Combining Gal4p-mediated expression enhancement and directed evolution of isoprene synthase to improve isoprene production in Saccharomyces cerevisiae

Author’s Accepted Manuscript Combining Gal4p-mediated expression enhancement and directed evolution of isoprene synthase to improve isoprene production in Saccharomyces cerevisiae Fan Wang, Xiaomei Lv, Wenping Xie, Pingping Zhou, Yongqiang Zhu, Zhen Yao, Chengcheng Yang, Xiaohong Yang, Lidan Ye, Hongwei Yu

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S1096-7176(16)30201-4 http://dx.doi.org/10.1016/j.ymben.2016.12.011 YMBEN1199

To appear in: Metabolic Engineering Received date: 29 October 2016 Revised date: 14 December 2016 Accepted date: 26 December 2016 Cite this article as: Fan Wang, Xiaomei Lv, Wenping Xie, Pingping Zhou, Yongqiang Zhu, Zhen Yao, Chengcheng Yang, Xiaohong Yang, Lidan Ye and Hongwei Yu, Combining Gal4p-mediated expression enhancement and directed evolution of isoprene synthase to improve isoprene production in Saccharomyces c e r e v i s i a e , Metabolic Engineering, http://dx.doi.org/10.1016/j.ymben.2016.12.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Combining Gal4p-mediated expression enhancement and directed evolution of isoprene synthase to improve isoprene production in Saccharomyces cerevisiae Fan Wanga, Xiaomei Lva, Wenping Xiea, Pingping Zhoua, Yongqiang Zhua, Zhen Yaoa, Chengcheng Yanga, Xiaohong Yanga, Lidan Yea,b*, Hongwei Yua* a

Institute of Bioengineering, College of Chemical and Biological Engineering,

Zhejiang University, Hangzhou 310027, PR China b

Key Laboratory of Biomass Chemical Engineering of Ministry of Education,

Zhejiang University, Hangzhou 310027, PR China

* Corresponding author. Tel & Fax: +86-571-87951873 Email address: [email protected]; [email protected] 1

Abstract Current studies on microbial isoprene biosynthesis have mostly focused on regulation of the upstream mevalonic acid (MVA) or methyl-erythritol-4-phosphate (MEP) pathway. However, the downstream bottleneck restricting isoprene biosynthesis capacity caused by the weak expression and low activity of plant isoprene synthase (ISPS) under microbial fermentation conditions remains to be alleviated. Here, based on a previously constructed Saccharomyces cerevisiae strain with enhanced precursor supply, we strengthened the downstream pathway through increasing both the expression and activity of ISPS to further improve isoprene production. Firstly, a two-level expression enhancement system was developed for the PGAL1-controlled ISPS by overexpression of GAL4. Meanwhile, the native GAL1/7/10 promoters were deleted to avoid competition for the transcriptional activator Gal4p, and GAL80 was disrupted to eliminate the dependency of gene expression on galactose induction. The IspS expression was obviously elevated upon enhanced Gal4p supply, and the isoprene production was improved from 6.0 mg/L to 23.6 mg/L in sealed-vial cultures with sucrose as carbon source. Subsequently, a novel high-throughput screening method was developed based on precursor toxicity and used for ISPS directed evolution towards enhanced catalytic activity. Combinatorial mutagenesis of the resulting ISPS mutants generated the best mutant ISPSM4, introduction of which into the GAL4-overexpressing strain YXM29 achieved 50.2 mg/L of isoprene in sealed vials, and the isoprene production reached 640 mg/L and 3.7 g/L in aerobic batch and fed-batch fermentations, respectively. These results demonstrated the effectiveness of the proposed combinatorial engineering strategy in 2

isoprene biosynthesis, which might also be feasible and instructive for biotechnological production of other valuable chemicals. Keywords: isoprene; GAL4 regulation; directed evolution; isoprene synthase; Saccharomyces cerevisiae 1. Introduction Isoprene, as the simplest member of isoprenoids, is a key monomer for rubber production. In nature, isoprene emission is ubiquitous among plants, but it is difficult to harvest this volatile product from the extended canopy of leafy plant (Guenther et al., 2006). Therefore, plant-based isoprene production is economically unfeasible for commercial applications. At the moment, isoprene is mainly produced from petroleum-derived feedstocks. However, the unrenewable nature of petroleum resources and pollutions caused by the petrochemical industry have led to serious problems. With development of biotechnology, bio-based isoprene production employing microbial cell factories has become a sustainable and green solution (Ye et al., 2016). The biosynthesis pathway of isoprene can be decomposed into two modules: the upstream MEP or MVA pathway and the downstream isoprene-forming pathway. In the upstream pathway, dimethylallyl diphosphate (DMAPP) is produced from pyruvate or acetyl-CoA under catalysis of multiple enzymes. In the downstream pathway, isoprene synthase (ISPS) catalyzes the conversion of DMAPP to isoprene (Fig. 1). Isoprene synthases sourced from different plants have been introduced to Escherichia coli, Bacillus subtilis or cyanobacteria for isoprene biosynthesis through 3

