Regeneration of NADPH Coupled with HMG-CoA Reductase Activity

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Regeneration of NADPH coupled with HMG-CoA reductase activity increases squalene synthesis in Saccharomyces cerevisiae Kalaivani Paramasivan, and Sarma Mutturi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02945 • Publication Date (Web): 28 Aug 2017 Downloaded from http://pubs.acs.org on August 29, 2017

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Journal of Agricultural and Food Chemistry

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Regeneration of NADPH coupled with HMG-CoA reductase activity increases squalene

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synthesis in Saccharomyces cerevisiae

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Kalaivani Paramasivan†,‡ and Sarma Mutturi†,‡,*

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Research Institute, Mysore, India.

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Microbiology and Fermentation Technology Department, CSIR-Central Food Technological

Academy of Scientific and Innovative Research, Mysore, New Delhi, India.

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ABSTRACT

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Although overexpression of tHMG1 gene is a well-known strategy for terpene synthesis in

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Saccharomyces cerevisiae, the optimal level for tHMG1p has not been established. In the present

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study, it was observed that two copies of tHMG1 gene on a dual gene expression cassette

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improved squalene synthesis in laboratory strain by 16.8-fold in comparison to single copy

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expression. It was also observed that tHMG1p is limited by its co-factor (NADPH), as the

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NADPH regenerating genes’ viz., ZWF1 and POS5 (full length and without mitochondrial

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presequence) has led to its increased enzyme activity. Further, it was demonstrated that

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overexpression of full length POS5 has improved squalene synthesis in cytosol. Finally, when

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tHMG1 and full length POS5 were co-overexpressed there was a net 27.5-fold increase in

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squalene when compared to control strain. These results suggest novel strategies to increase

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squalene accumulation in S. cerevisiae.

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KEYWORDS: S. cerevisiae, Squalene, tHMG1, ZWF1, POS5

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INTRODUCTION:

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S. cerevisiae is a well-studied eukaryotic model organism for production of terpenes due to the

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presence of specific precursor molecules in its sterol pathway.1-3 S. cerevisiae is advantageous

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over its prokaryotic counterpart, E. coli, for production of terpenoids owing to its ability to

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express cytochrome P450 oxidase and presence of cognate reductase enzymes.4 S. cerevisiae has

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been a cell factory for biosynthesis of diverse terpene molecules.5 Squalene which is an

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intermediate of ergosterol pathway, is an unsaturated triterpene hydrocarbon (C30H50) and is a

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precursor for sterol molecules in S. cerevisiae. Squalene has a demand of >1200 tonnes per year6

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and has an estimated market size of 211 million USD by 2021.7 Squalene is used in cosmetic

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industry as a skin moisturizer and also acts as antioxidant. In pharmaceutical industry, it is used

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as adjuvant for vaccines and also used for drug delivery. Squalene has shown to exhibit tumor-

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suppressive activity by preventing breast cancer and acts as a cardio-protective agent by reducing

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serum cholesterol level8. Further, squalene has also been used as a dietary supplement (Naziri et

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al. 2011).6

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In most metabolic engineering studies of S. cerevisiae towards a terpene production,

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overexpression of HMG-CoA reductase has become imperative as it is a key regulatory enzyme

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in the ergosterol pathway.9 HMG-CoA reductase is present as Hmg1p and Hmg2p isomers in S.

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cerevisiae, among which Hmg1p contributes to at least 83% of net enzyme activity in the wild-

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type strain.10 Overexpression of a membrane bound Hmg1p leads to endoplasmic reticulum

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membrane stacks otherwise called as karmellae formation.11 To overcome the regulation caused

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due to the feedback inhibition by ergosterol, a truncated form of HMG-CoA reductase enzyme

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has been overexpressed.12 Overexpression of a truncated form of Hmg1p coding only for the



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catalytic domain of the protein to bypass its regulation has not increased ergosterol content as

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expected rather it improved squalene synthesis over 30 times in yeast cells.13 During production

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of a terpene molecule, different researchers have overexpressed tHMG1 either on an episomal

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plasmid or integrated single or multiple copies.14,15 However, the copy number of this gene

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during overexpression was not optimized.

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Reactions involving HMG-CoA reductase (HMG1) and squalene synthase (ERG9) in the sterol

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pathway utilizes NADPH as a cofactor for squalene synthesis. NADPH is one of the universal

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electron carrier in yeast cells.16 S. cerevisiae has three different NAD kinase enzymes namely

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Utr1p, Yef1p and Pos5p which phosphorylates NAD+ to form NADP+ in cytoplasm and

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mitochondria.17 NADP+ synthesized by NAD kinases is further reduced to NADPH by NADP+-

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dependent dehydrogenase.18 NADPH synthesis and regeneration in yeast depends on activities of

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NADH kinases and NADP+-dependent dehydrogenases. NADH kinase phosphorylates NADH to

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form NADPH while the dehydrogenases reduces NADP+ to form NADPH. Pos5p has the

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activity of both NAD kinase as well as NADH kinase, however the latter activity is observed

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more.19 NADPH regeneration in the oxidative part of pentose phosphate pathway is carried out

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by two key enzymes such as glucose-6-phosphate dehydrogenase (G6PD coded by ZWF1) and 6-

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phospho gluconate dehydrogenase. ZWF1 is a key target for increasing regeneration of cytosolic

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NADPH not only in S. cerevisiae but also in Corynebacterium glutamicum for amino acid

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production.20 Although ZWF1 and POS5 have been targeted in some studies for increased

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regeneration of NADPH, neither of these genes were studied exclusively for squalene

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improvement. Furthermore, overexpression of POS5 was carried by eliminating the

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mitochondrial presequence, assuming the full length expression of this gene would not enhance

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cytosolic NADPH.21 Hence the aim of the present study is to address the following questions:


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a) Is single copy tHMG1 overexpression optimal for improving squalene?

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b) Role of regeneration of cytosolic NADPH in squalene synthesis

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c) Will the Pos5p present in mitochondria affect the cytosolic squalene synthesis?

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MATERIALS AND METHODS:

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Chemicals: Squalene and ergosterol standards were obtained from Sigma-Aldrich (Bangalore,

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India). Other HPLC grade chemicals were procured from SRL (Mumbai, India) and Qualigens

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(Mumbai, India). Gene amplifications were performed using Phusion® high-fidelity DNA

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polymerase (New England Biolabs, Ipswich, MA, USA) and Taq polymerase (Sigma-Aldrich,

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Bangalore, India). All restriction and ligase enzymes were from obtained from New England

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BioLabs (New England Biolabs, Ipswich, MA, USA). GenElute™ Plasmid Miniprep Kit

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(#PLN70, Sigma-Aldrich) was used for plasmid DNA purification. PureLink® Quick Gel

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Extraction and PCR Purification Combo Kit (#K220001, Invitrogen) was used for gel extraction

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and DNA purification, respectively.

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Strain and vectors: The information on strains, vectors and primers used in the present study

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are shown in Table 1. S. cerevisiae laboratory strain BY4741 (ATCC 201388) (Euroscarf,

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Germany) was kindly gifted by Prof. Ram Rajasekharan (Lipid Science Dept., CSIR-CFTRI).

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BY4741 was maintained as glycerol stocks stored at -80oC and was subcultured on YPD (yeast

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extract, peptone, dextrose) agar plates.

