryotic microalga Chlamydomonas reinhardtii [21]. Nevertheless, the yield is still too low for industrial production. Therefore, significant engineering is required to achieve sufficient yields for industrial applications. Scientists see the use of environmental signals (light, temperature) and endogenous signals (quorum sensing, pressure) to establish tightly controlled patchoulol biological production systems as the future trend.

Promoters are crucial elements in controlling the expression of target genes, and the emergence of inducible promoters provides the possibility to accurately and flexibly regulate the expression of foreign genes, which has become one of the hot spots in the research and development of genetic engineering. [22] Therefore, in the present study, two metabolic engineering strategies were conducted to enhance the production of patchoulol in yeast. First, ERG20 (encoding FPP synthase from yeast) and PTS (encoding patchoulol synthase from P. cablin) were used to increase the utilization of the FPP precursor. Second, the constituted promoter and light-inducible promoter are respectively used to drive the overexpression of the critical gene FPS and PTS. In addition, studies have reported that high-level patchoulol production in tobacco plants was achieved by fusing FPS and PTS genes. [17] Hence, vectors consisting of a gene fusion of PTS and FPS (PTS-FPS) inserted downstream of the constitutive and light-inducible promoter were also constructed. Finally, we carried out fermentation in yeast and envisioned our final product by meteorological chromatography.

Reference

[1] Yao G, Drew B T, Yi T S, et al. Phylogenetic relationships, character evolution and biogeographic diversification of Pogostemon s.l. (Lamiaceae) [J]. Mol Phylogenet Evol, 2016, 98(184-200.

[2] Henke N A, Wichmann J, Baier T, et al. Patchoulol production with metabolically engineered Corynebacterium glutamicum [J]. Genes (Basel), 2018, 9(4): 219.

[3] Zhang R, Yan P, Li Y, et al. A pharmacokinetic study of patchouli alcohol after a single oral administration of patchouli alcohol or patchouli oil in rats [J]. European Journal of Drug Metabolism and Pharmacokinetics, 2016, 41(4): 441-8.

[4] Wu Z, Zeng H, Zhang L, et al. Patchouli alcohol: a natural sesquiterpene against both inflammation and intestinal barrier damage of ulcerative colitis [J]. Inflammation, 2020, 43(4): 1423-35.

[5] 闵琼, 曾海荣, 陆翠燕, et al. 广藿香醇通过调控上皮间质转化抑制人胃癌细胞HGC-27的侵袭和转移 [J]. 药学服务与研究, 2020, 20(01): 6-11.

[6] Van Beek T A, Joulain D. The essential oil of patchouli, Pogostemon cablin: A review [J]. Flavour and Fragrance Journal, 2018, 33(1): 6-51.

[7] Swamy M K, Sinniah U R. Patchouli (Pogostemon cablin Benth.): Botany, agrotechnology and biotechnological aspects [J]. Ind Crop Prod, 2016, 87(161-76.

[8] Ma B, Liu M, Li Z H, et al. Significantly enhanced production of patchoulol in metabolically engineered Saccharomyces cerevisiae [J]. J Agric Food Chem, 2019, 67(31): 8590-8.

[9] Hybertson B M. Solubility of the sesquiterpene alcohol patchoulol in supercritical carbon dioxide [J]. J Chem Eng Data, 2007, 52(1): 235-8.

[10] 徐雯, 吴艳清, 丁浩然, et al. 广藿香的药理作用及机制研究进展 [J]. 上海中医药杂志, 2017, 51(10): 4.

[11] Swamy M K, Mohanty S K, Sinniah U R, et al. Evaluation of patchouli (Pogostemon cablin Benth.) cultivars for growth, yield and quality parameters [J]. Journal of Essential Oil-Bearing Plants, 2015, 18(4): 826-32.

[12] Swamy M K, Sinniah U R. Patchouli (Pogostemon cablin Benth.): Botany, agrotechnology and biotechnological aspects [J]. Industrial Crops & Products, 2016, 87(161-76.

[13] Srikrishna A, Satyanarayana G. An enantiospecific total synthesis of ()-patchouli alcohol [J]. Tetrahedron Asymmetry, 2005, 16(24): 3992-7.

[14] Westfall P J, Pitera D J, Lenihan J R, et al. Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin [J]. P Natl Acad Sci USA, 2012, 109(3): 655-6.