the endogenous MEP pathway (Lindberg et al., 2010; Lv et al., 2016; Lv et al., 2013; Xue et al., 2011; Zhao et al., 2011). More recently, introduction of exogenous MVA pathway into cyanobacteria and E. coli has been proven as an efficient strategy towards enhanced isoprene production (Bentley et al., 2014; Yang et al., 2016; Zurbriggen et al., 2012). In contrast, there are relatively few studies on biosynthesis of isoprene in eukaryotes harboring the native MVA pathway such as S. cerevisiae, which is featured by low risk of contamination during fermentation, great potential for isoprenoids accumulation, and feasibility as possible animal feed (Chang et al., 2006; Hong et al., 2012; Lv et al., 2014; Lv et al., 2016b). Regardless of the host species, all studies about isoprene biosynthesis have laid emphasis on the regulation of precursor supply, whereas hardly any attention was paid to the downstream isoprene-forming pathway. After up-regulation of the upstream pathway, the conversion of DMAPP to isoprene becomes the bottleneck in the whole isoprene biosynthesis pathway, the insufficient efficiency of which largely limits isoprene production. Meanwhile, the cytotoxicity of DMAPP upon over-accumulation inhibits cell growth (Lu et al., 2014; Martin et al., 2003; Withers et al., 2007). Therefore, how to improve the strength of isoprene-forming pathway catalyzed by ISPS has become the key to further enhancement of microbial isoprene synthesis. Copy number adjustment is a frequently adopted approach to enhance the expression of exogenous genes, but it has the risk to cause metabolic burden on microbial growth, exerting negative effects on high density fermentation (Karim et al., 2013). Transcriptional-level regulation is an alternative means to increase gene 4

expression. Especially, inducible expression has become a very prevalent and efficient method for enhancing expression of exogenous genes (Guan et al., 2016; Nakajima et al., 2016). The galactose (GAL) regulatory network in S. cerevisiae is one of the most well-characterized transcriptional regulation systems whose induction and repression are tightly regulated by galactose and glucose (Johnston et al., 1994; Lohr et al., 1995). GAL promoters (PGAL) are activated by binding the transcriptional regulator Gal4p. However, in natural yeast strains, the expression level of GAL4 gene is quite low and the amount of Gal4p is the rate-limiting factor for transcriptional activity. In a previous attempt to enhance Gal4p level by constitutive overexpression of GAL4, the desired regulatable feature of the system was concomitantly lost (Johnston et al., 1982). In a more recent study, the genomic replacement of GAL1 with GAL4 (resulting in fusion of GAL4 with the natural GAL1 promoter) led to a 4.6-fold increase in the expression of the reporter GFP, demonstrating the potential of appropriate Gal4p overproduction in maximizing heterologous expression of proteins driven by galactose-inducible promoters (Stagoj et al., 2006). These results inspired us to examine whether GAL4 overexpression could also act as a valid regulatory solution to metabolic bottlenecks in biosynthesis pathways by up-regulation of the PGAL-driven rate-limiting pathway enzyme, here ISPS. Aside from metabolic regulation towards elevated expression level, protein engineering of the rate-limiting enzyme for enhanced catalytic activity is another approach to eliminate metabolic bottlenecks. Directed evolution and rational design are practical strategies in protein engineering. For rational design, deep understanding 5

on the structure and catalytic reaction mechanism of the enzyme is a prerequisite. The amino acids involved in the ISPS catalysis remain unknown, impeding its rational design, although the X-ray crystal structure of recombinant isoprene synthase from grey poplar leaves has been resolved and the reaction mechanism has been preliminarily revealed (Faraldos et al., 2012; Köksal et al., 2010). In contrast, the advantage of directed evolution lies in its independence on the structure and reaction mechanism. However, to construct an efficient high-throughput screening method is often a challenge. Generally, only distinguished phenotypes that produce color change or distinction in growth rate can be easily identified (Lee et al., 2015). As a colorless compound with a low boiling point (34 °C), isoprene is released as a gas in microbial fermentation, which makes it difficult to construct a suitable product-based high-throughput screening method for ISPS directed evolution. Alternatively, as the substrate of ISPS, DMAPP has been reported to be cytotoxic and cause cell growth inhibition in a number of studies about isoprenoids biosynthesis (Lu et al., 2014; Martin et al., 2003; Withers et al., 2007). Under catalysis of ISPS, the accumulated DMAPP could be converted to isoprene and the cytotoxicity would be relieved accordingly, leading to improved cell growth. Hence, it might be feasible to establish a high-throughput method based on DMAPP toxicity relief upon enhanced ISPS activity. In

the

present

study,

we

proposed

to

strengthen

the

downstream

isoprene-forming pathway by simultaneously enhancing the expression and catalytic activity of ISPS (Fig. 1). Based on the previously constructed isoprene-producing S. 6

cerevisiae strain YXM08-ISPS with enhanced precursor supply by up-regulation of MVA pathway and down-regulation of squalene synthesis pathway (Lv et al., 2014), we overexpressed GAL4 under control of different GAL promoters and meanwhile deleted the native GAL promoters in the yeast chromosome to enhance Gal4p supply for activation of IspS transcription under control of PGAL1. Meanwhile, GAL80 was disrupted to eliminate the dependency of IspS expression on galactose induction. In addition, a novel high-throughput screening method was developed based on DMAPP toxicity relief as visualized by increased cell growth, and used for directed evolution of ISPS, leading to successful selection of positive ISPS mutants with enhanced catalytic activity. Subsequent combinatorial mutagenesis further improved ISPS activity. Finally, the above strategies were combined to intensify the isoprene-forming pathway and its effect on isoprene production was examined in both seal-vial cultures and aerobic batch or fed-batch fermentations.

Fig. 1. Strategy of strengthening isoprene-forming pathway for high isoprene production in S. 7

cerevisiae. The strategy consists of two parts. Part 1: The isoprene-forming pathway was strengthened by the enhancement of IspS expression. GAL4 was overexpressed with PGAL1 and PGAL4 respectively to enhance the supply of available Gal4p in the cell. Meanwhile, the endogenous PGAL1/7/10 was deleted to provide more Gal4p for activation of the PGAL1 controlling IspS transcription. Part 2: The isoprene synthesis pathway was further strengthened through directed evolution of ISPS towards enhanced activity. BY4742-C-04 with excessive accumulation of DMAPP was used as the host for ISPS directed evolution. DMAPP could be converted to isoprene by ISPS catalysis, so the growth rate was positively correlated with ISPS activity.