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medium22 lacking uracil or with G418 wherever appropriate. Ampicillin and G418 were used at

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100 µg/mL and 200 µg/mL, respectively. Nitrogen source in the SD medium was either

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(NH4)2SO4 (5 g/L) or glutamic acid (1 g/L). E. coli DH5α was used for propagating the plasmids

Engineered yeast strains were grown either in SD



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before transformation into yeast strains. The following expression cassettes were used for

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transformation in yeast cells: pYES2 (URA3, 2µ, PGal1; Invitrogen, Bangalore); pCEV-G1-Km

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(kanMX, 2µ, PTEF1, PPGK1) was obtained from Lars Nielsen and Claudia Vickers (Addgene

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plasmid #46813).23

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Instrumental analysis: An isocratic RP-HPLC was performed using a semi preparative

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reversed-phase C-18 column (Phenomenox Kinetex, Hyderabad, India) (particle size 5 µm, 250

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X 4.6 mm i.d.). The column was mounted to a HPLC unit (Shimadzu Scientific instruments,

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Kyoto, Japan) and was maintained at 35°C. The pump flow rate was 1.5 mL/min with 100%

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acetonitrile as the mobile phase. The injection volume was fixed to 20 µL. The chromatograph

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was coupled to a UV-VIS diode array multiple wavelength detector. Detection and quantification

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of squalene was carried out at 195 nm. Peak identification was achieved by comparing the

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retention time with the authentic standards and confirmation of spectral data. The

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chromatograms were processed using LC solution software. Quantification was accomplished

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with the aid of standard curves calculated using five point linear regression analysis.

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LC-MS was performed using Waters Q-Tof Ultima system equipped with an electrospray

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ionization source (Waters, Milford, MA, USA). All the samples and standards were diluted in

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chloroform prior sampling. The samples were injected using the autosampler (Waters 2695

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separation module) into a semi preparative reversed-phase C-18 column (Phenomenex Kinetex,

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Hyderabad, India) (particle size 5 µm, 250 X 4.6 mm i.d.) using isocratic elution with 100%

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acetonitrile as a mobile phase with flow rate of 1.5 mL/min. The separated components were

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detected using photo diode array detector (Waters 2996 PDA detector). Electro spray ionization

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in positive mode was used for the metabolite fragmentation. The source temperature was held at


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120°C. High-resolution data was obtained in the mass range of 200-1000 Da with a scan time of

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1 s and interscan of 0.1 s using 4 GHz TDC detector. The mass spectrum was obtained at a cone

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voltage of 100V. For positive mode, the capillary voltage was set to 3.5 kV. The high-resolution

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data was analyzed by MassLynx 4.0 software (Waters, Milford, MA, USA). Nitrogen was used

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as collision gas in the collision cell. The analysis was carried out at a desolvation temperature of

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350 oC and a cone gas flow of 50 L/h and a desolvation gas flow of 500 L/h. The Q-tof was

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tuned in positive mode allowing the passage of [M (410.7) + H (1) + CH3CN (41)]+. The results

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from LC-MS are provided in supporting information (Figure S1).

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Gene cloning and construction of strains: Template genomic DNA from BY4741 for PCR

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reactions was isolated using the standard glass beads method.22 PCR amplification of the tHMG1

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gene from template was performed using oligonucleotides tHmg1-Gal-F and tHmg1-Gal-R with

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flanking restriction sites BamHI and EcoRI (Table 1). This fragment was restriction digested and

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ligated into pYES2/NT-C to obtain pYGH. All DNA sub cloning steps were performed with E.

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coli DH5α using standard methods as described24 and nucleotide sequences of cloned genes were

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verified by sequencing (ABI-310 DNA sequencer, Applied Biosystems, CA, USA). pYGH was

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then introduced into the parent strains BY4741 to obtain SK3. PCR amplification of the tHMG1

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gene from template was performed using oligonucleotides tHmg1-pTEF-F and tHmg1-pTEF-R

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with flanking restriction sites NotI and SpeI (Table 1). The fragment was restriction digested and

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ligated with pCEV-G1-km to obtain pCTH. PCR amplification of the tHMG1 gene from

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template was performed using oligonucleotides tHmg1-pPGK-F and tHmg1-pPGK-R with

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flanking restriction sites BamHI and SalI (Table 1). The fragment was restriction digested and

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ligated with pCEV-G1-km to obtain pCTH. PCR amplification of the ZWF1, POS5(wM: with



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Mitochondrial presequence), POS5(w/oM: without mitochondrial presequence), and tHMG1

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gene from the genomic DNA template of BY4741 were performed using oligonucleotides pairs

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Zwf1 – F, Zwf1 – R; Pos5 – F (wM), Pos5 – R; Pos5-F (w/oM), Pos5 – R and tHmg1-pPGK-F,

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tHmg1-pPGK-R, respectively with flanking restriction sites BamHI and SalI (Table 1). The

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fragments were restriction digested and ligated with pCEV-G1-km to obtain pCPZ, pCPP(wM),

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pCPP(w/oM) and pCPH. These fragments were also inserted into pCTH to obtain pCTHPZ,

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pCTHPP(wM), pCTHPP(w/oM), and pCTHPH, respectively. The developed plasmids were

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introduced into BY4741 strain using Sc easy Com transformation kit (Invitrogen, Bangalore,

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India). The plasmid maps of vectors used in the present study are provided in supplementary

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figure (Figure S2).

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Squalene extraction from S. cerevisiae: Positive transformants were picked and pre-cultured in

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50 mL of yeast synthetic medium at 30°C, 180 rpm for 16 h. The inoculum from pre-culture was

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used to cultivate yeast cells with an initial OD of 0.1 in production culture at 30°C, 180 rpm for

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24 h. Cells were harvested by centrifugation to obtain cell pellets and the supernatants were

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discarded. The cells were frozen at -20°C overnight and later subjected to lyophilisation at -50°C

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under vacuum for 4 h. The lyophilized cells were dispersed in chloroform/ methanol (2:1, v/v)

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mixture (~50mL). The dispersed cells were then subjected to sonication for a 3 s pulse for 10

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cycles at 70% amplitude using sonicator (Qsonica, Newtown, CT, USA). The lysed cells were

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incubated under shaking condition for extraction at 30˚C, 180 rpm. Subsequently, the extract was

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filtered and subjected to flash evaporation at 50°C. The samples were dispersed in chloroform

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solvent and subjected to high speed centrifugation to remove debris prior to RP-HPLC analysis.

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To obtain the dry cell weight (DCW), cells harvested by centrifugation from the 50 mL culture


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were oven-dried. We obtained the following relationship between OD600 and DCW per

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millilitre (g-DCW/mL): DCW=(OD*0.53429)-0.22216. All optical densities at 600nm (OD600)

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measurements were taken using a Shimadzu UV-2550 spectrophotometer. All the experiments

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were carried out in triplicates and the experimental data represents the averages of biological

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triplicates. Standard deviations of the data were less than 7% among the replicates.

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HMG-CoA reductase assay: The cells were broken by vortexing with glass beads in 100 mM

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phosphate buffer (pH – 7.0), 1% Triton X-100 and 1 mM phenyl methyl sulphonyl fluoride

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(PMSF) following the method described in Polakowski et al. (1998).12 The supernatant was

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collected and the cytosolic fraction was further used for determining protein concentration and

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HMG-CoA reductase activity. The enzyme activity was carried out using HMG-CoA reductase

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assay kit from Sigma-Aldrich (St. Louis, MO, USA). Reactions were set using 20 µL of

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reconstituted NADPH (to obtain a final concentration of 400 µM) and 60 µL of HMG-CoA

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substrate solution (to obtain a final concentration of 0.3 mg/mL). The reaction was initiated

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either by addition of HMG-CoA reductase (concentration of 0.5 – 0.7 mg/mL) provided with the

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kit or by addition of protein extracted from the sample. The samples were mixed thoroughly

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prior to spectrophotometric analysis. The activity was calculated according to the manufacturer’s

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protocol and expressed as µmol of NADPH oxidized/min/mg protein. The protein content of the

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cell extracts was calculated using Bradford assay with bovine serum albumin (BSA) as a

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standard. Absorbance was measured at 340 nm against the blank sample using Shimadzu UV-

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2550 UV/Vis spectrophotometer connected to a computer. The absorbance was measured every

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15 s up to 5 min using a kinetic program. The A340 value gradually decreased over the time-

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course due to the oxidation of NADPH.