[15] Wei W, Wang P, Wei Y, et al. Characterization of Panax ginseng UDP-glycosyltransferases catalyzing protopanaxatriol and biosyntheses of bioactive ginsenosides F1 and Rh1 in metabolically engineered yeasts [J]. Molecular Plant, 2015,

[16] Guo J, Zhou Y J, Hillwig M L, et al. CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts [J]. Pnas, 2013, 110(29): 12108-13.

[17] Shuiqin W, Michel S, Anthony C, et al. Redirection of cytosolic or plastidic isoprenoid precursors elevates terpene production in plants. Nature Biotechnology, 2006, 24: 1441-47.

[18] Tang Y, Zhong L, Wang X, et al. Molecular identification and expression of sesquiterpene pathway genes responsible for patchoulol biosynthesis and regulation in Pogostemon cablin［J］. Bot Stud, 2019, 60：11.

[19] Henke NA, Wichmann J, Baier T, et al. Patchoulol production with metabolically engineered Corynebacterium glutamicum［J］. Genes (Basel), 2018, 9(4): E219.

[20] Zhan X, Zhang YH, Chen DF, et al. Metabolic engineering of the moss Physcomitrella patens to produce the sesquiterpenoids patchoulol and α/β-santalene［J］. Front Plant Sci, 2014, 5 ：636.

[21] Lauersen KJ, Baier T, Wichmann J, et al. Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii［J］. Metab Eng, 2016, 38: 331-343.

[22] Xue L, Hong-Bin W, and Hong-Lei J. Light signaling-dependent regulation of PSII biogenesis and functional maintenance. Plant Physiology, 2020, 183, 1855-1868.

Engineering

Abstract

Patchoulol is one of the main active components of Pogostemon cablin, and it is also an important indicator for evaluating the medicinal materials of P. cablin in the Chinese Pharmacopoeia. Patchoulol can be obtained by natural extraction, chemical synthesis, and synthetic biology engineering. Due to the low content of patchoulol in P. cablin and the low yield of chemical synthesis, the study of combinatorial biosynthesis and molecular regulation can provide the basis for obtaining high-content patchoulol. In this study, on the basis of the biosynthetic pathway, molecular regulation mechanism, and synthetic biology engineering of patchoulol, the key genes for its biosynthesis were combined and expressed in heterologous host yeast to produce patchoulol, which provides the technical foundations of the development and application of patchoulol.

Experiment design

8 type of expression vector

We designed eight plasmids: The strong constitutive promoter (pGK1) was separately used to connect FPS (BBa_K517004), PTS (BBa_K4270004), and FPS-linker-PTS (BBa_K4270010), and plasmids containing pGK1, FPS, terminator, pGK1, PTS terminator were constructed .In the following four designs, all the above strong promoters were replaced with light-induced promoters HCF173, and then the test was carried out to verify (Figure 1).

Experiment structure and testing

PCR amplification and estriction enzyme cutting expression vector pSP-GM2

Amplified by PCR, the plasmids were double digestion by the restriction enzymes NotI and SacI. Plenty of strands of DNA were obtained. Plasmid DNA, restriction enzyme, reaction buffer, and ddH2O were added successively in a 1.5 mL sterilized centrifuge tube according to the table below. Mix and mark well. The mixture reacted at 37℃ for eight to ten hours. Agarose gel electrophoresis was used to detect the product size and determine whether the enzyme was successfully cut.

Table 1 Enzyme digestion reaction system

Reagent	Volume (μL)
10 x Cutsmart buffer	5
Not I	0.5
Sac I	0.5
pSP-GM2 vector	1
ddH2O	43

Likage of expression vector and chemical transformation of E.coli DH5α

In vitro assembly gene, including promoter, control of the corresponding enzyme synthesis gene, sequence of the terminator, DNA ligase access strip DNA, forming a circular recombinant vector. Add 10 ng vector recombinant DNA solution to 50 μL competent E.coli, mix well, and let stand in an ice bath for 30 min. By heat pulse method, the mixture was shocked in a water bath at 42℃ for 30 s, then placed on ice for 3-5 minutes. 1 mL fresh medium was added and cultured at 37℃ for 1 h for amplification. Add 500 μL to sterilized LB medium into a centrifuge tube, mix evenly, and culture for 1 h at 37℃ and 200 rpm. Adjust the speed to 5000rpm, centrifuge at room temperature for 1min, take out the supernatant and pour it out until there was 100 μL of thallus remaining.