2. Materials and Methods 2.1 Strains and media Escherichia coli Top10 was used for gene cloning in this study. All recombinant E. coli strains were cultured in LB medium with appropriate antibiotics (100 μg/mL ampicillin or 50 μg/mL of kanamycin). For isoprene production, all recombinant S. cerevisiae strains in this study were constructed from BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) or BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) (Brachmann et al., 1998). YPD medium (1% Yeast extract, 2% Peptone and 2% D-Glucose), YPS medium (1% Yeast extract, 2% Peptone and 2% D-Sucrose) and YPG medium (1% Yeast extract, 2% Peptone and 2% D-Galactose) were used for yeast cultivation. YPD medium containing 200 μg/mL geneticin (G418) was used for selection of KanMX marker during gene integration and knockout in yeast (Güldener et al., 1996). Synthetic complete drop-out medium (Gietz et al., 2006) with different carbon sources and without uracil, namely, SD-URA (D-Glucose), SS-URA (D-Sucrose) and 8

SG-URA (D-Galactose) were used for selection and cultivation of yeast carrying derivative plasmids of p416XWP01-URA (Xie et al., 2015) 2.2 Construction of plasmids and strains All plasmids and primers (synthesized by Sangon Biotech, Shanghai, China) used in this study are listed in Supplementary Table S1 and Supplementary Table S2, respectively. pUMRI derivative plasmids with different homologous arms were used for gene integration into yeast genome (Lv et al., 2016b; Xie et al., 2015). The p416XWP01-URA plasmid was used for expression of IspS. General DNA amplification from genome was conducted according to the standard protocol of PrimeStar DNA polymerase (Takara, Dalian, China). Error-prone PCR was conducted using EasyTaq polymerase (Transgene, Beijing, China) and the concentration of Mn2+ was 0.1 mM. The primers in this study were synthesized by Sangon Biotech (Shanghai, China). All restriction enzymes and T4 ligase were purchased from Takara (Dalian, China). Kits used in DNA manipulation were purchased from Axgeny (Suzhou, China). For strains construction, pUMRI plasmids were linearized with corresponding restriction enzymes and integrated into yeast genome by electroporation. Recombinant strains were selected by G418 or uracil. The p416XWP01-URA plasmids carrying IspS were transformed into corresponding yeast strains for isoprene production. All strains used in this study are listed in Table 1. 2.3 Isoprene production in sealed vials To avoid loss of isoprene during cultivation, yeast strains were cultivated in 9

sealed vials (17 mL) containing 3 mL of fresh SS-URA/SG-URA medium at 30 °C for 24 h, and then heated to 37 °C with shaking (200 rpm) for 30 min to vaporize isoprene for GC quantification. 2.4 Isoprene production in aerobic batch fermentations and fed-batch fermentations The medium used in batch fermentations was based on that described by van Hoek et al. (Hoek et al., 2000), consisting of 25 g/L sucrose, 15 g/L (NH4)2SO4, 8 g/L KH2PO4, 3 g/L MgSO4, 0.72 g/L ZnSO4•7H2O, 12 mL/L vitamin solution and 10 mL/L trace metal solution. Single colonies were picked into seed culture and incubated at 30 °C for 24 h, and then transferred to 500 mL flasks containing 100 mL of fresh fermentation medium to get an initial OD600 of 0.05. After 24 h cultivation to an OD600 of 4-8, three flasks of cultures were pooled and transferred to a 5 L stirred-tank bioreactor containing 2.5 L of fermentation medium. The fermentation was carried out at 30 °C with an agitation speed of 200-600 rpm and an air flow rate of 1-3 vvm. For fed-batch fermentation, the feeding solution contained 500 g/L glucose, 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. pH was controlled at 5.0 by automatic addition of 5 M ammonia hydroxide which was meanwhile used as an additional nitrogen source. The glucose in medium was depleted at 12th hour. Feeding at a rate of 0.37 mL/min was initiated at 15th hour and ceased at 60th hour of the fed-batch fermentation. Glucose concentration in the fermentation broth was determined by Glucose Detection Kit (Rongsheng, Shanghai, China) based on Glucose oxidase-Peroxidase 10

(GOD-POD) method. For sucrose quantification, 3 M HCl was added into the fermentation broth to hydrolyze sucrose to glucose and the sugar concentration was determined by Glucose Detection Kit after 30 min incubation at 70 ℃. 2.5 Isoprene quantification by GC analysis Vapor samples from headspace of the sealed vials were analyzed by GC (Fuli, Wenling, China) equipped with a flame ionization detector (FID). An HP-FFAP column (30 m × 0.25 mm, 0.25 μm film thickness) was used with nitrogen as the carrier gas. The temperatures of oven, detector and injector were 80 °C, 180 °C and 180 °C, respectively. For isoprene analysis during fermentation, off-gas was gathered with bags every three hours. In every sampling, four parallel samples were collected for quantification of isoprene production. 2.6 Extraction and analysis of squalene Strains were cultivated at 30 ºC for 24 h before squalene extraction. Cells were collected by centrifuge and then disrupted by automatic tissue lyser-24 (Jingxin, Shanghai, China) for 5 minutes in 2 ml tube containing 0.1 mL of distilled water. The cell debris was washed twice with distilled water. Finally, a suitable amount of acetone was used to extract squalene. The analysis of squalene was performed on an HPLC system (LC 20AT, Shimadzu) equipped with a Kromasil C18 column (4.6 mm × 150 mm) and the UV/VIS signals were detected at 195 nm. The mobile phase was 100% acetonitrile with a flow rate of 1 mL/min at 40 °C. The standard compound of squalene (Sigma, Aldrich, St. Louis, MO) was dissolved in acetone and used for 11

