Orthogonal Engineering of Biosynthetic Pathway for Efficient

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Letter

Orthogonal Engineering of Biosynthetic Pathway for Efficient Production of Limonene in Saccharomyces cerevisiae Si Cheng, Xue Liu, Guozhen Jiang, Jihua Wu, Jin-lai Zhang, Dengwei Lei, Jianjun Qiao, Ying-Jin Yuan, and Guang-Rong Zhao ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.9b00135 • Publication Date (Web): 07 May 2019 Downloaded from http://pubs.acs.org on May 8, 2019

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ACS Synthetic Biology

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Revised

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Letter

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Orthogonal Engineering of Biosynthetic Pathway for Efficient

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Production of Limonene in Saccharomyces cerevisiae

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Si Cheng†, Xue Liu†, Guozhen Jiang†, Jihua Wu†, Jin-lai Zhang†, Dengwei Lei†, Ying-

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Jin Yuan†,‡, Jianjun Qiao†,‡, and Guang-Rong Zhao*,†,‡

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†Frontier

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Bioengineering (Ministry of Education), School of Chemical Engineering and

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Technology, Tianjin University, Yaguan Road 135, Jinnan District, Tianjin, 300350,

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China

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‡SynBio

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Engineering (Tianjin), Tianjin University, Yaguan Road 135, Jinnan District, Tianjin,

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300350, China.

Science Center for Synthetic Biology and Key Laboratory of Systems

Research Platform, Collaborative Innovation Centre of Chemical Science and

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*Corresponding

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Tel: +86-22-85356580; Fax: +86-22-27403389

author: Guang-Rong Zhao ([email protected])

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ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

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Limonene, a plant-derived natural cyclic monoterpene, is widely used in

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pharmaceutical, food and cosmetics industries. Conventional limonene biosynthetic

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(CLB) pathway in engineered Saccharomyces cerevisiae consists of heterologous

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limonene synthase (LS) using endogenous substrate geranyl diphosphate (GPP) and has

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been suffering from poor production of limonene. In this study, we report on an

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orthogonal engineering strategy in S. cerevisiae for improving the production of

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limonene. We reconstructed orthogonal limonene biosynthetic (OLB) pathway

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composed of SlNDPS1 that catalyzes IPP and DMAPP to NPP (cis-GPP) and plant LS

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that converts NPP to limonene. We find that the OLB pathway is more efficient for

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production of limonene than the CLB pathway. When expression of competing gene

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ERG20 was chromosomally regulated by glucose-sensing promoter HXT1, the OLB

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pathway harboring strain produced 917.7 mg/L of limonene in fed-batch fermentation,

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6-fold increase of the CLB pathway, representing the highest titer reported to date.

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Orthogonal engineering exhibits great potential for production of terpenoids in S.

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cerevisiae.

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KEYWORDS: limonene biosynthesis, orthogonal engineering, synthetic biology,

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pathway and metabolic engineering, Saccharomyces cerevisiae

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Plant terpenoids are valuable natural products, such as paclitaxel, artemisinin,

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lycopene, and limonene, which have been used in pharmaceutical and food industries.1

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Production of terpenoids from plants is always inefficient for seasonal cycles of slow

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growth with low content and environmental concerns for the solid waste discharge from

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the chemical extraction process. Metabolic engineering and synthetic biology of

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industrial microbes facilitates an alternative route for the effective production of

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terpenoids.2,3

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Saccharomyces cerevisiae is considered as a promising cell factory for inexpensive

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production of terpenoids. Yeast possesses potential mevalonate (MVA) pathway to

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provide the building blocks isopentenyl pyrophosphate (IPP) and dimethylallyl

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pyrophosphate (DMAPP) for heterologous biosynthesis of diverse terpenoids with

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cheap carbon sources.4 ERG20 catalyzes IPP and DMAPP to endogenous geranyl

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diphosphate (GPP) and farnesyl diphosphate (FPP) for biosynthesis of monoterpenes

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and sesquiterpenes, respectively. Over the last decades, numerous efforts have been

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paid for the production of terpenoids. The first excellent example was to engineer S.

