Improved pinene production in a recombinant yeast by fusion linker optimization and chaperon coexpression

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https://doi.org/10.1007/s43393-021-00032-0 ORIGINAL ARTICLE

Improved pinene production in a recombinant yeast by fusion linker optimization and chaperon coexpression

Quanlu Ren^1,2 · Yaxi He^1,2 · Xinyao Lu^1,2 · Hong Zong^1,2 · Bin Zhuge^1,2

Received: 12 February 2021 / Revised: 16 April 2021 / Accepted: 20 April 2021 / Published online: 30 August 2021

Abstract

Pinene is an active natural monoterpene from plants and has important applications in flavorings, fragrances,and pesticides.

Especially, pinene dimers are regarded as renewable fuels with high density. However, the microbial pinene production was limited by the low activity pinene synthase. In this study, the pinene synthase activity was improved by fusion linker optimization and chaperon coexpression. To construct the pinene pathway in Saccharomyces cerevisiae, YPL062W gene was deleted to increase the MVA pathway precursor acetyl-CoA. Truncated 3-hydroxyl-3-methylglutaryl-CoA reductase (tHMG1), isopentenyl-diphosphate isomerase (IDI1), and farnesyl diphosphate synthase mutant (ERG20^F96W−N127W) were then integrated to improve the GPP pool. Pinene synthase tPt1 was expressed in the constructed engineered yeast, and the titer of pinene reached 0.166 mg/L. GPP is the direct precursor of pinene, ERG20^ww and tPt1 were fused by different linkers and orders to improve the accessibility of GPP. Pinene titer reached 9.94 mg/L by fusion these proteins in the order of ERG20^ww and tPt1 and with a flexible linker (G)₈. After that, several chaperons were coexpressed and the chaperon Sil1p improved the pinene titer to 10.2 mg/L with a yield of 1.63 mg/L·OD₆₀₀. The results presented here provide novel information on the applications of protein fusion and protein chaperons in microbial pinene production.

Keywords Pinene · Saccharomyces cerevisiae · Fusion protein · Oligopeptide linker · Chaperones

Introduction

Pinene is a natural monoterpene from plant with important applications in flavorings, fragrances and pesticides [1].

Pinene dimers are renewable jet fuel with high-volumetric energy and acts as an alternative of the aviation fuel JP-10 [2]. Synthetic biology provides an alternative route to get these high-value-added chemicals. Generally, monoterpenes (C₁₀) can be biosynthesized from the C₅ intermediates isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the MVA pathway (Fig. 1). As the first key node, many strategies have been performed to provide more acetyl-CoA flux [3–5]. Genetic truncated 3-hydroxyl- 3-methylglutaryl-CoA reductase gene (tHMG1) [6] or a

mutated HMG2^K6G [7] has been overexpressed to release the restriction on mevalonate node. The interconversion between IPP and DMAPP by isopentenyl-diphosphate isomerase (IDI1) was further enhanced to improve the formation of GPP [8].

In the pinene pathway, ERG20 catalyzes IPP and DMAPP to form GPP, the direct precursor of pinene, but it also further consumes GPP to generate FPP (Fig. 1). Rational design was then performed to change GPP affinity of the ERG20 to suppress the competitive consumption of GPP, leading to a significant increase in monoterpenes titer [9]. Relying on these strategies, Chen further introduced the pinene synthase (Pt30) into a recombinant Saccharomyces cerevisiae, leading to a 11.7 mg/L titer of pinene [10]. In contrast, the titer of microbial limonene (Fig. 1), another GPP-derivative monoterpene, reached 917.7 mg/L in shake-flask level [11].

Obviously, the transformation of GPP to pinene is the limiting step and improving pinene synthase (PS) activity is urgent in the microbial production of pinene.

