Engineering Saccharomyces cerevisiae for enhanced (–)-α-bisabolol production

(–)-α-Bisabolol is naturally occurring in many plants and has great potential in health products and pharmaceuticals. However, the current extraction method from natural plants is unsustainable and cannot fulfil the increasing requirement. This study aimed to develop a sustainable strategy to enhance the biosynthesis of (–)-α-bisabolol by metabolic engineering. By introducing the heterologous gene MrBBS and weakening the competitive pathway gene ERG9, a de novo (–)-α-bisabolol biosynthesis strain was constructed that could produce 221.96 mg/L (–)-α-bisabolol. Two key genes for (–)-α-bisabolol biosynthesis, ERG20 and MrBBS, were fused by a flexible linker (GGGS)3 under the GAL7 promoter control, and the titer was increased by 2.9-fold. Optimization of the mevalonic acid pathway and multi-copy integration further increased (–)-α-bisabolol production. To promote product efflux, overexpression of PDR15 led to an increase in extracellular production. Combined with the optimal strategy, (–)-α-bisabolol production in a 5 L bioreactor reached 7.02 g/L, which is the highest titer reported in yeast to date. This work provides a reference for the efficient production of (–)-α-bisabolol in yeast.


Introduction
Sesquiterpenoids are a class of natural organic compounds produced by condensing two isopentenyl pyrophosphate (IPP) units and a dimethylallyl pyrophosphate (DMAPP) unit [1]. Because of their strong aroma and anti-inflammatory effects, they have been widely used [2]. (-)-α-Bisabolol (C 15 H 26 O) is a monocyclic sesquiterpene alcohol that was isolated and extracted from chamomile [3]. Due to its anti-inflammatory, bactericidal, antibacterial, skin soothing and moisturizing properties [4], it has been used as an ingredient in pharmaceuticals and cosmetics. In the future, (-)-α-bisabolol may be applied to clinical practice because of its analgesic function [5]. Moreover, (-)-α-bisabolol has low physiology toxicity [6], and it has great potential to become a widely used health product.
Recently, considerable efforts have focused on increasing the production of (-)-α-bisabolol in microbial cell factories, but there still exist many limitations and challenges. Overexpression of the heterologous mevalonate pathway genes (mvaE, mvaS, mvaK1, mvaK2, mvaD and idi) and farnesyl pyrophosphate synthetase gene ispA in Escherichia coli could improve the (-)-α-bisabolol titer to 9.1 g/L [12]. The efficient (-)-α-bisabolol synthase CcBOS from C. cardunculus var. Scolymus was tested, which increased the titer to 23.4 g/L in E. coli [13]. In contrast to E. coli, fungi are less susceptible to phage infection. Optimization of the MVA pathway is the key to (-)-α-bisabolol production in yeast. Shi et al. overexpressed the entire MVA pathway genes in Y. lipolytica to increase the metabolic flux to FPP and optimize the copy number of MrBBS and tHMG1, resulting in the production of 4.4 g/L in a 5 L bioreactor. This was the highest production in yeast reported to date [14]. A recent study showed that overexpression of tHMG1, ERG10, and ACS1 lead to a 13-fold increase in (-)-α-bisabolol biosynthesis in S. cerevisiae, but the final production was only 124 mg/L [11]. Compared with Y. lipolytica, the highest titer of (-)-α-bisabolol in S. cerevisiae was markedly lower. Therefore, the bottleneck in the synthesis of (-)-α-bisabolol by S. cerevisiae needs to be solved.
To search for bottleneck steps of (-)-α-bisabolol biosynthesis in S. cerevisiae, a series of (-)-α-bisabolol product strains were constructed and verified. First, the heterologous gene MrBBS was introduced and the competitive pathway gene ERG9 was weakened. Then, a flexible linker was used to fuse the key genes ERG20 and MrBBS. Next, the effects of MVA pathway modulation and multi-copy integration of the key limiting genes were studied. Additionally, the effect of different transporters on the transport of (-)-α-bisabolol was tested. Finally, the production of (-)-α-bisabolol in a 5 L bioreactor reached 7.02 g/L. This is the highest titer of (-)-α-bisabolol in yeast reported to date. This sustainable method has the potential for industrial (-)-α-bisabolol production.

