Overview
In framework of our project Aroma Anchor, we are aiming to produce rare fragrance fixatives including santalol and ambrein, which are terpenoids depending on the same precursors, IPP and DMAPP (Figure 1) that are produced by MVA pathway in the cell.
Figure 1. Biosynthetic pathway for santalol (A) and ambrein (B) in engineered S. cerevisiae.
Notes: The red star indicates the overexpression of the corresponding gene, and the genes labeled in green or blue represent their heterologous expression in yeast. tHMG1: Truncated HMG1 with the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase; IDI1: IPP isomerase; IPP: Isopentenyl pyrophosphate; DMAPP: Dimethylallyl pyrophosphate; FPP: Farnesyl diphosphate; DPP1: Diacylglycerol pyrophosphate phosphatase 1; LPP1: Lipid phosphate phosphatase 1; ClSS: α-Santalene Synthase from Clausena lansium; SaCPR2: cytochrome P450 reductases found in S. album; CYP736A167: a cytochrome P450 from S. album; BmeTC: Tetraprenyl-β-curcumene Cyclase from Bacillus megaterium.
Synthetic Biology uses engineered design logic to give organisms purposed design, to remake and even to resynthesis them. It's developement procedure usually follows an iteration of the DBTL (design-build-test-learn) cycle.
Figure 2. Summary of the different engineering cycles completed for the production of santalol and ambrein (D=Design, B=Build, T=Test, L=Learn)
Therefore, based on the SynBio scientific logic, we designed three cycles of genetic editing of S. cerevisiae to produce proper strains for such terpenoids synthesis
Terpenoids are a class of naturally occurring organic chemicals derived from the IPP and DMAPP. Spotting on the biosyhthetic pathway for santalol and ambrein, it is clear that both our target products belong to the terpenoids class. Therefore, we decided to construct a terpenoids producing strain by overexpressing MVA pathway for higher yields of santalol and ambrein (Figure 2). Considering the different enzyme machineries for the biosynthesis of Ambrein and Santalol, we made cycle 2 and cycle 3 respectively to construct santalol and ambrein producing strains respectively. We also used different strategies to optimize the strains and improve the production of our target fixatives. In the following content, we will introduce the construction and development of each cycle in cells, and the enlightenment for the next targeting cycles. We would also like to share some thoughts about in vivo cycle establishment for other enthusiastic researchers.
Cycle 1. Construction of terpenoids producing strain
Design-Build
FPP is the main substrate for santalene production, and squalene is the substrate of ambrein production in cells. These two substrate compounds are both transformed from their precursor IPP and DMAPP. In order to maximize the production of the substrate molecules, IPP and DMAPP production will be enlarged in vivo to support the downstream molecular transformation.
In order to reach the goal, we over-expressed the MVA pathway. From previous studies, we found that biggest rate-limiting step is mediated by the enzyme HMGR, which catalyzes HMG-CoA into mevalonate (Figure 1). According to Liu's research, the knock-in of tHMG1 and IDI1 can improve improving the speed limiting effect of enzymes and the production of FPP (Liu et al., 2013). We optimized the tHMG1 and IDI1 codons, making them compatible with S. cerevisiae translation system and enabling them to be expressed under the control of pTDH3 and pPGK1, eventually knocking them into the HIS3 site through CRISPR/Cas9 system for further modification (Figure 3A).
After using PCR to amplify different donor DNA template along with plasmid pCRCT-HIS3-1, we transformed them into S. cerevisiae strains. After 4 to 5 days of growth under temperature of 30℃, we use gel electrophoresis to screen the strains with tHMG1 and IDI1 inserts (Figure 3B). After curing the plasmid, the sequencing result confirmed the successful engineered S.cerevisiae strain for synthesis of terpenes, which is named as strain Lv1. Such a success brings the experiment to the next stage (Figure 3C).
Figure 3. (A) Schematic strategy of tHMG1 and IDI1 integration into site HIS3. (B) After transformation, colony PCR was performed to screen strain Lv1. (C) Strain 1, 4 and 5 has been confirmed positive by sequencing.
Test-Learn
As we ensure the expression of the two genes in S. cerevisiae, we were also confirming if this strategy would be helpful to construct terpenoids producing strain.
In order to verify our hypothesis, we aim to detect and compare the endogenous FPP and squarene production of wildtype and strain Lv1. However, limited by current compound detection methods, quantitative detection of FPP is still difficult to achieve. According to previous reports, endogenous phosphorylases including DPP1 and LPP1 in yeast can convert FPP into its dephosphorylated derivative farnesol (FOH), and the detection methods for FOH are relatively mature (Rubat et al., 2017). Therefore, quantitative detection of FOH in yeast fermentation broth indirectly reflects the synthesis and accumulation of FPP in the strain, which has become an important way of FPP detection at present.