Reducing the spatial distance between enzymes by protein fusion is a common strategy in synthetic biology. In this process, the oligopeptide linker between the enzymes

* Bin Zhuge Bzhuge@163.com

1 The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China

2 Research Center of Industrial Microbiology, School of Biotechnology, Jiangnan University, Wuxi 214122, China

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209 Systems Microbiology and Biomanufacturing (2022) 2:208–216

impacts protein expression and folding. In previous reports, fusion key enzymes by different linkers showed dramatically different effects on the titers of epoxycarotenoid violaxanthin [12], limonene [13], and geraniol [14], indicating the importance of the oligopeptide linker. Protein fusion was also adapted to pinene production [10, 15]. However, it is still unclear how different linkers affect pinene production.

Improving protein folding efficiency is another alternative strategy to promote the activity of metabolic enzymes [16].

Molecular chaperones are a category of proteins that assist the correct folding of proteins efficiently [17]. These chaperones have been applied in some cases to improve antibody [18] and enzyme productions [19] and chemical titers [20].

In S. cerevisiae, Hsp70s proteins are the most versatile chaperones. They can collaborate with the Hsp40s proteins and

nucleotide-exchange factors (NEFs) [17] to assist de novo folding, protein trafficking, and degradation of misfolded proteins. Besides, folding enzymes including PPIases also participate in the folding of heterologous protein [18].

In this study, the MVA pathway was enhanced in a recombinant S. cerevisiae. To improve the pinene titer, two pinene synthases were screened and then fused with ERG20^ww. Finally, the optimization of the oligopeptide linker sequence was performed and several molecular chaperones were introduced to improve the pinene titer.

Materials and methods

Strains, media, and plasmids

S. cerevisiae CEN.PK2-1C and plasmids p424, p416, pUG6 were stored in our laboratory. Pt1 (UniProtKB: Q84KL6) and Pt30 (UniProtKB: Q84KL3) from Pinus taeda (Loblolly pine) were codon optimized and synthesized by GENEWIZ (Suzhou, China). Escherichia coli JM109 was used for plasmid construction and cultured with LB medium (yeast extract 5 g/L, tryptone 10 g/L, NaCl 10 g/L) at 37 °C. YPD medium (yeast extract 10 g/L, tryptone 20 g/L, glucose 20 g/L) was used for S. cerevisiae growth and SD medium (yeast nitrogen base 6.7 g/L, glucose 20 g/L) adding different amino acids as required (Leucine 60 mg/L, Tryptophan 20 mg/L, Histidine 20 mg/L, Uracil 20 mg/L) was used for transformation.

ChloroP (http:// www. cbs. dtu. dk/ servi ces/ Chlor oP/), PSORT (http:// wolfp sort. org/) programs, and Uniprot (https:// www. unipr ot. org/) were used for the prediction of chloroplast signal peptide. N-terminus (48 amino acids) truncated Pt1 and Pt30 were inserted into the BamH I site of p424, resulting in p424-tPt1 and p424-tPt30, respectively.

ERG20^ww was obtained by fusion PCR (6). Then, ERG20^ww-(GS)₄ and (GS)₄ -ERG20^ww fragments were cloned into the sites of BamH I and Not I of p424-tPt1 (Fig. 2a) to construct p424-ERG20^ww-(GS)₄-tPt1 and p424- tPt1-(GS)₄-ERG20^ww, respectively. Further ERG20^ww-(G)₈, ERG20^ww-(GGGS)₂, ERG20^ww-(GSG)₃, ERG20^ww-(PT)₄, ERG20^ww-(EAAAK)₂ fragments were into the BamH I site of the p424-tPt1 to construct p424-ERG20^ww-(G)₈- tPt1, p424-ERG20^ww-(GGGS)₂-tPt1, p424-ERG20^ww- (GSG)₃-tPt1, p424-ERG20^ww-(PT)₄-tPt1, p424-ERG20^ww- (EAAAK)₂-tPt1, respectively.