Strains, plasmids and gene
The SQ14 strain was constructed in previous work [15]. The strains constructed in this study are shown in Table 2. E. coli JM109 was used for propagation and plasmid construction. The plasmid pY26-TEF-GPD with URA3 selection markers was used for gene expression in S. cerevisiae. pMD19T-Simple (TaKaRa, Dalian, Liaoning, China) was used for gene cloning. The plasmids constructed in this study are shown in Table S1. The primers used to construct the plasmids were synthesized by Sangon Biotech (Shanghai, China). The linker sequences are listed in Table S2. The MrBBS gene was codon-optimized and synthesized by Azenta (Suzhou, Jiangsu, China), and the sequence is shown in Table S2.

Strains construction
A Vazyme (Nanjing, China) high-fidelity Phusion DNA polymerase was used for amplifying DNA fragments. DNA fragments were purified and Gibson assembly was used to complete plasmid construction [16]. A Sangon Biotech (Shanghai, China) Seamless Cloning Kit was used for Gibson assembly. After cloning, all extracted plasmids were verified by Sangon Biotech (Shanghai, China). This study used the CRISPR-Cas9 system for single-gene editing [17], and the sgRNAs were designed using the website (https://www.benchling.com/). Both the promoter and terminator used in this study used the endogenous genetic elements of S. cerevisiae. A Frozen-EZ Yeast Transformation II Kit (Zymo Research, Los Angeles, CA, USA) was used for plasmid transformation and genome integration in S. cerevisiae. After transformation, cells were plated on  Yeast Nitrogen Base (YNB)-defective plates (Sangon Biotech, Shanghai, China). and cultivated for 3-5 days at 30 • C. The engineered strains were verified by colony PCR. The correct single-colonies were inoculated in 10 mL of YPD medium for one day. 1% of the inoculated volume was transferred and incubated for another day, strewn in YPD plates containing 1 g/L 5-fluoroorotic acid.

Shake flask fermentation and bioreactor fermentation
Single-colonies of S. cerevisiae were selected from YNB plates without amino acids, and cultured into a 250 mL shake flask containing 25 mL of yeast extract peptone dextrose (YPD) medium (20 g/L tryptone, 10 g/L yeast extract, and 20 g/L glucose) for 24 h at 30 • C and 220 rpm. The seed solution was inoculated into 25 mL of YPD medium to give an initial optical density (OD) of approximately 0.2. After 120 h of fermentation, the titer was measured using GC-MS.
Fermentation was carried out in a 5 L bioreactor (T&J Bioengineering, Shanghai, China) containing 2.5 L of fermentation medium (30 g/L glucose, yeast extract 10 g/L, tryptone 20 g/L, magnesium sulfate heptahydrate 1.5 g/L, calcium carbonate 10 g/L, 0.01% defoamer). The initial agitation speed was 250 rpm, and the dissolved oxygen (DO) was controlled at 20%-40%. The feed medium (600 g/L glucose, 24 mL/L vitamin solution, and 20 mL/L trace metal solution) was streamed after 12 h in the fermentation process. Then the absolute ethanol was continuously added for 60 h. Trace metal solution and vitamin solution were prepared according to the formula of our previous work [18]. The formulations were also provided in the supplementary data.