Screening of transformants

Pick a single bacteria placement from the conversion plate and place it on the new LB solid plate containing antibiotics, culture it at 37℃ for 2-4 h and then pick up the colonies. Put them into the centrifuge tube of the corresponding PCR reaction solution. Take the colony as the template of PCR reaction and amplify the colony by T5 Super PCR Mix.

Table 2 Colony PCR reaction system

Reagent	Dosage
2×T5 Super PCR Mix	5 μL
M13F（10 μM）	0.4 μL
M13R（10 μM）	0.4 μL
ddH2O	4.2 μL

Table 3 Colony PCR reaction procedure

Temperature	Time	Cycle
98℃	10 min	1
98℃	10 s	35
50℃	10 s
72℃	30 s
72℃	2 min	1

Plasmids extraction and extract sequencing

The transformed E.coli was inoculated with 0.5% glycerin and ampicillin in sterilized TB culture medium. Culture at 220 rpm for 12 h at 37℃ until saturated. Adjust the speed to 10000 rpm, centrifuge at room temperature for 6 min, remove and pour off the supernatant. The centrifugation process was repeated until the plasmid was successfully extracted. After gene sequencing, verify whether the vector is successfully transformed and consistent with the design sequence.

Transformation and screening of yeast

About 16 colonies growing in LB solid medium containing ampicillin were selected and verified by 2x Easy Taq enzyme. The PCR system is shown below.

Table 4 PCR reaction (20 μL)

Reagent	Volume (μL)
2x Easy Taq Mix	10
colony	0
forward primer	1
Downstream primer	1
buffer	8

The reaction time and temperature were set according to the table below, and electrophoresis was performed after amplification.

Table 5 experimental set

Procedure	Temperature/℃	Time/S	Repeat
preprocessing	94	30	1 cycle
denaturation	94	10	35 cycles
annealing	55	30
extension	72	30
final extension	72	60	1 cycle
hold	4	∞	1 cycle

The monoclones were selected into 5 mL of the corresponding defective medium (BY4741 into YPD containing glucose). The culture was incubated overnight at 28℃ at 200-220 rpm until the OD600 value was 0.5-0.6. Take 500 μL of cultured bacterial solution into 1.5 mL EP tube, centrifuge at 300-500 rpm for 3-5 min, and pour out the supernatant. The monoclones were selected into 5mL of corresponding defective medium (BY4741 into YPD containing glucose). The culture was incubated overnight at 28℃ at 200-220 rpm until the OD600 value was 0.5-0.6. Take 500 μL of cultured bacterial solution into a 1.5 mL EP tube, centrifuge at 300-500 rpm for 3-5 min, and pour out the supernatant. Add 500 μL of EZ1 and resuspend thallus. Centrifuge 300-500 rpm for 3-5 min to remove the supernatant. Add 50μL of EZ2, flick, and resuspended the thallus to obtain a competent state.

Transform procedure

3-5 μL plasmid was added to 50 μL competent state, mixed evenly, then 500 μL EZ3 was added, then evenly and lightly beaten with a blue gun since EZ3 is sticky. Incubated in a water bath at 30℃ for 45 min-1 h. After centrifugation at 500 rpm for 5 min, flocculent accumulation of thalli could be seen near the bottom of the tube. After centrifugation, 400 μL centrifuged EZ3 was sucked off, leaving 100 μL resuspended thallus and applied directly to the plate.

The vector was transformed into yeast and screened for nutritional deficiency. Successfully transformed recipient cells can be grown on nutritionally deficient media. The successfully transformed yeast cells were taken and examined again.

Fermentation of modified strains

Provide suitable nutrients and environment for fermentation, so that the successful transformation of yeast fermentation at room temperature.

Extraction and determining of metabolite

The content of patchoulol, a volatile sesquiterpene, was analyzed by gas chromatography.

Learning and design again:

We further optimized the original improved carrier. On this basis, the experimental scheme was further optimized.

Experiments

Protocol

1. The enzyme digestion reaction of pSP-GM2 vector

Material: pSP-GM2 vector, restriction enzyme, 10 x Cutsmart buffer, ddH2O.

Reagent	Volume (μL)
10 x Cutsmart buffer	5
restriction enzyme 1	0.5
restriction enzyme 2	0.5
pSP-GM2 vector	1
ddH2O	43

Procedure：

1. Add ingredients in 1.5 mL test tubes at a time according to the above table.

2. Mix and mark well.

3. 37℃ for 8-10 h.

