Enhancing linalool production by engineering oleaginous yeast Yarrowia lipolytica

Accepted Manuscript Short Communication Enhancing Linalool Production by Engineering Oleaginous Yeast Yarrowia lipolytica Xuan Cao, Liu-Jing Wei, Jia-Yu Lin, Qiang Hua PII: DOI: Reference:

S0960-8524(17)31009-X http://dx.doi.org/10.1016/j.biortech.2017.06.105 BITE 18342

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

31 March 2017 16 June 2017 17 June 2017

Please cite this article as: Cao, X., Wei, L-J., Lin, J-Y., Hua, Q., Enhancing Linalool Production by Engineering Oleaginous Yeast Yarrowia lipolytica, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech. 2017.06.105

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Enhancing Linalool Production by Engineering Oleaginous Yeast Yarrowia

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lipolytica

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Xuan Caoa# ∙ Liu-Jing Weia #∙ Jia-Yu Lina ∙ Qiang Huaa,b*

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a

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Technology, 130 Meilong Road, Shanghai 200237, PR China

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b

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Meilong Road, Shanghai 200237, China

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Qiang Hua, [email protected]

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# These two authors contributed equally to this work

State Key Laboratory of Bioreactor Engineering, East China University of Science and

Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130

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* Corresponding Author

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E-mail: [email protected]

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Address:

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State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China

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Abstract

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In this study, stepwise increases in linalool production were obtained by combining

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metabolic engineering and process optimization of an unconventional oleaginous yeast

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Yarrowia lipolytica. The linalool synthetic pathway was successfully constructed by

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heterologously expressing a codon-optimized linalool synthase gene from Actinidia

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arguta in Y. lipolytica. To enhance linalool productivity, key genes involved in the

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mevalonate pathway were overexpressed in different combinations. Moreover, the

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overexpression of mutant ERG20F88W-N119W gene resulted in further linalool production.

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A maximum linalool level of 6.96 ± 0.29 mg/L was achieved in shake flasks, which was

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the highest linalool production ever reported in yeasts.

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Keywords: linalool, Yarrowia lipolytica, linalool synthase, MVA pathway

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1. Introduction Linalool, an acyclic monoterpene alcohol, is one of the main compositions of many

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essential oils. Nearly 70% of the terpenoids of floral scents are represented by linalool.

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Linalool is used as a fragrance and flavour agent to be added to cosmetic products and

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household detergent as well as processed food and beverages. In addition, it is also a

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critical precursor for producing vitamin E, vitamin A, farnesol, citronellol and ionones.

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Besides, it has antifungal, antimicrobial and insecticidal properties (Herman et al., 2016;

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Aprotosoaie et al., 2014; Beier et al., 2014).

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At present, microorganisms have been successfully used for heterologous

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biosynthesis of terpenoids, such as lycopene produced in Escherichia coli, artemisinic

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acid biosynthesis in Saccharomyces cerevisiae and so on (Yoon et al., 2006; Ro et al.,

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2006). Linalool biosynthesis was also reported in several studies. Fusion expression of

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LIS (linalool synthase) and FPPS (FPP synthase) in S. cerevisiae and cultured in a 3L

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fermenter could resulted in an enhanced linalool production to 240 μg/L (Deng et al.,

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2016). Additionally, overexpression of tHMG1 and down regulation of ERG9 improved

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linalool titers 3-fold to 95 μg/L in S. cerevisiae (Amiri et al., 2016).

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Y. lipolytica is a dimorphic, non-pathogenic ascomycetous yeast (Bouchedja et al.,

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2017). It is a potential candidate host for terpenes production. In Y. lipolytica,

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monoterpenes can be synthesized through the mevalonate pathway (MVA), from

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dimethylallyl diphosphate (DMAPP) and its isomer isopentenyl diphosphate (IPP).

