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Mitochondrial acetyl-CoA utilization pathway for terpenoid productions
Author’s Accepted Manuscript Mitochondrial acetyl-CoA utilization pathway for terpenoid productions Jifeng Yuan, Chi-Bun Ching
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To appear in: Metabolic Engineering Received date: 11 March 2016 Revised date: 25 June 2016 Accepted date: 25 July 2016 Cite this article as: Jifeng Yuan and Chi-Bun Ching, Mitochondrial acetyl-CoA utilization pathway for terpenoid productions, Metabolic Engineering, http://dx.doi.org/10.1016/j.ymben.2016.07.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mitochondrial acetyl-CoA utilization pathway for terpenoid productions Jifeng Yuan1,2,4* and Chi-Bun Ching1,3* 1
Department of Chemical and Biomolecular Engineering, National University of
Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore 2
Temasek Laboratories, National University of Singapore, T-Lab Building 5A,
Engineering Drive 1, Singapore 117411, Singapore 3
Singapore Institute of Technology, 10 Dover Drive, Singapore 138683, Singapore
4
Present address: Biotransformation Innovation Platform, Agency for Science,
Technology and Research (A*STAR), Singapore 138673 * Corresponding author address: Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore; Tel: +65 66013179; Fax: +65 67764382; Email address: [email protected] (J. Yuan) or [email protected] (C.B. Ching)
ABSTRACT
Acetyl-CoA is a central molecule in the metabolism of the cell, which is also a precursor molecule to a variety of value-added products such as terpenoids and fatty acid derived molecules. Considering subcellular compartmentalization of metabolic pathways allows higher concentrations of enzymes, substrates and intermediates, and bypasses competing pathways, mitochondrion-compartmentalized acetyl-CoA utilization pathways might
1
offer better pathway activities with improved product yields. As a proof-of-concept, we sought to explore a mitochondrial farnesyl pyrophosphate (FPP) biosynthetic pathway for the biosynthesis of amorpha-4,11-diene in budding yeast. In the present study, the eightgene FPP biosynthetic pathway was successfully expressed inside yeast mitochondria to enable high-level amorpha-4,11-diene production. In addition, we also found the mitochondrial compartment serves as a partial barrier for the translocation of FPP from mitochondria into the cytosol, which would potentially allow minimized loss of FPP to cytosolic competing pathways. To our best knowledge, this is the first report to harness yeast mitochondria for terpenoid productions from the mitochondrial acetyl-CoA pool. We envision subcellular metabolic engineering might also be employed for an efficient production of other bio-products from the mitochondrial acetyl-CoA in other eukaryotic organisms.
Key words: Subcellular compartment; mitochondria; compartmentalization; acetyl-CoA; pathway relocalization; Saccharomyces cerevisiae
1. Introduction Microbial production of chemicals has been emerging as an alternative to petroleumbased chemical synthesis. By using genetically tractable host organisms such as Escherichia coli and Saccharomyces cerevisiae, products-of-interest can be synthesized from inexpensive starting materials by large-scale fermentation processes (Ajikumar et al., 2010; Chang and Keasling, 2006; Westfall et al., 2012). In order to achieve microbial
2
production of these chemicals at an industrial level, it is essential to engineer the central carbon metabolism in order to ensure the provision of adequate precursor levels for the synthesis of compounds-of-interest. Acetyl-CoA is a precursor molecule to a variety of bio-products, including fatty acids, 1-butanol, and terpenoids (Kocharin et al., 2012; Steen et al., 2008; Tehlivets et al., 2007; Xu et al., 2013; Xu et al., 2014). For this reason, there is a rising interest in engineering the central carbon metabolism of microbial cell factories for efficient conversion of sugars into acetyl-CoA to achieve high-level production of these industrially relevant compounds (Krivoruchko et al., 2015; Nielsen, 2014). In S. cerevisiae, the TCA cycle plays a very important role in catabolism and acetyl-CoA generated by the pyruvate dehydrogenase (PDH) complex is the key substrate for this process (Kresze and Ronft, 1981). Another important source for acetyl-CoA is acetylCoA synthetase (ACS) (van den Berg et al., 1996). ACS forms part of the PDH bypass in S. cerevisiae, consisting of pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD) and ACS. The reaction catalyzed by ACS consumes ATP to convert acetate to acetyl-CoA, forming AMP in two steps. Currently, researchers mainly focus on manipulating the cytosolic pool of acetyl-CoA by overexpressing the ACS pathway or a cytosol-based PDH system (Kozak et al., 2014; Shiba et al., 2007). There are limited reports on harnessing the mitochondrial acetyl-CoA for the synthesis of value-added chemicals in budding yeast. Based on the fact that subcellular compartmentalization of metabolic pathways allows higher concentrations of enzymes, substrates and intermediates, and bypasses competing pathways mitochondrion-compartmentalized acetyl-CoA utilization pathways may offer better pathway activities with improved 3
product yields (Avalos et al., 2013; Blumhoff et al., 2013; Li et al., 2015). In the present study, we sought to harness the subcellular mitochondrial compartment for producing acetyl-CoA derived chemicals from the mitochondrial acetyl-CoA pool. To enable the mitochondrial compartment as a reaction chamber for the production of acetylCoA derived chemicals, the final products have to be diffusible between mitochondria and the cytosol. Alternatively, mitochondrial transporters to translocate these compounds to the cytosol will be required. Considering terpenes are lipophilic compounds (Sikkema et al., 1995), it would be possible to freely diffuse across membranes. Therefore, utilizing a mitochondrion-compartmentalized pathway might enhance the terpenoid production in budding yeast. As a proof-of-concept, we examined a mitochondrial farnesyl pyrophosphate (FPP) biosynthetic pathway in S. cerevisiae for the biosynthesis of amorpha-4,11-diene (Fig. 1), which is a precursor for antimalarial drug (Paddon et al., 2013; Ro et al., 2006; Yuan and Ching, 2015a). In addition, we also investigated the effect on amorpha-4,11-diene production by different subcellular localizations of amorpha-4,11-diene synthase (ADS). 2. Materials and methods
2.1. Plasmid construction
Oligonucleotides used for plasmid constructions are listed in Supplementary Table 1. For constructing the mitochondrion-based orthogonal FPP biosynthetic pathway, a new δintegration platform was constructed as follows. The previously constructed pδBLE2.0 (Yuan and Ching, 2015b) was served as the template. The DNA sequence encoding the mitochondrial targeting signal (MTS) from Cox4 (Maarse et al., 1984) was PCR 4
amplified
from
the
genomic
DNA
of
S.
cerevisiae
using
F_NCox4_BamHI/R_NCox4_SalI. The PCR fragment was cut with BamHI/SalI, and inserted into pδBLE2.0 cut with BglII/SalI, to yield an intermediate plasmid. Next, the MTS of Cox4 was PCR amplified from the genomic DNA of S. cerevisiae using F_NCox4_BglII/R_NCox4_XhoI. The PCR fragment was cut with BglII/XhoI, and inserted into the above intermediate plasmid cut with BamHI/XhoI, to yield pδBLE2.0m. Subsequently, all FPP biosynthetic pathway genes were stepwise inserted into pδBLE2.0m in a similar way to the previous study (Yuan and Ching, 2015b). The resulting plasmids were designated as pδBLE2.0m-ERG13/ERG10, pδBLE2.0mERG12/tHMG1, and pδBLE2.0m-ERG19/ERG8 (as listed in Supplementary Table 2). Next, these plasmids were served as the template and genome integration cassettes were subsequently PCR amplified using primer pair Delta_IntF/Delta_IntR for constructing the FPP biosynthetic pathway.
