Metabolic Engineering 28 (2015) 8–18

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Sequential control of biosynthetic pathways for balanced utilization of metabolic intermediates in Saccharomyces cerevisiae Wenping Xie a, Lidan Ye a,b, Xiaomei Lv a, Haoming Xu a, Hongwei Yu a,n a b

Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang University, Hangzhou 310027, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 7 May 2014 Received in revised form 24 October 2014 Accepted 20 November 2014 Available online 2 December 2014

Balanced utilization of metabolic intermediates and controllable expression of genes in biosynthetic pathways are key issues for the effective production of value-added chemicals in microbes. An inducer/ repressor-free sequential control strategy regulated by glucose concentration in the growth environment was proposed to address these issues, and its efficiency was validated using heterologous betacarotenoid biosynthesis in Saccharomyces cerevisiae as an example. Through sequential control of the downstream, upstream, and competitive pathways of farnesyl diphosphate (FPP), the crucial metabolic node in the biosynthesis of terpenoids, in a predetermined order, a carotenoid production of 1156 mg/L (20.79 mg/g DCW) was achieved by high-cell density fermentation. Quantitative PCR analysis of the regulated genes demonstrated that the transcription patterns were controlled in a sequential manner as expected. The inducer/repressor-free nature of this strategy offers a both practical and economically efficient approach to improved biosynthetic production of value-added chemicals. & 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Keywords: Sequential control Biosynthetic pathway Metabolic engineering Carotenoids

1. Introduction Carotenoids are a diverse class of tetraterpenes widely used as food colorants, feed additives, nutrient supplements, cosmetic and pharmaceutical (Negi and Namitha, 2010). Generally, studies on microbial production of natural carotenoids are focused on optimizing the fermentation conditions of carotenogenic microorganisms such as Blaskeslea trispora, Xanthophyllomyces dendrorhous and Dunaliella salina (Nanou et al., 2012; Raja et al., 2007; Schmidt et al., 2011). Recently, Saccharomyces cerevisiae has become a frequently used platform for heterologous biosynthesis of terpenoids including carotenoids due to its genetic tractability, biosafety and robustness in fermentation (Ajikumar et al., 2008; Reyes et al., 2014; Verwaal et al., 2007). All sesquiterpenes, diterpenes, triterpenes and tetraterpenes in S. cerevisiae share a common precursor farnesyl diphosphate (FPP) (Fig. 1A). Therefore, how to redirect FPP flux efficiently toward a target pathway is crucial for improving the production of terpenoids. Generally, three types of strategies have been employed for this purpose. In earlier studies, the sole expression of the downstream genes of FPP led to the formation of only low amounts of target products (type I in Fig. 1B) (Dejong et al., 2006; Yamano et al., 1994). After 3-hydroxy-3-methylglutaryl-coenzyme-A reductase n

Corresponding author. Fax: þ86 571 8795 1873. E-mail address: [email protected] (H. Yu).

(HMG-CoA) was identified as a major rate-limiting enzyme in the mevalonate (MVA) pathway (Polakowski et al., 1998), its catalytic domain (tHMG1) was overexpressed together with the downstream genes of FPP to enhance the flux of the MVA pathway to its products (type II in Fig. 1B) (Dai et al., 2013; Verwaal et al., 2007; Zhou et al., 2012). However, part of FPP was converted to squalene since the sterol pathway also shares FPP as precursor (Polakowski et al., 1998). Since deletion of squalene synthase gene (ERG9) is lethal, to divert the flux from the synthesis of squalene to the target terpenoids, a number of alternative strategies were introduced, including decreasing the activity of ERG9 via the addition of inhibitors such as zaragozic acid (Kuranda et al., 2010) or downregulating the expression of ERG9 by replacing the original promoter with a methionine repressible MET3 promoter (type III in Fig. 1B) (Asadollahi et al., 2008; Westfall et al., 2012). In most of above studies, the strategies of expressing genes under strong promoters or increasing gene copy number are used to improve the production of target terpenoids (Dai et al., 2013; Verwaal et al., 2007; Zhou et al., 2012). However, a strong and early overexpression of heterologous pathways, especially for products like carotenoids which can only be stored in the membrane system due to their high hydrophobicity, may exert metabolic stress on cells (Krivoruchko et al., 2011; Verwaal et al., 2010). In addition, although down-regulation of ERG9 could enhance the supply of FPP, early repression of ERG9 might also impair the cell growth (Asadollahi et al., 2008) and stimulate the

http://dx.doi.org/10.1016/j.ymben.2014.11.007 1096-7176/& 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

W. Xie et al. / Metabolic Engineering 28 (2015) 8–18

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Fig. 1. Strategies for terpenoid production in S. cerevisiae. (A) The terpenoid biosynthetic pathway. By setting FPP as a metabolic node, we divided the pathway into four parts. Part 1: the upstream of FPP, containing the MVA pathway; part 2: the sterol pathway, where FPP is accumulated and mostly converted through the squalene biosynthetic pathway; part 3: other branching metabolic pathways derived from FPP; part 4: heterologous pathways introduced for synthesis of target products, with the beta-carotene biosynthetic pathway presented as an example, CrtE: geranylgeranyl pyrophosphate synthase, CrtYB: phytoene synthase/lycopene cyclase, CrtI: phytoene desaturase. (B) Comparison of strategies used in previous studies (Type I, II, III in the table) and the sequential control strategy proposed in this study. The part 1, 2 and 4 here refer to the pathways in (A). N: Normal gene expression level, O: Overexpression, R: Repression, I: Induction. (C) A simplified expression pattern for genes of the desired and undesired pathways in a sequential control strategy. The dynamics of induction and repression is accompanied with/responds to the change of a specific factor in cell growth environment during the cultivation process.

