Vitamin A production by engineered Saccharomyces cerevisiae from

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Vitamin A production by engineered Saccharomyces cerevisiae from xylose via two-phase in situ extraction Liang Sun, Suryang Kwak, and Yong-Su Jin ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.9b00217 • Publication Date (Web): 02 Aug 2019 Downloaded from pubs.acs.org on August 3, 2019

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ACS Synthetic Biology

Submitted to ACS Synthetic Biology

Vitamin A production by engineered Saccharomyces cerevisiae from xylose via two-phase in situ extraction

Liang Sun, 1,2 Suryang Kwak1,2, Yong-Su Jin1,2*

1Department

of Food Science and Human Nutrition, and 2Carl R. Woese Institute for Genomic

Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801

* Corresponding

author: Yong-Su Jin

Tel: 217-333-7981, Fax: 217-333-0508, Email: [email protected] Mailing address: 1206 W. Gregory Drive, Carl R. Woese Institute for Genomic Biology, Urbana, IL 61801, United States

1

ACS Paragon Plus Environment

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ABSTRACT Vitamin A is an essential human micronutrient and plays critical roles in vision, reproduction, immune system, and skin health. Current industrial methods for the production of vitamin A rely on chemical synthesis from petroleum-derived substrates, such as acetone and acetylene. Here, we developed a biotechnological method for production of vitamin A from an abundant and nonedible sugar. Specifically, we engineered Saccharomyces cerevisiae to produce vitamin A from xylose—the second most abundant sugar in plant cell wall hydrolysates—by introducing a βcarotene biosynthetic pathway, and a gene coding for β-carotene 15, 15’-dioxygenase (BCMO) into a xylose-fermenting S. cerevisiae. The resulting yeast strain produced vitamin A from xylose at a titer four-fold higher than from glucose. When a two-phase in situ extraction strategy with dodecane, or olive oil as an extractive agent was employed, vitamin A production improved additional two-fold. Furthermore, a xylose fed-batch fermentation with dodecane in situ extraction achieved a final titer of 3,350 mg/L vitamin A, which consisted of retinal (2,094 mg/L) and retinol (1,256 mg/L). These results suggest that potential limiting factors of vitamin A production in yeast, such as insufficient supply of isoprenoid precursors, and limited intracellular storage capacity, can be effectively addressed by using xylose as a carbon source, and two-phase in situ extraction. The engineered S. cerevisiae and fermentation strategies described in this study might contribute to sustainable and economic production of vitamin A, and vitamin A-enriched bioproducts from renewable biomass.

KEYWORDS: Vitamin A, β-carotene, xylose, Saccharomyces cerevisiae, in situ extraction

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Vitamin A refers to a group of natural retinoids including retinal, retinol, retinoic acid and retinyl esters that belong to the isoprenoid superfamily.1,2 Vitamin A is an essential micronutrient required for various biological functions in vision, reproduction, immune system, and differentiation of epithelial cells. Dietary vitamin A supplementation is crucial to prevent vitamin A deficiency that can cause childhood blindness, and increase vulnerability to infectious diseases.3–5 Additionally, vitamin A has received wide attention for applications in animal feed fortification, anti-aging cosmetics, and pharmaceuticals for skin diseases. Therefore, the vitamin A market continues to increase as the market size is expected to reach USD 864 million worldwide by 2021.6 Current industrial processes for producing vitamin A are based on chemical synthesis that starts from petroleum-derived substances, such as acetone and acetylene.7 Those manufacturing procedures might not be ideal for safe and sustainable production due to expensive reagents, complex purification steps and generation of considerable pollutants.8 Alternatively, microbial fermentation of sugars from renewable biomass into vitamin A could be more cost-effective and environmentally friendly than the petrochemical-based manufacturing processes. β-carotene is a main precursor for vitamin A synthesis in nature. In general, the conversion of β-carotene into vitamin A involves two enzymes: β-carotene 15, 15’-monooxygenase (BCMO), and retinol dehydrogenase. The first enzyme catalyzes the cleavage of β-carotene (C40) into two molecules of retinal (C20) which is then reduced to retinol by the second enzyme (Fig. 1). Therefore, production of vitamin A in non-carotenogenic microbes requires a heterologous βcarotene biosynthetic pathway consisting of phytoene synthase, phytoene desturase and lycopene cyclase and additional expression of β-carotene 15, 15’-monooxygenase (BCMO).