For the downstream conversion of FPP to amorpha-4,11-diene, we constructed a plasmidbased expression system for ADS gene. For the cytosolic expression of ADS gene, ADS gene amplified from pRS425ADS (Ro et al., 2006) using primer pair F_ADSc_BamHI/ R_ADS_XhoI was cut with BamHI/XhoI, and inserted into pYES2 cut with BamHI/XhoI, to yield pYES2-ADS. For the mitochondrial expression of ADS gene, ADS gene amplified using primer pair F_ADSm_BamHI/R_ADS_XhoI was cut with BamHI/XhoI, and inserted into pYES2m cut with BamHI/XhoI, to yield pYES2m-ADS. Here, plasmid pYES2m was derived from pYES2 with a pre-inserted MTS from Coq3.
2.2. Genetic modification in budding yeast
5
Pathway assembly in budding yeast was performed as previously described (Yuan and Ching, 2015b). Briefly, 50 μL of yeast cells together with approximately 8 μg mixture of equimolar individual integration cassette was electroporated in a 0.2 cm cuvette at 1.6 kV. After electroporation, cells were immediately mixed with 6 mL 1:1 mix of 1 M sorbital:YPD medium and recovered a rotary shaker for 3 h. Following that, cells were collected by centrifugation at 3000 rpm for 5 min on a centrifuge, washed and resuspended in 200 μL ddH2O. Next, 100 μL cells were plated on SC plate supplemented with 240 μg/mL of phleomycin (InvivoGen, San Diego, USA). Colonies were randomly picked from the plate and subjected to the diagnostic PCR analysis of genome integration events.
For the verification of genome integration events, colonies were first picked from the plate and streaked on phleomycin-containing plates to eliminate false positives. Cells were lysed by 20 mM NaOH for 30 min in 100°C water bath. PCR program was set as follows: 1 cycle of 95°C for 5 min; amplification, 30 cycles of 95°C for 15 s, 50°C for 30 s and 68°C for 90 s; 1 cycle of 68°C for 3 min. Universal primer pair of F_GAL1_Scr and R_CYC1_Scr was used to verify the genome integration event of ERG10, tHMG1, ERG8 and ERG20. Universal primer pair of F_GAL10_Scr and R_ADH1_Scr was used for the PCR verification of ERG13, ERG12, ERG19 and IDI1. Only colonies with the four-band pattern with the correct size corresponding to each gene were considered to have the orthogonal FPP biosynthetic pathway assembled into yeast chromosomes.
2.3. Amorpha-4,11-diene production by engineered yeast cells
6
Strains used in the present study are listed in Supplementary Table 3. Experiments were carried out in 250 mL shake-flasks. Flasks containing 20 mL SC-URA medium supplemented with 1.8% (w/v) galactose + 0.2% (w/v) glucose were inoculated to an initial OD600 of 0.05 with overnight cultures. 20% of dodecane was added to perform two-phase fermentation and to minimize the vaporization of amorpha-4,11-diene. Each time, 100 μL of cell culture was taken for measuring OD600 by microplate reader, and 10 μL dodecane layer was sampled and diluted in 490 μL ethyl acetate for the determination of product levels by gas chromatography-mass spectrometry (GC-MS). During the GC-MS analysis, 1 μL of diluted sample was injected into Shimadzu QP2010Ultra system equipped with a DB-5ms column (Agilent Technologies, USA). Helium was used as a carrier gas at a flow rate of 1.0 mL/min. For the amorpha-4,11diene detection, the oven temperature was first kept constant at 80°C for 2 min, and then increased to 190°C at a rate of 5°C/min, and finally increased to 300°C by 20°C/min. Both the injector and mass detector were set at 250°C. Scan mode was used to detect the mass range 40-240 m/z. Caryophyllene was used for plotting the standard curve, and amorpha-4,11-diene levels in the present study were presented as caryophyllene equivalents.
2.4. RNA extraction and quantitative real-time PCR
Fresh overnight cell culture was inoculated into 2 mL SC medium to an initial OD600 of 0.05. During the early-log growth phase, 1 × 107 cells were collected for total RNA extraction using RNeasy Mini Kit (QIAGEN, Germany). Genomic DNA contamination was eliminated by in-column digestion with DNase I (QIAGEN, Germany). 7
Approximately 200 ng of RNA was converted to cDNA using iScriptTM Reverse Transcription Supermix from Biorad. Oligonucleotides for qPCR studies of pathway genes and actin (ACT1, internal reference gene) were designed using the ProbeFinder (https://www.roche-applied-science.com),
and
oligonucleotides
used
for
qPCR
experiments are listed in Supplementary Table 4. Quantitative PCR analysis was performed using LightCycler 96 real-time machine with FastStart Essential DNA Green Master according to the manufacturer’s instructions. To correct for differences in the amounts of starting materials, ACT1 was chosen as a reference housekeeping gene. The results were presented as ratios of gene expression between the target gene (gene of interest) and the reference gene, ACT1 (Pfaffl, 2001). All assays were performed in triplicate, and the reaction without reverse transcriptase was used as a negative control.
3. Results 3.1. Expression of amorpha-4,11-diene synthase inside yeast mitochondria
Considering the fact that FPP is required for the synthesis of heme A and ubiquione inside mitochondria such as Cox10, Coq1 and Bts1 (Ashby and Edwards, 1990; Glerum and Tzagoloff, 1994; Jiang et al., 1995), there may exit an unknown mitochondrial transporter to translocate FPP from the cytosol into yeast mitochondria (Farhi et al., 2011). Previously, Morgan Farhi et al. have harnessed yeast mitochondria for the production of plant sesquiterpenes and sesquiterpene synthases have been successfully targeted into yeast mitochondria using the mitochondrial targeting signal (MTS) from subunit IV of cytochrome c oxidase (encoded by Cox4) (Farhi et al., 2011). According to the literature, another MTS from the O-methyltransferase (Coq3) has also been 8
extensively used for targeting heterologous proteins into yeast mitochondria (Gurvitz, 2009; Hsu et al., 1996). In the present study, the MTS from Coq3 was examined for relocalizing amorpha-4,11-diene synthase into yeast mitochondria. As shown in Fig. 2, strains with a mitochondrion-targeted ADS using the MTS from Coq3 produced approximately 26.4 mg/L amorpha-4,11-diene after 96 h, which is an additional 63% improvement over that of the reference strain with a cytosolic expression of ADS. These findings indicated that ADS could be functionally expressed inside mitochondria using the MTS from Coq3. In addition, our work also suggested that the mitochondrial matrix might provide a favorable environment for the ADS activity (Blumhoff et al., 2013; Farhi et al., 2011).
3.2. FPP translocation from the cytosol into mitochondria is a limiting step for the amorpha-4,11-diene overproduction In budding yeast, FPP is synthesized from the cytosolic acetyl-CoA by eight-enzymatic steps (Kuzuyama and Seto, 2012): ERG10, ERG13, HMG1/2, ERG12, ERG8, ERG19, IDI1 and ERG20. Several studies have successfully demonstrated to metabolically engineer the FPP biosynthetic pathway in S. cerevisiae for enhanced production of terpenoids, based on manipulation of genes related to the endogenous yeast pathway such as activation of the rate-limiting step of HMG1/HMG2 encoding for 3-hydroxy-3methylglutaryl-coenzyme A reductase (HMGR) and the transcriptional regulator UPC2 (Ro et al., 2006) or overexpression of the entire FPP biosynthetic pathway (Westfall et al., 2012; Yuan and Ching, 2015b).