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RESULTS AND DISCUSSION

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Effect of promoters and copy number on HMG-CoA overexpression during squalene

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synthesis

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In the present study, effect of different promoters (both inducible and constitutive) on tHMG1

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gene overexpression towards squalene synthesis has been evaluated. Towards this, three of the

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widely used promoters PGAL1, PTEF1 and PPGK1 were selected using a 2µ episomal plasmid vector.

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The expression level of tHMG1 gene from these plasmids has been indirectly measured by

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quantifying squalene. Quantification was carried out with HPLC and the peak was verified using

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LC-MS. The LC-MS results are provided in supporting information (Figure S1). The strains

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SK3, SK10 and SK12 harboring tHMG1 gene with PGAL1, PTEF1 and PPGK1 promoters,

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respectively, have significantly improved both squalene concentration and squalene yield (cf.

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Figure 1A) when compared to that of base strain, BY4741. A maximum squalene concentration

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of 6.1, 6.2 and 4.8 mg/L was observed in SK3, SK10 and SK12, respectively. A fold increase of

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3.0, 3.1 and 2.3 in squalene concentration was observed in these strains when compared to that of

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the base strain, substantiating the efficacy of HMG-CoA reductase on sterol pathway flux (Table

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2). The maximum squalene yield of 6.5 (mg/g DW) was observed in SK3, where PGAL1 was used

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as the promoter. The relative promoter efficiency ranking in terms of squalene titer observed to

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follow PGAL1=PTEF1>PPGK1. This result is in accordance with Vickers et al. (2013)23 where they

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have observed that expression strength of PPGK1 was poor compared to PTEF1. Partow et al.

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(2010)25 used lacZ as reporter gene to evaluate efficiency of seven different promoters and have

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observed that PTEF1 and PPGK1 performed similarly in glucose consumption phase and the

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performance of PTEF1> PPGK1 during ethanol consumption phase. These results also indicates that


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the performance of constitutive promoter (PTEF1) is as good as strong inducible promoter (PGAL1).

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The main advantage of having constitutive promoter such as PTEF1, is that the carbon source

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could be a simple sugar such as glucose instead of expensive galactose for large scale

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cultivations.

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Polakowski et al. (1998)12 and several other researchers have established that truncated HMG-

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CoA (tHMG1) overexpression improved the flux towards the ergosterol synthesis in S.

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cerevisiae. Since then overexpression of tHMG1 has more or less become a ubiquitous target for

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producing terpene compounds from S. cerevisiae. In order to assay whether the overexpression

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of single copy of tHMG1 (in an episomal plasmid) is optimal or sub-optimal, two copies of this

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gene was cloned into pCEV-G1-Km to generate pCTHPH for expression studies. Therefore,

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strain SK16 has been generated which harbors tHMG1 under PTEF1 and PPGK1 promoters (cf.

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Figure 1). From Figure (1A) it can be observed that there is a significant increase in the squalene

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concentration as well as squalene yield in SK16 strain, suggesting enhanced activity of Hmg1p.

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A maximum squalene titer of 28.1 mg/L and yield of 35.7 (mg/g DW) has been achieved with

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SK16 strain. There was 4.5- and 14-fold increase in squalene concentration in SK16 compared to

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that of SK10 and BY4741 strains, respectively (Table 2). Prior investigations have overexpressed

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tHMG1 gene either using episomal plasmids26 or by integrating this gene at a specific loci in the

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chromosome.27,28 Generally, S. cerevisiae harboring an episomal plasmid will usually have 10-40

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plasmid copies per cell.29 In case of commercial cultivations, expression of genes on plasmid is

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not preferred due to instability issues. Hence, key target genes are integrated into chromosome.

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For instance, tHMG1 gene has been integrated at three different loci to enhance the rate-limiting

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step in the terpene biosynthesis pathway27. In the current study, the strain harboring two copies



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of tHMG1 gene has led to a 4.5-fold increase in squalene yield over the strains harboring a single

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copy of the same gene. This suggests that even an episomal plasmid carrying a single copy of

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tHMG1 is sub-optimal for increasing the flux towards squalene or any other terpene precursor.

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Hence there is a scope for further improvement of terpene synthesis in S. cerevisiae if copy

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number of tHMG1 gene is targeted either by multiple integrations or by expressing multiple

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copies on an episomal plasmid to achieve results closer to optimum. This result also shows that

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the overexpression of HMG-CoA reductase using two copies scavenges the NADPH in cytosol,

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and might lower the fluxes of other anabolic reactions which require NADPH as cofactor. It has

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been proved that increasing copy number of the episomal plasmid will increase the protein levels

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translated from it.30 Likewise, it can be concluded that expression of the same gene as multiple

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copies in a single cassette could also increase the protein levels.

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Effect of ZWF1 and POS5 overexpression for cofactor improvement on squalene synthesis

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The iMM904 genome-scale metabolic model of S. cerevisiae has been screened for NADPH

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synthesizing reactions which led to the identification of 18 enzymes carrying irreversible

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reactions for NADPH regeneration and 7 reactions involved in reversible reactions (Table S1).31

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Among the 18 enzymes, one is present in peroxisome, six in mitochondria and eleven enzymes

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are present in cytoplasm. The model iMM904 has 1575 reactions among which 91 reactions

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involve NADPH. Out of 91 reactions, 18 reactions (irreversible) produce NADPH while 75

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reactions consume NADPH which implies that NADPH is one of the important cofactor in the

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yeast cells (cf. Table S1). Enzymes such as cytosolic Zwf1p, Ald6p and Idp2p contribute to the

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regeneration of NADPH from NADP+ in the cytosol of S. cerevisiae (cf. Table S1).32 The

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NADP+ required for these dehydrogenases is supplied by two key NAD kinases, Yef1p and


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Utr1p in the cytosol. In case of mitochondria, the NADPH is regenerated using Pos5p which

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executes both NADH kinase and NAD kinase activities, however the former activity was found

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to be higher.32,33 Hence, in the present study ZWF1 and POS5 which regenerate NADPH pool in

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cytoplasm and mitochondria, respectively, have been overexpressed to see the effect on squalene

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synthesis. ZWF1 and POS5 (with mitochondrial presequence) and POS5 (without mitochondrial

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presequence) were overexpressed in BY4741 to generate strains SK13, SK14 and SK15,

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respectively. Significant increase in squalene concentration and squalene yield was observed

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when compared to BY4741 (Figure 2A). The squalene yield obtained from SK13, SK14 and

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SK15 were found to be 14.9, 9.0 and 7.5 (mg/g DW), respectively (Table 2). Owing to the stable

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expression of the HMG-CoA reductase under PTEF1 promoter, the PTEF1-tHMG1 cassette was

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combined with PPGK1- ZWF1 and PPGK1-POS5 gene cassettes to form the strains, SK17, SK18

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and SK19 respectively. When these strains were analysed for squalene improvement, squalene

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yield of 47.4, 58.6 and 33 mg/g DCW was observed in SK17, SK18 and SK19 strains,

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respectively (Figure 2B). There was 2 and 3.7-folds increase in squalene concentration in SK17

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and SK18 strains compared to that of SK13 and SK14, respectively. The increase in

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concentration and yield with respect to BY4741 was 12.2 and 22.2 folds in case of SK17 strain,

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and 14 and 27.4 folds in case of SK18 strain (Table 2). Interestingly, the dry cell weight (DCW)

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of SK16, SK17 and SK18 were observed to be lower than all other strains (cf. Table 2). This

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suggests that the flux towards biomass formation is lowered whenever squalene flux was

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increased. Moreover, Donald et al. (1997)34 also suggests that increased squalene or

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farnesyldiphosphate (a presqualene intermediate) due to overexpression of tHMG1 could be

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cytotoxic to the cell and hence the reduced biomass.