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To date, only a few attempts of microbial vitamin A production have been reported and all of them were conducted using Escherichia coli as a host.9–12 Using a two-phase culture system with dodecane, Jang et al. produced up to 136 mg/L of vitamin A in an engineered E.coli harboring exogenous mevalonate (MVA) and vitamin A producing pathways.11 However, E. coli might not be a desired host for industrial production of vitamin A as drugs, food supplements and cosmetics ingredients because of possible endotoxin contamination in final products. Also, bacteriophage infection is a concern for a large-scale fermentation of E. coli. On the contrary, Saccharomyces cerevisiae has superior traits for the industrial production of isoprenoids including the ease of genetic manipulation, robust large-scale fermentation13, and the native MVA pathway with relatively high flux towards isoprenoids.14 Various isoprenoids 15 16 17, and ginsenosides 18 have been successfully produced by engineered S. cerevisiae. This study therefore aimed to overproduce vitamin A by engineered S. cerevisiae. Ensuring the availability of the key precursor, isopentenyl pyrophosphate (IPP), is necessary for the overproduction of isoprenoids.19 In S. cerevisiae, the MVA pathway producing IPP starts from cytosolic acetyl-CoA. Recent metabolic engineering efforts have therefore been focused on engineering central carbon metabolism to enhance the supply of cytosolic acetylCoA16,20,21 and optimizing expression levels of the enzymes in the MVA pathway for efficient generation of IPP.22–24 Despite intensive genetic perturbations to shunt carbon flux towards isoprenoids production, ethanol remains a major product from glucose due to the Crabtree effect of S. cerevisiae,25 which limits isoprenoids production from glucose. Even after introducing the metabolic designs allowing production of a targeted isoprenoid, S. cerevisiae displayed limited capacities for the production of hydrophobic isoprenoids, especially those accumulate intracellularly, such as polyketides and carotenoids, due to the 4


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restricted intracellular storage space for hydrophobic products. As such, attempts to expand the intracellular storage capacity have been made for improved production of squalene,26 and lycopene.27 To address the above-mentioned limiting factors for the high-level production of vitamin A, this study implemented two simple, but effective strategies. Specifically, we utilized a xylosefermenting S. cerevisiae strain SR828 as a host strain for the production of vitamin A. Xylose, a major sugar component of lignocellulosic biomass and non-edible sugar to human, was used as a carbon source in substitution of the conventional substrate glucose. In contrast to glucose fermentation, xylose utilization by engineered S. cerevisiae does not exert the Crabtree effect, facilitating ample supply of cytosolic acetyl-CoA. Therefore, enhanced production of acetyl-CoA derived products, such as squalene, and amorphadiene was achieved using xylose as a carbon source.29 Additionally, to overcome the limited intracellular storage capacity, we adopted an in situ extraction strategy using dodecane or olive oil as an extractive agent. Interestingly, β-carotene accumulation in the vitamin A producing strain was substantially reduced due to the removal of vitamin A from the cells via in situ extraction. To the best of our knowledge, this is the first report of vitamin A biosynthesis in an engineered yeast, and the titer of vitamin A was among the highest of high-value hydrophobic compounds produced in engineered yeast. This study presents an efficient, sustainable and environmentally friendly strategy for industrial production of vitamin A, and vitamin A enriched bioproducts using a sugar from renewable biomass.

RESULTS AND DISCUSSION

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Construction of an engineered S. cerevisiae capable of producing vitamin A from xylose. Complete vitamin A synthetic pathways exist in a group of proteorhodopsin-exploiting bacteria and archaea in the ocean’s photic zone.9,30 As these marine microorganisms synthesize trace amounts of retinal they are not apt for the large-scale production of vitamin A. In this study, the GRAS yeast S. cerevisiae was engineered to functionally express the whole heterologous vitamin A synthetic pathway and produce vitamin A from xylose as well as glucose (Fig. 1). Specifically, crtE, crtI, crtYB genes coding for phytoene synthase, phytoene desaturase and lycopene cyclase in the β-carotene biosynthesis pathway along with a codon optimized gene Blh coding for BCMO were introduced into a previously engineered xylose-fermenting S. cerevisiae strain SR8.28 As expected, the SR8A strain produced vitamin A as a mixture of retinal and retinol but the SR8B strain did not produce vitamin A (Fig. 2). Considering promiscuous retinol dehydrogenase activity of human (ADH3) and E.coli (ybbo) alcohol dehydrogenases,12,31 innate alcohol dehydrogenases (ADHs) might catalyze the production of retinol from retinal in SR8A . Comparison of vitamin A production patterns on glucose and xylose. SR8A produced substantial amount of ethanol on glucose even under aerobic conditions due to the Crabtree effect (Fig. 3a) but exhibited marginal ethanol production from xylose under aerobic conditions. As a result, cell growth on xylose was 57% higher than on glucose (Fig. 3b). The SR8A strain produced 60.90 mg/L of β-carotene, and 20.74 mg/L of vitamin A from 44 g/L of xylose. These levels are approximately three-fold and five-fold as much as the levels produced from 41 g/L glucose (20.36 mg/L of β-carotene and 4.51 mg/L of vitamin A) (Fig. 3c, Fig. 3d). Correspondingly, specific β-carotene and vitamin A production from xylose were 8.25 mg βcarotene /g dry cell weight (DCW) and 2.81 mg vitamin A/g DCW, respectively. These values are 94% and 181% higher than those from glucose (4.25 mg β-carotene /g cell and 0.94 mg vitamin 6