2. PCR and electropherosis

Material: DNA template、primer、dNTPs、5 × Q5 reaction buffer、Q5 DNA polymerase.

Procedure: (1) Mix up the recipe into 1.5 mL test tubes according to the following table.

Reagent	Volume (μL)
Q5 DNA polymerase	0.5
5 × Q5 reaction buffer	10
10 mM dNTPs	1
forward primer	2.5
downstream primer	2.5
DNA template	1
ddH2O	32.5

2. The reaction time and temperature were set according to the table below. Run the PCR program.

procedure	temperature/℃	time/s	repeat
preprocessing	98	30	1 cycle
denaturation	98	30	35 cycle
annealing	55	30
extension	72	60
Final extension	72	300	1 cycle
Hold	4	∞	1 cycle

3. DNA gel electrophoresis

(1) Preparation of 1% DNA gel Weigh 1 g of agarose into a cone-shaped flask, and then add 100 mL of 10xTAE buffer into this cone-shaped flask. Transfer the cone to the microwave and heat until boiling (2-3 times). Cooling the cone-shaped flask to about 50℃, and then add the nucleic acid dye into it. Mixing agarose and nucleic acid well and pour them into the rubber rack. After 1 hour later, 1% DNA gel is ready.

(2) point sample Mixing the DNA sample and the adding buffer (4:1). Then adding the mixture to the sample tank with a micropipette gun. Each tank addes 20 μL mixture. Recording the order of spot sampling and the amount of adding sample.

(3) Gel electrophoresis After installing the electrode wire, connect one end of the sample hole to the negative electrode and the other end to the positive electrode. Then switching on the power supply and adjust the voltage to 120 V. After electrophoresis for 20-30 min, stop electrophoresis.

4. Transformation of plasmid to E.coli

1. Take 50 µL of E.coli cells and defrost E.coli with ice bath for 25 min. Mixing E.coli gently with 10 µL target recombinant plasmid.

2. Heat shock at 42℃ for 90 s, then place it immediately in the ice bath for another 3-5 min.

3. Add 500 µL LB broth (not containing antibiotics), mix upside down, and put it in the shaker to recover at 200 rpm, 37℃ for 60 min.

4. Centrifuge at 5,000 rpm, room temperature, for 1 min. Keep 100 µL of supernatant and resuspend the bacteria. Coating on the LB broth containing ampicillin.

5. Place upside down to incubate at 37℃ for 10-12 h in the incubator.

5. Selecting colonies

Colony PCR

1. Inoculate BL21 cells using a pipette tip，use 2x Easy Taq enzyme for colonies selecting：

Reagent	Volume (μL)
2x Easy Taq Mix	10
colony	0
forward primer	1
Downstream primer	1
ddH2O	8

2. The reaction time and temperature were set according to the table below. Run the PCR program.

Procedure	Temperature/℃	Time/S	Repeat
preprocessing	94	30	1 cycle
denaturation	94	10	35 cycles
annealing	55	30
extension	72	30
final extension	72	60	1 cycle
hold	4	∞	1 cycle

6. DNA sequencing

Expand and culture the positive colonies in LB liquid medium containing ampicillin at 37℃ for 10-12 h. Extract the plasmid by using the plasmid extraction kit. Sent 10 μL plasmid to sequencing company for sequencing. The correctly sequenced plasmid was stored at -20°C and the corresponding bacterial solution was stored at -80°C.

7. Yeast transformation procedure

1. selecte the monoclones into 5mL corresponding defective medium (containing glucose)).

2. Incubate at 28℃ overnight at 200-220 rpm until OD600 value is 0.5-0.6.

3. Take 500 μL cultured yeast solution into 1.5mL EP tube, and centrifuge at 300-500 rpm for 3-5 min. Remove the supernatant.

4. Add 500 microliters of EZ1 into 1.5mL EP tube and resuspend yeast cells.

5. Centrifuge at 300-500 rpm for 3-5 min and remove the supernatant.

6. Add 50 μL EZ2 into 1.5mL EP tube, flick, and resuspend yeast cells.

7. Transformation: add 3-5 μL plasmid into 50μL yeast cells, mix evenly, then add 500μL EZ3 into it. Incubate yeast cells in a water bath at 30℃ for 45 min-1 h.