As we expected, there is a significant difference in the concentrations of farnesol and squalene in the extract of wildtype and strain Lv1 (Figure 4A-B). Finally, 2.21 mg/L of farnesol and 0.945 mg/L of squalene were detected in the fermentation broth of strain Lv1. However, both farnesol and squalene were not detected in the wildtype strain (Figure 4 C-E). Therefore, the insertion of tHMG1 and IDI1 helps to increase the substrate required for terpenoid synthesis, such as the supply of FPP and squalene, and may help to increase the production of terpenoid products.
Figure 4. Analysis of FOH and squalene accumulated in wildtype and strain Lv1 by GC-MS. (A) GC results of farnesol in different samples and standard. (B) GC results of squalene in different samples and standard. (C) Standard curve of farnesol. (D) Standard curve of squalene. (E) The quantitative results of farnesol and squalene.
Cycle 2-1. Construction of Santalene Production Cycle
Design-Build
Currently, it has been reported that S. cerevisiae can synthesize santalene by
the inserted santalene synthase, and then converts santalene to santalol by CYPs (cytochrome P450 enzymes). Also, sandalwood oil is composed of up to 54.2% a-santalol (Chonglong W. et al., 2015). Therefore, it urges us to choose the santalene synthase from Clausena lansium which is capable of producing the most a-santalene (Jiachen Z. et al, 2022). Besides, instead of overexpressing ERG20, we found out that the insertion of ERG20-F96W is helpful to improve production of santalene. As a result, we decided to insert ERG20_F96W and ClSS into the genome of strain Lv1.
In first round of transformation we tried to knock out DPP1 along with the insertion, because DPP1 has the ability to dephosphorylate FPP into farnesol (Chambon et al, 1990). Nevertheless, after two attempts and replacement of different experimenters, we still failed to screen the positive strain, so we chose another commonly used 106 site (Figure 5A). And finally, we successfully screened one positive strain out of 10 strains, which is named as Lv2s-1 (Figure 5B).
Meanwhile, Jiachen Z.'s research also suggests that ClSS with mutation of F441V can produce different structures of santalene with a ratio closer to that in sandalwood oil. And Xun Li's research indicates that S532A mutation can significantly improve ClSS's ability to produce santalene. Thus, we inserted ERG20_F96W and ClSS_F441V, S532A into the genome of strain Lv1, and successfully constructed strain Lv2s-2 (Figure 5C).
Figure 5. (A) Schematic strategy of ERG20_F96W and ClSS and ClSS_F441V, S532A the integration into site 106. (B) Colony PCR results performed to screen strain Lv2s-1 and Lv2s-2.
Test-Learn
Strain Lv2s-1 and Lv2s-2 possesses ClSS and ClSS_F441V, S532A respectively. According to the GC results, it is apparent that both strains can produce santalene(Figure 6A). However, using humulene as an internal standard, the yield of a-santalene in strains Lv2s-1 and Lv2s-2 was quantified as 2.513 mg/L and 1.148 mg/L, respectively. The double site mutation of ClSS not only failed to achieve the desired increase in yield, but also decreased the yield of a-santalene by about 54%. Therefore, it is more suitable to choose Lv2s-1 as our chassis cell for modification towards a santalol producer.
Figure 6. (A) GC results of santalene accumulated in strain Lv2s-1 and Lv2s-2 by GC-MS. (B) MS results at 10.13 s and 10.549 s. (C) The yield comparison of santalene in different strains.
Cycle 2-2. Construction Santalol Production Cycle
Design-Build
The odor character of sandalwood oil mainly comes from the Z-santalols rather than E-isomers (Jiachen Zi et al, 2020). Out of hundreds of CYPs, CYP736A167 is reported to specifically produces the Z-type santalols, and it should collaborate with SaCPR2 to oxidize santalene. Then we insert CYP736A167 and SaCPR2 into the genome of strain Lv2s-1. We chose two different site as our insertion site, one of which is a common used site, YPRCd15c. For the concern that LPP1 encodes the lipid phosphate phosphatase degrading FPP, gene LPP1 is also our target to knock-out while carrying out insertion (Figure 7A).
Finally, Lv2s-1's genome was inserted with gene CYP736A167 and SaCPR2 at the site YPRCd15c and LPP1, leading to successful construction of strain Lv3s-YP and Lv3s-LP respectively.(Figure 7B)
Figure 7. (A) Schematic strategy of CYP736A167 and SaCPR2 integration into the site YPRCd15c or LPP1. (B) After transformation, strain 1-4, 7-8 of Lv3s-YP appeared the successful integration into site YPRCd15c. (C) These two genes were also confirmed to be inserted into site LPP1 in the strain 7 of Lv3s-LP.
Test-Learn
As the GC resluts shown in Figure 8A, both strain Lv3s-YP and Lv3s-LP can produce a-santalol. Also, there are some santalene can be detected in the fermentation broth (Fugure 8A). As a result, refer to the standard curve of santalol (Figure 8B), the yields of santalol in Lv3s-YP and Lv3s-LP are 40.65 mg/L and 21.21 mg/L respectively. And the yields of santalene in Lv2s-1, Lv3s-YP and Lv3s-LP are 2.51 mg/L, 24.23 mg/L and 24.73 mg/L respectively.