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A/g cell). Additionally, the SR8A strain showed a higher content of ergosterol (21.48 vs. 17.32 mg ergosterol /g DCW) from xylose than glucose (Fig. 4), indicating that cytosolic acetyl-CoA supply, and metabolic fluxes through the MVA pathway might be higher under xylose conditions than glucose conditions. However, the lower accumulations of vitamin A than β-carotene under both glucose and xylose conditions (Fig. 3c, Fig. 3d) implies that the BCMO catalytic activity in the SR8A strain might not be enough to convert β-carotene and overproduce vitamin A in the engineered yeast. Improved vitamin A production by two-phase in situ extraction. Despite the positive effect of xylose utilization, the vitamin A titer by our engineered yeast was still low. We hypothesized that not only the insufficient activity of the heterologous BCMO but also the limited intracellular storage capacity for lipophilic compounds19 might be major hindrances to vitamin A production in our engineered yeast. As such, a two-phase in situ extraction strategy was adopted to remove the lipophilic end-product from cells. In order to examine the effects of two-phase in situ extraction on vitamin A production by engineered yeast, dodecane as an extractive solvent was added into yeast cultures at a volumetric ratio of 1:1 (Fig. S5). Dodecane did not deter cell growth or sugar consumption under both glucose and xylose conditions (Fig. 3a, Fig. 3b, Fig. 5a, Fig. 5b). With dodecane in situ extraction, the SR8A strain produced vitamin A at a final titer of 23.55 mg/L from 41 g/L of glucose and 55.31 mg/L from 43 g/L of xylose. These levels were about 4 fold and 2 fold higher than those of glucose and xylose cultures without dodecane (Fig. 8). Interestingly, vitamin A (instead of β-carotene) was the major product when two-phase cultures were conducted in contrast to the one-phase cultures (Fig. 5, Fig. 3, Fig. S6). These results demonstrate conversion of βcarotene into vitamin A can be facilitated if vitamin A is removed from cells via in situ extraction. 7



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According to the altered product distribution, around 90% of vitamin A was found in the dodecane phase. On the contrary, over 90% of β-carotene remained in the cell (Table S2). This indicates that only vitamin A can be effectively extracted into dodecane due to its shorter hydrophobic chain length (C20) than that (C40) of β-carotene. By removing the end-product from the cells, the conversion of β-carotene into vitamin A improved even though the BCMO activity was not augmented. We speculate that the vitamin A accumulation might cause inhibition on BCMO activity so that vitamin A production by the same strain in the one-phase fermentation was much lower than in the two-phase fermentation. There was no significant change on ergosterol accumulation with dodecane in situ extraction as compared to the cultures without dodecane (Fig. 4), which indicates that ergosterol production was not responsive to the two-phase culture system because ergosterol could not be extracted by dodecane. These results collectively suggest that the strategy of two-phase in situ extraction can effectively improve the production of vitamin A in engineered yeast by overcoming the limitation of intracellular storage capacity and alleviating possible feedback inhibition of an end-product. Considering the low economic feasibility and possible safety concerns of using dodecane for the industrial production of vitamin A, we chose olive oil as an alternative extractant which is more suitable than dodecane for nutraceutical, pharmaceutical and cosmetic products. Olive oils naturally contain small amount of carotenoids which contribute to the color and nutritional quality of this food.32 The olive oil used in this study contains β-carotene at a concentration of 0.52 mg/L. We examined the efficacy of olive oil as an extracting agent under xylose culture conditions by adding olive oil into the culture at a volumetric ratio of 1:1. The fermentation profiles and product formation patterns by the engineered strain with olive oil in situ extraction were similar to when we used dodecane (Fig. 6, Fig. 5). As a result, the SR8A strain produced 31.18 mg/L β-carotene, 8


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and 57.61 mg/L vitamin A under this condition (Fig. 6), demonstrating the feasibility of olive oil as an alternative to dodecane for effective in situ extraction of vitamin A. At the end of fermentation, the oil layer contained 2.15 mg/L of β-carotene, 29.82 mg/L of retinal, and 19.07 mg/L of retinol (Table S2, Fig. S7), indicating 85% of the produced vitamin A was enriched in the olive oil. Thanks to the GRAS status of S. cerevisiae, the vitamin A enriched olive oil (Fig. S7b) can be potentially exploited as fortified food product, similar to the concept of ‘Golden Rice’33, to prevent vitamin A deficiency. By exploring different extractive agents with specific properties, our engineered yeast S. cerevisiae might serve as a versatile platform for producing vitamin A enriched products for various fields. For example, mineral oil can be used for in situ extraction of vitamin A and the resulting vitamin A enriched mineral oil can be applied as a cosmetic ingredient for skin improvement without further purifications steps. The main technical barrier when including vitamin A in formulations is a poor solubility in aqueous solution and susceptibility to degradation.34 Scientists reported microencapsulation of vitamin A using various encapsulating agents, such as liposomes, cochleates and cyclodextrins, to improve dispersibility and stability.35 As such, we are examining the feasibility of β-cyclodextrin cavity for in situ extraction and encapsulation of vitamin A from an engineered yeast culture, which might be interesting for food and pharmaceutical industries to economically produce high-stability and bioavailable vitamin A as a food supplement or a drug. Fed-batch fermentation for the production of vitamin A by engineered yeast. As cell concentrations in the bioreactor are important for maximizing titers of intracellular products, we conducted a fed-batch fermentation to produce vitamin A by the SR8A strain. A high cell density culture of an engineered yeast can be easily achieved when xylose is used as a carbon source.29 As 9