expression of other competitive pathways such as farnesol synthesis (Kuranda et al., 2010; Song, 2006). Considering the potential metabolic stress of heterologous terpenoid biosynthetic pathways, the supply-consumption balance of FPP, and the distribution of metabolic flux between the desired and undesired pathways derived from FPP, we proposed a strategy to control the expression order of genes by a two-stage process for improving the production of terpenoids. In the first stage, genes in the MVA pathway and the squalene biosynthetic pathway expressed at a normal level or above to sustain cell growth. After the cells grow to a relatively high density, the second stage of metabolic control entails the overexpression of rate-limiting gene in the MVA pathway and the terpenoid biosynthetic pathway as well as downregulation of the competing ERG9 gene to ensure the efficient transformation of the metabolic flux from the MVA pathway to the target product. Herein, we refer to such a dynamic gene expression strategy as sequential control (Fig. 1B). This concept is similar to a mechanically controlled system where the on/off switch of individual units is controlled in a sequential manner. If this strategy is introduced and realized in metabolic engineering, then the metabolic pathways of the organisms would be finely controlled. Dynamic gene expression has been demonstrated to be a viable strategy for improving or controlling the production of target compounds in Escherichia coli and mammalian cells (Le et al., 2013; Meng et al., 2012; Zhang et al., 2012), but few studies have focused on the construction of dynamic biosynthetic pathways in S. cerevisiae. Generally, to construct a sequential control strategy, inducible promoters should be employed. Furthermore, to minimize the production cost, an inducer/inhibitor-free strategy should be designed with the ability to sense the change of a specific factor in the culture environment (such as a variation in nutrient availability) and to adjust the gene expression levels correspondingly (Holtz and Keasling, 2010; Keasling, 2012).

In the present paper, to improve the production of carotenoids, we established an inducer/inhibiter-free sequential control strategy in S. cerevisiae by combining a modified GAL regulation system (Xie et al., 2014) and a HXT1 promoter-controlled squalene synthetic pathway, which were repressed and induced by glucose, respectively. Using this strategy, the expression of genes in carotenoid pathway, MVA pathway, and competitive squalene pathway was sequentially controlled in response to the variation of glucose concentration in the culture environment (Fig. 1C). Transcriptional analysis was conducted to investigate the dynamic change of the key genes during cell cultivation. Moreover, a high-density fermentation process was developed in bioreactor to further demonstrate the applicability and effectiveness of this strategy.

2. Materials and methods 2.1. DNA manipulation and plasmid construction All plasmids used in this study are listed in Supplementary Table S1. Primers used for plasmids construction are listed in Supplementary Table S2. Cloning procedures were carried out in E. coli strain DH5α (Invitrogen). Standard DNA manipulation (Joseph and David, 2001) and Seamless Cloning and Assembly Kit (Invitrogen) were used for plasmid construction. DNA fragments of promoters, terminators, upstream and downstream regions of homologous arms were PCR amplified from the genomic DNA of S. cerevisiae BY4741. PrimeSTAR HS DNA polymerase kit (Takara, China), T4 DNA ligase (Fermentas) were used for DNA amplification and ligation. Plasmid DNA and PCR products were purified using Axygen plasmid miniprep kit and AxyPrep DNA gel extraction kit (Axygen Biosciences, Hangzhou, China). A p416XWP01-CrtE plasmid (Supplementary Fig. S1) was constructed as basic plasmid for compared the strength of

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Table 1 Yeast strains used in this study. Stain name

Parent strain

Genotypea

Reference

BY4741 YXWP-42 YXWP-53

S288C BY4741 BY4741

Boeke et al. (1998) This study Xie et al. (2014)

YXWP-60( þ) YXWP-65( þ) YXWP-67(þ) YXWP-74

YXWP-53 YXWP-53 YXWP-65( þ) BY4741

YXWP-82( þ) YXWP-83( þ) YXWP-101( þ)

YXWP-74 YXWP-74 YXWP-82( þ)

MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0 BY4741, Δlpp1::TPGK1-CrtYB-PHXT7-PTEF1-CrtI-TTPS1, HMG1::TADH1-CrtE-PGAL10-PGAL1-tHMG1-TCYC1, Δho::TADH1-CrtYB-PGAL10-PGAL1-CrtI-TCYC1, ΔYPRCtau3::TADH1-CrtYB-PGAL10-PGAL1-CrtI-TCYC1, Δgal80::URA3 ΔPERG9::PBTS1 ΔPERG9::PHXT1 Δlpp1, Δdpp1 Δdpp1::TADH1-CrtE-PGAL10-PGAL1-tHMG1-TCYC1, Δho::TADH1-CrtYB-PGAL10-PGAL1-CrtI-TCYC1, ΔYPRCtau3::TADH1-CrtYB-PGAL10-PGAL1-CrtI-TCYC1, Δgal80::URA3 Δlpp1::TPGK1-CrtYB-PHXT7-PTEF1-CrtI-TTPS1, ΔPERG9::PHXT1-PTEF1-CrtE-TCYC1 Δlpp1::TPGK1-CrtYB-PHXT7-PTEF1-CrtI-TTPS1, ΔPERG9::PBTS1-PTEF1-CrtE-TCYC1 his3::HIS3, met15::MET15, leu2::LEU2

a

This This This This

study study study study

This study This study This study

The description of genotype in the table is based on the parent strain.