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cerevisiae for production of artemisinic acid,5 the precursor for the antimalarial drug

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artemisinin. Bisabolene, another cyclic sesquiterpene with potential alternative to

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petroleum-derived diesel, achieved high titer in engineered S. cerevisiae.6 For

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monoterpene production, the GPP overproducing S. cerevisiae produced geraniol,7 an

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acyclic

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synthesized in yeast to expand the chemical space of terpenoids.8

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monoterpenol.

Additionally,

noncanonical 11-carbon

terpenes

were

Limonene is a well-famous cyclic monoterpene, and applied in pharmaceutical, 3



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pesticide, flavor, perfume, jet fuel, and other chemical industries.9,10 A few efforts have

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been made to improve the production of limonene in yeast. The conventional limonene

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biosynthetic (CLB) pathway of plants is composed of GPP synthase (GPPS) and

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limonene synthase (LS). Coexpression of GPPS mutated from yeast ERG20 and Citrus

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lemon LS (ClLS1) in S. cerevisiae resulted in minor limonene (0.12 mg/L) after

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headspace trapping of limonene, which is highly volatile and toxic to microbial

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growth.11 Enriched amino-nitrogen medium could increase biosynthesis of limonene to

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1.48 mg/L.12 Degradative regulation of ERG20 achieved 76 mg/L of limonene in

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engineered yeast strain.13 However, compared with other terpenoid pathways, the CLB

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pathway seems to be intractable in engineered S. cerevisiae. The reasons remain to be

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elucidated.

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Recently, alternative monoterpene biosynthetic pathway in tomato trichomes was

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reported.14 Several monoterpenes including limonene, α- and β-phellandrene, α- and γ-

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terpinene, and 2-carene in the glandular trichomes of tomato are synthesized from a

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neryl diphosphate (NPP) rather than GPP. NPP, the cis-isomer of GPP, is synthesized

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by NPP synthase (NPPS) from IPP and DMAPP. Considering the diverse effects of

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different configuration of substrate NPP from GPP on limonene biosynthesis, we

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proposed that engineering an orthogonal limonene biosynthetic (OLB) pathway

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composed of NPPS and corresponding LS using substrate NPP would open a possibility

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for efficient production of limonene in yeast.

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In this study, we firstly obtained the candidate limonene synthases by blast

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analysis using ClLS1 as the query sequence, and reconstructed the CLB pathway 4


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composed of yeast ERG20WW (ERG20F96W-N127W) and plant LS using substrate GPP, as

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well as the OLB pathway comprised of plant NPPS and plant LS using substrate NPP,

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respectively, in S. cerevisiae (Fig. 1). We then compared the capacity of the CLB and

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OLB pathways for limonene production by engineering glucose-sensing promoter

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HXT1 (PHXT1) of the competing gene ERG20, the branching node of precursors IPP and

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DMAPP fluxed to biomass and limonene biosynthesis. We find that the OLB pathway

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is competent to yeast cellular metabolism and more efficient for production of limonene

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with 917.7 mg/L in fed-batch shake-flask fermentation, 6-fold higher than that of the

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CLB pathway.

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Fig.1. Orthogonal strategy for production of limonene in Saccharomyces cerevisiae.

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Two artificial synthetic pathways to limonene from IPP and DMAPP were

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reconstructed, respectively. The conventional limonene biosynthetic (CLB) pathway is

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composed of yeast ERG20WW and plant tLS (A), while the orthogonal biosynthetic (OLB)

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pathway is composed of plant NPPS and plant tLS (B). The native promoter of

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chromosomal ERG20 was replaced with the HXT1 promoter. The endogenous genes

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tHMG1 and IDI1 were chromosomally overexpressed to supply sufficient precursors 5



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IPP and DMAPP in the starting strain.

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A-CoA, Acetyl coenzyme A; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A;

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MVA, mevalonate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl

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pyrophosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; NPP, neryl

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diphosphate; tHMG1, truncated HMG-CoA reductase; IDI1, IPP isomerase; ERG20WW,

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mutated farnesyl diphosphate synthase; LS, limonene synthase; ERG20, FPP synthase;

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NPPS, neryl diphosphate synthase; ERG9, squalene synthase; PGAL7, GAL7 promoter;

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PGAL10, GAL10 promoter; PHXT1, HXT1 promoter.