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there is no Crabtree effect from xylose, xylose can be fed intermittently to achieve a high celldensity culture. As such, we used xylose for a fed-batch fermentation to produce vitamin A at high titers. The SR8A strain was cultured in a 3-L bioreactor containing 1 L of a defined medium with intermittent xylose feedings (Fig. S8a). Noticeably, large amounts of glycerol (31.61 g/L) accumulated at the end of fermentation, indicating yeast cells might suffer from NADH/NAD+ redox imbalance. A previous study also reported high-level glycerol accumulation in a xylose fedbatch fermentation.29 The redox imbalance in xylose metabolism was known to be caused by the different cofactor dependences of XR (xylose reductase) and XDH (xylitol dehydrogenase) in xylose assimilation pathway.36 The final isoprenoid products in the cells consisted of 319.60 mg/L of β-carotene, and 953.08 mg/L of vitamin A (581.38 mg/L retinal and 371.70 mg/L retinol) (Fig. S9b). Interestingly, 149.82 mg/L of vitamin A (18.65% of the produced vitamin A) was detected in the medium supernatant which formed double layers of a cloudy water phase and a lipophilic layer with yellow solids after centrifugation of the fermentation broth. In contrast, negligible amounts of β-carotene detected in the medium (Table S2, Fig. S8a). The partial secretion of vitamin A implies that the accumulation of the heterologous hydrophobic products (β-carotene and vitamin A) summing up to 2.34% of the dry cell weight might have reached the ceiling of intracellular storage capacity in yeast. To further improve vitamin A production through alleviating the storage limitation problem, in situ extraction with dodecane was also implemented into a fed-batch fermentation with xylose. The two-phase culture system with 500 ml of a minimal medium and 500 ml of dodecane was homogenized by agitation into an emulsion. During a xylose fed-batch fermentation, well-mixed emulsions of cells, culture medium, and dodecane were sampled and separated by centrifugation for analyzing cell density, β-carotene, vitamin A and byproducts. The 10


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titers of each product were calculated based on the volume of an aqueous phase (500 ml). The amount of glycerol was drastically reduced (16.32 g/L vs. 31.61 g/L) as compared to the onephase fed-batch fermentation (Fig. 7a). The final cell concentration (79.36 g cell/L) was 46% higher than that of a fed-batch culture without dodecane (54.40 g cell/L) (Fig. 7a). The two-phase fed-batch culture with dodecane retained high concentrations of dissolved oxygen (DO) because oxygen dissolved better in dodecane.37 This might be a reason for the reduced glycerol production and increased cell concentration because surplus NADH can be re-oxidized through respiratory metabolism pathways in the presence of sufficient oxygen, and thus reducing aerobic glycerol formation and promoting cell growth.38 The two-phase xylose fed-batch fermentation produced 3,432 mg/L of vitamin A (2,094 mg/L of retinal and 1,338 mg/L of retinol) (Fig. 7b). The titer of vitamin A was among the highest of high-value hydrophobic compounds produced by engineered yeast.39 The vitamin A titer of the two-phase fed-batch fermentation was 3.6 folds higher than one-phase fed-batch fermentation and the ratio of vitamin A of total products (β-carotene + vitamin A) increased from 75% to 92% (Fig. 8, Fig. 7). As the conversion of β-carotene to retinal is an oxygen-consuming reaction (Fig. 1), the elevated DO by dodecane might be another contributing factor of the enhanced vitamin A proportion in addition to the alleviation of the storage limitation in two phase system. The dodecane layer with intense orange color consisted of 2,864 mg/L vitamin A and 11.33 mg/L β-carotene which comprised 83% of total vitamin A and 4% of total β-carotene, respectively (Fig. S8b, Table S2). The 500 ml dodecane layer from this 160-hour fed-batch fermentation contains an overall vitamin A activity of 4.8 million IU, which can supply the nutritional need of an adult man for over 4 years based on the Recommended Dietary Allowance (RDA) of 3000 IU/day40. 11



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Through fed-batch bioreactor operations, the advantages of xylose utilization—providing ample supply of isoprenoid precursors—and two-phase in situ extraction strategy—effectively overcoming the limited intracellular storage—were combined for maximizing vitamin A production in engineered S. cerevisiae. The high-level vitamin A production by the GRAS S. cerevisiae from xylose—the second most abundant sugar in plant cell wall hydrolysate—makes our strategy a cost-effective alternative to current chemical synthesis for the production of vitamin A as food supplements, cosmetic ingredients and drugs. Furthermore, the two-phase system for vitamin A production can potentially simplify downstream purification steps and eliminate the time and cost for large-scale operations. The energy-demanding extraction process might not be necessary because 92% of the produced vitamin A existed in dodecane layer.