promoters and a series of pMRI plasmids were constructed for pathway assembly. The detailed construction procedures of all plasmids construction are provided in Supplementary Table S3. 2.2. Strain construction Yeast strains used in this study are listed in Table 1. All strains in this work were constructed by the decentralized assembly strategy as described previously (Xie et al., 2014). Briefly, functional modules were integrated into genomic DNA in a stepwise manner. Geneticin (Sangon Biotech, China) was used for kanMX marker selection (Guldener et al., 1996). Synthetic complete selection media supplemented omitting corresponding amino acids were used for selection of nutritional markers (Gietz and Woods, 2006). The flowchart of yeast strains construction in this study are provided in Supplementary Fig. S2. To complement the auxotroph markers (MET15, HIS3 and LEU2) in a auxotrophic strain, the expression cassettes were amplified from corresponding strain or plasmid. MET15 was amplified from BY4742 genomic DNA (Brachmann et al., 1998), HIS3 was amplified from PUG23 plasmid, and the LEU2 marker was amplified from pESC-LEU plasmid (Stratagene). DNA segments were purified and co-transformed into auxotrophic strain, and the transformants were selected on SD-MET-LEU-HIS (synthetic defined medium with glucose as carbon source and methionine, leucine and histidine omitted) agar plates. 2.3. Analysis of carotenoid, squalene and farnesol The carotenoids and squalene were co-extracted using hot HClacetone (Xie et al., 2014). The concentration of total carotenoids was calculated using an extinction coefficient of 2500 (A1% ¼2500) at 450 nm (Scott, 2001). The analyses of β-carotene and lycopene were performed on a HPLC system (LC 20AT) equipped with a Kromasil C18 column (4.6 mm  150 mm) and the UV/VIS signals were detected at 450 nm. The mobile phase consisted of acetonitrile-methanol-isopropanol (50:30:20 v/v) with a flow rate of 1 mL/min at 40 1C. The same HPLC equipment was used for squalene analysis. UV/VIS signals were detected at 195 nm and acetonitrile was used as mobile phase with a flow rate of 1 mL/min at 40 1C (Lu et al., 2004). Because squalene was easily photooxidized during the process of extraction and storage (Mountfort et al., 2007), standard curves of squalene and oxidized squalene were established for calculating overall squalene production (Supplementary Fig. S3). Farnesol in the media and cells were extracted using methanol, hexane, and whole culture broth (1:1:1 v/v/v) (Song, 2009). The mixture was vortexed for 5 min and centrifuged. 500 μl of the top hexane layer was transferred into a new tube. Hexane in the

extracts was evaporated in a vacuum tank and 500 μl of methanol was added into the tube to redissolve farnesol. The same HPLC equipment as described above was used. Signals of farnesol was detected at 206 nm and a methanol/water mixture (90:10 v/v) was used as mobile phase with a flow rate of 1 mL/min at 40 1C. The standard compounds of β-carotene, lycopene, squalene and farnesol (Sigma, Aldrich, St. Louis, MO) were dissolved in acetone and used to prepare standard curves. The contents of carotenoids, squalene and farnesol were expressed as mg per g dry cell weight (mg/g DCW) and mg per liter (mg/l). 2.4. Cultivation in shaking flask The strains for promoter strength comparison were precultured in 5 ml SD-URA (synthetic defined medium with glucose as carbon source and uracil omitted) at 30 1C, 220 rpm for 24 h. Precultures were inoculated to an initial OD600 of 0.05 in 50 ml of SD-URA and SG-URA (synthetic defined medium with galactose as carbon source and uracil omitted) in 250 ml flasks and grown under the same condition for 72 h. The same method was applied for cultivating strains in YPD (1% yeast extract, 2% peptone, and 2% glucose), YPDG (1% yeast extract, 2% peptone, 1% glucose and 1% glycerol) or YPG (1% yeast extract, 2% peptone, and 2% galactose). 2.5. Fed-batch fermentation The media for fed-batch fermentation were composed of 25 g/L glucose, 15 g/L (NH4)2SO4, 8 g/L KH2PO4, 3 g/L MgSO4, 0.72 g/L ZnSO4.7H2O, 10 ml/L trace metal solution, and 12 ml/L vitamin solution (van Hoek et al., 2000). The seed culture was prepared by inoculating several colonies into a 500 ml flask containing 125 ml YPD and culturing at 30 1C and 230 rpm for 12 h to an OD600 of 8–12. Two flasks of seed cultures were transferred to a 5 L stirredtank bioreactor (Shanghai Huihetang Bioengineering equipment CO. Ltd, China) containing 2.5 L fermentation media. Fermentation was carried out at 30 1C with an agitation speed of 200 to 500 rpm and an airflow rate of 1 vvm to 2 vvm. pH was controlled at 5.0 by automatic addition of 5 M ammonia hydroxide. A two-stage fed-batch strategy was employed in this study. In the first stage, a feeding solution containing 500 g/L glucose, 9 g/L KH2PO4, 2.5 g/L MgSO4, 3.5 g/L K2SO4, 0.28 g/L Na2SO4, 10 ml/L trace metal solution and 12 ml/L vitamin solution was used to achieve fast cell growth. In the second stage, the feeding solution contained the same mineral salts and trace elements as described above, but was supplemented with 250 g/L glucose and 250 g/L glycerol as a mixed carbon source, along with 10 g/L yeast extract and 20 g/L peptone as a nitrogen source. This concentrated medium was used for inducing the accumulation of carotenoids.