Although expression of a mitochondrion-targeted ADS, as compared to its cytosolic 9
counterpart, strongly enhanced amorpha-4,11-diene production (Fig. 2), we suspected that the translocation of FPP from the cytosol into yeast mitochondria might become a bottleneck for amorpha-4,11-diene overproduction when the FPP formation rate is above a certain threshold. In the present study, we attempted to construct an orthogonal cytosolbased FPP biosynthetic pathway under the control of strong promoters to improve the metabolic flux towards the FPP biosynthesis to test the above-mentioned hypothesis. As shown in Fig. 3, the mitochondrion-targeted expression of ADS in strains with an orthogonal cytosolic FPP biosynthetic pathway showed a significantly reduced level of amorpha-4,11-diene over the cytosol-targeted expression of ADS in the same strains, suggesting the translocation of FPP from the cytosol into mitochondria would be a limiting factor for amorpha-4,11-diene overproduction. Engineered strains with a cytosolic expression of ADS (OCP1C, OCP2C and OCP3C) resulted in approximately 290~380 mg/L of amorpha-4,11-diene under shake-flask conditions after 96 h cultivation (Fig. 3a). In contrast, strains with a mitochondrial expression of ADS (OCP1M, OCP1M and OCP3M) only yielded 160~240 mg/L of amorpha-4,11-diene under the same experimental condition (Fig. 3b). As shown in Fig. 3c, further quantitative real-time PCR (qRT-PCR) analysis revealed that mRNA abundance of individual FPP pathway genes was highly expressed in these variants over the reference strain (Supplementary Fig. 1), indicating the cytosolic FPP pathway under the control of strong promoters is successfully constructed.
3.3. Exploring a mitochondrion-based orthogonal FPP biosynthetic pathway for amorpha-4,11-diene biosynthesis As acetyl-CoA concentrations within mitochondria are estimated to be 20–30-fold greater 10
than that of the cytosol (Weinert et al., 2014), the mitochondrial acetyl-CoA pool may offer an alternative source for high-level production of terpenoids. In the present study, we sought to sequester the entire eight-gene FPP biosynthetic pathway into yeast mitochondria for the production of amorpha-4,11-diene. As shown in Fig. 1, the mitochondrion-based FPP biosynthetic pathway would not only overcome the low acetylCoA concentration in the cytosol, but also avoid the access of many cytosolic competing pathways such as ergosterol biosynthesis, farnesol, and prenylated proteins. Therefore, subcellular metabolic engineering would potentially offer an even better FPP pathway activity towards the synthesis of terpenoids.
Although there are numerous MTSs to be used for targeting foreign enzymes into yeast mitochondria, it is desirable that MTSs could be cleaved upon the translocation of foreign enzymes into mitochondria, to prevent unnecessary interruptions to the enzymatic activity. To enable a functional mitochondrion-relocalized FPP biosynthetic pathway, all FPP pathway enzymes were fused with the MTS from Cox4 (Maarse et al., 1984). Noteworthy,
the
MTS
from
Cox4
comprises
26
amino
acids
(MTSLRQSIRFFKPATRTLCSSRYLLQ), and it is cleaved at the Leu-Gln site by protease upon the translocation of targeted proteins into the yeast mitochondrial matrix (Maarse et al., 1984). In addition, as the membrane-associated domain is not required for the HMGR activity (Donald et al., 1997), the truncated version of HMGR was used to avoid any interference to the mitochondrial translocation process. As galactose is a partly fermentable sugar which is preferably catabolized fermentatively to ethanol and contributes a moderate respiration to cellular energy production during the initial phase (Fendt and Sauer, 2010), strain OMP1M~OMP3M only produced small amounts of 11
amorpha-4,11-diene at 48 h, with a titer around 120 mg/L (Fig. 4a). In the following phase, ethanol was converted to acetyl-CoA through the PDH bypass, and further transported into mitochondria via the carnitine/aceyl-carnitine shuttle (Schmalix and Bandlow, 1993). As shown in Fig 4a, at a later stage around 96 h, amorpha-4,11-diene levels in strain OMP1M~OMP3M rapidly reached 370~430 mg/L. These findings clearly indicated that the mitochondrion-targeted FPP biosynthetic pathway could be functionally expressed inside yeast mitochondria. More importantly, we also noticed that the amorpha-4,11-diene productivity was much higher during the respiratory metabolism than the fermentative growth. Therefore, in order to take full advantage of yeast mitochondria for the production of acetyl-CoA derived chemicals, nonfermentable carbon sources with acetyl-CoA mainly generated from PDH might be more suitable for the optimal pathway activity of mitochondrion-compartmentalized pathways.
3.4. Mitochondrial compartment serves as a partial barrier for the metabolite translocation
To further confirm that amorpha-4,11-diene was synthesized in yeast mitochondria (Fig. 2 and Fig. 4a), we next introduced a cytosol-targeted expression of ADS into strains with the mitochondrial FPP biosynthetic pathway for amorpha-4,11-diene productions. As shown in Fig. 4b, strain OMP1C~OMP3C only yielded approximately 20% amorpha4,11-diene of the same strains with the mitochondrion-targeted ADS, confirming that amorpha-4,11-diene was synthesized from the mitochondrial amorpha-4,11-diene biosynthetic pathway. Notably, there were still small amounts of amorpha-4,11-diene produced by strain OMP1C~OMP3C, which might be due to the targeted proteins being
12
not fully relocalized into yeast mitochondria (Avalos et al., 2013) and/or the slight leakiness of the mitochondrial compartment to retain the FPP pool. Nevertheless, our findings clearly suggested that the mitochondrial compartment could serve at least a partial barrier for the translocation of FPP from mitochondria into the cytosol and it would potentially allow minimized loss of the mitochondrion-generated FPP pool to other cytosolic competing pathways such as squalene synthase step involved in the ergosterol biosynthesis (Paradise et al., 2008).
Furthermore, considering that the accumulation of DMAPP, IPP and FPP and other toxic intermediates was reported to be toxic to the host cells (Dahl et al., 2013; Yuan and Ching, 2015a), we decided to investigate whether the mitochondrion-compartmentalized FPP biosynthetic pathway could prevent the toxic effect to the host cells. As shown in Supplementary Fig. 2, overexpression of either the cytosolic or the mitochondrial FPP biosynthetic pathway resulted in a significant growth inhibition in all these variants, suggesting the accumulation of pathway intermediates inside yeast mitochondria is still toxic to the cells. Noteworthy, the toxic effect caused by the accumulation of these intermediates was mitigated upon the overexpression of the downstream FPP consumption step.
As shown in Fig. 4c, further qRT-PCR analysis revealed that mRNA abundance of individual FPP pathway genes was highly expressed in these variants, which is much higher than that of the reference strain (Supplementary Fig. 1). As combinatorial assembly of the mitochondrial FPP biosynthetic pathway genes using the antibioticassisted δ-integration platform is expected to balance the pathway expression level (Yuan
13
and Ching, 2014), variants with a better FPP pathway activity might be isolated from the library, and further qRT-PCR analysis of these variants would provide useful information for the rational design of metabolic pathways in the future.