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Naziri et al. (2011)35 observed maximum squalene yield of 10.02 mg/g DCW using terbinafine

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and methyl jasmonate as selection pressure during cultivation of wild-type S. cerevisiae.

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Whereas, Mantzouridou and Tsimidou (2010)36 achieved maximum yield of 18.5 mg/g DCW

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when HMG2 was overexpressed along with K6R mutation in Hmg2p.

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resulted in lesser squalene yield than the present study. Zhuang and Chappell (2015)37 reported a

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maximum squalene titer of 250 mg/L in ZXB yeast strain at end of 12 days, which is higher than

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maximum titer obtained in present study (46 mg/L in SK19 at the end of 24 h). ZXB was derived

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from parental BY4741 strain having mutations for sterol uptake enhancement (EMS treatment

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and selection), overexpression of hamster tHMG1, deletion of ERG1 and ERG9, and finally

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heterologous expression of squalene synthase from Botryococcus braunii.

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NADPH is tightly regulated in the cell in comparison to NADH.38 Studies have been conducted

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to manipulate the levels of NADP/NADPH ratio in the cytosol.39 Reducing equivalents in form

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of NADPH are essential for several metabolic reactions involved in the biosynthesis of cellular

309

macromolecules.40 NADPH is a major cofactor involved in ergosterol biosynthesis and is

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required by HMG-CoA reductase (HMG1) and squalene synthase (ERG9) for squalene

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synthesis. Increased squalene content in SK-13, SK-14 and SK-15 can be correlated to the

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increased NADPH regeneration by overexpressing ZWF1 and POS5 genes. When

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overexpression of these NADPH regenerating genes was coupled to tHMG1 overexpression, as

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seen in strains SK-17, SK-18 and SK-19 there was a synergistic effect on squalene synthesis.

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This indicates that either of Hmg1p or Erg9p, or both may be are limited by NADPH supply and

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hence improving regeneration of NADPH significantly improved squalene yield. Overexpression

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of POS5 gene was more effective during such coupled co-expression and has shown 1.2 fold

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higher squalene yield when compared to that of ZWF1 overexpression. This could be due to the

Both these studies


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fact that NADPH regeneration is higher in POS5 overexpressed strain owing to the dual

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functionality of Pos5p as a NAD+ and NADH kinase. The mechanism through which it improves

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squalene flux has been discussed subsequently.

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Overexpression of ZWF1 was proven to improve lysine production in C. glutamicum. 41 In case

323

of S. cerevisiae, ZWF1 overexpression has improved tolerance to furfural, which could be

324

exploited for lignocellulose- based bioethanol production.42 There are very few reports on its

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overexpression for terpene production in S. cerevisiae. Brown et al. (2015)43 has overexpressed

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ZWF1 for strictosidine production and Zhao et al. (2015)21 studied its effect on carotene

327

production. Kwon et al. (2006)44 observed there was 6.0 fold increase in Zwf1p activity in S.

328

cerevisiae strain when ZWF1 was overexpressed. However NADPH levels were not elevated

329

indicating rapid consumption of this co-factor. In their study the regenerated NADPH was

330

utilized by NADPH- dependent xylose reductase for converting xylose to xylitol as there was

331

significant improvement in xylitol (86 g/L) when compared to control (71 g/L). In contrast to

332

Zwf1p, Pos5p is localized in mitochondrial matrix and plays a key role in protection of

333

mitochondria from oxidative stress.45 Hou et al. (2009)46 overexpressed both full length Pos5 and

334

Pos5∆17 (lacking the mitochondrial target signal) in S. cerevisiae to generate cNDK (equivalent

335

to SK15 and SK19 in present study) and mNDK (equivalent to SK14 and SK18 in present study)

336

strains. They observed improved NADH kinase activity in mNDK and cNDK strains when

337

compared to reference strain and no significant changes in NAD kinase activity when cultivated

338

aerobically using glucose as carbon source.

339

The rationale behind improved squalene due to POS5 overexpression is not straightforward as in

340

case of ZWF1. The mitochondrial membrane is impermeable to NADH and NADPH. Hence the

341

cofactors are restricted to their respective organelles.33 The utr1yef1 double mutant S. cerevisiae



Page 16 of 39

342

strain showed viability implying that cytosolic NADP+ can also be supplied by Pos5p.32 Thus

343

supplied NADP+ can be reduced to NADPH by cytosolic dehydrogenases (Zwf1p, Ald6p and

344

Idp2p). However, the mechanism of NADP+ transport between cytosol and mitochondria has not

345

been well characterized and the function of several mitochondrial carrier proteins are yet to be

346

uncovered42. Furthermore, the mitochondrial transport protein Yhm2p which is involved in

347

citrate transport to cytosol has also enhanced the NADPH in cytosol via citrate-oxoglutarate

348

NADPH redox shuttle between mitochondria and cytosol.45 These two findings suggest that the

349

regeneration of NADPH (via NADH kinase) and NADP+ (via NAD kinase) inside the

350

mitochondria could possibly enhance the NADPH pool in the cytosol. Thus improved NADPH

351

regeneration in the cytosol in turn could have possibly increased the rates of HMG-CoA

352

reductase (Hmg1p) and squalene synthase (erg9p) thereby increasing the squalene flux. This

353

could be the plausible rationale for improved squalene during overexpression of mitochondrial

354

POS5 either in isolation or when coupled with tHMG1. Zhao et al. (2015)21 overexpressed ZWF1

355

and POS5 to observe improvement in carotenoid biosynthesis, however, the POS5 gene has been

356

overexpressed without the mitochondrial presequence. Hence the POS5 gene product was

357

restricted only to cytoplasm. Moreover the fold increase in the β-carotene yield was only 1.8 and

358

in lycopene it was 1.6 when compared to that of the control strain, however significant. NAD

359

kinase activity was found to be significantly improved in the strain overexpressing POS5 in their

360

study. However, NADH kinase activity was not assayed which is supposedly seen more when

361

compared to NAD kinase activity of Pos5p. Shi et al. (2013)47 overexpressed POS5 and ZWF1

362

from S. cerevisiae in C. glutamicum spp. lactofermentum to observe increased isoleucine

363

production. They have extended this study to proteomic analysis in Shi et al. (2015)48 to observe

364

24 differentially expressed genes in case of POS5 overexpression and 7 differentially expressed


Page 17 of 39


365

in case of ZWF-ppnk overexpression. Prior investigations were focused on the expression of

366

truncated POS5 gene and its influence in biochemical production.21,48 Strand et al. (2003)19 has

367

reported that even the full length POS5 overexpression has yielded much higher protein when

368

compared to truncated POS5 and the protein from full length expression has accumulated only in

369

mitochondria. To the best of our knowledge this is the first study where we report full length

370

POS5 overexpression has significantly improved squalene in S. cerevisiae. If we carefully

371

observe, the squalene levels were higher in the case of strains harboring plasmid containing both

372

HMG1 and Zwf1/Pos5, i.e. SK17, SK18 & SK19 in comparison to only Zwf1/Pos5, i.e. SK13,

373

SK14 & SK15. This indicates that overexpression of HMG1 has served as an additional sink for

374

the regenerated NADPH. Similar result was observed by Kwon et al. (2006)44 where expression

375

of NADPH- requiring xylose reductase has improved xylitol production when NADPH was

376

regenerated using overexpression of Zwf1. Hou et al. (2009)46 observes that NADPH

377

regeneration using oxidative pentose phosphate pathway is coupled with CO2 production

378

whereas NADPH regeneration via POS5 is a decoupled process. Probably this could be the

379

reason for higher concentration of squalene in SK18 and SK19 than SK17 (Table 2). From the

380

biomass data it observed that the strains SK14 and SK18 had lower dry cell values (DCW) in

381

comparison to strains SK15 and SK19. Especially, SK18 had the lowest biomass (0.48 g/L)

382

where maximum squalene yield was observed. Here we hypothesize that in strains SK15 and

383

SK19, the regenerated NADPH was involved in the biosynthesis of cellular macromolecules

384

thereby contributing to biomass. Moreover as mentioned in Hou et al. (2009)46, as the redox

385

cofactors are impermeable across mitochondrial membrane, the phenotypes could vary when

386

cytosolic and mitochondrial redox are perturbed.