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2.6. Quantitative PCR (qPCR)analysis of the transcription levels of integrated genes Total RNA was isolated from the harvested yeast cells by using the RNAiso Plus Kit (TakaRa, Dalian, China) according to the manufactures’ protocol. Genomic DNA contamination in RNA samples was eliminated using a DNase I treatment (Fermentas). The treated total RNA was reversely transcribed using a PrimeScript™ 1st Strand cDNA Synthesis Kit (TaKaRa Bio, Dalian, China) with a random 6-mer primer. qPCR was performed using SYBRs Premix Ex Taq™ (Tli RNaseH Plus) (TakaRa, Dalian, China) on a Mastercyclereprealplex (Eppendorf). The ACT1 gene was chosen as the internal control gene to normalize the different samples. The relative gene expression analysis was performed using the 2  △△CT method (Livak and Schmittgen, 2001).

3. Results 3.1. Construction of a colorimetric-based promoter strength comparison system Selection of appropriate promoters is of upmost importance for productivity optimization when designing heterologous pathways in an engineered strain (Alper et al., 2005; Sun et al., 2012). To reduce the metabolic flux from FPP to squalene by repressing the expression of the ERG9 gene, promoters with weaker strength than PERG9 should be identified. To develop a facile promoter strength comparison method, a beta-carotene biosynthetic pathway consisting of CrtYB and CrtI was constructed in the yeast strain YXWP42 as a colorimetric reporter (Fig. 2A). Four promoters related to terpenoid biosynthesis (PIDI1, PERG20, PERG9, PBTS1), three promoters related to glycometabolism (PGAL1, PHXT7, PHXT1), and three frequently-used promoters (PTEF1, PACT1, PCYC1) from previous studies (Partow et al., 2010; Sun et al., 2012), were compared in glucose (fermentable carbon source) or galactose (non-fermentable carbon source) containing synthetic media. According to the color phenotypes, an overall ranking of promoter strength was established on the SD-URA agar plate as follows: PHXT7  PTEF1 4PACT1 4PIDI1 4PERG9  PHXT1  PERG20 4PCYC1  PBTS1 4PGAL10, while on the SG-URA agar plate, the order was PGAL10  PHXT7  PTEF1 4PACT1 4PIDI1 4PERG9 4PERG20 4 PCYC1  PBTS1 4PHXT1. To quantitatively evaluate the strength of these promoters in glucose or galactose containing media, the total carotenoids in SD-URA or SG-URA shake-flask cultures were extracted and measured, and up to 20-fold variation was found among the ten promoters (Fig. 2C). In the ten promoters tested, PBTS1 was the weakest constitutive promoter in both SD-URA and SG-URA, while PHXT1 was induced in SD-URA but repressed in SG-URA. The absolute transcription activities of PHXT1, PERG9 and PBTS1 further confirmed the constitutive lower activity of PBTS1 and the dynamic change of PHXT1 at different glucose concentrations. Meanwhile, PERG9 was shown as a “static” moderate promoter that was not influenced by glucose concentration (Supplementary Fig. S4). Consequently, PBTS1 and PHXT1 could be used as candidates for down-regulation of ERG9. 3.2. Down-regulation of ERG9 by constitutive PBTS1 In our previous report, a yeast strain YXWP-53 was constructed for production of beta-carotene in which the key genes in the pathway including tHMG1 in the upstream MVA pathway were overexpressed using PGAL1 and PGAL10 promoters (Xie et al., 2014). The overexpression of tHMG1 was reported to result in the accumulation of squalene (Polakowski et al., 1998), which shares the precursor FPP with the carotenoid pathway. In this work, the