The gene of chaperons Cpr5p, Jem1p, Kar2p, Sil1p, Scj1p, Ssa1p and Ydj1p were obtained from the S. cerevi- siae genome by PCR, and then were inserted into the BamH I site of p416-PTEF1 (Fig. 2b) to generate p416-Cpr5p, p416- Jem1p, p416-Kar2p, p416-Sil1p, p416-Scj1p, p416-Ssa1p and p416-Ydj1p, respectively.

Fig. 1 Overview of pinene biosynthetic pathway in S. cerevisiae.

tHMG1 truncated 3-hydroxyl-3-methylglutaryl-CoA reductase, IDI1 isopentenyl-diphosphate isomerase, ERG20^ww farnesyl diphosphate synthase mutant sites of F96W-N127W, PS Pinene synthase, LS Limonene synthase

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Engineering S. cerevisiae for pinene production All promoters (PTDH3, PTEF1, PGAP), terminators (TCYC1, T_PGK1, TADH1) and genes (tHMG1, IDI1) were derived from the genome of S. cerevisiae and obtained by PCR. KanMX was cloned from pUG6. The P_TEF1-tHMG1-T_CYC1, P_GAP- IDI1-TPGK1, P_TDH3-ERG20^ww-TADH1, and YPLup-KanMX- YPL_dn expression cassettes were constructed by fusion PCR [6, 21]. S. cerevisiae transformations were performed via the LiAc/PEG method [22]. The termini of the gene expression cassettes had at least 400 bp of homologous arms with the insertion sites of S. cerevisiae genome and overlaps between the gene expression cassettes were at least 50 bp.

The detailed construction process was illustrated in Fig. 3 and all strains used in this study were presented in Table 1, all primers were presented in Online Resource_2.

Pinene production in shake‑flask cultures

The recombinant S. cerevisiae strains were cultured in 10 mL SD medium with suitable nutrition deficiency at 30 °C for 24 h, and the culture was then transferred to a fresh 10 mL SD medium for another 24 h. Cultivation (1%) was then inoculated into a 50 mL SD medium with the addition of 10% dodecane and cultivated at 30 °C for 5 days. Parallel fermentation experiments were performed in triplicate.

Pinene extraction, identification, and quantification Ten milliliters of medium (the mixture of dodecane and medium) was sampled and centrifuged at 10,000×g for 10 min, and then 500 μL of the dodecane layer was transferred to another tube. Anhydrous sodium sulfate was added to remove water. The collected dodecane was

filtered by 0.22 μm organic membrane for gas phase and mass spectrometry analysis. Then, 1μL dodecane was ana- lyzed by GC–MS using an Agilent System 6890 gas chro- matograph (GC) with an Agilent 5975 quadrupole mass selective detector (MSD) equipped with an HP-5 column (30 m × 0.25 mm × 0.25 μm, Agilent, Santa Clara, CA, USA). The injector and FID detector temperature were set to 260 °C. The oven temperature was as follows: 70 °C for 3 min and sequentially increased at the rate of 5 °C/min to 100 °C for 3 min and 40 °C/min to 250 °C for 3 min. The split ratio was 20:1. Pinene standard (purchased from Sigma Aldrich) was used for quantification.

Results and discussion

Increasing GPP pool by enhancing the MVA pathway and precursor acetyl‑CoA

Codon optimized tPt1 and tPt30 (with the chloroplast target- ing peptide removed) from Pinus taeda were introduced into S. cerevisiae CEN.PK2-1C to construct the pinene pathway.

Amino acid-deficient medium was selected as the fermentation medium for two-phase fermentation. Only stable pas- sage of plasmids in the strain could ensure the survival of the cell in the amino acid-deficient medium. Even though no pinene peak signal was detected in the upper dodecane of the broth by GC-FID, the presence of pinene was verified by GC–MS (Fig. 4). These results indicate the successful construction of the pinene pathway in the recombinant S.

cerevisiae but the titer of pinene was still rare.

The intracellular GPP pool of S. cerevisiae is a common limiting factor in the production of monoterpenes [6]. A sufficient supply of acetyl-CoA, the precursor of GPP, is

Fig. 2 The plasmid maps of p424-tPt1 and p416-PTEF1

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a precondition to the high-level production of terpenoids.