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RESULTS AND DISCUSSION

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Reconstruction of the CLB and OLB pathways

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In order to reconstruct artificial limonene biosynthetic pathway via metabolic

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engineering and synthetic biology, we searched the UniProt database to obtain

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annotated limonene synthases. Seven LSs from genus Citrus, ShLS from Solanum

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habrochaites that synthesizes limonene in vitro15 and ArLS from Agastache rugosa that

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functions for limonene production in Yarrowia lipolytica16 were chosen as candidate

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enzymes. The amino acid sequence identities among nine LSs were ranged from 17%

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to 96% (Fig. S1 and S2). All of those LSs have the conserved aspartate-rich DDXXD

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motif which is crucial for divalent cation (typically Mg2+ or Mn2+)-substrate complex

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binding.17 The presumptive N-terminal transit peptide of plant LSs targeting plastid

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membrane is detrimental to express in microbes and removed, generating the truncated

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LSs (tLSs). A rooted phylogenetic tree of the tLSs by using the neighbor-joining 6


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method was illustrated in Fig. 2A.

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To reconstructed the CLB pathway, we recruited a mutant variant of the yeast

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ERG20 enzyme ERG20WW which has the preferred activity to catalyze the formation

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of GPP7,18. The expression vectors of the ERG20WW and nine heterologous codon-

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optimized tLSs were constructed and introduced into the isoprene high-producing yeast

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strain yJGZ1,7 generating strains LimY1 to LimY9 (Table S1), respectively. After 48 h

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fermentation, as shown in Fig. 2B, the gas chromatography (GC) analysis exhibited that

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nine tLSs from six plant species were able to synthesize limonene in S. cerevisiae, and

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titers were quite divergent, in spite of no obvious difference in the cell growth (Fig.

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S3). CltLS1 exhibited the highest limonene production with titer of 15.5 mg/L, while

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the rest eight tLSs gave small amount of limonene (0.35-0.83 mg/L). Despite high

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identities between CltLS1 with CltLS2, CutLS1 and CstLS (Fig. 2A), respectively,

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none of CltLS2, CutLS1 and CstLS yielded better titer of limonene. Strain LimY3

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harboring the CLB pathway of ERG20WW and CltLS1 was used in following study.

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Analogously, we reconstructed the OLB pathway by employing SlNDPS1 gene14

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which encodes NPPS to convert yeast IPP and DMAPP to NPP. Nine tLSs genes were

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coexpressed with SlNDPS1 in strain yJGZ1, respectively, generating strains LimY10

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to LimY18. As shown in Fig. 2C, all of nine engineered strains produced limonene

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without difference of cell growth (Fig. S4). CutLS1, CutLS2 and CltLS3 gave the least

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effects on titers of limonene (from 1.37 to 2.52 mg/L), while ArtLS and ShtLS were

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better than CutLS1, CuLtS2 and CltLS3. CltLS2 did the best performance on

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production of limonene with titer of 28.9 mg/L, followed by CstLS with 26.4 mg/L of 7



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limonene. As expected, compared to the endogenous substrate GPP, all of the nine tLSs

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using NPP as designer substrate produced higher titers of limonene, 1.7- to 40-fold

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increase (Fig. 2D). These results illustrated that the OLB pathway is more efficient than

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the CLB pathway for limonene biosynthesis in S. cerevisiae. It is possible that the tLSs

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might have higher enzymatic activities towards NPP than GPP. Strain LimY13

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harboring the SlNDPS1 and CltLS2 was used in following study.

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Fig.2. Heterologous biosynthesis of limonene in engineered S. cerevisiae. (A)

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Phylogenetic analysis of nine tested tLSs proteins by using the neighbor-joining method.

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Analysis was conducted in MEGA7. (B) Biosynthesis of limonene in strains harboring

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the CLB pathway. (C) Biosynthesis of limonene in strains harboring the OLB pathway.

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(D) Comparison of limonene biosynthesis between the CLB and OLB pathways. The 8


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relative amount of limonene was calculated based on the unit of the titer of limonene in

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strain LimY8.