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squalene content in YXWP-53 was measured (22.43 mg/g DCW in YPD and 18.75 mg/g DCW in YPDG), and found to be significantly higher than the production of carotenoids (7.25 mg/g DCW in YPD and 9.64 mg/g DCW in YPDG) (Fig. 3A), suggesting that most of the metabolic flux from the enhanced MVA pathway was lost to the undesired sterol synthetic pathway. To divert the flux away from the sterol pathway, PBTS1, the weakest constitutive promoter screened from the ten promoters, was used to replace the original PERG9 for down-regulation of ERG9. As shown in Fig. 3A, comparing with YXWP-53, the squalene production of the newly constructed yeast strain YXWP-60( þ) was decreased by 11.5% and 46.5% in YPD and YPDG shake flasks, respectively. Unexpectedly, the carotenoid production of YXWP-60 (þ) was decreased by 65% in YPD but increased by 23.8% in YPDG. The decline of squalene production did not lead to corresponding improvement of the final carotenoid production. Especially, in YXWP-60( þ), the production of carotenoids was decreased to 2.54 mg/g DCW in YPD, and the accumulation of farnesol was detected (Fig. 3B and Supplementary Fig. S5). The formation of farnesol may be attributed to the unbalanced utilization of FPP, which triggered endogenic pathways for FPP hydrolysis (Kuranda et al., 2010; Song, 2006). In addition, although constitutive repression of ERG9 by PBTS1 improved the carotenoid production to 11.93 mg/g DCW in YPDG, the production of squalene was still as high as 10.03 mg/g DCW. Comparing the squalene production of YXWP-53 and YXWP-60 (þ) (Fig. 3A) and the promoter strength of PBTS1 and PERG9 (Fig. 2), the decrease of the squalene production was not linear with the promoter strength. One possible explanation is that ERG9 was able to transform most of the accumulated FPP into squalene due to its high catalytic activity (Asadollahi et al., 2010) even though its expression was down-regulated. Therefore, PBTS1 might not be weak enough for the down-regulation of squalene pathway, and it would thus be necessary to find another way to down regulate ERG9. 3.3. Construction of a sequential control system in yeast PHXT1 is a high-glucose-induced and low-glucose-repressed promoter, and weaker than PBTS1 in low-glucose condition (Supplementary Fig. S4). Meanwhile, in a GAL80 disrupted yeast GAL regulation system, the activity of GAL promoters was repressed by glucose and induced by non-fermentable carbon sources or glucose-limiting conditions (Xie et al., 2014). Thus, if the glucose-inducible PHXT1 is combined with the glucoserepressible modified GAL regulation system, a sequential control strategy as proposed in Fig. 1C could be achieved for the sequential expression of ERG9 gene and carotenoid pathway genes in response to the change of glucose concentration in culture media (Fig. 4). We expect this strategy could help to balance the utilization of FPP and improve the production of carotenoids. For this purpose, the yeast strain YXWP-65( þ) was constructed from the parent strain YXWP-53 by replacing the native PERG9 promoter with PHXT1 (Supplementary Fig. S2). 3.4. Sequential control of gene expression by glucose Theoretically, the initial glucose concentration in shake flask could decide the expression time of the GAL promoter-controlled carotenoid pathway and the PHXT1-controlled squalene pathway. Balancing the trade-off between the two pathways would be a key issue for the balanced utilization of FPP and improvement of carotenoid production. Therefore, we measured the squalene and carotenoids production of YXWP-65( þ) cultured in YPD, YPDG and YPG, regarded as high glucose, moderate glucose and glucosefree conditions, respectively.

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Fig. 2. The schematic of the promoter strength comparison system. (A) Geranylgeranyl diphosphate synthase (GGPPS) is a key rate-limiting enzyme in the carotenoid biosynthetic pathway. CrtE is a GGPPS gene from Xanthophyllomyces dendrorhous. Only very low level of carotenoids can be accumulated if only the CrtYB and CrtI genes were overexpressed in the yeast stain. Therefore, there is a positive correlation between the expression level of CrtE and the accumulation of carotenoids in cells. For this reason, the production of carotenoids can be used to indicate the strength of the promoter controlling the expression of CrtE. (B) Qualitative analysis of promoter strength on SG-URA and SD-URA agar plate. The different transformants were spotted on the same SG-URA and SD-URA agar plates. After 3 days of incubation at 30 1C, the plates was photographed. (C) Quantitative analysis of promoter strength was performed by measuring the carotenoid production (mg/g DCW) in yeast cells after 72 h growth in SG-URA and SD-URA shaking flasks. The activity of PERG9 was used as the reference for normalization, and error bars represent the standard deviations of two flasks.

Compared to YXWP-53 cultured in YPD, although the production of squalene was decreased 10.1 mg/g DCW in YXWP-65( þ), the carotenoid production was not increased as expected, contrarily, it was decreased by 1.54 mg/g DCW. Interestingly, when YXWP-65( þ) was cultured in YPDG, the production of squalene was decreased to 2.56 mg/g DCW, meanwhile, the carotenoid production was increased to 14.4 mg/g DCW, which was about 1.5 times higher than that of YXWP-53 (Fig. 3). However, YXWP-65 (þ ) cultured in YPG showed an obvious elongation of the lag phase and a lower biomass, only reaching an OD600 of 1.96 after

48 h of culture (Supplementary Fig. S6), indicating that early repression of ERG9 or induction of the carotenoid pathway can be toxic to cell growth. According to these results, the initial glucose concentration in YPDG appeared to be suitable for balancing the squalene and carotenoid biosynthetic pathways. In order to investigate whether the induction/repression of gene expression was in accordance with the theoretical model in Fig. 1C, the transcription levels of tHMG1, ERG9, CrtE, CrtYB and CrtI of YXWP-65( þ) cultured in YPDG were investigated by qPCR (Fig. 5A). Concurrently, the time