387



Page 18 of 39

388

In-vitro HMG-CoA reductase assay

389

HMG1 contributes about 83% of the HMG-CoA reductase activity in wild-type cells while its

390

isomer, HMG2, contributes the remaining activity thus indicating the significance of HMG1.10

391

HMG-CoA is transported to endoplasmic reticulum where it is reduced to mevalonate by

392

membrane bound glycoprotein HMG-CoA reductase. The reductive cleavage of HMG-CoA to

393

mevalonate and CoA requires two molecules of NADPH. HMG-CoA reductase activity was

394

determined based on the reduction in absorbance at 340 nm which in turn is due to the NADPH

395

oxidation by the catalytic subunit of HMG-CoA reductase in the presence of the substrate HMG-

396

CoA. In the present study, the strains conferring HMG-CoA reductase activity was ascertained

397

based on the estimation of NADPH changes as described earlier in methods section. When

398

compared to BY4741, the crude cell extracts of all the transformants displayed significantly

399

higher enzyme activity (cf. Figure 3). Recombinant strains, SK10 and SK12 attributed similar

400

activity indicating that TEF1 and PGK1 promoter performance was almost same. Whereas SK3

401

strain, which harbors tHMG1 under PGAL1 showed slightly higher activity when compared to that

402

of SK10 and SK12. Maximum increase in the reductase activity was observed in the strains

403

SK17 and SK18 with fold increase of 10 and 11, respectively, when compared to that of BY4741

404

(Table 2). Strains SK17 and SK18 harbors tADH1-tHMG1-PTEF1-PPGK1-ZWF1-tCYC1 and

405

tADH1-tHMG1-PTEF1-PPGK1-POS5-tCYC1 cassettes, respectively, indicating the tHMG1

406

overexpression and NADPH regenerating ZWF1/POS5 improves the HMG-CoA reductase

407

activity significantly in comparison to other recombinant strains. Interestingly, the SK16 strain

408

harboring tADH1-tHMG1-PTEF1-PPGK1-tHMG1-tCYC1 increased the reductase activity by 1.4-

409

fold in comparison to SK10/SK12 and 1.2- fold in comparison to SK3 indicating that presence of

410

two tHMG1 gene copies on a single cassette will improve its activity when compared to single


Page 19 of 39


411

copy. Hence the strain SK16 produced more squalene in comparison to SK3, SK10 and SK12.

412

Interestingly, ZWF1 (strain SK13) and POS5 (strain SK14) overexpression improved the HMG-

413

CoA reductase activity by 6.5- and 5.5- folds in comparison to BY4741 suggesting the HMG1

414

up-regulation. Moreover, this fold increase is alike to that of strains SK10 and SK12 indicating

415

same effect as that of tHMG1 overexpression. Thus the enzyme activity results corroborates the

416

squalene estimation results found in the earlier section. Polakowski et al. (1998)12 has estimated

417

the HMG-CoA reductase activity in the tHMG1 overexpressed strains. In their study it was

418

observed that the episomal plasmid- based overexpression had significantly higher activity in

419

comparison to the single copy integration of tHMG1. In case of episomal expression the specific

420

activity obtained in their study was 0.18 U/mg protein whereas in the present study it ranged

421

between 0.13-0.15 U/mg protein. The specific activity values obtained in the present study is in

422

agreement with Polakowski et al. (1998)12 for both wild-type and tHMG1 overexpressed strains.

423

A maximum specific activity of 0.218 U/mg protein was obtained in the strain SK18 harboring

424

tADH1-tHMG1-PTEF1-PPGK1-POS5-tCYC1 cassette.

425 426

Effect of nitrogen sources on G418 – based vector for tHMG1 overexpression

427

Aminoglycoside 3′-phosphotransferase coded by aphA1 gene from E. coli confers

428

aminoglycoside, G418 (Geneticin®) resistance in S. cerevisiae and is a commonly implemented

429

selection marker during transformation in S. cerevisiae.23,49,50 The kanMX module on a G418

430

selection cassette contains the aphA1 gene under a specific promoter and terminator. The

431

plasmid pCEV-G1-Km used in the present study harbors kanMX module for selection of

432

transformants using G418 antibiotic. It has been established that if ammonium sulphate is used

433

as nitrogen source in the growth medium for cultivation of S. cerevisiae strain harboring kanMX



Page 20 of 39

434

cassette, the resistance towards G418 is lost.23,50 The exact reason for such loss is not established,

435

however, it was suggested that the ammonium sulphate assimilation might downregulate the

436

reactions involved in import and activity of G418.50 So when selective pressure is lost during

437

successive generations of cell cycle, the percentage of cells harboring plasmid will be lowered as

438

the cells which are free of plasmids will have higher growth rate due to selective advantage. This

439

in turn lowers the expression of genes present on the plasmid. The very role of selective pressure

440

(addition of G418 to the medium for present study) is to maintain the plasmid in cell population.

441

It was also established that if ammonium sulphate is substituted by glutamate (1 g/L) as the

442

nitrogen the resistance to G418 was retained and therefore the selection pressure was

443

maintained.23,50 To corroborate this reasoning experimentally, it was observed that in the strain

444

SK10 squalene content was meagre 1.42 mg/L when (NH4)2SO4 was used as nitrogen source

445

whereas substituting with glutamate the squalene synthesis was improved to 6.3 mg/L (cf. Figure

446

4A). Squalene yield and titers improved significantly indicating the positive effect of glutamate

447

as nitrogen source on G418- based gene expression (cf. Figure 4A). Hence, the SD medium used

448

for cultivation has been modified by replacing (NH4)2SO4 with glutamic acid (1 g/L) for the

449

strains harboring plasmids with geneticin as a selection marker for all shake-flask experiments.

450

Vickers et al. (2013)23 provided comprehensive information on factors affecting the G418

451

selection, and medium pH and its composition were suggested to be key factors.

452

Similarly the carbon sources, glucose and galactose, have been evaluated for squalene production

453

when constitutive promoter PTEF1 was utilized. When strain SK10 was cultivated on 2% glucose

454

or 2% galactose using glutamic acid (1 g/L) as the nitrogen source, the squalene production

455

levels were slightly higher in case of 2% glucose when compared to 2% galactose (Figure 4B).

456

Partow et al. (2010) reported that TEF1 is a truly constitutive promoter is not affected by change


Page 21 of 39


457

in the carbon source. Therefore, the results obtained are also in accordance with Partow et al.

458

(2010).25

459 460

In conclusion, we demonstrate that single copy episomal overexpression of tHMG1 is suboptimal

461

for producing squalene in S. cerevisiae. We also show that NADPH regenerating enzymes can

462

improve squalene synthesis in S. cerevisiae when overexpressed either in isolation or in tandem

463

with tHMG1. In case of latter, the effect was highly synergistic. We also observed that full length

464

POS5 overexpression improved squalene in cytosol even though its protein, pos5p, localizes in

465

mitochondria. Although further studies are needed to elucidate the exact reason for this

466

observation. Finally we conclude that the strategies employed in present study could be

467

potentially applied for engineering S. cerevisiae for squalene improvement and also for other

468

industrially relevant terpenes.

469

470

471

AUTHOR INFORMATION

472

*Corresponding author

473

Microbiology and Fermentation Technology Department, CSIR-Central Food Technological

474

Research Institute, Mysore, India.