8. Centrifugate at 500 rpm for 5 min.

9. After centrifugation, remove 400μL supernate. Resuspend the yeast cells and coate them on a solid medium plate.

Implementation

Background

Patchoulol is one of the most abundant components in patchouli oil[1, 2] and is also considered the main component contributing to the pharmacological effects and aroma of patchouli oil[3], which is widely used in medicine, daily chemicals, food, and other industries, with broad market prospects. However, there is a serious imbalance between the supply and demand of patchoulol and patchouli oil in the world. There are three ways to prepare patchouli so far. Typically, patchoulol is extracted from the leaves of Pogostemon cablin. However, the low content of patchoulol in P. cablin is the bottleneck of its large-scale application [4]. Although the chemical synthesis method can support patchoulol with high purity, it has many drawbacks, such as too many steps, complex operation, high synthesis cost, and low yield, so it is may not suitable for large-scale industrial production [5]. Compared with plant extraction and chemical synthesis, microbial fermentation synthesis exhibits several advantages in producing patchoulol, such as fast-growing, land-saving, and controllable culture conditions. With the development of synthetic biology, patchoulol has been produced in yeast under the control of a constitutive promoter. [6] However, the production of patchoulol is uncontrollable and yeast cells may produce too much patchoulol at a time, which is toxic to yeast cells. Herein, our team has innovatively applied the light-induced promoter in the biosynthesis of patchoulol in yeast, which makes the production of patchoulol controllable. When the light is on, yeast cells produce patchoulol. Otherwise, it is not produced patchoulol. We believe the increasing production of patchoulol will lower the price of patchoulol. Furthermore, the lower price of patchoulol can effectively help more people particularly those in outlying poverty-stricken areas.

Target customers

1. Patchouli essential oil factory

Patchouli essential oil is in great demand in the market because of its long history of use, wide range of uses, and good fragrance fixing effect. Patchouli essential oil is beneficial to the body and mind. For example, it can be mentally refreshing, relieve anxiety, and make people feel excited. Physiologically, it has a good bactericidal effect, promotes skin regeneration and reduces inflammation, and so on. These properties have made patchouli popular. As we all know, patchoulol is one of the main ingredients in patchouli oil. If our project can produce patchoulol on a large-scale industrial production, it will generate a lot of business value.

2. Pharmaceutical factory

Patchoulol is also considered the main component contributing to the pharmacological effects of patchouli oil. It is often used as raw materials for drugs because of its anti-tumor, anti-inflammatory, antibacterial, antioxidant, and other pharmacological effects[7]. Nowadays, The market demand for patchoulol is increasing. For example, Huoxiang Zhengqi Shui (KangShengTang), a kind of medicine that is always available in every household in China, contains a huge amount of patchoulol. Therefore, when our project achieves the efficient production of patchoulol, we can sell patchoulol to pharmaceutical factories and profit from it.

3. Customers who need environment-friendly pesticides

Because of its insecticidal biological activity, patchoulol can be made into insecticides to control some specific species of pests. And because it has the characteristics of low toxicity, easy degradation, safety, and environmental friendliness, it is a good choice to use as an insecticide. Therefore, we can also consider producing a large amount of patchoulol by ourselves, preparing it into an environment-friendly insecticide, and selling it to customers who need it in this regard.[8]

Possible challenges

Although our project has achieved initial results in the laboratory environment, considering the difference between the laboratory environment and the actual large-scale production environment, we are worried that there will be some problems during the large-scale production. For example, the accumulation of too much yeast may lead to an untimely and/or insensitive response of the light-induced prompter in yeast to light conditions, and even cause the light-induced promoter to lose its original function, making the whole biosynthesis process uncontrollable. These accidents that may occur in the mass production process will greatly affect the mass production of patchoulol, which may limit the availability of our product to the market. So for us, problems that arise during mass production can be a challenge.

Reference

[1] Van Beek T A, Joulain D. The essential oil of patchouli, Pogostemon cablin: A review [J]. Flavour and Fragrance Journal, 2018, 33(1): 6-51.

[2] Hybertson B M. Solubility of the sesquiterpene alcohol patchoulol in supercritical carbon dioxide [J]. J Chem Eng Data, 2007, 52(1): 235-8.

[3] 徐雯, 吴艳清, 丁浩然, 等. 广藿香的药理作用及机制研究进展 [J]. 上海中医药杂志, 2017, 51(10): 4.

[4] Henke N A, Wichmann J, Baier T, et al. Patchoulol production with metabolically engineered corynebacterium glutamicum [J]. Genes (Basel), 2018, 9(4): 219.