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Fig. 3. Analysis of products of YXWP-53, YXWP60( þ ) and YXWP-65(þ ). (A) Squalene and carotenoid production by different yeast strains in YPD and YPDG. (B) Farnesol production by different yeast strains in YPD and YPDG. The cultivations were carried out in 100 ml shake-flasks containing 50 ml medium for 72 h. Error bars represent the standard deviations of three flasks.

courses of the glucose concentration in medium, carotenoid accumulation, and biomass were also determined. The cells at 6 h with a glucose concentration of about 9 g/L were used as the control for qPCR because the cells were entering the log phase. With the depletion of glucose, the PGAL1/GAL10-controlled betacarotene pathway was up-regulated by 8–13 folds while the ERG9 gene under the control of PHXT1 was down-regulated by about 10 folds (Fig. 5B). This trend of transcription was essentially consistent with the model described in the introduction (Fig. 1C), except for a slight lag in the induction of GAL promoter compared to the repression of PHXT1. 3.5. Improving the sequential control strategy for further enhanced carotenoid production Although the production of carotenoids was increased by adjusting the induction/repression time of the carotenoid and squalene synthetic pathway, compared to YXWP-53, the decrease in squalene production (16.98 mg/g DCW) was still not linear with the increase in carotenoid production (4.76 mg/g DCW) in YXWP65( þ) (Fig. 3A). The slight lag between the induction of GAL promoter and the repression of PHXT1 during glucose depletion as indicated by the qPCR results (Fig. 5A) might lead to accumulation of FPP, and subsequently trigger other endogenous FPP-consuming pathways. To better balance the utilization of FPP, we introduced an extra copy of constitutive beta-carotene synthetic pathway in to the original sequential control system to partially switch on the downstream pathway of FPP at the beginning (Fig. 4).

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Fig. 4. Simplified glucose-dependent sequential control strategy. In a culture environment with high concentration of glucose, the GAL promoter controlled beta-carotene pathway was repressed via the protein kinase Snf1 and the transcription factor Mig1 (Xie et al., 2014), while the expression of ERG9 was activated via the membrane receptors Snf3 and Rgt2 and the transcription factor Rgt1 (Greatrix and van Vuuren, 2006). Under glucose-limiting conditions, the induction and repression of these two pathways would be reversed. A constitutive carotenoid biosynthetic pathway was used to make up the time lag between the overexpression of the MVA pathway and the downstream beta-carotene pathway, and to further balance the utilization of FPP. PTEF1 and truncated PHXT7 were two strong promoters identified in our study as well as in previous studies (Hauf et al., 2000; Sun et al., 2012).

Furthermore, in our previously constructed strain YXWP-53, the tHMG1 gene was integrated into the HMG1 site. Such an integration event could lead to possible intergenic deletions with a frequency of 1 out of 800 (Xie et al., 2014). To eliminate this issue, all plasmids were integrated into different genomic loci without intergenic repeated sequences. Based on the above considerations, two yeast strains, YXWP-74 and YXWP-82( þ ) were constructed to investigate whether an extra copy of constitutive beta-carotene synthetic pathway had a positive effect on carotenoid production. YXWP-74 shared a similar genotype with YXWP-53 except for the genomic locations of tHMG1 and CrtE. The production analysis demonstrated that the squalene and carotenoid contents in YXWP-74 were similar to those in YXWP-53 (Fig. 6A), suggesting that the altered integration loci did not affect the expression of CrtE and tHMG1. Compared with YXWP-65( þ ) in YPD, the squalene and farnesol contents were decreased while the carotenoid production was enhanced in YXWP-82( þ), which could be attributed to the additional copy of the constitutive beta-carotene biosynthetic pathway. Excitingly, when YXWP-82( þ) was cultured in YPDG, the carotenoid production reached to 19.71 mg/g DCW, approximately two-fold those of YXWP-53 and YXWP-74, while the squalene content was decreased to 2.25 mg/g DCW, and only trace amount of farnesol was detected (Fig. 6A and B). This carotenoid production is the highest ever reported among the shake flask cultures of carotenogenic S. cerevisiae strains (Reyes et al., 2014; Verwaal et al., 2007; Yamano et al., 1994; Yan et al., 2012). In addition, we constructed the YXWP-83( þ) in which the overexpression gene was the same as YXWP-82( þ), except that ERG9 was repressed by PBTS1. Although the best carotenoid

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Fig. 5. Analysis of YXWP-65(þ ) cultured in YPDG shaking flask (A) The dynamic gene transcription of the PGAL1/GAL10-controlled carotenoid pathway and the PHXT1controlled ERG9. The samples at time points of 6 h, 12 h, 30 h, 48 h and 60 h were investigated. (B) Time courses of glucose concentration, biomass, and carotenoid accumulation of YXWP-65( þ) in YPDG shaking flask. Data are average values of three replicas.