475

E-mail: [email protected]; [email protected]

476

Telephone: +91-821-2517539

477

ORCID: 0000-0002-3251-8643



Page 22 of 39

478

Author contributions:

479

Funding: The financial support was provided by the Department of Biotechnology, India (award

480

of research fellowship) and by the Science Engineering and Research Board (SERB), India

481

(YSS/2014/000565).

482

Notes:

483

The authors declare no competing financial interest

484

485

ACKNOWLEDGEMENTS

486

The authors thank Mr. Sandeep Kumar, RGNF-JRF for his useful comments and suggestions on

487

cloning experiments and Director Prof. Ram Rajasekharan (Lipid Science Dept., CSIR-CFTRI)

488

for providing us the BY4741 strain and the plasmid, pYES2/NTC. The authors also acknowledge

489

Mr. P Mukund Lakman, Central Instrumentation Facilities, CSIR-CFTRI for and HPLC and LC-

490

MS studies.

491

492

Supporting information

493

Figure S1: Shows the LC-MS profile of squalene against a standard

494

Figure S2: Shows the plasmid maps constructed for the current study

495

Table S1: Provides the list of reactions in Saccharomyces cerevisiae involved in NADPH

496

synthesis based on iMM904 in-silico model

497


Page 23 of 39


498

REFERENCES

499

(1)

500

Horning, T.; Tsuruta, H.; Melis, D. J.; Owens, A.; Fickes, S.; Diola, D.; Benjamin, K. R.;

501

Keasling, J. D.; Leavell, M. D.; McPhee, D. J.; Renninger, N. S.; Newman, J. D.; Paddon, C. J.,

502

Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to

503

the antimalarial agent artemisinin. Proc. Natl. Acad. Sci. U S A. 2012, 109, E111-8.

504

(2)

505

terpenoids. Comput. Struct. Biotechnol. J. 2012, 3, e201210006.

506

(3)

507

Makris, A. M.; Roussis, V.; Kampranis, S. C., Reconstructing the chemical diversity of labdane-

508

type diterpene biosynthesis in yeast. Metab. Eng. 2015, 28, 91-103.

509

(4)

510

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

511

Sarpong, R.; Keasling, J. D., Production of the antimalarial drug precursor artemisinic acid in

512

engineered yeast. Nature. 2006, 440, 940-3.

513

(5)

514

of Saccharomyces cerevisiae. Crit. Rev. Biotechnol., 2017,

515

DOI:10.1080/07388551.2017.1299679

516

(6)

517

potential of biotechnology. Lipid. Technol. 2011, 23, 270-273.

518

(7)

519

(Accessed on 24th August 2017)

Westfall, P. J.; Pitera, D. J.; Lenihan, J. R.; Eng, D.; Woolard, F. X.; Regentin, R.;

Kampranis, S. C.; Makris, A. M., Developing a yeast cell factory for the production of

Ignea, C.; Ioannou, E.; Georgantea, P.; Loupassaki, S.; Trikka, F. A.; Kanellis, A. K.;

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

Paramasivan, K.; Mutturi, S., Progress in terpene synthesis strategies through engineering

Naziri, E.; Mantzouridou, F.; Tsimidou, M. Z., Squalene resources and uses point to the

http://www.marketsandmarkets.com/Market-Reports/squalene-market-542345.html



Page 24 of 39

520

(8)

Spanova, M.; Daum, G., Squalene - biochemistry, molecular biology, process

521

biotechnology, and applications. Eur. J. Lipid. Sci. Tech. 2011, 113, 1299-1320.

522

(9) Rodwell, V. W.; Nordstrom, J. L.; Mitschelen, J. J., Regulation of HMG-CoA reductase. Adv.

523

Lipid Res. 1976, 14, 1-74.

524

(10) Basson, M. E.; Thorsness, M.; Rine, J., Saccharomyces cerevisiae contains two functional

525

genes encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Proc. Natl. Acad.

526

Sci. U.S.A. 1986, 83, 5563-67.

527

(11)

528

A proliferation of stacked membrane pairs surrounding the yeast nucleus. J. Cell Biol. 1988,

529

107, 101-114.

530

(12)

531

glutaryl-CoA reductase leads to squalene accumulation in yeast. Appl. Environ. Microbiol.1998,

532

49, 66-71.

533

(13)

534

cerevisiae. i-ACES 2014, 1, 57-63.

535

(14)

536

gene encoding HMG-CoA reductase in Saccharomyces cerevisiae for production of prenyl

537

alcohols. Appl. Microbiol. Biotechnol. 2009, 82, 837–845.

538

(15)

539

precursor supply in Saccharomyces cerevisiae. J. Biotechnol. 2013, 168, 446–451.

540

(16)

541

of NADP. Redox Rep. 2010, 15, 2-10.

Wright, R.; Basson, M., Increased amounts of HMG-CoA reductase induce "Karmellae":

Polakowski, T.; Stahl, U.; Lang, C., Overexpression of a cytosolic hydroxyl methyl

Thompson, A.; Kwak, S.; Jin, Y.-s., Squalene Production using Saccharomyces

Ohto, C.; Muramatsu, M.; Obata, S.; Sakuradani, E.; Shimizu, S. Overexpression of the

Liu, J.; Zhang, W.; Du, G.; Chen, J.; Zhou, J. Overproduction of geraniol by enhanced

Agledal, L.; Niere, M.; Ziegler, M., The phosphate makes a difference: cellular functions


Page 25 of 39


542

(17)

Shi, F.; Kawai, S.; Mori, S.; Kono, E.; Murata, K., Identification of ATP-NADH kinase

543

isozymes and their contribution to supply of NADP(H) in Saccharomyces cerevisiae. FEBS. J.

544

2005, 272, 3337-3349.

545

(18)

546

in the respiratory chain activity of Saccharomyces cerevisiae. Acta. Biochim. Biophys. Sin. 2011,

547

43, 989-995.

548

(19)

549

Copeland, W. C., POS5 gene of Saccharomyces cerevisiae encodes a mitochondrial NADH

550

kinase required for stability of mitochondrial DNA. Eukaryot. Cell. 2003, 2, 809-820.

551

(20)

552

flux engineering of L-lysine production in Corynebacterium glutamicum: over expression and

553

modification of G6P dehydrogenase. J Biotechnol. 2007, 132, 99–109.

554

(21)

555

biosynthesis in recombinant Saccharomyces cerevisiae. Lett. Appl. Microbiol. 2015, 61, 354-

556

360.

557

(22)

558

Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold

559

Spring Harbor, NY 2005.

560

(23)

561

vectors with antibiotic selection markers for engineering in Saccharomyces cerevisiae. Microb.

562

Cell. Fact. 2013, 12, 96.

563

(24)

564

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 2001.

Shi, F.; Li, Z.; Sun, M.; Li, Y., Role of mitochondrial NADH kinase and NADPH supply

Strand, M. K.; Stuart, G. R.; Longley, M. J.; Graziewicz, M. A.; Dominick, O. C.;

Judith, B.; Corinna, K.; Andrea, H.; Oskar, Z.; Christoph, J.B.; Christoph, W., Metabolic

Zhao, X.; Shi, F.; Zhan, W., Overexpression of ZWF1 and POS5 improves carotenoid

Amberg, D. C.; Burke, D. J.; Dawson, D.; Stearns, T., Methods in Yeast Genetics: A

Vickers, C. E.; Bydder, S. F.; Zhou, Y.; Nielsen, L. K., Dual gene expression cassette

Sambrook, J., and D. E. Russell., Molecular cloning: a laboratory manual, No. Ed. 2.



Page 26 of 39

565

(25)

Partow, S.; Siewers, V.; Bjørn, S.; Nielsen, J.; Maury, J., Characterization of different

566

promoters for designing a new expression vector in Saccharomyces cerevisiae. Yeast 2010, 27,

567

955-64.

568

(26)

569

cerevisiae by metabolic engineering. FEMS. Microbiol. Lett. 2013, 345, 94-101.

