Production and quantification of sesquiterpenes in Saccharomyces cerevisiae, including extraction, detection and quantification of terpene products and key related metabolites.

protocol

Production and quantification of sesquiterpenes in Saccharomyces cerevisiae, including extraction, detection and quantification of terpene products and key related metabolites Sarah Rodriguez1,2, James Kirby1,3, Charles M Denby1,3 & Jay D Keasling1–5 1Joint BioEnergy Institute, Emeryville, California, USA. 2Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. 3California Institute of

Quantitative Biosciences (QB3), University of California, Berkeley, California, USA. 4Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, USA. 5Department of Bioengineering, University of California, Berkeley, California, USA. Correspondence should be addressed to J.D.K. ([email protected]).

© 2014 Nature America, Inc. All rights reserved.

Published online 24 July 2014; doi:10.1038/nprot.2014.132

The procedures described here are designed for engineering Saccharomyces cerevisiae to produce sesquiterpenes with an aim to either increase product titers or to simply generate a quantity of product sufficient for identification and/or downstream experimentation. Engineering high-level sesquiterpene production in S. cerevisiae often requires iterations of strain modifications and metabolite analysis. To address the latter, the methods described here were tailored for robust measurement of metabolites that we have found to be fundamental indicators of pathway flux, using only gas chromatography and mass spectrometry (GC-MS) instrumentation. Thus, by focusing on heterologous production of sesquiterpenes via the mevalonate (MEV) pathway in S. cerevisiae, we detail procedures for extraction and detection of the key pathway metabolites MEV, squalene and ergosterol, as well as the farnesyl pyrophosphate (FPP)-derived side products farnesol and nerolidol. Analysis of these compounds is important for quality control, because they are possible indicators of pathway imbalance. As many of the sesquiterpene synthase (STS) genes encountered in nature are of plant origin and often not optimal for expression in yeast, we provide guidelines for designing gene expression cassettes to enable expression in S. cerevisiae. As a case study for these protocols, we have selected the sesquiterpene amorphadiene, native to Artemisia annua and related plants. The analytical steps can be completed within 1–2 working days, and a typical experiment might take 1 week.

INTRODUCTION Development of the protocol Isoprenoids constitute one of the most structurally diverse groups of natural products, with applications ranging from medicine, agriculture and nutrition to the specialty chemical and energy sectors. The sesquiterpene amorphadiene and its acid derivatives have attracted much interest since they were discovered to be precursors to the antimalarial drug artemisinin. Recent improvements in the titers of amorphadiene1 and artemisinic acid2,3 in S. cerevisiae demonstrate the impressive capacity of yeast as a host for sesquiterpene production. However, despite the capacity to produce large quantities of the sesquiterpene precursor FPP in yeast, many STSs are severely limited by their kinetics, and their expression in engineered yeast strains often results in accumulation of FPP and related prenyl phosphate precursors to toxic levels. Therefore, a combination of STS expression optimization coupled with pathway balancing may be required to achieve the desired in vivo production levels. In the Experimental design section, we briefly address factors related to host engineering and gene expression. The PROCEDURE describes the routine extraction, detection and quantification of sesquiterpene products, common pathway intermediates and side products that can be used to monitor product titers, as well as pathway imbalances during an iterative engineering and optimization project. Applications of this method Amorphadiene is used as an example of a representative sesquiterpene in this protocol; these methods may be applied to monitor 1980 | VOL.9 NO.8 | 2014 | nature protocols

the production of any sesquiterpene olefin (C15H24) in yeast, and we have used them to quantify the production of bisabolene, germacrene A, α-humulene, δ-guaiene and vetispiradiene, to name a few4,5. Detection of oxidized sesquiterpenes (alcohols, acids, aldehydes, ketones and lactones) is also often possible with this protocol, but a trial run with an authentic standard, if available, is advised. In this protocol, caryophyllene is used as an internal standard. Although we do not expect caryophyllene and amorphadiene to ionize with equal efficiency, caryophyllene is a good sesquiterpene to use as a standard if no authentic standard is available, as is the case for amorphadiene, because it is not produced naturally by yeast and it has distinctive GC-MS characteristics. In this situation, the target sesquiterpene titer may be expressed as caryophyllene equivalents. The protocols describing extraction, detection and quantification of a sesquiterpene product (amorphadiene), a major pathway intermediate (MEV), and FPP-derived side products (farnesol, nerolidol, squalene and ergosterol) may be applied to any S. cerevisiae engineering endeavors that aim to produce compounds derived from the MEV pathway, and therefore these ancillary methods are not limited to sesquiterpene production. Comparison with other methods for sesquiterpene detection GC for sesquiterpene detection. Analytical methods applied to the separation of plant essential oils often use chromatographic separation methods such as GC and liquid chromatography