CONCLUSIONS In this study, we constructed an engineered S. cerevisiae strain capable of producing vitamin A from xylose—the second most abundant and non-edible sugar in nature. By xylose utilization and two-phase in situ extraction, the limiting factors, such as insufficient supply of isoprenoid precursors and restrained intracellular storage capacity, for the production of vitamin A were simply and effectively addressed without sophisticated genetic perturbations and enzyme engineering. The engineered strain produced 3,432 mg/L of vitamin A as a mixture of retinal (2,094 mg/L) and retinol (1,338 mg/L) in a two-phase xylose fed-batch fermentation with dodecane in situ extraction. Olive oil was also assessed to be an effective extractive agent in a two-phase xylose culture, and the acquired olive oil containing 166 IU/ml of vitamin A after fermentation can be potentially used as a vitamin A enriched food product. These findings confirm the feasibility of producing lipophilic chemicals, especially isoprenoids, in S. cerevisiae. 12


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Our engineered S. cerevisiae strain and strategies described here form the basis of a sustainable bioprocess for industrial production of vitamin A and vitamin A-enriched bioproducts.

MATERIALS AND METHODS

Plasmids and strain construction. The strains, plasmids, PCR primers and gBlock used in this study are listed in Table S1. Standard molecular biology procedures were followed as described previously.41 The integration plasmid YIplac211YB/I/E* was kindly provided by Verwaal et al.,42 which carries carotenoid biosynthetic genes from Xanthophyllomyces dendrorhous including crtYB (encodes a bifunctional phytoene synthase and lycopene cyclase), crtI (phytoene desaturase) and crtE (GGPP synthase). The genes crtYB, crtI and crtE are regulated a strong constitutive TDH3 promoter and CYC1 terminator. URA3 was disrupted as an auxotrophic marker in the SR8 strain,28 using CRISPR-Cas9 technology.43 Donor DNA was amplified using primers URA3donor-U and URA3donor-D. The plasmid CAS9-NAT (Addgene#64329) was transformed into the SR8 strain followed by the guide RNA plasmid gRNA-ura-HYB and donor DNA transformation. Cells were selected on YPD plate supplemented with 120 µg/mL nourseothricin and 300 µg /mL Hygromycin B (YPDNH). The positive colonies with URA3 deletion were confirmed by sequencing using primers URA3-Seq-U and URA3-Seq-D and designated as the SR8U- strain. The plasmid YIplac211YB/I/E* was linearized by StuI and transformed into the SR8U- strain. Cells were plated onto a SC-ura plate and grew for color development. The most red colony was picked as the SR8B strain. For constructing a vitamin A producing strain, the Blh protein from the uncultured marine bacterium 66A039 was used as BCMO to catalyze the conversion of β-carotene to retinal (Fig. 1). 13



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A strong constitutive CCW12 promoter was chosen to control the expression of Blh gene as it showed nearly identical transcription levels (P = 0.61) in the middle of glucose and xylose utilization as TDH3 promoter (P = 0.39, Student’s t-test) exhibited (Fig. S1). The amino acid sequence of Blh gene obtained from NCBI GeneBank (accession number AAY68319) was codon optimized and synthesized using the service of IDT (Integrated DNA Technologies, USA). The acquired gBlock coBlh was amplified using primers Blh-Amp-U and Blh-Amp-D and cloned into the XhoI and BamHI sites of the pRS426-pCCW12 plasmid, resulting in the plasmid pRS426-Blh. The Blh expression cassette flanked by a strong constitutive CCW12 promoter and CYC1 terminator was amplified from the plasmid pRS426-Blh using the primers CS8donor-U and CS8donor-U to be used as a donor DNA. The CS8 loci44 was selected as an intergenic region for integration of the Blh expression cassette via Cas9-based genome editing. The plasmid CAS9NAT was first transformed into the SR8B strain. The plasmid pRS42H-CS8 coding for a guide RNA which targets CS8 loci was then co-transformed with the donor DNA. Cells were selected on YPDNH plate. Positive colonies were confirmed by diagnostic PCR and designated as the SR8A strain. Batch and fed-batch culture for vitamin A production. To compare vitamin A production profiles by engineered yeast on glucose and xylose, engineered yeast cells were pre-cultured for 2-3 days in 5 mL of the modified Verduyn medium45 with 20 g/L glucose, or 20 g/L xylose as a carbon source. The Verduyn medium contained per liter: (NH4)2SO4, 15 g; KH2PO4, 8 g; MgSO4, 3 g; trace element solution, 10 mL; vitamin solution, 12 mL. The trace element solution contained per liter: EDTA, 15 g; ZnSO4, 5.75 g; MnCl2, 0.32 g; CuSO4, 0.50 g; CoCl2, 0.47 g; Na2MoO4, 0.48 g; CaCl2, 2.90 g; FeSO4, 2.80 g. The vitamin solution contained per liter: biotin, 0.05 g; calcium pantothenate, 1 g; nicotinic acid, 1 g; myoinositol, 25 g; thiamine hydrochloride, 1 g; 14