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Metabolic superiority of the OLB pathway to the CLB pathway

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The metabolite conversion at the node of IPP and DMAPP is the crossroad to

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generate and distribute substrates GPP and NPP. GPP is further used for limonene

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biosynthesis by LS or cell growth by ERG20, and NPP is solely used for limonene

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biosynthesis. Direct static downregulation of the competing gene ERG20 using

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conventional inducible or constitutive promoters seems unsuitable for heterologous

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production of monoterpene, which was demonstrated by the example that the

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expression of ERG20 driven by weak BTS1 promoter or the copper repressible CTR3

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promoter greatly decreased the final biomass and simultaneously impaired production

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of acyclic monoterpene.19 Applying the dynamic regulation could allow the engineered

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cell to flexibly control expression of heterologous pathway and reasonably distribute

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the metabolic flow. Degron tagging ERG20 paired with a sterol responsive promoter

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led to the accumulation of limonene with the CLB pathway after the stationary stage,

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but the lag stage was prolonged.13 The glucose-sensing promoter PHXT1 that is stronger

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in the presence of glucose and weaker in the absence of glucose would be a promising

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candidate to fit the diauxic yeast fermentation process,20 in which glucose is used in

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first stage of cell growth with simultaneous production of ethanol that is then used in

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second stage of cell growth and product biosynthesis of interest. Therefore, for the CLB

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pathway, the chromosomal native ERG20 promoter of strain LimY3 was replaced with 9



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promoter PHXT1, generating strain LimY26. Due to sufficient supply of glucose, the cell

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growth between strains LimY26 and LimY3 was not obviously different, strain

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LimY26 produced 43.3 mg/L of limonene (Fig. 3A). tHMG1 (truncated 3-hydroxy-3-

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methylglutaryl coenzyme reductase) is a rate-limiting enzyme in the MVA pathway.

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The overexpression of tHMG1 could result in the accumulation of squalene because of

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the high catalytic activities of ERG20 and ERG9 (squalene synthase) towards DMAPP

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to squalene,21 which is a competing product of limonene. We measured the content of

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squalene, representing the amount of carbon flux to the cell growth pathway. Surely,

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the large amounts of squalene were accumulated in the tHMG1 and IDI1 (encoding

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isopentenyl diphosphate isomerase) overexpressing strain yJGZ1 and strain LimY3,

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with 32.3 mg/g DCW and 28.1 mg/g DCW (Fig. 3C), respectively. Employing PHXT1

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decreased the squalene accumulation to 17.5 mg/g DCW in strain LimY26 (Fig. 3C). It

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indicated that the PHXT1 controlling ERG20 could reallocate the carbon flux from growth

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pathway to limonene synthetic pathway. In order to test the adaptability of the CLB

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pathway to the fermentation process, 13 g/L of glucose was fed to the culture at 20 h

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after initial glucose was exhausted. Unexpectedly, the production of limonene was not

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increased by feeding with glucose in strain LimY26, while the biomass and squalene

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were slightly increased to some extent (Fig. 3B). The combination of dynamic

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expression of ERG20 controlled by the PHXT1 and constitutive expression of ERG20WW

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in yeast strain could lead to promising production of monoterpene geraniol by geraniol

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synthase using substrate GPP,19 while it was less effective for the limonene production

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(Fig. 3C), implying that limonene synthase CltLS1 in the CLB pathway would not adapt 10


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to yeast cellular environment and become the limiting factor for limonene production.

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The similar phenomenon was observed in the production of taxadiene using taxadiene

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synthase in S. cerevisiae.22 In addition, expression of ERG20WW with remaining partial

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FPPS activity18 would allow the crosstalking of the CLB pathway to FPP pathway, and

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at least when LS was uncompetitive, lead to more GPP distribution for biosynthesis of

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squalene than that of limonene.

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Fig.3. Fermentation of strains LimY3 and LimY26 harboring the CLB pathway without 12


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glucose feeding (A) and with glucose feeding at 20 h (B). (C) Titer of limonene and

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amount of squalene at 48 h.

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In comparative parallel, the CLB pathway was replaced by the OLB pathway, and

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the production of limonene and squalene was different. Regulation of ERG20 by PHXT1

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was beneficial for the OLB pathway and led to 118.5 mg/L of limonene in strain

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LimY28 (Fig. 4A), a 1.7-fold increase of titer, compared to strain LimY26 harboring

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the CLB pathway. It suggested that NPP synthase SlNDPS1 and limonene synthase

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CltLS2 in the OLB pathway worked well together to biosynthesize limonene. Squalene

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in strain LimY28 was decreased to 4.1 mg/g DCW, one fourth of that in strain LimY26

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(Fig. 4C). When glucose feeding was carried out, strain LimY28 continuously produced

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limonene to 178.7 mg/L (Fig. 4B), a 3-fold increase than that of strain LimY26, and

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squalene further decreased to 2.3 mg/g DCW (Fig. 4C). The results confirmed that

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SlNDPS1 and CltLS2 of the OLB pathway were more adaptive to yeast cellular

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metabolic environment than ERG20WW and CltLS1 of the CLB pathway. Taking

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together, the OLB pathway would be independent of endogenous squalene pathway and

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benefits limonene biosynthesis.