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Geranyl diphosphate (GPP) is a direct precursor for monoterpene production. However,

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Y. lipolytica cannot endogenously produce linalool for lacking LIS, which converts GPP

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to linalool. Here, we demonstrated that engineering linalool biosynthesis by

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overexpressing LIS gene from Actinidia arguta in Y. lipolytica. Additionally, metabolic

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engineering and process optimization were combined to further improve linalool

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production. Finally, a linalool-overproducer strain was developed with approximately

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76-fold improvement (up to 6.96 ± 0.29 mg/L) in comparison with the reference strain,

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which provides a good example to increase monoterpene production in Y. lipolytica.

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2. Materials and methods

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2.1. Strains, vectors, chemicals and culture media

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The Y. lipolytica strain Po1f (Leu-, Ura-) was utilized for target genes expressing in

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this study. Po1f strain and the plasmids pINA1269 and pINA1312 were depicted

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previously (Madzak et al., 2004). The chemicals and culture media used were based on

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those described in Cao et al. (2016).

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2.2. Mutagenesis

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Site-directed mutagenesis of ERG20 was conducted by using the

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KOD-Plus-Mutagenesis Kit from TOYOBO Co., Ltd. (Osaka, Japan).

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2.3. Plasmids construction

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The gene encoding linalool synthase (LIS, GenBank ID: GQ338153.1) from A. arguta was codon optimized and synthesized by GeneRay Biotech. The methods for

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plasmid construction were well described in the Supplementary file. All the vectors

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were constructed using the One Step Cloning Kit from Vazyme Biotech Co., Ltd.

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(Nanjing, China). The vectors used in this study are summarized (Supplement Table 1).

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2.4. Strain construction

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The methods for construction were described in the Supplementary file. The strains

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constructed in this study are listed (Supplement Table 2).

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2.5. Yeast cultivation

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Seed medium and fermentation medium used in the shake flask and fermenter

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culture were based on those described in Cao et al. (2016). Strains were cultured at

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30°C and 220 rpm for 2 days. All the flask fermentation results represented the means ±

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S.D. of three independent experiments. At the same time, glucose, glycerol, fructose

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and citrate were chosen as sole carbon source or mixed carbon sources to investigate the

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influence of different carbon sources on linalool production.

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2.6. Analysis

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OD600 and dry cell weight (DCW) were detected according to Cao et al. (2016).

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The method for linalool analysis was described in the Supplementary file.

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3. Results and discussion

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3.1. Heterologously overexpressing LIS gene in Y. lipolytica for (S)-linalool

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biosynthesis

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To heterogeneously synthesize linalool, codon-optimized LIS gene from A. arguta was integrated into the genome of Y. lipolytica, resulting in an

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(S)-(+)-linalool-producing strain CXY01. This engineered strain was cultured in YPD

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medium with an overlay of 1 mL dodecane in shake flasks for three days and the final

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linalool concentrations were quantified by HPLC. The data showed that albeit very low,

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CXY01 strain could successfully synthesize linalool at about 0.09 mg/L (Fig. 1). This

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linalool production level was to some extent higher than that in a recently reported S.

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cerevisiae strain harbouring the same LIS gene (60 μg/L) (Deng et al., 2016).

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In this study, Y. lipolytica strain with the introduced LIS gene was confirmed to be

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able to accumulate linalool from geranyl diphosphate, which is consistent with other

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works done with yeasts. In our previous work, interestingly, neryl diphosphate (NPP)

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instead of GPP was found to serve as the precursor for limonene synthesis in Y.

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lipolytica (Cao et al., 2016). The different results on precursors used for linalool or

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limonene biosynthesis could also partly confirm the hypothesis that GPP was the

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precursor of acyclic monoterpene whereas NPP was the precursor of monocyclic

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monoterpene in yeasts (Liu, 2013).

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3.2. Overexpression of endogenous HMG1 gene enhanced linalool production

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To improve linalool production, the incremental overexpression of the rate limiting

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enzymatic step in the MVA pathway, namely HMG1, was conducted. The HMG1 gene

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encodes 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and its

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overexpression is commonly considered to be beneficial for the isoprenoid production

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in yeast. For example, overexpression of tHMG1 in S. cerevisiae increased the linalool

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production by more than 50% (Amiri et al., 2016). Similarly, overexpression of the

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HMG1 gene in Y. lipolytica led to a significant improvement in the yields of lycopene

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and limonene (Matthäus et al., 2014; Cao et al., 2016). Herein, the endogenous HMGR

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was successfully overexpressed based on the construction of plasmid p1269-HMG1 and

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introduction of this plasmid into the genome of strain CXY01. As expected, the

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resulting strain of CXY21 showed significant increase in linalool production (0.52 ±

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0.01 mg/L), approximately 4.7-fold improvement over the linalool level observed in the

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reference strain CXY01 (Fig. 1).