YPL062W is a core promoter for the aldehyde dehydroge- nase 6 (ALD6), and ALD6 catalyzes acetaldehyde to ace- tate in the acetyl-CoA pathway [5]. Deletion of YPL062W suppresses the ALD6 activity, upregulating the mevalonate pathway and improving the titer of terpenoids [5]. Therefore, the YPL062W was deleted. 3-hydroxyl-3-methylglutaryl- CoA reductase (HMGR) catalyzes the conversion of HMG- CoA to mevalonate which is regulated at the transcriptional and posttranscriptional levels [23]. HMGR mutant without the N-terminal regulatory domain (tHMG1) can release the steroid-based negative feedback of the MVA pathway [6, 24]. The overexpression of tHMG1 has a significant effect in broadening the flux of the MVA pathway and increasing the synthesis of terpenes [11, 14, 24–26], which is beneficial to the synthesis of pinene. The isomerase IDI1 catalyzes the isomerization of IPP to DMAPP [8, 10]. To balance the supply of the crucial metabolic intermediate IPP/DMAPP in the MVA pathway, the rate-limiting genes tHMG1 and IDI1 were integrated into the δ sites (multiple copy integration sites). ERG20 mutant ERG20^ww with low GPP affinity can suppress the competitive consumption of GPP to unleash

more GPP was benefit to the titer of monoterpenes [9]. The copy number of IDI1 appeared to be important to the titer of geraniol [8]. Therefore, another copy IDI1 and ERG20^ww were integrated into the NTS sites (multiple copy integration sites). These modifications on the MVA pathway and introduction of tPt1 improved the titer of pinene to 0.166 mg/L (Fig. 5a).

Expression of ERG20^ww and tPt1 fusion protein boost the pinene production

The accessibility of GPP has a huge impact on the microbial productions of geraniol [14] and terpineol [6]. The fusion of ERG20^ww and PS minimizes losses of GPP through dif- fusion, degradation, or utilization by rival enzymes, leading to an improvement of pinene titer [10]. However, the fusion orders of ERG20^ww and PS exhibited opposite results in some reports [15, 27]. Here, ERG20^ww and tPt1 were fused by the flexible linker (GS)₄ with different orders (ERG20^ww- tPt1 or tPt1-ERG20^ww). As shown in Fig. 5a, the order of ERG20^ww-tPt1 showed a better pinene titer of 7.6 mg/L.

Fig. 3 Schematic diagram of MVA pathway enhanced strain construction. a tHMG1 and IDI1 were integrated into δ sequences. b ERG20^ww and IDI1 were integrated into rDNA sequences. c YPL062W was substituted by KanMX

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Oligopeptide linker impacts enzyme expression and activity, showing dramatically different effects on the titer of limonene [13] and geraniol [14]. However, the impact of different linkers on pinene production was rarely reported [10, 15]. Two rigid and three flexible linkers with similar length were selected and tried to improve the pinene titer.

Rigid linkers, like (PT)n [28] and (EAAAK)n [29], normally prevent unfavorable interaction and guarantee a certain distance forming between domains [30]. In contrast, flexible linkers give more space to domains for acting [31]. Here, the flexible linkers, rich in glycine, showed gradual improve- ments in the titer of pinene (Fig. 5b). These results indicate that the flexibility of the ERG20^ww-tPt1 is important for their folding and activity. Besides, glycine has a smaller

side chain than serine, which may provide more flexibility to the fusion enzymes and then improves the titer of pinene.

The fusion of ERG20^ww-(G)₈-tPt1 showed the highest pinene titer to 9.94 mg/L (Fig. 5b).