standard curve preparation. 2.7 Real-time Quantitative PCR (qPCR) analysis of the transcriptional level of IspS Total RNA was isolated from yeast cells by the RNAiso Plus Kit (Takara, Dalian, China) according to the protocol. Genomic DNA contamination in RNA samples was eliminated by DNase I (Takara, Dalian, China). Reverse transcription was conducted by PrimeScriptTM 1st Strand cDNA Synthesis Kit (Takara, Dalian, China) with Oligo dT primer. Real-time Quantitative PCR was performed by SYBR® Premix Ex TaqTM (Tli RNaseH Plus) (TakaRa, Dalian, China) on Mastercycler ep Realplex2 (Eppendorf, USA). The ACT1 gene was used as the internal control to normalize different samples. The transcriptional level of IspS was analyzed with the 2-△△CT method (Livak et al., 2001). 3. Results 3.1 GAL4 overexpression to enhance IspS expression In our previous work, a recombinant strain YXM08 with enhanced precursor supply was constructed based on BY4741 by overexpressing tHMG1, substituting PERG20 with a weaker promoter and regulating acetyl-CoA supply (Lv et al., 2014). Introduction of p416XWP01-ISPS into YXM08, generating YXM08-ISPS, achieved 6.2 mg/L isoprene production in sealed vials with galactose as carbon source. However, the high cost of galactose makes it unsuitable for industrial purposes. Deletion of GAL80 (encoding a repressor which prevents Gal4p from binding to GAL promoters in response to galactose) has been proposed to switch the regulatory sugar from galactose to glucose (Lv et al., 2014; Torchia et al., 1984; Xie et al., 2014). 12

When the concentration of glucose becomes low in media, transcription of exogenous genes controlled by GAL promoters is activated (Xie et al., 2014). In the present study, YXM13-ISPS was constructed from YXM08-ISPS by GAL80 knockout. The result that isoprene production of YXM13-ISPS in SS medium (6.0 mg/L) was comparable to that of YXM08-ISPS in SG medium (6.2 mg/L) suggested that the dependency on galactose was eliminated in YXM13-ISPS as anticipated and that sucrose could be used as a suitable carbon source for isoprene production in the following research (Fig. S1). Considering the enhanced precursor supply and relatively weak downstream pathway in YXM13-ISPS, expression of IspS should be strengthened in order to accelerate the conversion of DMAPP to isoprene. For this purpose, GAL4 was overexpressed in YXM13-ISPS to establish a two-level regulatory system to amplify the IspS expression by providing more activation protein Gal4p for the PGAL1 controlling IspS transcription. Meanwhile, in order to weaken the competition for Gal4p by other GAL promoters, the expression cassette of GAL4 was integrated to the original site of native PGAL1/7/10 in yeast chromosome so as to knock out these promoters (Fig. 1). To select a suitable promoter, GAL4 was overexpressed in YXM13-ISPS with PGAL1 and PGAL4 respectively, generating YXM28-ISPS and YXM29-ISPS (Fig. S2). Cultivation of these three strains in sealed vials containing SS-URA medium showed that YXM29-ISPS had the highest isoprene production, which was about 3.8-fold higher than that of YXM13-ISPS (Fig. 2A). Meanwhile, the squalene production 13

decreased from 4.13 mg/L to 2.82 mg/L upon GAL4 overexpression. These results suggested that the isoprene-forming pathway was strengthened and the competing pathway was weakened by enhancement of IspS expression. In addition, growth experiments excluded the adverse effects of PGAL1/7/10 deletion and GAL4 overexpression on biomass (Fig. S3). As a matter of fact, YXM29-ISPS showed even slightly better growth than YXM13-ISPS. These results demonstrated that GAL4 overexpression successfully enhanced isoprene production without sacrificing cell growth. A

B

14

Fig. 2 Enhancement of IspS expression. (A) Isoprene and squalene production of YXM13-ISPS, YXM28-ISPS and YXM29-ISPS. All strains were cultivated in SS-URA medium at 30 °C for 24 h in sealed vials. (B) Transcriptional level of IspS. Samples collected at 18 h, 24 h, 48 h and 72 h during the fermentation of the three strains in flasks were used for qPCR to determine the transcriptional level of IspS. The IspS transcriptional level of YXM13-ISPS at 18 h was set to 1.

To further verify whether the improvement in isoprene production was indeed resulted from enhanced IspS expression, real-time quantitative PCR was conducted to monitor the transcriptional level of IspS in the process of flask fermentation. As shown in Fig. 2B, the IspS transcription of YXM13-ISPS was kept at a low level during fermentation. In contrast, YXM29-ISPS and YXM28-ISPS achieved about 6fold and 4-fold higher transcription level respectively within the first 24 h. Despite the decline in the late stage of fermentation, the overall expression levels in the GAL4-overexpressing strains were obviously higher than that in YXM13-ISPS. The results unambiguously suggested that the improvement of isoprene production in YXM29-ISPS and YXM28-ISPS should be attributed to the enhanced transcriptional level of IspS upon GAL4 overexpression. Meanwhile, GAL4 overexpression was validated as an efficient regulatory strategy for expression enhancement of the rate-limiting pathway enzyme in isoprene biosynthesis. 3.3 Development of high-throughput screening method based on DMAPP toxicity To further strengthen the isoprene-forming pathway, engineering of ISPS via directed evolution towards elevated catalytic activity would be a promising solution, for which a suitable high-throughput screening method is required. In the MVA 15

pathway, acetyl-CoA is converted to DMAPP by seven steps of enzyme catalysis. Integrating the genes of the entire MVA pathway including ERG10, HMGS, tHMG1, ERG12, ERG8, MVD1, and IDI1 is expected to enhance the MVA pathway flux and thus the precursor supply for isoprene synthesis. In our previous work (Lv et al., 2016), we have constructed BY4742-C-04 from BY4742 by overexpressing two copies of tHMG1 and one copy each of the other MVA pathway genes in the chromosome, and observed severe growth inhibition (Fig. 3A). We speculated that the strengthened upstream pathway and the weak downstream pathway in BY4742-C-04 resulted in excessive accumulation of DMAPP, which was previously reported to be cytotoxic (Lu et al., 2014; Martin et al., 2003; Withers et al., 2007). To examine whether the growth inhibition was indeed associated with DMAPP accumulation, BY4742-C-04-A (BY4742-C-04