[5] Srikrishna A, Satyanarayana G. An enantiospecific total synthesis of (-)-patchouli alcohol [J]. Tetrahedron Asymmetry, 2005, 16(24): 3992-7.

[6] Albertsen L, Chen Y, Bach L S, et al. Diversion of flux toward sesquiterpene production in Saccharomyces cerevisiae by fusion of host and heterologous enzymes [J]. Appl Environ Microbiol, 2011, 77(3): 1033-40.

[7] 魏晓露, 彭成, 万峰. 广藿香醇体外抗呼吸道病毒作用研究 [J]. 中药药理与临床, 2013, 29(1): 4.

[8] 陈义娟, 范能能, 蒋杰贤, et al. 百秋李醇的用途及杀虫剂 [M]. 2020.

Notebook

Week 1 (6.20-6.26)

We inoculated and cultured Escherichia coli containing the target vector in a liquid LB growth medium. Then we waited for the cultures to grow and develop. We extracted the target vector and designed primers needed for the construction of our vectors and sent them to the company for synthesis.

We purchased the reagents needed for our experiments.

Week 2 (6.27-7.3)

Part one First attempt to digest target vector.

We used the target pSP-GM2 vector, restriction enzyme, reaction buffer, and ddH2O to digest the target vector. The reaction system is as follows:

Reagent	Volume (μL)
10 x Cutsmart buffer	5
restriction enzyme 1	0.5
restriction enzyme 2	0.5
pSP-GM2 vector	1
ddH2O	43

Procedures to digest target vector: (1) Add ingredients in 1.5 mL test tubes at a time according to the above table.

2. Mix and mark well.

3. 37℃ for 8-10 h.

Part teo PCR amplification of corresponding parts.

PCR amplification for PST gene, FPS gene, and the HCF173 promoter. However, promoter HCF173 first was not successfully amplified. After the second test, we successfully amplified HCF173 promoter.

Procedure for PCR amplification: (1) Mix up the recipe into 1.5 mL test tubes according to the following table.

Reagent	Volume (μL)
Q5 DNA polymerase	0.5
5 × Q5 reaction buffer	10
10 mM dNTPs	1
forward primer	2.5
downstream primer	2.5
DNA template	1
ddH2O	32.5

2. The reaction time and temperature were set according to the table below. Run the PCR program.

procedure	temperature/℃	time/s	repeat
preprocessing	98	30	1 cycle
denaturation	98	30	35 cycle
annealing	55	30
extension	72	60
Final extension	72	300	1 cycle
Hold	4	∞	1 cycle

Week 3 (7.4-7.10)

Part one Construction of target vector.

We construct the target vector by mixing pSP-GM2 vector with the genes we cloned. These plasmids were later transformed into E.coli BL21.

Part two Transformation of the plasmid to E.coli

Procedure: (1) Take 50 µL of E.coli cells and defrost E.coli with an ice bath for 25 min. Mixing E.coli gently with 10 µL target recombinant plasmid.

2. Heat shock at 42℃ for 90 s, then place it immediately in the ice bath for another 3-5 min.

3. Add 500 µL LB broth (not containing antibiotics), mix it upside down, and put it in the shaker to recover at 200 rpm, 37℃ for 60 min.

4. Centrifuge at 5,000 rpm, room temperature, for 1 min. Keep 100 µL of supernatant and resuspend the bacteria. The coating on the LB broth containing ampicillin.

5. Place upside down to incubate at 37℃ for 10-12 h in the incubator.

Week 4 (7.11-7.17)

Selecting colonies to inoculate and culture

Colony PCR was used to select positive colonies, which was verified by sequencing and then in liquid LB.

General procedures:

1. Inoculate BL21 cells using a pipette tip, use 2 x Easy Taq enzyme for colonies selecting：

Reagent	Volume (μL)
2x Easy Taq Mix	10
colony	0
forward primer	1
Downstream primer	1
ddH2O	8

2. The reaction time and temperature were set according to the table below. Run the PCR program.

Procedure	Temperature/℃	Time/S	Repeat
preprocessing	94	30	1 cycle
denaturation	94	10	35 cycles
annealing	55	30
extension	72	30
final extension	72	60	1 cycle
hold	4	∞	1 cycle

Week 5 (7.18-7.24)

Transformation of plasmid to Yeast

Procedure:

1. select the monoclones into 5mL corresponding defective medium (containing glucose)).