The effects of chaperones on pinene production Chaperons assist protein folding and translocations in cells [32]. To further improve ERG20^ww-tPt1 activity and the titer of pinene, seven different chaperones were coexpressed with ERG20^ww-(GS)₄-tPt1. As shown in Fig. 5c, the co-expression of Sil1p, Cpr5p, Jem1p, or Scj1p with ERG20^ww-(GS)₄- tPt1 showed positive effects on the pinene titer. The titer of

Fig. 4 GC–MS analysis of the upper dodecane of fermentaton broth and the pinene standard. a Chromatogram for the fermentaton broth and the standard of pinene. b GC–MS spectra of the peak strain

(RT = 3.86 min) produced by engineered yeast strain. c GC–MS spectra for the pinene standard

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pinene of the recombinant harboring ERG20^ww-(GS)₄-tPt1 and Kar2p was not changed.

Kar2p is the main HSP70 chaperone protein in the endoplasmic reticulum with multiple functions in protein folding [33] and unfolded protein response (UPR) regulation [34]. It has promoted the titer of single-chain antibodies (scFv) by improving protein folding [35]. In S. cerevisiae, high-level expression of heterologous proteins may cause the accumulation of unfolded protein, thereby inducing the UPR to reduce the amount of unfolded protein [36]. In this process, unfolded protein binds Kar2p competitively to release Ire1p from the Kar2p/Ire1p complex, leading to the oligomeriza- tion of Ire1p and the induction of UPR [37]. Overexpression of Kar2p may inhibit the release and activation of Ire1p [38], further suppressing the induction of UPR. Therefore, overexpression of Kar2p showed a negative impact on the production of human IgG [18]. In other cases, high-level Kar2p has no significant effect on the production of human granulocyte colony-stimulating factor (GCSF), acid phosphatase (PHO), and bovine pancreatic trypsin inhibitor (BPTI) [39], which was similar to this study.

Kar2p also participates in the polypeptide chain binding and release cycle via an ATP-dependent manner [40]. Sil1p

functions as NEFs in the cycle of Kar2p to promote ADP release [41] and has been applied in promoting the production of human IgG [18]. The intracellular amount of Sil1p is much lower than Kar2p, bring about that overexpression of Sil1p was more effective to improve the reaction cycle of Kar2p [18]. Here, overexpression of Sil1p resulted in the optimal titer of pinene to 10.2 mg/L, which was improved by 33.5% (Fig. 5c).

Overexpression of Jem1p has significantly increased the production of recombinant human albumin, but Scj1p has no obvious effect on it [16]. Jem1p is an endoplasmic reticulum membrane protein [42], whereas Scj1p is located in the lumen of the endoplasmic reticulum [43]. The difference in the location of Jem1p and Scj1p may contribute to their performance in improving the titer of pinene [16]. PPIases involved in the rearrangement of peptidyl-prolyl bonds in unfolded proteins [44]. They have been applied in the productions of immunoglobulin domains [45] and some spe- cific antibodies [18]. Cpr5p is a PPIases and showed a 20%

improvement in the titer of pinene through co-expressing with the ERG20^ww-(GS)₄-tPt1 (Fig. 5c).

However, these positive effects were not observed in the co-expression of the ERG20^ww-(G)₈-tPt1 with these

Table 1 Strains used in this

study Strains Descriptions Source

E.coli JM109 General cloning host TaKaRa

S. cerevisiae PK2-1C MATa ura3-52 trp1-289 leu2-3_112 his3Δ1 MAL2-8c SUC2 Lab stored Sc.MPS S. cerevisiae PK2-1C YPL062WΔ::KanMX δ::PTEF1-