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3.3. Enhancement of linalool production by co-overexpressing HMG1 with other MVA

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pathway genes or the IDI1 gene

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Our previous study suggested that the co-overexpression of HMG1 with some other

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MVA pathway genes could result in further enhancement of limonene production in Y.

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lipolytica (Cao et al., 2016). Similar attempts were also made in this study to investigate

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the effects of the co-overexpression of the HMG1 gene together with

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phosphomevalonate kinase-encoding gene ERG8, acetoacetyl-CoA thiolase-encoding

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gene ERG10, mevalonate kinase-encoding gene ERG12, or mevalonate diphosphate

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decarboxylase-encoding gene ERG19. Increases in final linalool concentrations, ranging

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from 27% to 62%, were observed for the resulting strains of CXY31, CXY32, CXY33,

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and CXY34 compared to the CXY21 strain (Fig. 1), among which the overexpression of

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both the HMG1 and ERG12 genes showed the highest linalool titer of 0.84 ± 0.06 mg/L.

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Interestingly, only a combination of HMG1 and ERG12 overexpression enhanced the

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production of limonene, a monocyclic monoterpene, whereas the co-overexpression of

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HMG1 with ERG8/ERG10/ERG19 gene only resulted in insignificant changes in

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limonene production (Cao et al., 2016).

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Isopentenyl diphosphate isomerase (encoded by the IDI1 gene) catalyzes the

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isomerization of IPP to DMAPP, and acts crucially in the distribution of GPP and FPP

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fluxes. It is an effective strategy to enhance the expression of this enzyme for improved

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terpenoid production (Zhao et al., 2016). To investigate the effect of IDI1

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overexpression on linalool production in Y. lipolytica, the CXY01 strain was engineered

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by introducing 1-3 additional copies of the IDI1 gene, resulting in strains CXY22,

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CXY23, and CXY24, respectively. The linalool productions in both the CXY22 and

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CXY23 strains reached approximately 0.43 mg/L (4.6-fold of the CXY01 strain). The

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final linalool concentration was, however, increased to 0.75 mg/L in CXY24 harboring

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three additional IDI1 genes, implying that balancing the IPP and DMAPP pools might

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play an important role in enhancing carbon flux to monoterpene biosynthesis. It is also

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very interesting to study the effect of co-overexpressed HMG1 and IDI1 genes in the

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reference strain CXY01. Surprisingly, the simultaneous overexpression of these two

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genes in strain CXY35 significantly improved linalool level to 1.44 ± 0.04 mg/L, almost

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16-fold of that of the reference strain CXY01 and 2.8-fold of that of CXY21 strain with

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overexpressed HMG1 alone (Fig. 1). Again, this result was inconsistent with limonene

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production in Y. lipolytica with the same genetic manipulation (Cao et al., 2016),

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probably due to the different precursor responsible for the biosynthesis of linalool or

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limonene.

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3.4. Investigation of carbon sources for linalool production in Y. lipolytica

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The effects of different carbon sources on linalool synthesis in strain CXY35

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were investigated to obtain a favorable medium composition of carbon source for future

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fermentation optimization. Glucose, glycerol, fructose, or citrate (20 g/L each) was

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employed as the sole carbon source to test the cell growth and linalool production. The

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culture with citrate exhibited the highest linalool titer and content of 2.52 ± 0.17 mg/L

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and 356 ± 27 μg/g DCW, respectively, which might be attributed to the enhancement of

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cytosolic acetyl-CoA as well as the MVA pathway flux. Glycerol was the second

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favorable carbon source for linalool production in this study (Fig. 1). To overcome the

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limitation of low cell density when cultivated on citrate, a mixed carbon source of 10

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g/L citrate together with 10 g/L of glucose, glycerol, or fructose was investigated and a

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significant increase in cell growth was observed (i.e. 12.1 g/L of final cell concentration

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in citrate-glucose medium in comparison with 7.1 g/L cell concentration in citrate

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medium). Accordingly, an average increase of 75% in final linalool concentration was

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obtained for all three cases of mixed carbon sources, whereas linalool contents kept

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almost unchanged compared to the culture with 20 g/L citrate as the sole carbon source

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(Fig. 1).