(BY4742-C-04 overexpressing

overexpressing ERG20

and

ERG20),

ERG9)

and

BY4742-C-04-B BY4742-C-04-C

(BY4742-C-04 overexpressing IspS) were constructed by integrating corresponding genes into the genome of BY4742-C-04. All recombinant strains were cultivated in YPS medium to compare the growth. BY4742-C-04-B showed the best growth among all these strains (Fig. 3A). As two key enzymes in squalene synthesis pathway, ERG20 and ERG9 efficiently converted excessive DMAPP to squalene, the precursor of ergosterol which is responsible for structural features of cell membrane (Polakowski et al., 1998). Consequently, the growth inhibition by diphosphate intermediates was eliminated. In contrast, serious inhibition was still observed in growth of BY4742-C-04-A because overproduction of farnesyl pyrophosphate (FPP) as another 16

diphosphate metabolite could also cause toxicity to the cells. For the strain BY4742-C-04-C with overexpression of IspS, the growth was only slightly better than the parent strain, ascribed to the low activity of wild-type IspS which failed to alleviate the toxicity. If IspS activity could be improved to comparable levels to those of ERG20 and ERG9, DMAPP would be more efficiently converted to isoprene and the strain would grow much faster, as observed for BY4747-C-04-B. The above results positively correlated DMAPP cytotoxicity relief (displayed as better cell growth) with downstream enzyme activity, which could serve as the basis for a high-throughput screening method for ISPS evolution. 3.4 Directed evolution of ISPS towards improved activity For ISPS directed evolution, the mutant library was generated by error-prone PCR, ligated to p416XWP01, and transformed into BY4742-C-04. Colonies showing fast growth were picked from agar plates, and cultivated in liquid medium for further confirmation. Among the about 10,000 colonies screened, four mutants showed constantly better growth than the wild-type strain (Fig. S4A). For these strains, the plasmids were isolated and re-transformed into BY4742-C-04 to further verify whether the mutants also led to improvement in isoprene production. As a result, two positive mutants ISPSM212 and ISPSM63 were obtained, with 1.6-fold and 1.8-fold higher isoprene-forming activity than the wild type, respectively (Fig. S4B). Sequencing of these mutants revealed two mutation sites in ISPSM212, F340L (close to the active domain) and I478V (on the periphery of the active domain), whereas ISPSM63 had a single mutation A570T which locates at the entrance of the 17

active pocket. To evaluate the influence of each mutation on the activity of ISPS and to achieve an optimal mutant for maximized isoprene production, single site mutagenesis and combinatorial mutagenesis were performed by overlap PCR. All constructed mutant plasmids (Table 2) were transformed to BY4742-C-04, and the isoprene production was comparatively analyzed. The combinatorial mutant ISPSM4 (A570T/F340L) showed the highest production (3-fold higher than the wild type) among all mutants, whereas IspSM3 (A570T) had the best performance in all single-site mutants (Fig. 3B). The results indicated the site 570 residue had a greater influence on ISPS activity than other mutation sites. A

B

18

C

D

Fig. 3 Directed evolution and combinatorial mutagenesis of ISPS. (A) Toxicity verification of DMAPP. All strains were cultivated in YPS medium at 30 °C to measure the growth curves. (B) Isoprene production of the mutant strains. BY4742-C-04-ISPS was used as the control. All strains were cultivated in SS-URA medium at 30 °C for 24 h in sealed vials. (C) Growth curves of BY4742-ISPS, BY4742-C-04-ISPS and BY4742-C-04-ISPSM4. All strains were cultivated in 100 ml SS-URA medium at 30 °C in shaking flasks. OD600 was measured every 12 hours. (D) ISPS and ISPSM4 were transformed into YXM29 to compare isoprene production, squalene production and dry cell weight. Both strains were cultivated in SS-URA medium at 30 °C for 24 h in sealed vials. 19

To validate the proposed high-throughput screening method and to confirm whether the improvement of ISPS activity could indeed lead to relief of cell growth inhibition, the mutant strain BY4742-C-04-ISPSM4 (BY4742-C-04 carrying p416XWP01-ISPSM4) was cultivated in SS-URA medium to measure the growth curve using BY4742-ISPS and BY4742-C-04-ISPS as the controls. The obviously faster growth and higher biomass of BY4742-C-04-ISPSM4 compared with BY4742-C-04-ISPS evidenced the positive correlation between cell growth and ISPS activity (Fig. 3C). However, the growth of BY4742-C-04-ISPSM4 did not fully recover to the normal level of BY4742-ISPS, suggesting the toxicity was still not completely eliminated. 3.5 Further enhancement of isoprene production by combining GAL4 regulation and ISPS directed evolution. To further accelerate the conversion of DMAPP to isoprene, the positive mutant ISPSM4 was transformed into the GAL4-overexpressing YXM29 to integrate Gal4p-mediated expression enhancement and directed evolution of ISPS. Isoprene and squalene production of YXM29-ISPSM4 were analyzed and compared to those of YXM29-ISPS (Fig. 3D). After replacing the wild-type ISPS with ISPSM4, the isoprene production was increased from 23.6 mg/L to 50.2 mg/L, whereas the squalene production dropped from 2.82 mg/L to 2.2 mg/L. The results indicated further enhancement of the isoprene-forming pathway. Meanwhile, the biomass of YXM29-ISPS and YXM29-ISPSM4 (2.1 mg/mL and 2.25 mg/mL dry cell weight) did not display significant difference. 20

3.6 High density fermentation for isoprene production A

B

C

21

Fig. 4 Aerobic batch fermentation of the strains YXM13-ISPS, YXM29-ISPS and YXM29-ISPSM4. (A) Isoprene production; (B) Growth curves. (C) Sucrose concentration in fermentation broth.