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pyridoxol hydrochloride, 1 g; p-aminobenzoic acid, 0.20 g. Pre-cultured cells grown on glucose or xylose were inoculated into glucose or xylose main fermentations, respectively, at an initial optical density (OD600nm) of around 1.0. Main fermentations were conducted with 50 mL Verduyn medium containing 40g/L glucose, or xylose in 250 mL baffled flasks at 30 ℃ and 300 rpm. Fermentation media were buffered with 50 mM potassium hydrogen phthalate at pH of 5.5. The two-phase batch cultures were performed at the same conditions mentioned above in 25 mL Verduyn medium with additional 25 mL dodecane (Sigma-Aldrich, St. Louis, US), or olive oil (Mild Olive Oil, Meijer, Urbana, US) as an extractive solvent. For a xylose fed-batch fermentation, the engineered yeast was pre-cultured for 48 hours in 200 mL Verduyn medium containing 40 g/L xylose at 30 ℃ and 300 rpm. Cells were harvested and washed with sterile deionized water before inoculation. The fed-batch cultures were performed in a 3-liter bioreactor (New Brunswick Scientific-Eppendorf, Enfield, CT) containing 1 L Verduyn medium with an initial xylose concentration of 80 g/L. Additional xylose was added to the bioreactor up to 80 ± 10 g/L each time upon its depletion. The pH was maintained at 5.5 automatically by pumping in 4 M NaOH, or 4 M HCl. The temperature was held constant at 30 ℃ with agitation adjusted to maintain aerobic conditions according to the cell densities. Antifoam 204 (Sigma-Aldrich, St. Louis, US) was added to prevent the formation of foam. In order to guarantee sufficient supply of oxygen, the ventilation quantity was adjusted as the cell density increased and turned to the maximum (5 Lpm) when OD600 reached 100. In the xylose fed-batch fermentation with dodecane in situ extraction, a mixture of 500 mL dodecane and 500 mL Verduyn medium was used as a two-phase culture system. The xylose feeding pattern and fermentation conditions were kept the same as one-phase fed-batch culture.

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Analytical methods. Cell growth was monitored by measuring OD600 using a spectrophotometer (BioMate 5; Thermo Fisher Scientific, Waltham, USA). Dry cell weight (DCW) was determined from a plot of optical density and dry cell weight. To calibrate the plot, engineered yeast was grown in Verduyn medium, harvested by centrifugation at 10,000 rpm, and washed two times with distilled water. Washed cell pellets were resuspended in distilled water to various optical densities and filtrated via dried cellulose acetate membrane filters. After cell filtration, membrane filters were dried to constant weight in an 80°C convection oven and then weighed. The conversion factor to obtain dried cell weight from OD600 was 4.1. The extracellular concentrations of glucose, xylose, xylitol, glycerol, acetate and ethanol were detected by high-performance liquid chromatography (HPLC, Agilent 1200 Series, Agilent Technologies, Wilmington, US) equipped with a refractive index detector and the Rezex ROA-Organic Acid H+ (8%) column (Phenomenex Inc, Torrance, CA). The diluted culture supernatants were loaded and analyzed at 50 ℃ with 0.005 M H2SO4 as the mobile phase. The flow rate was set at 0.6 mL/min. To identify vitamin A produced in the engineered strain, cell pellets were crushed by a beat beater and hydrophobic components in the cell extract were extracted using acetone as previously described.46 The resulting extracts of the SR8B and SR8A strains were analyzed using gas chromatography/mass spectrometry (GC/MS) after chemical derivatization by methoxyamination and trimethylsilylation. Specifically, acetone extracts were dried in a centrifugal vacuum concentrator. The dried samples were then methoxyaminated by mixing with 10 μL of 40 mg/mL methoxyamine chloride in pyridine (Sigma-Aldrich, St. Louis, US) and incubating for 90 min at 30 ℃ and 300 rpm. For trimethylsilylation, 45 μL of N-methyl-N(trimethylsilyl)-trifluoroacetamide (Sigma-Aldrich, St. Louis, US) was added and inocubated for 30 min at 37 ℃ and 300 rpm. The derivatized samples were analyzed using an Agilent 7890A 16


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GC/5975C MSD system (Agilent Technologies, Wilmington, US) equipped with a RTX-5Sil MS capillary column (30 m × 0.25 mm, 0.25 μm film thickness; Restek, Bellefonte, US) and an additional 10 m guard column. The derivatized sample (1 μL) was injected into GC in a splitless mode. The oven temperature was initially set at 150 ℃ for 1 min and then ramped to 330 ℃ at 20 ℃/min, where it was held for 5 min. Electron ionization was performed at 70 eV and the temperatures of the ion source and transfer line were 230 ℃ and 280 ℃, respectively. The mass spectra were recorded in a scan range of 85-700 m/z. To measure the concentration of β-carotene and vitamin A produced by the engineered strain, cells, culture media, dodecane layers and olive oil layers were harvested by centrifugation and then analyzed separately. The cell extracts, culture media, dodecane layers and olive oil layers were analyzed with spectrophotometer (BioMate 5; Thermo Fisher Scientific, Waltham, US) at 453 nm and HPLC (LC-20A, Shimadzu, Kyoto, Japan) at 352 nm for the detection of β-carotene and vitamin A, respectively. Specifically, for β-carotene measurement, the optical density (OD453nm) values of the β-carotene containing cell extracts, culture media, dodecane layers and olive oil layers were read in spectrophotometer using acetone, Verduyn medium, dodecane and olive oil as blank solution, respectively. β-carotene concentration was calculated by the equation: concentration (β-carotene) = 6.7824*OD453nm -1.1107, which was obtained by plotting OD453 of serially diluted of β-carotene standard (Cat. No. C4582, Sigma, USA) solutions with the corresponding concentrations. The method of β-carotene measurement by spectrophotometer was validated using HPLC (Fig. S2). For the vitamin A analysis, the vitamin A containing cell extracts, culture media, dodecane layers and olive oil layers were diluted using methanol and then injected into HPLC. Vitamin A was separated using a C18 column (Phenomenex Kinetex 5 μL C18, Phenomenex Inc, Torrance, CA) and detected using a UV detector (Shimadzu SPD-20A, 17