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Supplementation with auxiliary carbon source such as pyruvate is generally

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favorable for the production of monoterpene in Y. lipolytica, which was also evidenced

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by marked increases in linalool synthesis in this study. Although the addition of 2 g/L

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pyruvate did not change the performance of linalool production, both final linalool

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concentration and linalool content increased significantly when 4 g/L or more pyruvate

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was supplemented. When the amount of pyruvate added was 8 g/L, linalool

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concentration and content reached 4.60 ± 0.17 mg/L and 580 ± 33 μg/g, approximately

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1.8-fold and 1.6-fold respectively of those obtained in citrate-only medium, suggesting

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a noticeable effect of pyruvate supplementation on linalool production in Y. lipolytica.

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3.5 Effect of overexpression of ERG20F88W-N119W in CXY35 strain

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GPP is the direct precursor of linalool and the availability of GPP in yeast was

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usually limited. In fact, the enzyme ERG20p (encoded by ERG20) functions as both

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GPPS (GPP synthase) and FPPS, which causes difficulty in separating both synthases

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and overexpressing GPPS only. Recently, researchers have been working on modifying

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ERG20 gene for high GPP pool in S. cerevisiae. Ignea et al. reported that the mutations

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in ERG20p (i.e. F96W and N127W) significantly affected the production phenotype in

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S. cerevisiae (Ignea et al., 2014). To ensure adequate availability of GPP, a similar

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strategy was also employed to modify ERG20p in Y. lipolytica. Based on amino acid

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alignment analysis of Y. lipolytica ERG20p (GI: CAG79180.1) and S. cerevisiae

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ERG20p (GI: CAA89462.1), amino residues F88 and N119 of Y. lipolytica ERG20p

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were identified to create double mutations of F88W and N119W (Fig. 2). Introducing

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mutant ERG20F88W-N119W into Y. lipolytica strain CXY01 resulted in strain CXY25 with

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about 0.56 mg/L of linalool production. However, linalool production was notably

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elevated when three genes of HMG1, IDI1, and ERG20F88W-N119W were simultaneously

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overexpressed in Y. lipolytica. The resulting strain CXY36 could yield 5.34 ± 0.19 mg/L

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linalool (334 ± 10 μg/g DCW) in YPD medium, which was further increased to 6.96 ±

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0.29 mg/L (939 ± 60 μg/g DCW), the highest linalool production in yeasts to our

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knowledge, when cultivated in shake flask containing YP medium with 20 g/L citrate

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and 8 g/L pyruvate as carbon sources.

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4. Conclusions

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In summary, efforts have been made to enhance linalool production in Y. lipolytica

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with focuses on the overexpression of critical pathway enzymes and the optimization of

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carbon sources. The final linalool production of 6.96 mg/L (939 μg/g DCW) in shake

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flask culture is the highest titer ever reported on linalool biosynthesis by the engineered

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yeast strains. The results obtained in this study therefore demonstrated that Y. lipolytica

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provides a compelling platform for the overproduction of linalool and other acyclic

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monoterpenes.

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Declarations

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Competing interests

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The authors declare that they have no competing interests.

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Funding

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This work was financially supported by National Natural Science Foundation of China

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(21576089).

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Acknowledgements

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We thank Prof. Catherine Madzak (Institut National de la Recherche

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Agronomique/AgroParisTech, France) for the auxotrophic Y. lipolytica strain Po1f and

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the plasmids pINA1269 and pINA1312.