4. Discussion Acetyl-CoA serves as a central molecule in the metabolism of the cell for energy generation, and it is also a precursor molecule to a variety of value-added products such as terpenoids and fatty acid derived molecules. Considering acetyl-CoA concentrations within mitochondria are estimated to be 20–30-fold greater than that of the cytosol (Weinert et al., 2014), harnessing the mitochondrial acetyl-CoA pool offers an alternative strategy for high-level production of acetyl-CoA derived chemicals (Avalos et al., 2013). Moreover, subcellular compartmentalization of metabolic pathways would allow higher concentrations of enzymes, substrates and intermediates, and bypass competing pathways (Avalos et al., 2013; Blumhoff et al., 2013; Li et al., 2015). Besides the abovementioned merits, relocalization of the entire biosynthetic pathway into mitochondria would also allow bypassing the control mechanism at the post-translational level such as phosphorylation in its native compartment (Tripodi et al., 2015). In the present work, we successfully demonstrated that the FPP biosynthetic pathway could be functionally expressed inside yeast mitochondria for a relatively high level production of amorpha4,11-diene. Moreover, we also noticed that the mitochondria might offer at least a partial barrier for cytosolic pathways to access the mitochondrial FPP pool, suggesting compartmentalization of metabolic pathways would also allow minimized loss of intermediates to many other cytosolic competing pathways (Fig. 1). In the present study, the best engineered yeast strain with a mitochondrion-compartmentalized FPP 14
biosynthetic pathway yielded 427 mg/L amorpha-4,11-diene, which is approximately 21.5 mg per g of hexose. In the future, in order to take full advantage of yeast mitochondria for synthesis of acetylCoA derived chemicals, work will be focused on redirecting the flux towards respiration and to avoid the ethanol-producing phase. Unlike majority of microbes in which the switch between the fermentative and respiratory pathway is controlled by the oxygen level, S. cerevisiae controls fermentation versus respiration primarily in response to the sugar level. Although it was reported that S. cerevisiae relies on the respiratory catabolism in a glucose-limited chemostat (Postma et al., 1989), it will be desirable to generate a fully respiratory metabolism at high glucose levels for the optimal productivity of the mitochondrion-compartmentalized pathway. Previously, S. cerevisiae with a fully respiratory metabolism was generated by applying a chimeric hexose transporter (designated as TM6*) to mediate the reduced sugar uptake rate (Henricsson et al., 2005; Otterstedt et al., 2004). Such strategy could be similarly implemented to redirect the flux towards respiration, which would greatly improve the amorph-4,11-diene production in our engineered yeast strains. In budding yeast, the mitochondrial pyruvate is not only used for generating acetyl-CoA to provide the ATP formation to maintain the cell growth, but also for lipoic acid, and branched-chain amino acid biosynthesis (Dickinson, 2000; Hiltunen et al., 2010; Pronk et al., 1996). Hence, the availability of the mitochondrial pyruvate might become a bottleneck for the production of acetyl-CoA derived chemicals in yeast mitochondria. In yeasts, several strategies, involving manipulation of the cofactor level to selectively open the node of cytosolic pyruvate (Liu et al., 2007) and increasing the expression level of the mitochondrial pyruvate carriers (Li et al., 2015), had been 15
applied to redistribute the carbon flux of pyruvate into mitochondria. Such strategies might be similarly implemented to improve the translocation of pyruvate into mitochondria,
which
would
potentially further
improve
the
productivity of
mitochondrion-compartmentalized metabolic pathways. As fatty acid derived chemicals such as fatty alcohols, alkanes, alkenes, methyl-ketone can be freely diffusible between subcellular compartments, it will be interesting to see whether de novo fatty acid biosynthetic pathway (Runguphan and Keasling, 2014; Steen et al., 2010) and reversal β-oxidation pathway (Dellomonaco et al., 2011; Lian and Zhao, 2015) can be similarly transplanted inside yeast mitochondria. In addition, 1-butanol biosynthetic pathway (Steen et al., 2008) may also be compartmentalized into yeast mitochondria. Moreover, future work will also be dedicated to examine the compartmentalization strategy in other eukaryotes with a respiratory metabolism for an enhanced production of acetyl-CoA derived chemicals. ACKNOWLEDGEMENT
This work was supported by National University of Singapore.
AUTHOR CONTRIBUTIONS
J.Y. conceived and designed the project. J.Y. performed the experiments and collected the data. J.Y. and C.B.C interpreted the data. J.Y. wrote the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests. 16
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Tripodi, F., Nicastro, R., Reghellin, V., Coccetti, P., 2015. Post-translational modifications on yeast carbon metabolism: Regulatory mechanisms beyond transcriptional control. Biochimica et biophysica acta. 1850, 620-7. van den Berg, M. A., de Jong-Gubbels, P., Kortland, C. J., van Dijken, J. P., Pronk, J. T., Steensma, H. Y., 1996. The two acetyl-coenzyme A synthetases of Saccharomyces cerevisiae differ with respect to kinetic properties and transcriptional regulation. The Journal of biological chemistry. 271, 28953-9. Weinert, B. T., Iesmantavicius, V., Moustafa, T., Scholz, C., Wagner, S. A., Magnes, C., Zechner, R., Choudhary, C., 2014. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. Molecular systems biology. 10, 716. Westfall, P. J., Pitera, D. J., Lenihan, J. R., Eng, D., Woolard, F. X., Regentin, R., Horning, T., Tsuruta, H., Melis, D. J., Owens, A., Fickes, S., Diola, D., Benjamin, K. R., Keasling, J. D., Leavell, M. D., McPhee, D. J., Renninger, N. S., Newman, J. D., Paddon, C. J., 2012. Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin. Proceedings of the National Academy of Sciences of the United States of America. 109, E111-8. Xu, P., Gu, Q., Wang, W., Wong, L., Bower, A. G., Collins, C. H., Koffas, M. A., 2013. Modular optimization of multi-gene pathways for fatty acids production in E. coli. Nature communications. 4, 1409. Xu, P., Li, L., Zhang, F., Stephanopoulos, G., Koffas, M., 2014. Improving fatty acids production by engineering dynamic pathway regulation and metabolic control. Proceedings of the National Academy of Sciences of the United States of America. 111, 11299-304. Yuan, J., Ching, C. B., 2014. Combinatorial engineering of mevalonate pathway for improved amorpha-4,11-diene production in budding yeast. Biotechnology and bioengineering. 111, 608-17. Yuan, J., Ching, C. B., 2015a. Dynamic control of ERG9 expression for improved amorpha-4,11-diene production in Saccharomyces cerevisiae. Microbial cell factories. 14, 38. Yuan, J. F., Ching, C. B., 2015b. Combinatorial Assembly of Large Biochemical Pathways into Yeast Chromosomes for Improved Production of Value-added Compounds. ACS synthetic biology. 4, 23-31. List of figures Figure 1 Schematic diagram of mitochondrion-based orthogonal FPP biosynthetic pathway for amorpha-4,11-diene production. Genes from the FPP biosynthetic pathway in S. cerevisiae are shown in blue; heterologous expression of ADS gene is shown in green; and side-pathways that lead to side-product formations are shown in red. The pathway intermediates OAA, IPP, DMAPP, and FPP are defined as oxaloacetate,
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isopentenyl pyrophosphate, dimethylallyl pyrophosphate and farnesyl diphosphate, respectively. ADH, alcohol dehydrogenase; PDC, pyruvate decarboxylase; PDH, pyruvate dehydrogenase; ALD, acetaldehyde dehydrogenase; ACS, acetyl-CoA synthetase.
Figure 2 Expression of amorpha-4,11-diene synthase inside yeast mitochondria using the mitochondrial targeting signal from Coq3. Amorpha-4,11-diene production by different strains. Strain CEN.PK2-1C was transformed with plasmid pYES2-ADS or pYES2mADS. All engineered yeast strains were cultivated under shake-flasks supplemented with 1.8% (w/v) galactose + 0.2% (w/v) glucose. Data represented the average and standard deviation of three independent experiments.
Figure 3 The transportation of FPP from the cytosol into mitochondria limits the amorpha-4,11-diene production. (a) Amorpha-4,11-diene production by different strains with an orthogonal cytosolic FPP biosynthetic pathway coupled with the cytosolic expression of ADS. (b) Amorpha-4,11-diene production by different strains with an orthogonal cytosolic FPP biosynthetic pathway coupled with the mitochondrial expression of ADS. Three randomly isolated variants (OCP1, OCP2 and OCP3) with an orthogonal cytosolic FPP biosynthetic pathway were transformed with plasmid pYES2ADS or pYES2m-ADS. (c) Quantitative real-time PCR analysis to determine the expression profile of FPP biosynthetic pathway genes in the engineered strains. The results are presented as the relative abundance of FPP biosynthetic pathway genes in each strain with respect to that of ACT1. Data represent the average and standard deviation of three independent experiments.