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Fig.4. Fermentation of strains LimY13 and LimY28 harboring the OLB pathway

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without glucose feeding (A) and with glucose feeding at 20 h (B). (C) Titer of 14


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limonene and amount of squalene at 48 h.

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Efficiency of the OLB pathway in fed-batch fermentation

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To validate the long term effectiveness of the OLB pathway for limonene

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production, we performed the fed-batch fermentation in shake-flask using strain

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LimY28 with ethanol as the feeding carbon source, which was better than glucose used

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(Fig. S5). As shown in Fig. 5A, during the initial 12 h when glucose was present, the

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cells grew exponentially with the rate of 0.885 OD/h while limonene was scarcely

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produced. Entering the ethanol consumption stage, cell growth slowed down at lower

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rate of average 0.3 OD/h and reached to the stationary stage at 96 h. Accordingly,

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limonene was greatly accumulated from 12 h to 120 h at the production rate of 8.29

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mg/ (L·h) with the ﬁnal titer of 917.7 mg/L, 12-fold improvement than the previous

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reported titer.13

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On the other hand, regardless of similar growth pattern of strain LimY26 with

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strain LimY28, a disparate production behavior of the CLB pathway was observed.

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Limonene staggered slowly to the final titer of 141.6 mg/L at 120 h (Fig.5 B). The

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inferior performance of the CLB pathway in fed-batch culture was consistent with batch

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fermentation (Fig.3), further confirming that the CLB pathway of ERG20WW and

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CltLS1 was inherently unsuitable to production of limonene in S. cerevisiae.

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Fig. 5. Ethanol fed-batch fermentation of strains LimY28 harboring the OLB pathway

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(A) and LimY26 harboring the CLB pathway (B) in shake-flask. Ethanol was fed at

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20 h and 40 h for limonene production.

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Conclusion

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We report for the first time that the orthogonal engineering for efficient production

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of limonene in S. cerevisiae. The OLB pathway composed of SlNDPS1 and CltLS2 was

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reconstructed and the competing gene ERG20 for consumption of precursors IPP and

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DMAPP was regulated by glucose-sensing PHXT1. This strategy achieved 917.7 mg/L

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limonene at shake-flask level, 6-fold increase than the CLB pathway composed of

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ERG20WW and CltLS1. We show the OLB pathway is robust to fermentation

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environment and compatible to the cellular metabolisms in S. cerevisiae, exhibiting

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great potential for efficient production of limonene. In contrast, the CLB pathway

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crosstalking squalene pathway at least partially contributed to biomass biosynthesis and

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impaired the limonene production. Moreover, the activity of GPP-specific limonene

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synthase fitting in with the cellular environment would be a key factor for limonene

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production using the CLB pathway in S. cerevisiae. 16


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The orthogonal biodevices have been used in metabolic engineering and synthetic

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biology, such as RNA switches23 and genetic circuits.24 Recently, orthogonality

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principles of metabolic pathway design was proposed25 and show significant

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advantages in production of desired chemicals including muconic acid,26 and fatty acid

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and its ester.27,28 Here, we showed that orthogonal secondary metabolic pathway for

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production of limonene is more sufficient and robust than the conventional pathway

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which crosstalks with biomass synthesis at node GPP. Like most of organisms in nature,

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yeast does not possess cis-GPP and derived terpenoids. Cis-prenyltransferases and cis-

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terpenoids have been discovered from plants29-31 and insects32,33. It would be the great

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potential of the orthogonal pathways for efficient production of heterologous valuable

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terpenoids such as artemisinin, taxol and nature rubber in S. cerevisiae in future.