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Appendix A. Supplementary data

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References

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1. Amiri, P., Shahpiri, A., Asadollahi, M.A., Momenbeik, F., Partow, S., 2016.

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Metabolic engineering of Saccharomyces cerevisiae for linalool production.

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Biotechnol Lett. 38: 503.

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2. Aprotosoaie, A. C., Hăncianu, M., Costache, I. I., & Miron, A. (2014). Linalool: a

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review on a key odorant molecule with valuable biological properties. Flavour and

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fragrance journal. 29(4), 193-219.

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3. Beier, R.C., Byrd, J.A., II, Kubena, L.F., Hume, M.E., McReynolds, J.L., Anderson,

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R.C., Nisbet, D.J., 2014. Evaluation of linalool, a natural antimicrobial and

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insecticidal essential oil from basil: Effects on poultry. Poult Sci. 93 (2): 267-272.

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5. Cao, X., Lv, Y.B., Chen, J., Imanaka, T., Wei, L.J., Hua, Q., 2016. Metabolic

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engineering of oleaginous yeast Yarrowia lipolytica for limonene overproduction.

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Biotechnol Biofuels. 9:214.

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6. Deng, Y., Sun, M.X., Xu, S., Zhou, J.W., 2016. Enhanced (S)-linalool production by

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fusion expression of farnesyl diphosphate synthase and linalool synthase in

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Saccharomyces cerevisiae. J Appl Microbiol. 121(1): 187-195.

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7. Herman, A., Tambor, K., Herman, A., 2016. Linalool affects the antimicrobial efficacy of essential oils. Curr Microbiol. 72: 165.

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8. Ignea, C., Pontini, M., Maffei, M.E., Makris, A.M., Kampranis, S.C., 2014.

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Engineering Monoterpene Production in Yeast Using a Synthetic Dominant

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Negative Geranyl Diphosphate Synthase. ACS Synth. Biol. 3 (5): 298–306.

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9. Liu, J.D., 2013. Key issues in the metabolic engineering of Saccharomyces

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cerevisiae for monoterpene production [D]. Jiangnan University. 10. Madzak, C., Gaillardin, C., Beckerich, J.M., 2004. Heterologous protein expression

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and secretion in the non-conventional yeast Yarrowia lipolytica: a review. J.

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Biotechnol. 109 (1-2): 63-81.

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11. Matthäus, F., Ketelhot, M., Gatter, M., Barth, G., 2014. Production of lycopene in

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the non-carotenoid producing yeast Yarrowia lipolytica. Appl. Environ. Microbiol.

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12. Ro, D.K., Paradise, E.M., Ouellet, M., Fisher, K.J., Newman, K.L., Ndungu, J.M.,

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Ho, K.A., Eachus, R.A., Ham, T.S., Kirby, J., Chang, M.C.Y., Withers, S.T., Shiba,

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Y., Sarpong, R., Keasling, J.D., 2006. Production of the antimalarial drug precursor

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artemisinic acid in engineered yeast. Nature. 440: 940-943.

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13. Yoon, S.H., Lee, Y.M., Kim, J.E., Lee, S.H., Lee, J.H., Kim, J.Y., Jung, K.H., Shin,

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Y.C., Keasling, J.D., Kim, S.W., 2006. Enhanced lycopene production in

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Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl

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diphosphate from mevalonate. Biotechnol Bioeng. 94 (6): 1025-1032.

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14. Zhao, J.Z., Bao, X.M., Li, C., Shen, Y., Hou, J., 2016. Improving monoterpene

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geraniol production through geranyl diphosphate synthesis regulation in

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Saccharomyces cerevisiae. Applied Microbiology and Biotechnology. 100(10):

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4561-4571.

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Figures captions

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Fig. 1 Quantitative analysis of linalool production in engineered Y. lipolytica strains and

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effects of different carbon sources on linalool production in shake flask culture.