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Figure 4 Orthogonal mitochondrial FPP biosynthetic pathway for amorpha-4,11-diene productions. (a) Amorpha-4,11-diene production by different strains with an orthogonal mitochondrial FPP biosynthetic pathway coupled with the mitochondrial expression of ADS. (b) Amorpha-4,11-diene production by different strains with an orthogonal mitochondrial FPP biosynthetic pathway coupled with the cytosolic expression of ADS. Three randomly isolated variants (OMP1, OMP2 and OMP3) with an orthogonal mitochondrial FPP biosynthetic pathway were transformed with plasmid pYES2m-ADS or pYES2-ADS. (c) Quantitative real-time PCR analysis to determine the expression profile of FPP biosynthetic pathway genes in the engineered strains. The results are presented as the relative abundance of FPP biosynthetic pathway genes in each strain with respect to that of ACT1. Data represent the average and standard deviation of three independent experiments.
Highlights
Amorpha-4,11-diene synthase is functionally expressed inside yeast mitochondria. An eight-gene FPP biosynthetic pathway is transplanted into yeast mitochondria. The mitochondrion-targeted FPP biosynthetic pathway is functional in yeast. The mitochondrial compartment serves as a barrier for the metabolite transportation. Subcellular metabolic engineering offers an alternative way for terpenoid synthesis.
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PII: DOI: Reference:
S1096-7176(16)30063-5 http://dx.doi.org/10.1016/j.ymben.2016.07.008 YMBEN1135
To appear in: Metabolic Engineering Received date: 11 March 2016 Revised date: 25 June 2016 Accepted date: 25 July 2016 Cite this article as: Jifeng Yuan and Chi-Bun Ching, Mitochondrial acetyl-CoA utilization pathway for terpenoid productions, Metabolic Engineering, http://dx.doi.org/10.1016/j.ymben.2016.07.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mitochondrial acetyl-CoA utilization pathway for terpenoid productions Jifeng Yuan1,2,4* and Chi-Bun Ching1,3* 1
Department of Chemical and Biomolecular Engineering, National University of
Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore 2
Temasek Laboratories, National University of Singapore, T-Lab Building 5A,
Engineering Drive 1, Singapore 117411, Singapore 3
Singapore Institute of Technology, 10 Dover Drive, Singapore 138683, Singapore
4
Present address: Biotransformation Innovation Platform, Agency for Science,
Technology and Research (A*STAR), Singapore 138673 * Corresponding author address: Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore; Tel: +65 66013179; Fax: +65 67764382; Email address: [email protected] (J. Yuan) or [email protected] (C.B. Ching)
ABSTRACT
Acetyl-CoA is a central molecule in the metabolism of the cell, which is also a precursor molecule to a variety of value-added products such as terpenoids and fatty acid derived molecules. Considering subcellular compartmentalization of metabolic pathways allows higher concentrations of enzymes, substrates and intermediates, and bypasses competing pathways, mitochondrion-compartmentalized acetyl-CoA utilization pathways might
1
offer better pathway activities with improved product yields. As a proof-of-concept, we sought to explore a mitochondrial farnesyl pyrophosphate (FPP) biosynthetic pathway for the biosynthesis of amorpha-4,11-diene in budding yeast. In the present study, the eightgene FPP biosynthetic pathway was successfully expressed inside yeast mitochondria to enable high-level amorpha-4,11-diene production. In addition, we also found the mitochondrial compartment serves as a partial barrier for the translocation of FPP from mitochondria into the cytosol, which would potentially allow minimized loss of FPP to cytosolic competing pathways. To our best knowledge, this is the first report to harness yeast mitochondria for terpenoid productions from the mitochondrial acetyl-CoA pool. We envision subcellular metabolic engineering might also be employed for an efficient production of other bio-products from the mitochondrial acetyl-CoA in other eukaryotic organisms.
Key words: Subcellular compartment; mitochondria; compartmentalization; acetyl-CoA; pathway relocalization; Saccharomyces cerevisiae
1. Introduction Microbial production of chemicals has been emerging as an alternative to petroleumbased chemical synthesis. By using genetically tractable host organisms such as Escherichia coli and Saccharomyces cerevisiae, products-of-interest can be synthesized from inexpensive starting materials by large-scale fermentation processes (Ajikumar et al., 2010; Chang and Keasling, 2006; Westfall et al., 2012). In order to achieve microbial
2
production of these chemicals at an industrial level, it is essential to engineer the central carbon metabolism in order to ensure the provision of adequate precursor levels for the synthesis of compounds-of-interest. Acetyl-CoA is a precursor molecule to a variety of bio-products, including fatty acids, 1-butanol, and terpenoids (Kocharin et al., 2012; Steen et al., 2008; Tehlivets et al., 2007; Xu et al., 2013; Xu et al., 2014). For this reason, there is a rising interest in engineering the central carbon metabolism of microbial cell factories for efficient conversion of sugars into acetyl-CoA to achieve high-level production of these industrially relevant compounds (Krivoruchko et al., 2015; Nielsen, 2014). In S. cerevisiae, the TCA cycle plays a very important role in catabolism and acetyl-CoA generated by the pyruvate dehydrogenase (PDH) complex is the key substrate for this process (Kresze and Ronft, 1981). Another important source for acetyl-CoA is acetylCoA synthetase (ACS) (van den Berg et al., 1996). ACS forms part of the PDH bypass in S. cerevisiae, consisting of pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD) and ACS. The reaction catalyzed by ACS consumes ATP to convert acetate to acetyl-CoA, forming AMP in two steps. Currently, researchers mainly focus on manipulating the cytosolic pool of acetyl-CoA by overexpressing the ACS pathway or a cytosol-based PDH system (Kozak et al., 2014; Shiba et al., 2007). There are limited reports on harnessing the mitochondrial acetyl-CoA for the synthesis of value-added chemicals in budding yeast. Based on the fact that subcellular compartmentalization of metabolic pathways allows higher concentrations of enzymes, substrates and intermediates, and bypasses competing pathways mitochondrion-compartmentalized acetyl-CoA utilization pathways may offer better pathway activities with improved 3
product yields (Avalos et al., 2013; Blumhoff et al., 2013; Li et al., 2015). In the present study, we sought to harness the subcellular mitochondrial compartment for producing acetyl-CoA derived chemicals from the mitochondrial acetyl-CoA pool. To enable the mitochondrial compartment as a reaction chamber for the production of acetylCoA derived chemicals, the final products have to be diffusible between mitochondria and the cytosol. Alternatively, mitochondrial transporters to translocate these compounds to the cytosol will be required. Considering terpenes are lipophilic compounds (Sikkema et al., 1995), it would be possible to freely diffuse across membranes. Therefore, utilizing a mitochondrion-compartmentalized pathway might enhance the terpenoid production in budding yeast. As a proof-of-concept, we examined a mitochondrial farnesyl pyrophosphate (FPP) biosynthetic pathway in S. cerevisiae for the biosynthesis of amorpha-4,11-diene (Fig. 1), which is a precursor for antimalarial drug (Paddon et al., 2013; Ro et al., 2006; Yuan and Ching, 2015a). In addition, we also investigated the effect on amorpha-4,11-diene production by different subcellular localizations of amorpha-4,11-diene synthase (ADS). 2. Materials and methods
2.1. Plasmid construction
Oligonucleotides used for plasmid constructions are listed in Supplementary Table 1. For constructing the mitochondrion-based orthogonal FPP biosynthetic pathway, a new δintegration platform was constructed as follows. The previously constructed pδBLE2.0 (Yuan and Ching, 2015b) was served as the template. The DNA sequence encoding the mitochondrial targeting signal (MTS) from Cox4 (Maarse et al., 1984) was PCR 4
amplified
from
the
genomic
DNA
of
S.