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Shimadzu, Kyoto, Japan) at 40 ℃. The mobile phase was 95% methanol and 5% acetonitrile at a flow rate of 1 ml/min. The standard curves of retinal (Cat. No. R2500, Sigma-Aldrich, St. Louis, US) and retinol (Cat. No. R7632, Sigma-Aldrich, St. Louis, US) were prepared for the quantification of vitamin A (Fig. S3). For analysis of ergosterol, cell pellets were saponified and extracted using n-heptane for HPLC measurement as described in previous studies.47 Briefly, cells from 2 mL fermentation broth were mixed with 0.6 mL extraction solution (50% KOH: C2H5OH =2: 3) and incubated in 85 °C water bath for 2 hours. The saponified mixtures were then extracted with 0.6 mL n-heptane. The n-heptane extracts were dried and redissolved in 0.5 mL of acetonitrile for the analysis using Shimadzu HPLC system equipped with UV detector (Shimadzu SPD-20A) and C18 column (Phenomenex Kinetex 5 μL C18). The mobile phase was 100% acetonitrile at a flow rate of 2 mL/min. Ergosterol was detected by UV absorbance at 280 nm. The standard curves of retinal (Cat. No. 45480, Sigma-Aldrich, St. Louis, US) were prepared for the quantification of vitamin A (Fig. S4).

ASSOCIATED CONTENT Supporting Information Table S1. Strains, plasmids, primers and gBlock used in this study. Table S2. Distribution of products in different phases of culture. Figure S1. Transcription levels of TDH3, CCW12, and ACT1 (reference) on glucose and xylose. Figure S2. Validation of β-carotene concentrations by HPLC. Figure S3. Standard curve for calculating retinal and retinol concentrations. Figure S4. Standard curve for calculating ergosterol concentration. Figure S5. Pictures of SR8A batch cultures with dodecane in situ extraction. Figure S6. The final compositions of the products after 18


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batch fermentations of the SR8A strain. Figure S7. Pictures of xylose batch cultures with olive oil in situ extraction. Figure S8. Pictures of xylose fed-batch bioreactor cultures. Figure S9. Xylose feb-batch fermentation of the SR8A strain without dodecane in situ extraction. (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Present Addresses: 1206 West Gregory Drive, Urbana, IL 61801 ORCID: Yong-Su Jin: 0000-0002-4464-9536 Liang Sun: 0000-0001-8826-7276 Suryang Kwak: 0000-0002-5202-8326 Author Contributions Y.-S.J., L.S. developed the idea of this work. Y.-S.J., L.S. designed the experiments. L.S., S.K. performed the experiments. L.S., S.K. and Y.-S.J. wrote the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was funded by the DOE Center for Advanced Bioenergy and Bioproducts Innovation (U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under Award Number DE-SC0018420). Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the author(s) and do 19



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not necessarily reflect the views of the U.S. Department of Energy. L. S. would like to thank the China Scholarship Council (CSC) for financial support (File No. 201606350094). The authors thank Christine Atkinson for her diligent proofreading of this paper.

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Figure captions

Fig. 1. Biosynthetic pathways of vitamin A from glucose and xylose in engineered S. cerevisiae. A heterogolous xylose assimilation pathway contains xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK). A heterologous vitamin A biosynthetic pathway consists of GGPP synthase (CrtE), phytoene desturase (CrtI), bifunctional phytoene synthase, lycopene cyclase (CrtYB), and an endogenous enzyme exhibiting a promiscuous activity of retinol dehydrogenase. Xylose assimilation pathway is connected with lower glycolytic pathway via the pentose phosphate pathway (PPP). Pyruvate produced from glucose and xylose is converted into cytosolic acetyl-CoA. Yeast synthesizes farnesyl pyrophosphate (FPP) from cytosolic acetyl-CoA via the mevalonate pathway (MVA) consisting of Erg10, Erg13, Hmgr, Erg12, Erg8, Erg19, IDI1 and Erg20. FPP is the common precursor for the biosynthesis of ergosterol and heterogolous vitamin A. G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; GAP, glyceraldegyde-3-phosphate; X5P, xyllulose-5-phosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate.

Fig. 2. Identification of the vitamin A produced in the forms of retinal and retinol from batch fermentation in the engineered yeast Saccharomyces cerevisiae SR8A using GC/MS. (a) The overlaid GC/MS chromatograms of the engineered yeast (SR8A) and control (SR8B) strains. (b) MS spectrum of retinal produced by the SR8A strain which was identical to that of retinal standard. (c) MS spectra of retinol produced by strain SR8A which was identical to that of retinol standard. 28


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Fig. 3. Batch fermentation profiles of the engineered S. cerevisiae SR8A on glucose (a) and xylose (b), and the corresponding β-carotene and vitamin A production profiles from glucose (c) and xylose (d). Experiments were performed in tripilicate, and error bars represent standard deviations.