production of YXWP-83( þ) reached 15.52 mg/g DCW, it was still lower than that of YXWP-82( þ). This result further demonstrated that the dynamic control strategy was superior to overall low expression of ERG9 controlled by a static PBTS1. 3.6. High-density fermentation for carotenoid production YXWP-82( þ) was genetically modified from BY4741 (derived from S. cerevisiae S288C) (Brachmann et al., 1998). Though the URA3 marker was complemented during the construction of YXWP-82( þ), it remained auxotrophic to leucine, methionine and histidine. To meet the demand of large-scale fermentation, the MET15, LEU2 and HIS3 markers were integrated into YXWP-82 (þ ), generating a new strain YXWP-101( þ ). Based on the sequential control strategy described above, a two-stage fed-batch strategy was adopted (Fig. 7A). At the first stage, 500 ml of feeding solution containing 250 g glucose was fed to assist fast cell growth. By the completion of the first feeding stage, the OD600 reached 140 while the carotenoid content remained at a low level (2.5 mg/g DCW). Another 500 ml of inducing feeding solution which contained 125 g glucose and 125 g glycerol was subsequently supplied. At the second feeding stage, the production of carotenoid was vastly improved while the cell growth rate was decreased. In the end, 1156 mg/L of carotenoid was obtained, and the content in cells reached 20.79 mg/g

Fig. 6. Products analysis of YXWP-74 and YXWP-82( þ). (A) Squalene and carotenoid production in YPD and YPDG. (B) Farnesol production in YPD and YPDG. Error bars represent the standard deviations of three flasks.

DCW after 120 h of fermentation, which consisted of 32.56% of beta-carotene, 28.32% of lycopene and 39.06% of other carotenes (Fig. 7B and C). Our work is the first case reporting carotenoid production by engineered S. cerevisiae strains under high celldensity fermentation conditions.

4. Discussion Although the expression of genes could be controlled at transcriptional, post-transcriptional and translational levels, transcriptional control is the most frequently used strategy for construction and optimization of new pathways in metabolic engineering (Keasling, 2012; Seo et al., 2013). In this study, using a simple colorimetric screening method, carotenoid production was employed as a reporter for promoter strength in fermentable and non-fermentable carbon sources. Compared with the traditional GFP or LacZ-based promoter characterization methods (Partow et al., 2010; Sun et al., 2012), the strategy presented in this work is more facile because it is intuitive and no special equipment is required. Fine-tuning of gene expression has been demonstrated to be very important for pathway optimization (Alper et al., 2005; Kashiwagi et al., 2009), so the promoters with

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Fig. 7. High-density fermentation for carotenoid production. (A) Feeding profile and residual glucose in the two-stage feeding process. Feeding rates are displayed as milliliter feeding solution per fermentation volume per hour. The residual glucose in medium at time points of 3 h, 9 h, 14 h, 43 h and 60 h were investigated. (B) Time courses of cell growth and carotenoid production of YXWP-101( þ ) during the fed-batch fermentation. (C) The red liquid in the bottle is the fermentation broth at 120 h. The pie chart represents the composition of the carotenoid fermentation product.

a strength range of about 20 times difference characterized in this work will provide guidance for promoter selection when multiple promoters of different strengths are needed for pathway assembly in future studies. To develop a highly efficient carotenogenic yeast strain, two problems have to be solved. First, carotenoids can only be stored in the membrane system due to its high hydrophobicity, while accumulation of too many carotenoids has been found toxic to

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cells (Klein-Marcuschamer et al., 2007; Verwaal et al., 2010). Consequently, separating the cell growth stage from the product accumulation stage would be helpful for enhancing carotenoid production. Second, finding a suitable ERG9 down-regulation strategy to minimize the squalene production while not impairing cell growth is a key issue for further improving carotenoid production in the recombinant yeast. To address these issues, we employed the sequential control strategy which combined the modified GAL regulation system-controlled beta-carotene biosynthetic pathway and the PHXT1-controlled squalene synthetic pathway. There were several advantages of this strategy: (i) the tradeoff of the two glucose-responsive pathways guaranteed the fast cell growth at an early stage followed by repression of the squalene biosynthesis and induction of the carotenoid pathway, respectively; (ii) more importantly, this strategy was inducer and repressor-free, so all the induction and repression of pathways were switched automatically in response to the glucose concentration in media. Several previous studies in E. coli shown dynamic control to be optimal in controlling metabolic flux, and that the bioprocess productivity could be enhanced by separating the stages of cell growth and product accumulation (Anesiadis et al., 2008; Gadkar et al., 2005; Solomon et al., 2012). Keasling et al. also proposed that creating synthetic pathways that could flexibly adapt to the changing environments would be the next frontier in metabolic engineering (Holtz and Keasling, 2010; Keasling, 2012). The sequential control strategy in our work could be regarded as a practical implementation of the ideas mentioned above. The constitutive weak promoter PBTS1 was also tested to for ERG9 down-regulation, but was found to be not weak enough to repress the synthesis of squalene (Fig. 3).Turning PHXT1 into a relevant “static” state in a glucose-free medium for further repression of ERG9 led to an obvious elongation of the lag phase and a lower biomass (Supplementary Fig. S6), which was similar to the ERG9 repression by a MET3 promoter (Asadollahi et al., 2008). In addition, although the supply of FPP was increased and cells could grow well when YXWP-60( þ ) and YXWP-65( þ) were cultured in YPD, the production of carotenoids was decreased and a new by-product farnesol was detected. LPP1 and DPP1 were reported to be responsible for the formation of farnesol (Faulkner et al., 1999). Therefore, to further reduce the possible loss of precursors, we constructed the strain YXWP-67( þ ) by deleting these two genes, but no obvious influence on carotenoid production was observed (Tab. S4). Since FPP is also the precursor of dolichols, heme A, quinones and farnesylated mating factors, the accumulated FPP might be lost through other pathways rather than the formation of farnesol (Grabinska and Palamarczyk, 2002). In previous studies on cubebol and casbene production in S. cerevisiae, it was found that enhanced supply of FFP failed to increase the production of target terpenoids (Asadollahi et al., 2010; Kirby et al., 2010), which was similar to the observations in our study. Consequently, we speculate the unbalanced utilization of FPP might be the major cause for carotenoid production decrease when YXWP-60( þ) and YXWP-65( þ) were cultured in YPD. Hence, the timing and strength of ERG9 repression and carotenoid pathway induction is extremely important for the accumulation of both the biomass and the target products. Fortunately, we could control the two pathways dynamically in the YXWP-65( þ ) strain to further balance the supply and utilization of FPP, which was hard to achieve by a static control strategy. Although the carotene production of YXWP-65( þ) was improved by adjusting the initial glucose supply, it was still found that a slight lag existed between the response of the PHXT1controlled squalene pathway and the PGAL1/GAL10-controlled carotenoid and MVA pathways, which might led to the accumulation of FPP and triggered expression of other competitive pathways. Therefore, we introduced an additional constitutive beta-carotene