To achieve higher isoprene production and meanwhile to further demonstrate the effectiveness of strengthening the downstream isoprene-forming pathway under aerobic condition, the culture condition was switched from sealed vials to bioreactor, and batch fermentation was performed with YXM13-ISPS, YXM29-ISPS and YXM29-ISPSM4. It was found that the rates of both sugar uptake and isoprene production of YXM29-ISPS were much faster than those of YXM13-ISPS (Fig. 4C and Fig. S5A). Consistent with the results of sealed vials, YXM29-ISPS (346 mg/L) showed great increase in isoprene production compared with YXM13-ISPS (95 mg/L) (Fig. 4A). This result proved Gal4p-mediated expression enhancement of ISPS as a powerful means to strengthen the isoprene-forming pathway so as to improve isoprene production under both favorable aerobic condition and anaerobic condition. Surprisingly, the OD600 in stationary phase of YXM29-ISPS was approximately 2.6-fold higher than that of YXM13-ISPS during batch fermentation, showing much more obvious growth advantage than in sealed vials (Fig. 4B). Subsequently, by substituting the wild-type ISPS in YXM29 with the best mutant ISPSM4, the isoprene production of YXM29-ISPSM4 was further improved to 640 mg/L and the growth rate was also slightly increased, demonstrating further elimination of metabolic bottleneck by improvement of ISPS activity. Considering both the high yield

(Table S3) and excellent growth,

YXM29-ISPSM4 was selected for high density fermentation. To meet the demand of 22

large-scale fermentation and save production cost, the His3 and Met15 markers were complemented into the genome of YXM29-ISPSM4. The carbon source in fed-batch fermentation was switched to glucose, since it was difficult to achieve high cell density with sucrose as the carbon source. At the early stage of fermentation, glucose in the medium was consumed for cell growth while isoprene production remained low. Feeding at a rate of 0.37 mL/min was initiated at 15th hour when both the biomass and isoprene production started to increase dramatically. During the feeding stage, the glucose concentration in the fermentation broth was kept low, which was conducive to isoprene synthesis. From the 34th hour to the 51th hour, the isoprene production rate was greater than 100 mg/L/h (Fig. S5B). From the 63th hour on, the strain stepped into stationary phase of growth and continued to produce isoprene at a very low productivity. At the end of fermentation, the OD600 reached 168 and 3.7 g/L of isoprene was produced (Fig. 5).

Fig. 5 Fed-batch fermentation of YXM29- ISPSM4. Fermentation was performed in a 5 L fermentor 23

containing 2.5 L of fermentation medium at 30 °C with an airflow rate of 1-3 vvm. pH was controlled automatically at 5.0 with the addition of 5 M NH4OH. Feeding at a rate of 0.37 mL/min was initiated at 15th hour and ceased at 60th hour of the fed-batch fermentation. The isoprene concentration in the off-gas was determined by GC every three hours.

4. Discussion In biosynthesis, enhancement of precursor supply is a common strategy adopted to improve the production of the target metabolite and its effectiveness has been demonstrated in many studies (Lv et al., 2014; Ohto et al., 2009; Polakowski et al., 1998; Xie et al., 2013). However, sometimes excessive enhancement of the pathway runs counter to our desire. Imbalance of the metabolism and accumulation of the intermediate may cause damage to cells and eventually reduce the metabolite production. It was reported that excessive overexpression of the MEP pathway caused accumulation of toxic pyrophosphate intermediates such as DMAPP and isopentenyl pyrophosphate (IPP) which synergistically resulted in decreased cell growth and overall underproduction of the target compound (Lu et al., 2014). Similarly, in the present study, BY4742-C-04 gave a low biomass and isoprene production, which could be ascribed to the overproduction of DMAPP leading to impaired cell health. Indeed, sufficient precursor supply facilitated by enhanced MVA flux is required for efficient isoprene biosynthesis in yeast. However, the weak downstream pathway constitutes a metabolic bottleneck due to the insufficient conversion of the accumulated precursor to the product. Though the ISPS sourcing from Populus alba has relatively higher activity than ISPS of other sources, its expression level and 24

catalytic activity in microorganisms is generally low (Sasaki et al., 2005). In our starting strain YXM08-ISPS, considering the low competitiveness of the isoprene-forming pathway in comparison with the squalene synthesis pathway, ERG9 expression had been down-regulated to divert more precursor (DMAPP) flux to isoprene synthesis (Lv et al., 2014). However, the isoprene-forming pathway was too weak to convert the abundant DMAPP to isoprene, resulting in inhibition on cell growth. This result also pointed to the necessity of strengthening the isoprene-forming pathway. In the present study, we combined metabolic engineering and protein engineering to strengthen the downstream section of isoprene synthesis pathway by simultaneously enhancing the expression and catalytic activity of ISPS. Although the expression of genes could be controlled at transcriptional, post-transcriptional and translational levels, transcriptional control is the most commonly used strategy in metabolic engineering due to its convenience and diversification (Keasling, 2012; Seoa et al., 2013). GAL regulatory system is one of the most well-studied transcriptional control systems in S. cerevisiae, where Gal4p acts as a transcriptional activator for the GAL promoters. Considering the generally low concentration of Gal4p available in yeast cells, which limits the transcriptional activity of GAL promoters and thus the expression of the PGAL-driven target gene, enhancement of its supply was proposed as a regulatory strategy to enable two-level amplification of ISPS expression. More activation protein Gal4p was provided for PGAL1-IspS through GAL4 overexpression together with chromosomal GAL1/7/10 promoters deletion, 25