Standards and analytical methods
The standard (-)-α-bisabolol was purchased from Sigma-Aldrich (Saint Louis, MO, USA). The mother liquor of (-)-α-bisabolol at a concentration of 10 g/L was obtained by dissolving 1 g of (-)-α-bisabolol standard in 100 mL of ethanol. For analysis, the stock solution of 10 g/L   (-)-α-bisabolol in ethanol was diluted into a series of solutions from 5 mg/L to 500 mg/L as the standards.
To extract extracellular (-)-α-bisabolol, 500 μL fermentation culture was mixed with 500 μL methanol. The mixture was vortexed for 1 min and centrifuged. The supernatant was used for analysis after filtration through a 0.22 μm nylon membrane. To extract intracellular (-)-α-bisabolol, cells of 500 μL culture was collected through centrifuging, washed twice with distilled water, and then resuspended into 1 mL ethanol. The cells were then analyzed using a glass bead homogenizer (FastPrep-24 5G). After centrifuging, the supernatant was collected and filtered using a 0.22 μm nylon membrane for the analysis.
To quantify (-)-α-bisabolol, the samples were analyzed by using a Shimadzu's Gas Chromatograph Mass Spectrometer QP2010 (Kyoto, Japan). An RTX-5 capillary column (30 m × 0.25 mm × 0.25 μm) was used under the following conditions: initial column temperature at 90 • C for 0.5 min, a heating rate of 20 • C/min to 280 • C for 10 min. The injector temperature was 280 • C. The ion source and interface temperatures were 200 • C and 170 • C respectively. The carrier air helium velocity was 1.6 mL/min, and the injection volume was 1 μL. Total ion chromatographs of the fermentation sample and (-)-α-bisabolol standard in this study are shown in Fig. S1.
Promoter strength is directly related to gene transcription level. To further improve the efficiency of Erg20-(GGGS) 3 -MrBBS. The gradient promoters P INO1 , P TDH3 , P ERG1 and P RPS5 were selected, and their strengths ranged from strong to weak [19]. Three promoters of the GAL series, P GAL10 , P GAL7 and P GAL2 were also selected. Among them, P GAL7 had the best performance, although its intensity was not the highest. The production of (-)-α-bisabolol further increased to 639.34 mg/L (Fig. 3B).

Improving precursor supplementation by modulating the MVA pathway
When metabolite flow of the MVA pathway was intensified to (-)-α-bisabolol, FPP may become insufficient. Dpp1 is a competitive enzyme that converts FPP to farnesol [21]. Knockout of DPP1 increased the titer but not significantly. In addition, a key gene ERG20 was Fig. 2. Effect of ERG9 weakening and the copy number of MrBBS on the titer of (-)-α-bisabolol. A. Comparison of peak time of (-)-α-bisabolol standard and peak time of fermentation broth. The gas chromatogram of fermentation broth was marked by the red line, and the (-)-α-bisabolol standard was marked by the blue line. B. Cell growth and production of (-)-α-bisabolol after weakening of ERG9 promoter in strains BS2 and BS3. C. Cell growth and production of (-)-α-bisabolol in strains BS3 and BS3-MrBBS. The values are the average of three biological replicates. Error bars represent standard deviations. A. Production of (-)-α-bisabolol and cell growth after fusion expression of ERG20 and MrBBS. The numbers '1, 2, 3' represent the copy number of linkers. The yellow arrow represents the GAL10 promoter and the green arrow represents the TEF1 promoter. The far left of the gene is the start codon, and the start codon linked to the linker sequence was deleted. The green arrow represents P TEF1 and the yellow arrow represents P GAL10 . The red 'T' represents T TAT1 and the blue 'T' represents T ADH1 . These gene cassettes were constructed in the pY26 plasmid and expressed in BS3. B. Production of (-)-α-bisabolol and cell growth under the control of different strength promoters. The values are the average of three biological replicates. Error bars represent standard deviations. overexpressed at the DPP1 site, which increased the titer by 3.5% compared with the control (Fig. 4). Overexpression of ERG10, another key gene in the MVA pathway, was not ideal for (-)-α-bisabolol production (Fig. 4). We speculated that overexpression of a single-gene in the MVA pathway may have caused an accumulation of intermediates. Transcription factor regulation is another solution. Upc2 G888A is a global activator of MVA pathway, and its overexpression had been shown to promote the synthesis of trichodermol [22]. Rox1 and Ypl062w were proven to be negative regulators of MVA pathway [23]. Knockout of the ROX1 showed good results, the titer of (-)-α-bisabolol increased to 795.06 mg/L (Fig. 4). In addition, the metabolism of ethanol was enhanced by replacing the wild-type promoter of ADH2 with P TEF1 . The growth of the resulting strain improved, but (-)-α-bisabolol production was significantly reduced (Fig. 4).