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pathway to convert any possible FPP accumulation resulted from ERG9 repression into the target carotenoid before the inducible MVA and beta-carotenoid pathways were switched on. With the depletion of glucose in media, the expression of ERG9 was repressed, meanwhile, the induced expression of tHMG1 triggered the strong metabolic flow of the MVA pathway for FPP generation, and the inducible and constitutive carotene biosynthetic pathways worked along with the MVA pathway to maximize carotenoid production. Finally, in the YXWP-82( þ ) strain, this comprehensive sequential control strategy enhanced production of carotenoids to 19.71 mg/g DCW. Because the growth conditions of cells in shake flask and fed-batch fermentation were different, we also investigated whether the sequential control strategy could be applied for high-cell-density fermentation. In contrast with the static promoter-controlled biosynthetic pathways in which product accumulation synchronized with biomass accumulation (Dai et al., 2013; Zhou et al., 2012), the profile of fedbatch fermentation results in our work indicated the stages of cell growth and carotenoid accumulation were clearly separated (Fig. 7B), which was considered as a practicable fermentation pattern for production of cytotoxic products (Anesiadis et al., 2008; Keasling, 2012; Krivoruchko et al., 2011). During the feeding process, the residual glucose concentration was below the detection limit in the fermentation medium because the glucose added was quickly depleted by cells, but it was in fact different from the glucose-free condition in shake-flask cultures due to the continuous feeding of

glucose. Therefore, the expression of carotenogenic genes in fed-batch fermentation was not fully induced as in the shake flask (Fig. 5), but rather progressively induced. As shown in Fig. 8, the transcription levels analysis demonstrated the CrtE, CrtYB, CrtI and tHMG1 genes were 20–50-fold up-regulated while the ERG9 gene was about 50-fold down-regulated. Meanwhile, the accumulation of squalene was dramatically decreased when the cells were cultured in glucose-limiting conditions, which was consistent with the transcriptional variation of ERG9. These results demonstrated that the sequential control strategy could work equally well in the fed-batch mode. In fed-batch fermentation, despite the significantly increased carotenoids titer, no obvious increase was observed in carotenoid production compared to that obtained from the shaking flask. Unlike most monoterpenes and sesquiterpenes that can be secreted extracellularly during fermentation (Asadollahi et al., 2010; Westfall et al., 2012), carotenoids can only be stored in the membrane system due to its high hydrophobicity, which might be the major limiting factor restricting further enhancement of carotenoid production in the high-density fermentation. Improvement of the membrane storage capacity might be a future solution to this problem.

5. Conclusion Constructing preprogrammed biosynthetic pathways is one of the directions for efforts on pathway control and metabolic

Fig. 8. Sequential control in high-density fermentation. (A) Schematic showing the sequential control mode of the carotenoid pathway, squalene synthetic pathway and MVA pathway in the high-density fermentation process. (B) Samples at 9 h, 43 h, 60 h, 84 h, and 96 h during the fermentation of YXWP-101( þ ) were used for qPCR to determine the transcriptional change of the integrated genes. (C) Squalene production analysis during the fermentation process.

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engineering. The work presented here is the first implementation of constructing an inducer/repressor-free dynamic control strategy to balance two pathways which utilize the same precursor in S. cerevisisae. By using this strategy, production of 1156 mg/L (20.79 mg/g) of carotenoids was achieved. The applications of the sequential control strategy are however not limited to carotenoids production, but can also be applied in a multitude of pathways for the production of terpenoids or other chemical products. The inducer/repressor-free nature of this strategy offers a practical and economically efficient way to improve the biosynthesis of value-added chemicals in the fermentation industry.

Acknowledgments This work was financially supported by the Natural Science Foundation of China (Grant nos. 21176215 and 21406196), the Program for Zhejiang Leading Team of S&T Innovation (Grant no. 2011R50007), the Fundamental Research Funds for the Central Universities (Grant no. 2014QNA4025) and Zhejiang Provincial Natural Science Foundation of China (Grant no. LQ14B060005).

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