Apparently, the insertion site has a significant impact on the yield of santalol. However, Lv3s-YP can produces 1.92 fold of santalol than Lv3s-LP, which is totally out of our expectation. So far, it seems like the knock-out of LPP1 can't help improve the yield of santalol. Besides, further modifications have made the strain more capable of producing santalene. After all, Lv3s-YP is our favorite strain to produce santalol.
Figure 8. (A) Analysis of santalol and santalene accumulated in strain Lv3s-YP and Lv3s-LP by GC-MS. (B) The standard curve of santalol. (C) The yield comparison of santalene and santalol in different strains based on GC-MS.
Cycle 3. Construction of Ambrein Production Cycle
Design-Build
Researchers used Pichia pastoris and S. cerevisiae to produce the main compound in Ambergris-ambreinto to solve the problem of deficiency of rare ambergris using synthetic biology techniques in previous studies (Harald P. et al, 2018; Harald P. et al, 2019). They reported heterologously expression of the cyclase to form a monocycle and a bicycle at both ends of squalene to produce ambrien. Among all the cyclase that have been expressed in yeast to produce ambrein, BmeTC_D373C can achieve the highest yield. However, Tsutomu S.'s research team utilised cell-free system and found out that synergistic effect of the three cyclases BmeTC_Y167A, D373C and BmeTC can reach about 9.77-fold higher activity than solely BmeTC_D373C (Tsutomu S. et al, 2020).
Thus, utilising strain Lv1 as chassis cell, we tried to insert all three genes of BmeTC_Y167A, D373C and BmeTC into the 106 site to construct our strain Lv2a-1 (Figure 9A), and the successful integration were confirmed in strain as the positive PCR results shown in Figure 9C.
Based on this synergistic effect that can promote the biosynthesis of ambrein, we presumed that using flexible linker to construct a fuison protein of BmeTC_Y167A, D373C and BmeTC could remarkably improve the yield of ambrein. To verify our assumption, we integrated BmeTC-Flexible Linker-BmeTC_Y167A, D373C into 106 site (Figure 9B, D).
Figure 9. (A) Schematic strategy of BmeTC_Y167A, D373C and BmeTC integration into the site 106. (B) Schematic strategy of BmeTC_Y167A, D373C-Flexible Linker-BmeTC integration into the site 106. (C) These two genes were confirmed to be inserted into site 106 in the strain 6 of Lv2a-1.(D) These two genes were confirmed to be inserted into site 106 in the strain 2-8 of Lv2a-2.
Test-Learn
Unfortunately, we did not observe any peaks suspected of ambrein. But the reaction substrate, squalene, can be detected at 9.70-9.83 min. Compared with strain Lv1, the yield of squalene significantly decreased in different further modified strains. To be specific, the yield of squalene decrease up to 77.40% in strain Lv2a-1 and 90.69% in strain Lv2a-2. Although there's no ambrein can be detected, the decrease of squalene might indicate the synthesis of some potential intermediates. We will optimize our testing method and the specificity of cyclase to reach the goal of ambrein production.
Figure 10. (A) Analysis of ambrein and squalene, accumulated in strain Lv1, Lv2a-1 and Lv2a-2 by GC-MS. (B) The yield comparison of squalene in different strains based on GC-MS.
Discussion
This year, LINKS-China successfully obtain the yeast strain Lv3s-YP with the ability of producing 40.65 mg/L a-santalol and 24.23 mg/L a-santalene, in which sandalwood components components can reach up to 64.88 mg/L in 100 mL shake flask culture. In the future, we will continue optimizing our fermentation process in 1-10L pilot scale fermenter, in order to maximise production efficiency. We have interviewed a R&D director Mr. Zongjin Li from Link Spider Co., ltd. who is an experienced engineer in biological fermentation. We were informed that by stragety of precisely regulating pH, temperature, supplements, etc. it is potentially for us to raise the production efficiency up to 0.65g/L for current yeast strain.
In the construction of ambrein-producing strains, due to the lack of commercialised standard sample of ambrein molecules, the testing of ambrein is quite difficult. We haven't got information of the production yield of relevant molecules. However, we believe that our jobs have solidified a firm basis for the future industrialised production of ambrein. We will continue to optimise the metabolic pathway through different strategies, such as increase the production efficiency of the precursor Squalene, in order to better regulate the metabolic flows to the production of ambrein.
In the future, we will further construct our strains systematically, target to reach an production efficiency up to more than 1g/L. We have done research on companies which produce natural molecules by traditional fermentation. We have learned lots of professional knowledge about fermentation production, and enact a 5-year business plan based on our strain (details in 'Entrepreneurship' section). We believe, benefits from synthetic biology, we can produce gorgeous essence and fragrance materials for the public in a more economical, more sustainable, and more environmental-friendly way.
References
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