Fermentation optimization of high-yielding strains of (-)-α-bisabolol
Stable and appropriate culture conditions are beneficial to the growth of the strain. Moreover, the strategy of feeding affects the economic feasibility of the (-)-α-bisabolol production. Calcium carbonate can maintain the pH relative stability in the fermentation system. Fermentation with glucose and ethanol as the mixed carbon source contributed to the accumulation of terpenoids in S. cerevisiae [27]. Preliminary fermentation optimization in the shake flasks revealed that calcium carbonate and ethanol supplements had a positive effect on the synthesis of the product (Fig. S3). To further evaluate the fermentation performance of the strain, a scale-up experiment with a 5 L bioreactor was carried out based on the shake-flask results. First, the influence of calcium carbonate on fermentation was examined ( Fig. 6A and B). When calcium carbonate was added, the maximum OD 600 increased from 56.2 to 62.4, and the yield increased from 4.21 g/L to 6.07 g/L. Compared with the calcium carbonate-free control, the OD 600 and the yield of (-)-α-bisabolol increased by 11.03% and 44.18%, respectively. These results indicated that the addition of calcium carbonate was beneficial to cell growth and the accumulation of (-)-α-bisabolol. Next, the effects of different carbon sources in the fermentation were further compared. Compared with glucose feeds alone, ethanol significantly enhanced the accumulation of (-)-α-bisabolol, with a yield of 7.02 g/L ( Fig. 6B and C). Additionally, the proportion of intracellular production decreased from 31.30% to 21.79%. This result shows that ethanol may promote the transport of (-)-α-bisabolol.