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MATERIALS AND METHODS

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Strains and Media

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Escherichia coli trans-T1 (TransGen Biotech, Beijing, China) was used for plasmid

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construction and cultivated at 37 °C in Luria-Bertani (LB) medium (10 g/L tryptone, 5

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g/L yeast extract, and 10 g/L NaCl). When needed, 100 μg/mL of ampicillin was

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supplemented into the medium.

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Saccharomyces cerevisiae yJGZ17 was used as the starting strain for reconstruction

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of limonene-producing strains. S. cerevisiae strains without plasmid were cultivated in

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yeast extract-peptone-dextrose (YPD) medium (20 g/L glucose, 20 g/L tryptone, and

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10 g/L yeast extract) and strains with plasmid in synthetic complete (SC) drop-out 17



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medium (20 g/L glucose, 6.7 g/L yeast nitrogen base, and 2 g/L amino acid mixed

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powder), respectively.

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Construction of Plasmids and Strains

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The plasmids and yeast strains used in this study are listed in Table S1. DNA

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manipulating agents, including T4 DNA ligase and restriction endonucleases were

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purchased from Thermo Scientific (Beijing, China). The ClonExpress II One Step

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Cloning Kit, DNA Polymerase of Phanta Super Fidelity and Taq were obtained from

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Vazyme (Nanjing, China). The primers of polymerase chain reaction (PCR) used in this

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study were synthesized by GENEWIZ (Suzhou, China) and listed in Table S2.

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The ERG20WW gene encoding GPPS was obtained through PCR using plasmid

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pJGZ38 as the template.7 Nine LSs genes and SlNDPS1 from plants were synthesized

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by GenScript (Nanjing, China) with codon optimization for S. cerevisiae. The N-

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terminal sequences targeting the plastid of LSs and SlNDPS1 were removed through

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PCR. For CuLS1, CuLS2, ClLS1, ClLS2, ClLS3, CsLS, PtLS and ArLS, the N-terminal

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plastid-targeting sequences precede a tandem pair of arginines.34 For ShLS that lacks

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an obvious plastid-targeting sequence, the N-terminal plastid-targeting sequence was

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predicted by ChloroP (http://www.cbs.dtu.dk). SlNDPS1 was truncated according to

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previous report.16 The nucleic acid sequences of truncated versions of synthesized LSs

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(tLSs) and SlNDPS1 were listed in Table S3. TENO2-TGPD fragment was digested with

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NotI and inserted into plasmid pRS426, generating plasmid pCS. Nine different tLSs

317

were digested with BsaI and inserted into the same site of pJGZ1, generating pCSL1 to

318

pCSL9, respectively. ERG20WW and SlNDPS1 were digested with BsaI and inserted into 18


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319

the same site of pZJL1, generating pCSG1 and pCSN1, respectively. Then different

320

ERG20 WW-tLS fragments were obtained through OE-PCR with corresponding primers

321

and ligated into the pCS by ClonExpress II One Step Cloning Kit, generating pCS01 to

322

pCS09, respectively. Similarly, different NPPS-tLS fragments were ligated into the pCS,

323

generating pCS10 to pCS18, respectively. To replace native ERG20 promoter,

324

synthesized PHXT1 promoter and TRP marker as well as the up and down 600 bp

325

homologous arms of ERG20 promoter were amplified from the S. cerevisiae CEN.PK2-

326

1C genome DNA and assembled by OE-PCR. All fragments and plasmids were verified

327

by sequencing before yeast transformation.

328

Limonene-producing strains were constructed by transforming expression

329

plasmids or DNA fragment containing PHXT1 to yJGZ1 using the LiAc/PEG/ssDNA

330

method.

331

Fermentation Conditions

332

For shake-flask fermentation, a single colony was picked from the SC plate and

333

inoculated into 2 mL SC medium and cultivated at 30 ℃, 250 rpm for 20 h.

334

Subsequently, the preculture was transferred into 6 mL SC medium with initial OD600

335

of 0.2 and cultivated at 30 ℃, 250 rpm for 20 h. Then the seed culture was transferred

336

into 250 mL flasks containing 50 mL YPD medium with an initial OD600 of 0.2. The

337

two-phase shake-flask fermentation was started by adding 10% (v/v) isopropyl

338

myristate as organic extractant phase into flasks, which could relieve the toxicity caused

339

by limonene and then incubated at 30 ℃, 250 rpm for 2 days to produce limonene.