570

(27)

571

diphosphate pool as direct precursor of sesquiterpenes through metabolic engineering of the

572

mevalonate pathway in Saccharomyces cerevisiae. Biotechnol. Bioeng. 2010, 106, 86-96.

573

(28)

574

engineering applications in Saccharomyces cerevisiae. FEMS. Yeast. Res. 2012, 12, 197-214.

575

(29)

576

Yeast. 1992, 8, 423–488.

577

(30)

578

number of episomal plasmids in Saccharomyces cerevisiae for improved protein production.

579

FEMS Yeast Res. 2012, 12, 598-607.

580

(31) Mo, M. L.; Palsson, B. Ø.; Herrgård, M. J. Connecting extracellular metabolomic

581

measurements to intracellular flux states in yeast. BMC Syst. Biol. 2009, 3, 37.

582

(32)

583

Saccharomyces cerevisiae. J. Biol. Chem. 2009, 284, 7553-7560.

584

(33)

585

NADPH in Saccharomyces cerevisiae. EMBO. J. 2003, 22, 2015-2024.

Li, Q.; Sun, Z.; Li, J.; Zhang, Y., Enhancing beta-carotene production in Saccharomyces

Asadollahi, M. A.; Maury, J.; Schalk, M.; Clark, A.; Nielsen, J., Enhancement of farnesyl

Da Silva, N. A., and Srikrishnan, S., Introduction and expression of genes for metabolic

Romanos, M.A.; Scorer, C.A.; Clare, J.J., Foreign gene expression in yeast – a review.

Chen, Y., Partow, S., Scalcinati, G., Siewers, V., & Nielsen, J. Enhancing the copy

Miyagi, H.; Kawai, S.; Murata, K., Two sources of mitochondrial NADPH in the yeast

Outten, C. E.; Culostta, V. C., A novel NADH kinase is the mitochondrial source of


Page 27 of 39


586

(34) Donald, K. A. G.; Hampton, R. Y.; Fritz, I. B. Effects of overproduction of the catalytic

587

domain of 3-hydroxy-3- methylglutaryl coenzyme A reductase on squalene synthesis in

588

Saccharomyces cerevisiae. Appl. Environ. Microbiol. 1997, 63, 3341–3344.

589

(35) Naziri, E.; Mantzouridou, F.; Tsimidou, M. Z. Enhanced squalene production by wild-type

590

Saccharomyces cerevisiae strains using safe chemical means. J. Agric. Food Chem. 2011, 59,

591

9980–9989.

592

(36) Mantzouridou, F.; Tsimidou, M. Z. Observations on squalene accumulation in

593

Saccharomyces cerevisiae due to the manipulation of HMG2 and ERG6. FEMS Yeast Res. 2010,

594

10, 699–707.

595

(37) Zhuang, X.; Chappell, J. Building terpene production platforms in yeast. Biotechnol.

596

Bioeng. 2015, 112, 1854–1864.

597

(38) Moreira Dos Santos, M.; Raghevendran, V.; Kötter, P.; Olsson, L.; Nielsen, J. Manipulation

598

of malic enzyme in Saccharomyces cerevisiae for increasing NADPH production capacity

599

aerobically in different cellular compartments. Metab. Eng. 2004, 6, 352–363.

600

(39) Asadollahi, M. A.; Maury, J.; Patil, K. R.; Schalk, M.; Clark, A.; Nielsen, J., Enhancing

601

sesquiterpene production in Saccharomyces cerevisiae through in silico driven metabolic

602

engineering. Metab. Eng. 2009, 11, 328-34.

603

(40) Grabowska, D.; Chelstowska, A. The ALD6 gene product is indispensable for providing

604

NADPH in yeast cells lacking glucose-6-phosphate dehydrogenase activity. J. Biol. Chem. 2003,

605

278, 13984–13988.

606

(41) Becker, J.; Klopprogge, C.; Herold, A.; Zelder, O.; Bolten, C. J.; Wittmann, C., Metabolic

607

flux engineering of L-lysine production in Corynebacterium glutamicum—over expression and

608

modification of G6P dehydrogenase. J. Biotechnol. 2007, 132, 99-109.



Page 28 of 39

609

(42) Gorsich, S.; Dien, B.; Nichols, N.; Slininger, P.; Liu, Z.; Skory, C., Tolerance to furfural-

610

induced stress is associated with pentose phosphate pathway genes ZWF1, GND1, RPE1, and

611

TKL1 in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 2006, 71, 339-349.

612

(43) Brown, S.; Clastre, M.; Courdavault, V.; O’Connor, S. E., De novo production of the plant-

613

derived alkaloid strictosidine in yeast. Proc. Natl. Acad. Sci. U.S.A. 2015, 3205-10.

614

(44) Kwon, D.-H.; Kim, M.-D.; Lee, T.-H.; Oh, Y.-J.; Ryu, Y.-W.; Seo, J.-H. Elevation of

615

glucose 6-phosphate dehydrogenase activity increases xylitol production in recombinant

616

Saccharomyces cerevisiae. J. Mol. Catal. B Enzym. 2006, 43, 86–89.

617

(45) Castegna, A.; Scarcia, P.; Agrimi, G.; Palmieri, L.; Rottensteiner, H.; Spera, I.; Germinario,

618

L.; Palmieri, F., Identification and functional characterization of a novel mitochondrial carrier for

619

citrate and oxoglutarate in Saccharomyces cerevisiae. J. Biol. Chem. 2010, 285, 17359-17370.

620

(46) Hou, J.; Vemuri, G. N.; Bao, X.; Olsson, L. Impact of overexpressing NADH kinase on

621

glucose and xylose metabolism in recombinant xylose-utilizing Saccharomyces cerevisiae. Appl.

622

Microbiol. Biotechnol. 2009, 909–919.

623

(47) Shi, F.; Li, K.; Huan, X.; Wang, X., Expression of NAD(H) kinase and glucose-6-phosphate

624

dehydrogenase improve NADPH supply and L-isoleucine biosynthesis in Corynebacterium

625

glutamicum ssp. lactofermentum. Appl. Biochem. Biotechnol. 2013, 171, 504-521.

626

(48) Shi, F.; Li, K.; Li, Y., Comparative proteome analysis of global effect of POS5 and zwf-

627

ppnK

628

lactofermentum. Biotechnol. Lett. 2015, 37, 1063-1071.

629

(49) Webster, T. D.; Dickson, R. C., Direct selection of Saccharomyces cerevisiae resistant to

630

the antibiotic G418 following transformation with a DNA vector carrying the kanamycin-

631

resistance gene of Tn903. Gene. 1983, 26, 243-252.

overexpression

in

l-isoleucine

producing

Corynebacterium

glutamicum

ssp.


Page 29 of 39


632

(50) Cheng, T.H.; Chang, C.-R.; Joy, P.; Yablok, S.; Gartenberg, M. R., Controlling gene

633

expression in yeast by inducible site-specific recombination. Nucleic. Acids. Res. 2000, 28, e108-

634

e108.

635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654



655

Page 30 of 39

Figure Legends

656 657

Figure 1: Squalene synthesis in tHMG1 overexpressed S. cerevisiae strains. (A) The comparison

658

of BY4741 with respect to SK13, SK10 and SK12 represents the effect of single copy

659

overexpression of tHMG1 using promoters PGAL1, PTEF1 and PPGK1, respectively. Whereas, the

660

comparison of SK16 to other engineered strains (SK10, SK12 and SK13) reveals the effect of

661

plasmid harboring two copies tHMG1 to single copy tHMG1 on squalene. (B) Pictorial

662

representation of gene cassettes used in the strains.