Discussion and conclusion
(-)-α-Bisabolol has good prospects in health care products and cosmetics. The production of (-)-α-bisabolol by biosynthesis is attractive [28]. In the present study, heterologous production of (-)-α-bisabolol was achieved in S. cerevisiae. Weakening of ERG9 and improving the copy number of MrBBS were the keys to (-)-α-bisabolol accumulation. Fusion expression improved the utilization efficiency of key enzymes on substrates. Knockout of the repressive factor Rox1 significantly improved the titer of (-)-α-bisabolol. Overexpression of PDR15 effectively promoted the efflux of (-)-α-bisabolol. Finally, by optimizing the media composition and feed strategy, we have achieved a titer of 7.02 g/L in a 5 L bioreactor. (-)-α-Bisabolol production in yeast is summarized in Table 1. Our work has value for the study of economically viable (-)-α-bisabolol-producing S. cerevisiae.
The formation of efficient metabolic channels by co-localizing enzymes can greatly improve the catalytic activity of key enzymes [29]. Achieving protein assembly through protein ligation is a common strategy [30]. The type and length of the linkers are critical to the effect of protein ligation [31]. Excessively long linkers reduced the stability of fusion proteins, while excessively short linkers affected the correct translation and folding of proteins [32]. In this study, when a single-copy linker was used to connect Erg20 and MrBBS, the yield decreased, while a two-copy linker significantly increased the yield. This indicated that too short a linker may affect the correct expression of Erg20 and MrBBS. Guo et al. explored the effects of different linker lengths on the synthesis of resveratrol from 4CL and STS catalytic substrates, which greatly improved its catalytic efficiency [33]. Meanwhile, Fig. 5. Enhance production of (-)-α-bisabolol by transporter engineering. A. Intracellular and extracellular production of (-)-α-bisabolol after transporters (ΔPDR12, ΔPDR1, ΔPDR5, ΔSNQ2, ΔAUS1, ΔPDR15, ΔPDR11, ΔSTE6, ΔYOR1, ΔPDR10) and regulators (ΔWAR1, ΔMSN4, ΔMOT3, ΔPDR3, ΔARO80, ΔYRR1) were knocked out in BS5. B. Intracellular and extracellular production of (-)-α-bisabolol after overexpression of PDR15 and PDR3 in BS5. The values are the average of three biological replicates. Error bars represent standard deviations.
the direction of the linker also impacts protein expression. A suitable linker helped Erg20 and MrBBS maximize their catalytic activity. Jiang et al. designed the direction of the GES and Erg20 fusion protein with the help of surface electrostatic distribution analysis, which greatly increased the geraniol production [34]. In this work, a fusion of Erg20 and MrBBS improved the metabolic flux toward (-)-α-bisabolol synthesis.
A lack of precursor FPP supply would reduce production efficiency [35]. Main competitive gene ERG9 which is essential for yeast growth that can only be weakened [36]. Replacing the wild promoter of ERG9 with weak promoters such as P HXT1 or weakening the catalytic performance of Erg9 by reducing the half-life of the protein were proven feasible to weaken ERG9 [15,37]. The strategy of replacing the wild-type promoter of ERG9 with P HXT1 also guided our fermentation optimization. In our study, glucose supplementation helped to activate the expression of ERG9 during the early growth stage of the strain, while ethanol supplementation inhibited the expression of ERG9 and enhanced the (-)-α-bisabolol production. In addition, the MVA pathway is directly related to FPP production. HmgR catalyzes the conversion of HMG-CoA to mevalonic acid, which has been recognized as the first rate-limiting step of MVA pathway due to the irreversibility of the reaction. Erg20 catalyzes the synthesis of FPP from IPP and DMAPP, and its high expression is beneficial to improve the content of FPP. Meng et al. significantly increased the titer of the precursor valencene by the overexpression of tHMG1 and the fusion expression of ERG20 and CnVS, laying the foundation for the de novo synthesis of nootkatone [38]. The regulation of transcription factors is also an effective strategy. Knockout of ROX1 increased the transcription level of the whole MVA pathway to enhance the supply of FPP precursors [23], and the (-)-α-bisabolol production was improved significantly. Overexpressing a non-limiting gene alone was not helpful to production, whereas increasing the whole metabolic flux of the MVA pathway was the best choice for (-)-α-bisabolol production.
Extracellular transport engineering can reduce metabolic pressure on cells and significantly increase titer of the products [39]. S. cerevisiae contains powerful transport systems that transport a wide variety of toxic substances. ABC transporters have detoxifying properties in prokaryotic and eukaryotic cells and have been shown to export a wide variety of products. Xu et al. improved the efficiency of the extracellular transport of rubusoside by overexpressing the yeast endogenous transporter Pdr11 and transcriptional activator Msn4, thus improving the titer of rubusoside [26]. Jiao et al. simultaneously overexpressed PDR11 and YOL075C, which was favorable for the transport of tocotrienol [40]. Therefore, we tried to explore some highly efficient endogenous ABC transporters. PDR15 is a paralog of PDR5. Both are involved in multi-substrate resistance mechanisms [41]. PDR15 was shown to be involved in (-)-α-bisabolol transport, with significantly improved the titer after overexpression. The crystal structure and transport mechanism of PDR5 (the paralog of PDR15) have been resolved [42], while most of the other binding transport mechanisms between transporters and substrates have yet to be elucidated.
In summary, a high titer (-)-α-bisabolol-producing strain was constructed by expressing heterologous genes, fusion expression, modulating the MVA pathway, strengthening the transport mechanism and fermentation optimization. The production of (-)-α-bisabolol reached 7.02 g/L in a 5-L bioreactor. After our work, there are still some strategies to enhance (-)-α-bisabolol synthesis. First, S. cerevisiae has a strong ethanol biosynthesis pathway, and the supply of acetyl-CoA (precursor of the MVA pathway) may be insufficient. Systematic enhancement of the acetyl-CoA synthesis pathway is an effective approach to improve the production of (-)-α-bisabolol. Second, high expression of the MVA pathway gene consumes large amounts of NADPH, and the supply of cofactor NADPH may also be a limiting factor. Finally, intracellular accumulation of the product remains a problem. Two-phase fermentation may further promote product secretion [40]. Fig. 6. Fed-batch fermentation of (-)-α-bisabolol production in a 5 L bioreactor. Time courses of cell growth, (-)-α-bisabolol production, glucose consumption, and ethanol production are presented. A. Production of (-)-α-bisabolol by batch fermentation in a 5 L bioreactor. The concentration of dissolved oxygen was maintained at 20-30%, and the concentration of glucose was maintained at 0-1 g/L. B. (-)-α-Bisabolol was produced by batch fermentation in a 5 L bioreactor. The dissolved oxygen concentration was maintained at 20-30%, the glucose concentration was maintained at 0-1 g/L, and 10 g/L of calcium carbonate was initially added. C. Production of (-)-α-bisabolol by batch fermentation in a 5 L bioreactor. The dissolved oxygen concentration was maintained at 20-30%, the glucose concentration was maintained at 0-1 g/L, and 10 g/L of calcium carbonate was initially added. Ethanol was added at 60 h, and the concentration of ethanol was kept at 10-20 g/L. The values are the average of three biological replicates. Error bars represent standard deviations.

Declaration of competing interest
The authors declare that they do not have any financial or commercial conflict of interest in connection with the work submitted.