which led to enhanced IspS expression and thus efficiently accelerated the conversion of DMAPP to isoprene. This is the first application of GAL4 overexpression in metabolic pathway regulation, although replacement of GAL1 with GAL4 has been previously reported to enhance heterologous production of GFP (Stagoj et al., 2006). To be noted, although PGAL4 has a weaker strength than PGAL1, higher IspS transcriptional level was achieved in the recombinant strain YXM29-ISPS in which GAL4 was overexpressed with PGAL4, leading to obviously higher isoprene production (Fig. 2A and 2B). As found in the constitutive Gal4p overproduction (Johnston et al., 1982), more Gal4p in YXM28-ISPS would not necessarily result in higher transcriptional level of IspS. This result implied the importance of an appropriate expression level of GAL4 in metabolic regulation. In addition, upon the alleviation of cytotoxicity ascribed to the accelerated conversion of DMAPP by overexpressed ISPS, YXM29-ISPS showed better growth than YXM13-ISPS. To our surprise, the growth advantage of YXM29-ISPS was much more evident in the batch fermentation than the sealed-vial cultures (Fig. 4B). We speculated that aerobic condition was more favorable to carbon source uptake and GAL4 expression than the anaerobic sealed vials, where the cell health might be negatively influenced by limitation in nutrient transfer and oxygen supply (Ostergaard et al., 2000). The unfavorable condition in sealed vials might also explain the more than 10-fold difference in isoprene production between the sealed-vial culture and batch fermentation for all the strains. Besides the weak expression of ISPS, the limited catalytic activity of this enzyme also contributes to the low strength of the isoprene-forming pathway, causing 26

another barrier to high isoprene production. The fact that the amino acids involved in the catalytic reaction are not completely revealed makes the rational design of ISPS difficult. Directed evolution is effective to improve properties of enzymes, especially for those with insufficient background information. In order to achieve the goal of “you get what you screen for” (Kuchner et al., 1997), an efficient high-throughput screening method is the key. Accumulation of DMAPP, the precursor of isoprene, has been reported to be cytotoxic and could impair cell growth (Martin et al., 2003; Withers et al., 2007). Conversion of DMAPP to isoprene by ISPS catalysis can relieve the toxicity leading to better cell growth. The positive correlation between ISPS activity and cell growth makes it possible to develop a high-throughput screening method for ISPS directed evolution. In the terpenoids synthesis pathway, many pyrophosphate intermediates, such as IPP, DMAPP, FPP and GPP (geranyl pyrophosphate), can cause toxicity to cells (Withers et al., 2007). Thus, the novel high-throughput screening method developed in this study could also be applied for directed evolution of other enzymes using these toxic metabolites as substrates. Three positive mutation sites (F340L, I478V and A570T) were obtained through directed evolution in this paper (Fig. 6). The side chain benzyl group of F340 located at the active domain of ISPS was changed into isobutyl group. We speculated that the van der Waals force between this leucine and the threonine at position 342 was weaker than that between the original phenylalanine and the threonine, resulting in shorter distance between the hydroxyl group on the side chain of T342 and the substrate molecule. According to a previous study about the catalytic mechanism of 27

ISPS, isoprene formation occurs in two steps via an allylic carbocation intermediate, namely the leave of diphosphate group and the syn-periplanar elimination (Köksal et al., 2010). The nucleophilic hydroxyl group of T342 might contribute to the syn-periplanar elimination reaction, thus improving ISPS activity. The mutation A570T located at the entrance of the active pocket was hypothesized to change the conformation of DMAPP into the favorable chair-like binding conformation thus improving the catalytic activity, because the diphosphate leaving group itself could serve as the general base to assist the elimination reaction when DMAPP was in chair-like conformation. Taken together, the best mutant ISPSM4 which combined F340L and A570T was more conducive to the elimination reaction. Molecular docking was conducted to analyze the effect of F340L and A570T on catalysis of ISPS. As shown in Fig. 6, the comparison between the ISPS-DMAPP complex and the ISPSM4-DMAPP complex well demonstrated our speculation. In contrast, the I478V mutation located on the periphery of the active domain did not significantly improve ISPS activity. Since this residue is far away from the active domain, how it affects ISPS activity is temporarily unknown, and requires further investigation. In addition, the underperformance of the combinatorial triple mutant ISPSM6 in comparison with the double mutant ISPSM4 indicated that I478V might prevent DMAPP from forming the optimal conformation due to the decreased intermolecular force between sites 338 and 478. To the best of our knowledge, this is the first open publication about successful modification of ISPS. Our results have revealed A570 and F340 as two sites with evident influence on ISPS catalytic activity, which may be of great 28

significance for further study on the catalytic reaction mechanism of ISPS. Although the combination of F340L and A570T mutations led to evident improvement in the ISPS activity, introduction of this ISPS variant did not completely eliminate the growth inhibition of BY4742-C-04 (Fig. 3C). This result suggested that the ISPS activity was still not sufficiently high to catalyze the conversion of all excessive DMAPP to isoprene. In future research, saturated mutagenesis of these candidate sites would be attempted to further increase ISPS activity.

A

B

Fig. 6 Structure of the ISPS-DMAPP complex (A) and ISPSM4-DMAPP complex (B). F338 (green) and F485 (purple) are conserved residues in isoprene synthases. F340L (black), and A570T (blue) are the mutation sites in ISPSM4. T342 (red) locates close to DMAPP, which may contribute to the syn-periplanar elimination reaction.