cerevisiae
using
F_NCox4_BamHI/R_NCox4_SalI. The PCR fragment was cut with BamHI/SalI, and inserted into pδBLE2.0 cut with BglII/SalI, to yield an intermediate plasmid. Next, the MTS of Cox4 was PCR amplified from the genomic DNA of S. cerevisiae using F_NCox4_BglII/R_NCox4_XhoI. The PCR fragment was cut with BglII/XhoI, and inserted into the above intermediate plasmid cut with BamHI/XhoI, to yield pδBLE2.0m. Subsequently, all FPP biosynthetic pathway genes were stepwise inserted into pδBLE2.0m in a similar way to the previous study (Yuan and Ching, 2015b). The resulting plasmids were designated as pδBLE2.0m-ERG13/ERG10, pδBLE2.0mERG12/tHMG1, and pδBLE2.0m-ERG19/ERG8 (as listed in Supplementary Table 2). Next, these plasmids were served as the template and genome integration cassettes were subsequently PCR amplified using primer pair Delta_IntF/Delta_IntR for constructing the FPP biosynthetic pathway.
For the downstream conversion of FPP to amorpha-4,11-diene, we constructed a plasmidbased expression system for ADS gene. For the cytosolic expression of ADS gene, ADS gene amplified from pRS425ADS (Ro et al., 2006) using primer pair F_ADSc_BamHI/ R_ADS_XhoI was cut with BamHI/XhoI, and inserted into pYES2 cut with BamHI/XhoI, to yield pYES2-ADS. For the mitochondrial expression of ADS gene, ADS gene amplified using primer pair F_ADSm_BamHI/R_ADS_XhoI was cut with BamHI/XhoI, and inserted into pYES2m cut with BamHI/XhoI, to yield pYES2m-ADS. Here, plasmid pYES2m was derived from pYES2 with a pre-inserted MTS from Coq3.
2.2. Genetic modification in budding yeast
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Pathway assembly in budding yeast was performed as previously described (Yuan and Ching, 2015b). Briefly, 50 μL of yeast cells together with approximately 8 μg mixture of equimolar individual integration cassette was electroporated in a 0.2 cm cuvette at 1.6 kV. After electroporation, cells were immediately mixed with 6 mL 1:1 mix of 1 M sorbital:YPD medium and recovered a rotary shaker for 3 h. Following that, cells were collected by centrifugation at 3000 rpm for 5 min on a centrifuge, washed and resuspended in 200 μL ddH2O. Next, 100 μL cells were plated on SC plate supplemented with 240 μg/mL of phleomycin (InvivoGen, San Diego, USA). Colonies were randomly picked from the plate and subjected to the diagnostic PCR analysis of genome integration events.
For the verification of genome integration events, colonies were first picked from the plate and streaked on phleomycin-containing plates to eliminate false positives. Cells were lysed by 20 mM NaOH for 30 min in 100°C water bath. PCR program was set as follows: 1 cycle of 95°C for 5 min; amplification, 30 cycles of 95°C for 15 s, 50°C for 30 s and 68°C for 90 s; 1 cycle of 68°C for 3 min. Universal primer pair of F_GAL1_Scr and R_CYC1_Scr was used to verify the genome integration event of ERG10, tHMG1, ERG8 and ERG20. Universal primer pair of F_GAL10_Scr and R_ADH1_Scr was used for the PCR verification of ERG13, ERG12, ERG19 and IDI1. Only colonies with the four-band pattern with the correct size corresponding to each gene were considered to have the orthogonal FPP biosynthetic pathway assembled into yeast chromosomes.
2.3. Amorpha-4,11-diene production by engineered yeast cells
6
Strains used in the present study are listed in Supplementary Table 3. Experiments were carried out in 250 mL shake-flasks. Flasks containing 20 mL SC-URA medium supplemented with 1.8% (w/v) galactose + 0.2% (w/v) glucose were inoculated to an initial OD600 of 0.05 with overnight cultures. 20% of dodecane was added to perform two-phase fermentation and to minimize the vaporization of amorpha-4,11-diene. Each time, 100 μL of cell culture was taken for measuring OD600 by microplate reader, and 10 μL dodecane layer was sampled and diluted in 490 μL ethyl acetate for the determination of product levels by gas chromatography-mass spectrometry (GC-MS). During the GC-MS analysis, 1 μL of diluted sample was injected into Shimadzu QP2010Ultra system equipped with a DB-5ms column (Agilent Technologies, USA). Helium was used as a carrier gas at a flow rate of 1.0 mL/min. For the amorpha-4,11diene detection, the oven temperature was first kept constant at 80°C for 2 min, and then increased to 190°C at a rate of 5°C/min, and finally increased to 300°C by 20°C/min. Both the injector and mass detector were set at 250°C. Scan mode was used to detect the mass range 40-240 m/z. Caryophyllene was used for plotting the standard curve, and amorpha-4,11-diene levels in the present study were presented as caryophyllene equivalents.
2.4. RNA extraction and quantitative real-time PCR
Fresh overnight cell culture was inoculated into 2 mL SC medium to an initial OD600 of 0.05. During the early-log growth phase, 1 × 107 cells were collected for total RNA extraction using RNeasy Mini Kit (QIAGEN, Germany). Genomic DNA contamination was eliminated by in-column digestion with DNase I (QIAGEN, Germany). 7
Approximately 200 ng of RNA was converted to cDNA using iScriptTM Reverse Transcription Supermix from Biorad. Oligonucleotides for qPCR studies of pathway genes and actin (ACT1, internal reference gene) were designed using the ProbeFinder (https://www.roche-applied-science.com),
and
oligonucleotides
used
for
qPCR
experiments are listed in Supplementary Table 4. Quantitative PCR analysis was performed using LightCycler 96 real-time machine with FastStart Essential DNA Green Master according to the manufacturer’s instructions. To correct for differences in the amounts of starting materials, ACT1 was chosen as a reference housekeeping gene. The results were presented as ratios of gene expression between the target gene (gene of interest) and the reference gene, ACT1 (Pfaffl, 2001). All assays were performed in triplicate, and the reaction without reverse transcriptase was used as a negative control.
3. Results 3.1. Expression of amorpha-4,11-diene synthase inside yeast mitochondria
Considering the fact that FPP is required for the synthesis of heme A and ubiquione inside mitochondria such as Cox10, Coq1 and Bts1 (Ashby and Edwards, 1990; Glerum and Tzagoloff, 1994; Jiang et al., 1995), there may exit an unknown mitochondrial transporter to translocate FPP from the cytosol into yeast mitochondria (Farhi et al., 2011). Previously, Morgan Farhi et al. have harnessed yeast mitochondria for the production of plant sesquiterpenes and sesquiterpene synthases have been successfully targeted into yeast mitochondria using the mitochondrial targeting signal (MTS) from subunit IV of cytochrome c oxidase (encoded by Cox4) (Farhi et al., 2011). According to the literature, another MTS from the O-methyltransferase (Coq3) has also been 8
extensively used for targeting heterologous proteins into yeast mitochondria (Gurvitz, 2009; Hsu et al., 1996). In the present study, the MTS from Coq3 was examined for relocalizing amorpha-4,11-diene synthase into yeast mitochondria. As shown in Fig. 2, strains with a mitochondrion-targeted ADS using the MTS from Coq3 produced approximately 26.4 mg/L amorpha-4,11-diene after 96 h, which is an additional 63% improvement over that of the reference strain with a cytosolic expression of ADS. These findings indicated that ADS could be functionally expressed inside mitochondria using the MTS from Coq3. In addition, our work also suggested that the mitochondrial matrix might provide a favorable environment for the ADS activity (Blumhoff et al., 2013; Farhi et al., 2011).