2. Incubate at 28℃ overnight at 200-220 rpm until OD600 value is 0.5-0.6.

3. Take 500 μL cultured yeast solution into a 1.5 mL EP tube, and centrifuge at 300-500 rpm for 3-5 min. Remove the supernatant.

4. Add 500 microliters of EZ1 into a 1.5 mL EP tube and resuspend yeast cells.

5. Centrifuge at 300-500 rpm for 3-5 min and remove the supernatant.

6. Add 50 μL EZ2 into 1.5mL EP tube, flick, and resuspend yeast cells.

7. Transformation: add 3-5 μL plasmid into 50 μL yeast cells, mix evenly, then add 500 μL EZ3 into it. Incubate yeast cells in a water bath at 30℃ for 45 min-1 h.

8. Centrifugate at 500 rpm for 5 min.

9. After centrifugation, remove 400μL supernate. Resuspend the yeast cells and coat them on a solid medium plate.

Week 6 (8.1-8.7) and Week 7 (8.8-8.14)

Selecting colonies to inoculate and culture

Single yeast colonies were taken from stock plates and inoculated into 8 ml YPD. We carry out PCR on these colonies and plan to test them again. Results showed that some plasmids have been successfully transformed into Yeast.

Week 7 (8.15-8.27)

Cultivation of recombinant yeasts in a Shaking Flask.

The recombinant yeasts were precultured in 5 mL of YPD at 30℃, 220 rpm for 24 h. Precultures were inoculated to 50 mL of YPGD or YPGL in 250 mL flasks at an initial OD600 of 0.05 and were grown under the same condition. An overlay of 5 mL dodecane was added to the flasks after 24 h and the light was opened.

Analysis of Patchoulol

Samples from the organic layer were centrifuged for 15 min at 6000 rpm to determine the level of patchoulol during fermentation. The patchoulol was analyzed by gas chromatography-mass spectrometry using the HP-5 column. The sample (1 μL) was injected in splitless mode. The patchoulol standard was used to identify the substances.

Proof of concept

Method development

Two kinds of promoters were selected for gene expression, constitutive promoter PGK1 and light-inducible promoter HCF173 (BBa_K4270001). [1]

By fusing with FPS (farnesyl diphosphate synthase), PTS (patchoulol synthase), and FPS-linker-PTS genes via PCR, they were linked into pSP-GM2 vector. After the transformation of pSP-GM2 plasmids into E.coli, the DNA of each gene was amplified by PCR and sequenced for verification. The recombinant vectors were then transformed into yeast. Fermentation was then carried out and the target compounds were detected by meteorological chromatography. A qualitative and quantitative analysis was carried out for the production of patchoulol. The ones with the best performance were further applied to real fermentation.

Plasmid construction

The DNA sequence of PTS (encoding patchoulol synthase from P. cablin) was optimized according to the preference of yeast for amino acid codons. Then FPS (BBa_K517004), PTS (BBa_K4270004), and FPS-linker-PTS (BBa_K4270010) were fused with promoter PGK1 or light-inducible promoter HCF173 and constructed into pSP-GM2 plasmids separately, which contains ampicillin resistance genes for colony selection.

The eight plasmids: pSP-GM2-FPS, pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS, pSP-GM2-PTS-FPS, pSP-GM2-HCF173-FPS, pSP-GM2-HCF173-PTS, pSP-GM2-HCF173-PTS-linker-FPS, pSP-GM2-HCF173-PTS-FPS were transformed into E.coli according to the protocol. The colony PCR was conducted for verification of the correct plasmids (Figure 1 and Figure 2). Then the amplified DNAs were sequenced and compared with the designed DNA sequences for further verification.

Then these vectors are all transformed into yeast. The colony PCR was conducted for further verification of these recombinant vectors.

Figure 1. A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR results of pSP-GM2-PTS-FPS.

Figure 2. A. The colony PCR results of pSP-GM2-HCF173-FPS. B. The colony PCR results of pSP-GM2-HCF173-PTS. C. The colony PCR results of pSP-GM2-HCF173-PTS-linker-FPS. D. The colony PCR results of pSP-GM2-HCF173-PTS-FPS.

Patchoulol detection

After breeding and fermentation, Yeast fermentation broth was used for patchoulol detection via the Gas chromatography (GC) method. Unfortunately, we did not detect patchoulol in the supernatant but detected patchoulol in the standard substance containing patchoulol (Figure 3). The reason we did not detect patchoulol in the supernatant may due to the low yield of patchoulol and not enough fermentation.