Fig. 4. Ergosterol production by the engineered SR8A strain from glucose and xylose in batch fermentaion without dodecane (a) and with dodecane (b) in situ extraction. Cells were harvested at the end of fermentation for ergosterol extractiona and quantification. Experiments were performed in tripilicate, and error bars represent standard deviations.

Fig. 5. Batch fermentation of the engineered S. cerevisiae SR8A with dodecane in situ extraction on glucose (a) and xylose (b), and the corresponding β-carotene and vitamin A production profiles from glucose (c) and xylose (d). Experiments were performed in tripilicate, and error bars represent standard deviations.

Fig. 6. Xylose batch fermentation profiles of the engineered S. cerevisiae SR8A with olive oil in situ extraction (a), and the corresponding β-carotene and vitamin A production profiles (b). Experiments were performed in tripilicate, and error bars represent standard deviations.

Fig. 7. Xylose feb-batch fermentation of the engineered S. cerevisiae SR8A with dodecane in situ extraction. (a) and (b) are fermentaion profiles and the production profiles of β-carotene, retinal and retinol, respectively. The height of each aera at different time point represents the proportion of each product to the total product titer along culture time. 29



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Fig. 8. Summary of vitamin A titers and specific contents by engineered engineered S. cerevisiae SR8A in different culture conditions.

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Fig. 1

Fig. 1. Biosynthetic pathways of vitamin A from glucose and xylose in engineered S. cerevisiae. A heterogolous xylose assimilation pathway contains xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK). A heterologous vitamin A biosynthetic pathway consists of GGPP synthase (CrtE), phytoene desturase (CrtI), bifunctional phytoene synthase, lycopene cyclase (CrtYB), and an endogenous enzyme exhibiting a promiscuous activity of retinol dehydrogenase. Xylose assimilation pathway is connected with lower glycolytic pathway via the pentose phosphate pathway (PPP). Pyruvate produced from glucose and xylose is converted into cytosolic acetyl-CoA. Yeast synthesizes farnesyl pyrophosphate (FPP) from cytosolic acetyl-CoA via the mevalonate pathway (MVA) consisting of Erg10, Erg13, Hmgr, Erg12, Erg8, Erg19, IDI1 and Erg20. FPP is the common precursor for the biosynthesis of ergosterol and heterogolous vitamin A. G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; GAP, glyceraldegyde-3-phosphate; X5P, xyllulose-5-phosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate.

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Fig. 2

(b)

(a)

(c)

Fig. 2. Identification of the vitamin A produced in the forms of retinal and retinol from batch fermentation in the engineered yeast Saccharomyces cerevisiae SR8A using GC/MS. (a) The overlaid GC/MS chromatograms of the engineered yeast (SR8A) and control (SR8B) strains. (b) MS spectrum of retinal produced by the SR8A strain which was identical to that of retinal standard. (c) MS spectra of retinol produced by strain SR8A which was identical to that of retinol standard.

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Fig. 3

(a)

(b)

(c)

(d)

Fig. 3. Batch fermentation profiles of the engineered S. cerevisiae SR8A on glucose (a) and xylose (b), and the corresponding β-carotene and vitamin A production profiles from glucose (c) and xylose (d). Experiments were performed in tripilicate, and error bars represent standard deviations.

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Fig. 4

(a)

(b)

Fig. 4. Ergosterol production by the engineered SR8A strain from glucose and xylose in batch fermentaion without dodecane (a) and with dodecane (b) in situ extraction. Cells were harvested at the end of fermentation for ergosterol extractiona and quantification. Experiments were performed in tripilicate, and error bars represent standard deviations.

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Fig. 5

(a)

(b)

(c)

(d)

Fig. 5. Batch fermentation of the engineered S. cerevisiae SR8A with dodecane in situ extraction on glucose (a) and xylose (b), and the corresponding β-carotene and vitamin A production profiles from glucose (c) and xylose (d). Experiments were performed in tripilicate, and error bars represent standard deviations.

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Fig. 6

(a)

(b)

Fig. 6. Xylose batch fermentation profiles of the engineered S. cerevisiae SR8A with olive oil in situ extraction (a), and the corresponding β-carotene and vitamin A production profiles (b). Experiments were performed in tripilicate, and error bars represent standard deviations.

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Fig. 7

(a)

(b)

Fig. 7. Xylose feb-batch fermentation of the engineered S. cerevisiae SR8A with dodecane in situ extraction. (a) and (b) are fermentaion profiles and the production profiles of β-carotene, retinal and retinol, respectively. The height of each aera at different time point represents the proportion of each product to the total product titer along culture time.

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Fig. 8

Fig. 8. Summary of vitamin A titers and specific contents by engineered engineered S. cerevisiae SR8A in different culture conditions.

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Vitamin A production by engineered Saccharomyces cerevisiae from xylose via two-phase in situ extraction

Liang Sun, 1,2 Suryang Kwak1,2, Yong-Su Jin1,2*

For Abstract Graphic Use Only

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Vitamin A production by engineered Saccharomyces cerevisiae from

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