3.2. FPP translocation from the cytosol into mitochondria is a limiting step for the amorpha-4,11-diene overproduction In budding yeast, FPP is synthesized from the cytosolic acetyl-CoA by eight-enzymatic steps (Kuzuyama and Seto, 2012): ERG10, ERG13, HMG1/2, ERG12, ERG8, ERG19, IDI1 and ERG20. Several studies have successfully demonstrated to metabolically engineer the FPP biosynthetic pathway in S. cerevisiae for enhanced production of terpenoids, based on manipulation of genes related to the endogenous yeast pathway such as activation of the rate-limiting step of HMG1/HMG2 encoding for 3-hydroxy-3methylglutaryl-coenzyme A reductase (HMGR) and the transcriptional regulator UPC2 (Ro et al., 2006) or overexpression of the entire FPP biosynthetic pathway (Westfall et al., 2012; Yuan and Ching, 2015b).
Although expression of a mitochondrion-targeted ADS, as compared to its cytosolic 9
counterpart, strongly enhanced amorpha-4,11-diene production (Fig. 2), we suspected that the translocation of FPP from the cytosol into yeast mitochondria might become a bottleneck for amorpha-4,11-diene overproduction when the FPP formation rate is above a certain threshold. In the present study, we attempted to construct an orthogonal cytosolbased FPP biosynthetic pathway under the control of strong promoters to improve the metabolic flux towards the FPP biosynthesis to test the above-mentioned hypothesis. As shown in Fig. 3, the mitochondrion-targeted expression of ADS in strains with an orthogonal cytosolic FPP biosynthetic pathway showed a significantly reduced level of amorpha-4,11-diene over the cytosol-targeted expression of ADS in the same strains, suggesting the translocation of FPP from the cytosol into mitochondria would be a limiting factor for amorpha-4,11-diene overproduction. Engineered strains with a cytosolic expression of ADS (OCP1C, OCP2C and OCP3C) resulted in approximately 290~380 mg/L of amorpha-4,11-diene under shake-flask conditions after 96 h cultivation (Fig. 3a). In contrast, strains with a mitochondrial expression of ADS (OCP1M, OCP1M and OCP3M) only yielded 160~240 mg/L of amorpha-4,11-diene under the same experimental condition (Fig. 3b). As shown in Fig. 3c, further quantitative real-time PCR (qRT-PCR) analysis revealed that mRNA abundance of individual FPP pathway genes was highly expressed in these variants over the reference strain (Supplementary Fig. 1), indicating the cytosolic FPP pathway under the control of strong promoters is successfully constructed.
3.3. Exploring a mitochondrion-based orthogonal FPP biosynthetic pathway for amorpha-4,11-diene biosynthesis As acetyl-CoA concentrations within mitochondria are estimated to be 20–30-fold greater 10
than that of the cytosol (Weinert et al., 2014), the mitochondrial acetyl-CoA pool may offer an alternative source for high-level production of terpenoids. In the present study, we sought to sequester the entire eight-gene FPP biosynthetic pathway into yeast mitochondria for the production of amorpha-4,11-diene. As shown in Fig. 1, the mitochondrion-based FPP biosynthetic pathway would not only overcome the low acetylCoA concentration in the cytosol, but also avoid the access of many cytosolic competing pathways such as ergosterol biosynthesis, farnesol, and prenylated proteins. Therefore, subcellular metabolic engineering would potentially offer an even better FPP pathway activity towards the synthesis of terpenoids.
Although there are numerous MTSs to be used for targeting foreign enzymes into yeast mitochondria, it is desirable that MTSs could be cleaved upon the translocation of foreign enzymes into mitochondria, to prevent unnecessary interruptions to the enzymatic activity. To enable a functional mitochondrion-relocalized FPP biosynthetic pathway, all FPP pathway enzymes were fused with the MTS from Cox4 (Maarse et al., 1984). Noteworthy,
the
MTS
from
Cox4
comprises
26
amino
acids
(MTSLRQSIRFFKPATRTLCSSRYLLQ), and it is cleaved at the Leu-Gln site by protease upon the translocation of targeted proteins into the yeast mitochondrial matrix (Maarse et al., 1984). In addition, as the membrane-associated domain is not required for the HMGR activity (Donald et al., 1997), the truncated version of HMGR was used to avoid any interference to the mitochondrial translocation process. As galactose is a partly fermentable sugar which is preferably catabolized fermentatively to ethanol and contributes a moderate respiration to cellular energy production during the initial phase (Fendt and Sauer, 2010), strain OMP1M~OMP3M only produced small amounts of 11
amorpha-4,11-diene at 48 h, with a titer around 120 mg/L (Fig. 4a). In the following phase, ethanol was converted to acetyl-CoA through the PDH bypass, and further transported into mitochondria via the carnitine/aceyl-carnitine shuttle (Schmalix and Bandlow, 1993). As shown in Fig 4a, at a later stage around 96 h, amorpha-4,11-diene levels in strain OMP1M~OMP3M rapidly reached 370~430 mg/L. These findings clearly indicated that the mitochondrion-targeted FPP biosynthetic pathway could be functionally expressed inside yeast mitochondria. More importantly, we also noticed that the amorpha-4,11-diene productivity was much higher during the respiratory metabolism than the fermentative growth. Therefore, in order to take full advantage of yeast mitochondria for the production of acetyl-CoA derived chemicals, nonfermentable carbon sources with acetyl-CoA mainly generated from PDH might be more suitable for the optimal pathway activity of mitochondrion-compartmentalized pathways.
3.4. Mitochondrial compartment serves as a partial barrier for the metabolite translocation
To further confirm that amorpha-4,11-diene was synthesized in yeast mitochondria (Fig. 2 and Fig. 4a), we next introduced a cytosol-targeted expression of ADS into strains with the mitochondrial FPP biosynthetic pathway for amorpha-4,11-diene productions. As shown in Fig. 4b, strain OMP1C~OMP3C only yielded approximately 20% amorpha4,11-diene of the same strains with the mitochondrion-targeted ADS, confirming that amorpha-4,11-diene was synthesized from the mitochondrial amorpha-4,11-diene biosynthetic pathway. Notably, there were still small amounts of amorpha-4,11-diene produced by strain OMP1C~OMP3C, which might be due to the targeted proteins being
12
not fully relocalized into yeast mitochondria (Avalos et al., 2013) and/or the slight leakiness of the mitochondrial compartment to retain the FPP pool. Nevertheless, our findings clearly suggested that the mitochondrial compartment could serve at least a partial barrier for the translocation of FPP from mitochondria into the cytosol and it would potentially allow minimized loss of the mitochondrion-generated FPP pool to other cytosolic competing pathways such as squalene synthase step involved in the ergosterol biosynthesis (Paradise et al., 2008).
Furthermore, considering that the accumulation of DMAPP, IPP and FPP and other toxic intermediates was reported to be toxic to the host cells (Dahl et al., 2013; Yuan and Ching, 2015a), we decided to investigate whether the mitochondrion-compartmentalized FPP biosynthetic pathway could prevent the toxic effect to the host cells. As shown in Supplementary Fig. 2, overexpression of either the cytosolic or the mitochondrial FPP biosynthetic pathway resulted in a significant growth inhibition in all these variants, suggesting the accumulation of pathway intermediates inside yeast mitochondria is still toxic to the cells. Noteworthy, the toxic effect caused by the accumulation of these intermediates was mitigated upon the overexpression of the downstream FPP consumption step.