Figure 3 The detection of patchoulol in the supernatant. A-H. The detection of patchoulol in Yeast fermentation broth from Yeast transformed with pSP-GM2-FPS, pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS, pSP-GM2-PTS-FPS, pSP-GM2-HCF173-FPS, pSP-GM2-HCF173-PTS, pSP-GM2-HCF173-PTS-linker-FPS, pSP-GM2-HCF173-PTS-FPS plasmids, respectively. I. The detection of patchoulol in the standard substance containing patchoulol.

Reference

[1] Xue L, Hong-Bin W, and Hong-Lei J. Light signaling-dependent regulation of PSII biogenesis and functional maintenance. Plant Physiology, 2020, 183, 1855-1868.

Results

Overview

Our results consist of our gene cloning, colony PCR results and Gas chromatography results.

1. Codon Optimization

To improve the translation efficiency of PTS in yeast, we first optimized the Pogostemon cablin PTS gene according to the codon preference of yeast. During optimization, the amino acid sequence of the protein encoded by PTS remained unchanged (Figure 1) and the codon was adjusted.

Figure 1. The original PTS protein sequence and optimized PTS protein sequence.

2. DNA Preparation

The FPS (ERG20) gene was PCR-amplified from the genome of S. cerevisiae 4741. The fusion of FPS and PTS with a Gly-Ser-Gly (GSG) tag was constructed according to a previous work,[1] which generated a fusion protein called PTS-linker-FPS. The FPS (ERG20) gene was PCR-amplified from the genome of S. cerevisiae 4741. The light-inducible promoter HCF173 (HIGH CHLOROPHYLL FLUORESCENCE173) was PCR-amplified from the genome of wild-type Arabidopsis thaliana. [2] The results are shown in Figure 2.

Figure 2A. The PCR results of FPS. B. The PCR results of PTS. C. The PCR results of HCF173. D. The PCR results of PTS-linker-FPS.

After HCF173 and FPS, PTS, PTS-linker-FPS were separately amplified, HCF173 and other genes were overlapped and amplified using primers. The results are shown in Figure 3.

Figure 3A. The PCR results of HCF173-FPS. B. The PCR results of HCF173-PTS. C. The PCR results of HCF173- PTS-linker-FPS. D. The PCR results of PTS-FPS (namely HCF173-FPS- terminator -HCF173- PTS- terminator)

3. Construction of Plasmids

We constructed 4 plasmids with the constitutive promoter (pGK1) separately connecting FPS, PTS, FPS-linker-PTS. The plasmids containing promoter pGK1-FPS-terminator-pGK1-PTS-terminator were also built. We also constructed 4 plasmids with HCF173 as the promoter. The schematic diagram of these plasmids is shown in Figure 4.

Figure 4. The schematic diagram of plasmids. Pcp, constitutive promoter pGK1. Plip, light-induced promoter HCF173. Tns, T ADH1 or T CYC1 terminator.

The colony PCR was conducted for further verification of these recombinant vectors. The results are shown in Figure 5 and Figure 6.

Figure 5A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR results of pSP-GM2-PTS-FPS.

Figure 6A. The colony PCR results of pSP-GM2-HCF173-FPS. B. The colony PCR results of pSP-GM2-HCF173-PTS. C. The colony PCR results of pSP-GM2-HCF173-PTS-linker-FPS. D. The colony PCR results of pSP-GM2-HCF173-PTS-FPS.

4. Patchoulol detection

After breeding and fermentation, Yeast fermentation broth was used for patchoulol detection via the Gas chromatography (GC) method. Unfortunately, we did not detect patchoulol in the supernatant but detected patchoulol in the standard substance containing patchoulol (Figure 7). The reason we did not detect patchoulol in the supernatant may due to the low yield of patchoulol and not enough fermentation.

Figure 7. The detection of patchoulol in the supernatant. A-J. The detection of patchoulol in Yeast fermentation broth from Yeast transformed with pSP-GM2-FPS, pSP-GM2- FPS K197P, pSP -GM2- FPS K197H, pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS, pSP-GM2-PTS-FPS, pSP-GM2-HCF173-FPS, pSP-GM2-HCF173-PTS, pSP-GM2-HCF173-PTS-linker-FPS, pSP-GM2-HCF173-PTS-FPS plasmids, respectively. I. The detection of patchoulol in the standard substance containing patchoulol.