tHMG1,P_GAP-IDI1,LEU2 NTS::P_TDH3-ERG20^ww,P_PGK1- IDI1,HIS

This study

Sc.PS1 Sc.MPS, p424-tPt1 This study

Sc.PS2 Sc.MPS, p424-tPt1-(GS)₄-ERG20^ww This study

Sc.PS3 Sc.MPS, p424-ERG20^ww-(GS)₄-tPt1 This study

Sc.PS4 Sc.MPS, p424-ERG20^ww-(G)₈-tPt1 This study

Sc.PS5 Sc.MPS, p424-ERG20^ww-(GSG)₃ -tPt1 This study

Sc.PS6 Sc.MPS, p424-ERG20^ww-(GGGS)2-tPt1 This study

Sc.PS7 Sc.MPS, p424-ERG20^ww-(PT)₄-tPt1 This study

Sc.PS8 Sc.MPS, p424-ERG20^ww-(EAAAK)₂-tPt1 This study

Sc.PS9 Sc.PS3, p416-Sil1p This study

Sc.PS10 Sc.PS3, p416-Cpr5p This study

Sc.PS11 Sc.PS3, p416-Jem1p This study

Sc.PS12 Sc.PS3, p416-Scj1p This study

Sc.PS13 Sc.PS3, p416-Kar2p This study

Sc.PS14 Sc.PS3, p416-Ydj1p This study

Sc.PS15 Sc.PS3, p416-Ssa1p This study

Sc.PS16 Sc.PS4, p416-Kar2p This study

Sc.PS17 Sc.PS4, p416-Jem1p This study

Sc.PS18 Sc.PS4, p416-Sil1p This study

Sc.PS19 Sc.PS4, p416-Scj1p This study

Sc.PS20 Sc.PS4, p416-Cpr5p This study

Sc.PS21 Sc.PS4, p416-Ssa1p This study

Sc.PS22 Sc.PS4, p416-Ydj1p This study

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chaperons (Fig. 5d). Obviously, the slight structure differ- ences between ERG20^ww-(GS)₄-tPt1 and ERG20^ww-(G)₈- tPt1 were responsible for these unexpected results. There are many kinds of molecular chaperones and they have certain functional specificity [17, 46]. The fusion protein of pinene synthase (tPt1) and ERG20^ww, which has larger size and the structure changes, may cause the molecular chaperone to misbind and fold the protein incalculably, resulting in an inestimable effect.

Ssa1p is a chaperon in the cytoplasm, involved in the protein folding and trafficking [47]. Ydj1p is a cochaperone protein of Ssa1p and participates in the activity of Ssa1p [48].

At present, there are still no reports on their applications in improving protein folding. Here, they showed negative impacts on the titer of pinene production. Ssa1p decreased

the titer of pinene of the ERG20^ww-(GS)₄-tPt1 strain by 29%

(Fig. 5c). Coexpression of Ydj1p and ERG20^ww-(G)₈-tPt1 suppressed the titer of pinene by 50.5% (Fig. 5d). Besides, all of the chaperons tested here had no obvious effect on cell growth.

In conclusion, the MVA pathway and the supplement of the precursor acetyl-CoA were enhanced in a recombinant S. cerevisiae. Two pinene synthases from P. taeda were applied and the tPt1 was more conducive to the production of pinene. The pinene titer was improved by 58.9 times to 9.94 mg/L by optimizing the order and linker of the ERG20^ww and tPt1. Finally, several chaperons were coexpressed with ERG20^ww-(GS)₄-tPt1 and the pinene titer and yield were further improved to 10.2 mg/L and 1.63 mg/L·OD₆₀₀, respectively. The pinene titer obtained

Fig. 5 Pinene production of engineered yeast. a Effects of different fusion orders of ERG20^ww and tPt1 on the titer of pinene. b Effects of different linkers on the titer of pinene. c Effects of different chaper-

ones on the titer of pinene titer in Sc.PS3. d Effects of different chaperones on the titer of pinene titer in Sc.PS4. The data are expressed as the mean ± SD of three independent experiments

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here was similar to the highest microbial pinene level in S.

cerevisiae reported [10]. In contrast, the yield of pinene was obviously higher than ever reported [10]. Therefore, improving cell growth by condition optimizations will be benefit to the microbial pinene level in future (Fig. 5).

Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s43393- 021- 00032-0.

Funding This work was supported by the National Natural Science Foundation of China (No. 31970033).

Data availability All data generated or analysed in this study are included in this published article and its supplementary information files.

Declarations

Conflict of interest The authors declared that they had no conflict of interest.

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