As shown in Fig. 4c, further qRT-PCR analysis revealed that mRNA abundance of individual FPP pathway genes was highly expressed in these variants, which is much higher than that of the reference strain (Supplementary Fig. 1). As combinatorial assembly of the mitochondrial FPP biosynthetic pathway genes using the antibioticassisted δ-integration platform is expected to balance the pathway expression level (Yuan
13
and Ching, 2014), variants with a better FPP pathway activity might be isolated from the library, and further qRT-PCR analysis of these variants would provide useful information for the rational design of metabolic pathways in the future.
4. Discussion Acetyl-CoA serves as a central molecule in the metabolism of the cell for energy generation, and it is also a precursor molecule to a variety of value-added products such as terpenoids and fatty acid derived molecules. Considering acetyl-CoA concentrations within mitochondria are estimated to be 20–30-fold greater than that of the cytosol (Weinert et al., 2014), harnessing the mitochondrial acetyl-CoA pool offers an alternative strategy for high-level production of acetyl-CoA derived chemicals (Avalos et al., 2013). Moreover, subcellular compartmentalization of metabolic pathways would allow higher concentrations of enzymes, substrates and intermediates, and bypass competing pathways (Avalos et al., 2013; Blumhoff et al., 2013; Li et al., 2015). Besides the abovementioned merits, relocalization of the entire biosynthetic pathway into mitochondria would also allow bypassing the control mechanism at the post-translational level such as phosphorylation in its native compartment (Tripodi et al., 2015). In the present work, we successfully demonstrated that the FPP biosynthetic pathway could be functionally expressed inside yeast mitochondria for a relatively high level production of amorpha4,11-diene. Moreover, we also noticed that the mitochondria might offer at least a partial barrier for cytosolic pathways to access the mitochondrial FPP pool, suggesting compartmentalization of metabolic pathways would also allow minimized loss of intermediates to many other cytosolic competing pathways (Fig. 1). In the present study, the best engineered yeast strain with a mitochondrion-compartmentalized FPP 14
biosynthetic pathway yielded 427 mg/L amorpha-4,11-diene, which is approximately 21.5 mg per g of hexose. In the future, in order to take full advantage of yeast mitochondria for synthesis of acetylCoA derived chemicals, work will be focused on redirecting the flux towards respiration and to avoid the ethanol-producing phase. Unlike majority of microbes in which the switch between the fermentative and respiratory pathway is controlled by the oxygen level, S. cerevisiae controls fermentation versus respiration primarily in response to the sugar level. Although it was reported that S. cerevisiae relies on the respiratory catabolism in a glucose-limited chemostat (Postma et al., 1989), it will be desirable to generate a fully respiratory metabolism at high glucose levels for the optimal productivity of the mitochondrion-compartmentalized pathway. Previously, S. cerevisiae with a fully respiratory metabolism was generated by applying a chimeric hexose transporter (designated as TM6*) to mediate the reduced sugar uptake rate (Henricsson et al., 2005; Otterstedt et al., 2004). Such strategy could be similarly implemented to redirect the flux towards respiration, which would greatly improve the amorph-4,11-diene production in our engineered yeast strains. In budding yeast, the mitochondrial pyruvate is not only used for generating acetyl-CoA to provide the ATP formation to maintain the cell growth, but also for lipoic acid, and branched-chain amino acid biosynthesis (Dickinson, 2000; Hiltunen et al., 2010; Pronk et al., 1996). Hence, the availability of the mitochondrial pyruvate might become a bottleneck for the production of acetyl-CoA derived chemicals in yeast mitochondria. In yeasts, several strategies, involving manipulation of the cofactor level to selectively open the node of cytosolic pyruvate (Liu et al., 2007) and increasing the expression level of the mitochondrial pyruvate carriers (Li et al., 2015), had been 15
applied to redistribute the carbon flux of pyruvate into mitochondria. Such strategies might be similarly implemented to improve the translocation of pyruvate into mitochondria,
which
would
potentially further
improve
the
productivity of
mitochondrion-compartmentalized metabolic pathways. As fatty acid derived chemicals such as fatty alcohols, alkanes, alkenes, methyl-ketone can be freely diffusible between subcellular compartments, it will be interesting to see whether de novo fatty acid biosynthetic pathway (Runguphan and Keasling, 2014; Steen et al., 2010) and reversal β-oxidation pathway (Dellomonaco et al., 2011; Lian and Zhao, 2015) can be similarly transplanted inside yeast mitochondria. In addition, 1-butanol biosynthetic pathway (Steen et al., 2008) may also be compartmentalized into yeast mitochondria. Moreover, future work will also be dedicated to examine the compartmentalization strategy in other eukaryotes with a respiratory metabolism for an enhanced production of acetyl-CoA derived chemicals. ACKNOWLEDGEMENT
This work was supported by National University of Singapore.
AUTHOR CONTRIBUTIONS
J.Y. conceived and designed the project. J.Y. performed the experiments and collected the data. J.Y. and C.B.C interpreted the data. J.Y. wrote the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests. 16
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isopentenyl pyrophosphate, dimethylallyl pyrophosphate and farnesyl diphosphate, respectively. ADH, alcohol dehydrogenase; PDC, pyruvate decarboxylase; PDH, pyruvate dehydrogenase; ALD, acetaldehyde dehydrogenase; ACS, acetyl-CoA synthetase.
Figure 2 Expression of amorpha-4,11-diene synthase inside yeast mitochondria using the mitochondrial targeting signal from Coq3. Amorpha-4,11-diene production by different strains. Strain CEN.PK2-1C was transformed with plasmid pYES2-ADS or pYES2mADS. All engineered yeast strains were cultivated under shake-flasks supplemented with 1.8% (w/v) galactose + 0.2% (w/v) glucose. Data represented the average and standard deviation of three independent experiments.
Figure 3 The transportation of FPP from the cytosol into mitochondria limits the amorpha-4,11-diene production. (a) Amorpha-4,11-diene production by different strains with an orthogonal cytosolic FPP biosynthetic pathway coupled with the cytosolic expression of ADS. (b) Amorpha-4,11-diene production by different strains with an orthogonal cytosolic FPP biosynthetic pathway coupled with the mitochondrial expression of ADS. Three randomly isolated variants (OCP1, OCP2 and OCP3) with an orthogonal cytosolic FPP biosynthetic pathway were transformed with plasmid pYES2ADS or pYES2m-ADS. (c) Quantitative real-time PCR analysis to determine the expression profile of FPP biosynthetic pathway genes in the engineered strains. The results are presented as the relative abundance of FPP biosynthetic pathway genes in each strain with respect to that of ACT1. Data represent the average and standard deviation of three independent experiments.
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Figure 4 Orthogonal mitochondrial FPP biosynthetic pathway for amorpha-4,11-diene productions. (a) Amorpha-4,11-diene production by different strains with an orthogonal mitochondrial FPP biosynthetic pathway coupled with the mitochondrial expression of ADS. (b) Amorpha-4,11-diene production by different strains with an orthogonal mitochondrial FPP biosynthetic pathway coupled with the cytosolic expression of ADS. Three randomly isolated variants (OMP1, OMP2 and OMP3) with an orthogonal mitochondrial FPP biosynthetic pathway were transformed with plasmid pYES2m-ADS or pYES2-ADS. (c) Quantitative real-time PCR analysis to determine the expression profile of FPP biosynthetic pathway genes in the engineered strains. The results are presented as the relative abundance of FPP biosynthetic pathway genes in each strain with respect to that of ACT1. Data represent the average and standard deviation of three independent experiments.
Highlights
Amorpha-4,11-diene synthase is functionally expressed inside yeast mitochondria. An eight-gene FPP biosynthetic pathway is transplanted into yeast mitochondria. The mitochondrion-targeted FPP biosynthetic pathway is functional in yeast. The mitochondrial compartment serves as a barrier for the metabolite transportation. Subcellular metabolic engineering offers an alternative way for terpenoid synthesis.
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