340

The fed-batch fermentation in the shake-flask was conducted at 30 ℃, 250 rpm for 19



341

5 days. Ethanol was added at 20 h and 40 h as the carbon source with the final

342

concentration of 10 g/L for limonene production. All of the experiments were

343

performed twice, and average data were shown.

344

Biomass and Metabolite Analysis.

345

Cell optical density (OD) was measured at 600 nm with TU-1810

346

spectrophotometer. Residual glucose and residual ethanol of fermentation broth were

347

measured by a biosensor SBA-90 (Biology Institute of Shandong Academy of Sciences,

348

China). Squalene was extracted with ethyl acetate and quantified according to previous

349

study.34

350

For limonene analysis, the organic layer used was harvested by centrifugation of

351

fermentation broth at 8000 rpm for 10 min. After dehydration with anhydrous

352

magnesium sulfate, the isopropyl myristate phase was used for GC or GC-mass

353

spectrometer (GC-MS) analysis directly. An Agilent Technologies 7820A GC system

354

equipped with a HP-5 column (30 m × 0.25 mm × 0.25 µm). Sample (1 µl) was injected

355

in split mode (1:20) and the GC oven temperature program was applied with 3 mL/min

356

nitrogen as carrier gas: 100 °C for 1 min, 10 °C/min to 270 °C, an increase of 30 °C/min

357

to 320 °C, hold for 15 min. The injector temperature was 250 °C. The structure of

358

limonene was further analyzed using GC/MS-QP2010 Plus of Shimadzu (Japan) with

359

a quadrupole mass analyzer (QMA), HP-5MS column (30 m × 0.25 mm × 0.25 µm).

360

And the mass scan range was 50-900 m/z. Limonene standard (GC grade) was

361

purchased from TCL biotech Co., Ltd. (Shanghai, China). All of the GC analysis were

362

quantified using a seven-point calibration curve and the R2 coefficient for the 20


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363


calibration curve was higher than 0.99.

364 365

ASSOCIATED CONTENT

366

The Supporting Information is available free of charge via the Internet at

367

http://pubs.acs.org

368

Tables S1–S3; Figures S1–S5.

369

AUTHOR INFORMATION

370

Corresponding Author

371

*E-mail: [email protected]. Phone: +86-22-85356580. Fax: +86-22-27403389.

372

Author Contributions

373

S Cheng, X Liu, YJ Yuan, J Qiao and GR Zhao designed the experiments and analyzed

374

the data. S Cheng performed the experiments. J Wu and D Lei assisted in experiments

375

and analyzed the data. G Jiang and J Zhang provided strains and materials. S Cheng, X

376

Liu and GR Zhao drafted the manuscript. All of the authors have read and approved the

377

final manuscript.

378

Notes

379

The authors declare no competing financial interest.

380

ACKNOWLEDGMENTS

381

This study was financially supported by the National Key R&D Program of China

382

(2017YFD0201400).

383

ABBREVIATIONS

384

MVA, mevalonate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl 21



385

pyrophosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; NPP, neryl

386

diphosphate; LS, limonene synthase; CLB, conventional limonene biosynthesis; OLB,

387

orthogonal limonene biosynthesis.

388

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27



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For Table of Contents Use Only

513

Orthogonal Engineering of Biosynthetic Pathway for Efficient Production of Limonene

514

in Saccharomyces cerevisiae

515 516

Si Cheng†, Xue Liu†, Guozhen Jiang†, Jihua Wu†, Jin-lai Zhang†, Dengwei Lei†, Ying-

517

Jin Yuan†,‡, Jianjun Qiao†,‡, and Guang-Rong Zhao*,†,‡

518 519

†Frontier

520

Bioengineering (Ministry of Education), School of Chemical Engineering and

521

Technology, Tianjin University, Yaguan Road 135, Jinnan District, Tianjin, 300350,

522

China

523

‡SynBio

524

Engineering (Tianjin), Tianjin University, Yaguan Road 135, Jinnan District, Tianjin,

525

300350, China.

526

*Corresponding

527

Tel: +86-22-85356580; Fax: +86-22-27403389

Science Center for Synthetic Biology and Key Laboratory of Systems

Research Platform, Collaborative Innovation Centre of Chemical Science and

author: Guang-Rong Zhao ([email protected])

528

529

28


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Orthogonal Engineering of Biosynthetic Pathway for Efficient

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