Finally, to maximize the strength of isoprene-forming pathway so as to convert as much DMAPP as possible to isoprene, ISPSM4 was transformed into the GAL4-overexpressing strain YXM29. The resulting strain YXM29-ISPSM4 achieved both fast cell growth and obviously enhanced isoprene production in fed-batch fermentation. However, time course study during batch fermentation found that both 29

the strains YXM29-ISPS and YXM29-ISPSM4 entered stationary phase earlier than YXM13-ISPS (Fig. 4). This slightly shorter growing period in Gal4p-overproducing strain might be attributed to the cell lysis and transcriptional inhibition of certain Gal4p-regulated genes caused by GAL4 overexpression as previously reported (Gill et al., 2002; Martegani et al., 1993). In particular, when GAL4 was expressed intensely during the exponential phase in fed-batch fermentation, the Gal4p level might exceed the upper limit that the cells could afford (Fig. 5). Therefore, the strain could not further grow to achieve higher cell density and entered into the stationary phase at a relatively early time point. In spite of the slight negative impact on cellular lifespan, GAL4 overexpression played a decisive role in the improvement of biomass and isoprene production in the early and middle stages of fermentation. In the late stage of fermentation, appropriate reduction of GAL4 expression level through increasing glucose concentration by accelerating the feeding speed might be a feasible solution to the problem of short growth life resulted from overproduction of Gal4p. In addition, although the highest isoprene titer (3.7 g/L) ever reported in eukaryotic hosts was achieved in this research, there is still a large gap between the theoretical yield (251.9 mg isoprene/g glucose and 132.6 mg isoprene/g sucrose) and the actual yield (22.9 mg isoprene/g glucose and 25.6 mg isoprene/g sucrose) (Table S3). This could be probably resulted from the loss of acetyl-CoA flux to other pathways. The isoprene yield might be further improved by increasing the acetyl-CoA availability to the MVA pathway. 5. Conclusions 30

To further explore the capacity of isoprene production in S. cerevisiae, we enhanced both the expression and catalytic activity of ISPS to strengthen the downstream isoprene-forming pathway so that DMAPP could be more efficiently converted to isoprene. GAL4 overexpression was adopted as an efficient strategy to enhance ISPS expression, which might be widely applicable for regulation of key enzymes in biosynthesis under control of GAL promoters. In addition, the high-throughput screening method developed based on diphosphate cytotoxicity relief was proven to be feasible for ISPS directed evolution, and might also instructive for engineering of other terpene synthases which catalyze the conversion of pyrophosphate

intermediates.

Through

combinatorial

engineering

of

the

isoprene-forming pathway, the isoprene production reached 3.7 g/L, which is the highest titer ever reported in engineered eukaryotic hosts. The result demonstrates the efficiency of integrating metabolic engineering and protein engineering in regulation of isoprene biosynthesis. This strategy is not only limited to isoprene production, but can also be adapted for the bioproduction of other isoprenoids and chemical products. Acknowledgments This work was financially supported by the Natural Science Foundation of China (Grant Nos. 21406196 and 21576234), Zhejiang Provincial Natural Science Foundation of China (Grant No. LQ14B060005), and Qianjiang Talents Project.

31

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34

Table 1 List of strains and plasmids Strains

Genotype

Plasmids

References

BY4741

MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0

None

Boeke et al. (1998)

BY4742

MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0

None

Boeke et al. (1998)

BY4742-ISPS

BY4742, ΔGAL80::leu2,

None

This study

None

Lv et al. (2016)

ΔTy4:: PGAL10-IspS-TADH1 BY4724-C-04

BY4742, ΔLPP1::TCYC1-ERG10-PGAL1-PGAL10-HMGS-TADH1, ΔHO:: TTPS1-tHMG1-PGAL7-PGAL2-ERG12-TPGK, ΔDPP1::TCYC1-tHMG1-PGAL1-PGAL10-PMK-TADH1, ΔGAL80:: TTPS1-MVD1-PGAL7-PGAL2-IDI1-TPGK1

BY4742-C-04-A

BY4742-C-04, ΔTy4::TCYC1-ERG20-PGAL1

None

This study

BY4742-C-04-B

BY4742-C-04,

None

This study

ΔTy4::TCYC1-ERG9-PGAL1-PGAL10-ERG20-TADH1 BY4742-C-04-C

BY4742-C-04, ΔTy4:: PGAL10-IspS-TADH1

None

This study

BY4742-C-04-ISPS

BY4742-C-04

p416XWP01-ISPS

This study

BY4742-C-04-ISPSM4

BY4742-C-04

p416XWP01-ISPSM4

This study

YXM08

BY4741, ΔPERG9::PBTS1,

None

Lv et al. (2014)

ΔTy4::TCYC1-ACS2-PTEF1-PHXT7-ERG10-TADH1 YXM08-ISPS

YXM08

p416XWP01-ISPS

This study

YXM13-ISPS

YXM08, ΔGAL80::leu2

p416XWP01-ISPS

This study

YXM28-ISPS

YXM13, ΔGAL1/7/10:: PGAL1-GAL4 -TCYC1

p416XWP01-ISPS

This study

YXM29-ISPS

YXM13, ΔGAL1/7/10:: PGAL4-GAL4 -TCYC1

p416XWP01-ISPS

This study

YXM29-ISPSM4

YXM13, ΔGAL1/7/10:: PGAL4-GAL4 -TCYC1

p416XWP01-ISPSM4

This study

35

Table 2 List of the mutants Mutants

Mutation sites

M63

A570T

M212

F340L

M1

F340L

M2

I478V

I478V

M3 M4

A570T F340L

M5 M6

F340L

A570T I478V

A570T

I478V

A570T

Highlights 

Strengthening isoprene-forming pathway by improving expression and activity of isoprene synthase.



Adoption of GAL4 overexpression for metabolic regulation of isoprene biosynthesis.



Directed evolution of isoprene synthase based on DMAPP toxicity.



Highest isoprene production ever reported in engineered eukaryotes.

36