663

Figure 2: Squalene synthesis in S. cerevisiae strains with overexpression of NADPH

664

regenerating genes (A) Comparison among strains SK13, SK14 and SK15 indicates the effect of

665

overexpression of ZWF1, POS5 (with mitochondrial presequence) and POS5 (without

666

mitochondrial presequence), respectively. (B) Comparison between the strains SK17, SK18 and

667

SK19 indicates the synergistic effect of simultaneous expression of tHMG1 and NADPH

668

regenerating genes. Here SK17, SK18 and SK19 represents SK13, SK14 and SK15 strains,

669

respectively, with additional tHMG1 coexpression.

670

Figure 3: Comparison of HMG-CoA reductase activity in various S. cerevisiae strains used in

671

the present study

672

Figure 4: Effect of medium components on squalene synthesis (A) Effect of nitrogen source on

673

squalene synthesis using the strain SK10 harboring kanMX cassette for conferring G418

674

resistance when cultivated in (NH4)2SO4 and glutamate. (B) Effect of carbon source on squalene

675

synthesis using the SK10 having tHMG1 gene under constitutive promoter PTEF1 when cultivated

676

in glucose and galactose.

677


Page 31 of 39


Tables Table 1: Primers, plasmids and strains used in this study Name

Description

Reference

tHmg1-Gal-F

CGGGATCCGACCAATTGGTGAAAACTGAAG

This study

tHmg1-Gal-R

CGGAATTCGTAACACATGGTGCTGTTGT

This study

Primers

ATAAGAATAGGCGGCCGCATGGACCAATTGG tHmg1-pTEF - F

TGAAAACTGAAG

This study

CGGTCGACTAGTGTAACACATGGTGCTGTTG tHmg1 -pTEF- R

T

This study

CGGTCGGGATCCATGGACCAATTGGTGAAAA tHmg1-pPGK - F

CTGAAG

This study

ACGCGCGTCGACGTAACACATGGTGCTGTTG tHmg1 -pPGK- R

T

This study

Zwf1 – F

CGGTCGGGATCCATGAGTGAAGGCCCCGT

This study

ACGCGCGTCGACGCCGATAAATGAATGTGCT Zwf1 – R

TGC

This study

CGGTCGGGATCCACTTCCACAGTTCTCAACTC Pos5 – F(wM)

TTC

This study

CGGTCGGGATCCATGCACCATGAGTACGTTG Pos5 – F (w/oM)

GATTCACA

This study

ACGCGCGTCGACAGAGAATCTCATTGAATCT Pos5 – R

TTGCAT

This study

Yeast expression vector with URA selection,

Invitrogen

Plasmids pYES2-NTC

(Vickers et pCEV-G1-Km

Yeast expression vector with G418 selection

al. 2013)

pYGH

pYES2NT/C- PGAL1-tHmg1

This study

pCTH

pCEV-G1-Km-PTEF1-tHmg1

This study

pCPZ

pCEV-G1-Km-PPGK1-Zwf1

This study 31

ACS Paragon Plus Environment


Page 32 of 39

pCPP(wM)

pCEV-G1-Km-PPGK1-Pos5(wM)

This study

pCPH

pCEV-G1-Km-PPGK1-tHmg1

This study

pCPP(w/oM)

pCEV-G1-Km-PPGK1-Pos5 (w/oM)

This study

pCTHPZ

pCEV-G1-Km-PTEF1-tHmg1-PPGK1-Zwf1

This study

pCTHPP(wM)

pCEV-G1-Km-PTEF1-tHmg1-PPGK1-Pos5(wM)

This study

pCTHPH

pCEV-G1-Km-PTEF1-tHmg1-PPGK1-tHmg1

This study

pCTHPP(w/oM)

pCEV-G1-Km-PTEF1-tHmg1-PPGK1-Pos5(w/oM)

This study

MATa; his3∆ 1; leu2∆ 0; met15∆ 0; ura3∆ 0

Euroscarf,

Strains BY4741

SRD GmbH SK1

Yeast BY4741 harboring pYES2/NTC

This study

SK3

Yeast BY4741 harboring pYGH

This study

SK8

Yeast BY4741 harboring pCEV-G1-Km

This study

SK10

Yeast BY4741 harboring pCTH

This study

SK12

Yeast BY4741 harboring pCPH

This study

SK13

Yeast BY4741 harboring pCPZ

This study

SK14

Yeast BY4741 harboring pCPP(wM)

This study

SK15

Yeast BY4741 harboring pCPP(w/oM)

This study

SK16

Yeast BY4741 harboring pCTHPH

This study

SK17

Yeast BY4741 harboring pCTHPZ

This study

SK18

Yeast BY4741 harboring pCTHPP(wM)

This study

SK19

Yeast BY4741 harboring pCTHPP(w/oM)

This study


Page 33 of 39


Table 2: Fold improvement in squalene production for different S. cerevisiae strains Strain

Overexpressed protein

Yield (mg/g

Yield fold

Conc.

Titer fold

DW)

increase a

(mg/L)

increase a

BY4741

NA

2.1±0.36

NA

2±0.21

NA

SK3

tHmg1p (1X)

6.5±2.6

3

6.1±0.05

3

SK10

tHmg1p (1X)

3.5±0.13

1.6

6.3±0.03

3

SK12

tHmg1p (1X)

4.4±2.07

2

4.8±2.39

2.3

SK13

Zwf1p

14.9±0.32

6.9

12.6±1.72

6.1

SK14

Pos5p (wM)

9.05±0.47

4.3

7.6±1.05

3.8

SK15

Pos5p (w/oM)

7.5±1.48

3.6

10±3.48

5

SK16

tHmg1p (2X)

35.7±0.92

16.8

28.1±3

13.7

SK17

tHmg1p & Zwf1p

47.4±3.2

22.2

24.9±0.32

12.2

tHmg1p & Pos5p

58.6±1.43

27.5

28.4±1.08

13.9

33±2.96

16.5

46±4.08

23

SK18

(wM) tHmg1p & Pos5p

SK19

a

(w/oM)

With respect to BY4741

NA: Not Applicable



Page 34 of 39


Page 35 of 39


Figure 1. Squalene synthesis in tHMG1 overexpressed S. cerevisiae strains. (A) The comparison of BY4741 with respect to SK13, SK10 and SK12 represents the effect of single copy overexpression of tHMG1 using promoters PGAL1, PTEF1 and PPGK1, respectively. Whereas, the comparison of SK16 to other engineered strains (SK10, SK12 and SK13) reveals the effect of plasmid harboring two copies tHMG1 to single copy tHMG1 on squalene. (B) Pictorial representation of gene cassettes used in the strains. 352x142mm (96 x 96 DPI)



Figure 2. Squalene synthesis in S. cerevisiae strains with overexpression of NADPH regenerating genes (A) Comparison among strains SK13, SK14 and SK15 indicates the effect of overexpression of ZWF1, POS5 (with mitochondrial presequence) and POS5 (without mitochondrial presequence), respectively. (B) Comparison between the strains SK17, SK18 and SK19 indicates the synergistic effect of simultaneous expression of tHMG1 and NADPH regenerating genes. Here SK17, SK18 and SK19 represents SK13, SK14 and SK15 strains, respectively, with additional tHMG1 co-expression. 245x282mm (300 x 300 DPI)


Page 36 of 39

Page 37 of 39


Figure 3. Comparison of HMG-CoA reductase activity in various S. cerevisiae strains used in the present study 272x208mm (300 x 300 DPI)



Figure 4. Effect of medium components on squalene synthesis (A) Effect of nitrogen source on squalene synthesis using the strain SK10 harboring kanMX cassette for conferring G418 resistance when cultivated in (NH4)2SO4 and glutamate. (B) Effect of carbon source on squalene synthesis using the SK10 having tHMG1 gene under constitutive promoter PTEF1 when cultivated in glucose and galactose. 278x282mm (300 x 300 DPI)


Page 38 of 39

Page 39 of 39


TOC 324x144mm (96 x 96 DPI)


Regeneration of NADPH Coupled with HMG-CoA Reductase Activity

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