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Linalool productions were analyzed in the engineered strains Po1f, CXY01, CXY21,

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CXY22, CXY23, CXY24, CXY31, CXY32, CXY33, CXY34 and CXY35. The

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engineered strain CXY35 was cultured in YP medium with Glu: 20 g/L glucose; Gly: 20

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g/L glycerol; Fru: 20 g/L fructose; Cit: 20 g/L citrate; Cit-pyr-2: 20 g/L citrate and 2 g/L

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pyruvate; Cit-pyr-4: 20 g/L citrate and 4 g/L pyruvate; Cit-pyr-8: 20 g/L citrate and 8

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g/L pyruvate; Cit-Glu: 10 g/L citrate and 10 g/L glucose; Cit-Gly: 10 g/L citrate and 10

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g/L glycerol; Cit-Fru: 10 g/L citrate and 10 g/L fructose. The strain was grown in all the

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mediums for 48 h. Three repeats were conducted for each strain, and error bars

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represent standard deviations.

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Fig. 2 Amino acid alignment of Y. lipolytica ERG20p and S. cerevisiae ERG20p.

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Highlighted in deep blue are homologous residues. Amino acid sequence of ERG20p

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(CAG79180.1) from Y. lipolytcia and amino acid sequence of ERG20p (CAA89462.1)

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from S. cerevisiae were blasted by clustalx and the final format was generated by

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DNAMAN. The red frames represent the mutated sites.

279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311

Fig.1.

312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329

CAG79180.1 CAA89462.1 Consensus

......MSKAKFESVFPRISEELVQLLRDEGLPQDAVQWFSDSLQYNCVGGKLNRGLSVV MASEKEIRRERFLNVFPKLVEELNASLLAYGMPKEACDWYAHSLNYNTPGGKLNRGLSVV f vfp eel l g p a w sl yn ggklnrglsvv

54 60

CAG79180.1 CAA89462.1 Consensus

DTYQLLTGK..KELDDEEYYRLALLGWLIELLQAFFLVSDDIMDESKTRRGQPCWYLKPK DTYAILSNKTVEQLGQEEYEKVAILGWCIELLQAYFLVADDMMDKSITRRGQPCWYKVPE dty l k l eey a lgw iellqa flv dd md s trrgqpcwy p

112 120

CAG79180.1 CAA89462.1 Consensus

VGMIAINDAFMLESGIYILLKKHFRQEKYYIDLVELFHDISFKTELGQLVDLLTAPEDEV VGEIAINDAFMLEAAIYKLLKSHFRNEKYYIDITELFHEVTFQTELGQLMDLITAPEDKV vg iaindafmle iy llk hfr ekyyid elfh f telgql dl taped v

172 180

CAG79180.1 CAA89462.1 Consensus

DLNRFSLDKHSFIVRYKTAYYSFYLPVVLAMYVAGITNPKDLQQAMDVLIPLGEYFQVQD DLSKFSLKKHSFIVTFKTAYYSFYLPVALAMYVAGITDEKDLKQARDVLIPLGEYFQIQD dl fsl khsfiv ktayysfylpv lamyvagit kdl qa dvliplgeyfq qd

232 240

CAG79180.1 CAA89462.1 Consensus

DYLDNFGDPEFIGKIGTDIQDNKCSWLVNKALQKATPEQRQILEDNYGVKDKSKELVIKK DYLDCFGTPEQIGKIGTDIQDNKCSWVINKALELASAEQRKTLDENYGKKDSVAEAKCKK dyld fg pe igkigtdiqdnkcsw nkal a eqr l nyg kd e kk

292 300

CAG79180.1 CAA89462.1 Consensus

LYDDMKIEQDYLDYEEEVVGDIKKKIEQVDESRGFKKEVLNAFLAKIYKRQ IFNDLKIEQLYHEYEESIAKDLKAKISQVDESRGFKADVLTAFLNKVYKRS d kieq y yee d k ki qvdesrgfk vl afl k ykr

343 351

Fig.2.

330 331 332 333 334 335 336

1 Y. lipolytica was metabolically engineered to produce linalool. 2 Overexpression of genes in MVA pathway and IDI1 enhanced linalool production. 3 Overexpressing ERG20F88W-N119W gene further led to a high linalool yield. 4 Citrate was beneficial to linalool accumulation in Y. lipolytica.