protocol (LC) to separate individual components. Although LC may be a preferred technique for the analysis of terpenes that have been modified with acid or sugar groups owing to their increased boiling points and hydrophilicity, GC is by far the most commonly used method for detection of terpene olefins. Sesquiterpene olefins, with boiling points ranging from ~250 to 280 °C, are ideal candidates for gas-phase separation, and, importantly, the mass spectral ‘fingerprint’ generated when GC is coupled with MS is often crucial for identification. The fact that sesquiterpene olefins share the chemical formula C15H24 necessitates substantial ion fragmentation to differentiate similar molecules; GC-MS offers the key advantage of a standardized electron impact (EI) ionization energy that allows comparison of spectra across labs and to published libraries6 (see below). The first GC columns extensively used for terpene detection were nonpolar capillary columns with polydimethylsiloxane (methyl silicone) or polar capillary columns of type ‘Carbowax 20M’ (PEG). The popularity and usefulness of the columns resulted in a compendium of retention indices (Kovats retention index) for >400 individual monoterpene and sesquiterpene compounds7. The introduction of enantioselective capillary columns significantly aided the ability to separate the various terpene enantiomers often found together in essential oils8. Separation of enantiomers of both terpene olefins and alcohols was further improved with the introduction of columns containing various hydrophobic cyclodextrin derivatives 9. We typically use two columns for the detection of sesquiterpenes and associated metabolites. We use a DB-5MS column, a nonpolar capillary column containing polydimethylsiloxane and a phenyl arylene polymer, for the detection of high-molecular-weight components such as squalene and ergosterol. We use a CycloSil-B capillary column, which contains a cyclodextrin derivative, for MEV detection and separation of sesquiterpenes and FPP side products farnesol and nerolidol. The DB-5MS is also usually sufficient for the separation and detection of sesquiterpene olefins unless the extract contains a mixture of enantiomers that require the resolving power of the CycloSil-B. Coupled GC-MS for sesquiterpene detection. Although vast improvements to GC columns have increased the resolution of complex enantiomeric mixtures from oils and extracts, the retention time (RT, or retention index) alone is often not sufficient for positive confirmation of a particular terpene. The combination of RT and mass spectrum generated by a coupled GC-MS is generally considered sufficient for positive identification of a terpene when the sample is run alongside an authentic standard. The most commonly used instrument to enable identification of a sesquiterpene is a GC coupled to an MS operating in EI mode. Bombardment of the analyte with electrons results in fragmentation of the parent molecule, which generates a mass spectrum that is often unique to that compound. Owing to the cyclic nature of most sesquiterpenes, the fragmentation pattern of two independent compounds will be quite different despite their common molecular formula. Several compendiums cataloging spectral patterns can be used as references10,11 for comparison of fragmentation patterns. In addition, the standard use of a constant EI energy (70 eV) as a default on most instruments allows for comparison of spectra generated in different laboratories to each other and to mass spectral libraries. An extensive library of mass spectra has been compiled and

is regularly updated by the National Institute of Standards and Technology (NIST), available as the Wiley Registry 10th Edition NIST 2012 Mass Spectral Library12, and it can be purchased directly from GC-MS manufacturers. More targeted terpene MS libraries are also available, for example, the Mondello Mass Spectra of Flavors and Fragrances of Natural and Synthetic Compounds13. Several software options are available that use various matching algorithms to assign values and probabilities that express the similarity of a user-generated spectrum to a library spectrum. However, identification based on library match alone is considered equivocal, and the most accepted method for positive identification is to compare retention indices and mass spectra with authentic standards. When sesquiterpene standards are used, consideration of the purity and correct chirality of the compound is of vital importance for correct quantification and identification. Occasionally, a GC instrument is coupled with an MS instrument operating in chemical ionization (CI) mode. CI, considered a soft form of ionization, ionizes the molecules of interest through interaction with a gas, typically ammonia or methane. The greatest advantage of CI mode is the identification of an intact molecular species (e.g., a protonated ion) by its relatively unique mass14. However, owing to the plethora of sesquiterpene products with the same parent molecular weight, CI is rarely used as an ionization method for sesquiterpene detection and characterization. If CI is used, the issue of identical molecular weight can be circumvented by derivatization of a terpene containing nonconjugated double bonds with trioxo(tert-butylimido)osmium(VIII), which form vicinal monoamino alcohols, bisamino alcohols and aminotriols15. These newly formed derivatives may yield distinct mass spectra sufficient for identification. Experimental design Engineering strains for sesquiterpene production. The procedure described in this protocol provides a guide to quantification of sesquiterpene pathway intermediates and products in an engineered S. cerevisiae strain. In this section, we provide an overview of the isoprenoid biosynthetic pathways and outline general considerations when engineering strains for sesquiterpene production. Background. Isoprenoids constitute one of the oldest-known16 and largest classes of natural products17, comprising more than 30,000 known compounds. Not surprisingly, their structural diversity lends itself to a wide range of biological functions, including, but not limited to, electron transport, subcellular protein targeting, membrane fluidity, photosynthesis and plant defense. The precursor units for all isoprenoids are isopentenyl pyrophosphate (IPP) and its structural isomer dimethylallyl pyrophosphate (DMAPP). The IPP and DMAPP units can be synthesized via either of two biological routes, the MEV pathway or the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The MEV pathway begins with the condensation of two acetylCoA units (Fig. 1), whereas the first step of the DXP pathway entails conversion of pyruvate and glyceraldehyde 3-phosphate into DXP. Isoprenoid biosynthesis via one of these pathways takes place in virtually all independent life forms; as a general rule, the DXP pathway is prevalent in eubacteria and eukaryotic plastids, whereas the MEV pathway is prevalent in archaea and eukaryotes. nature protocols | VOL.9 NO.8 | 2014 | 1981

protocol generate the final product. Several reviews are available to provide more detail on these reaction mechanisms and to illustrate the diversity of STSs18. Often, sesquiterpene olefins are subsequently modified by a range of enzymes that can rearrange or oxidize the carbon skeleton, sometimes followed by acylation, acetylation or glycosylation of the oxidized product. These subsequent modifications of the sesquiterpene backbone by enzymes such as cytochrome P450 reductases build an even larger library of decorated sesquiterpenes (often called sesquiterpenoids), which can occur as alcohols, ketones, aldehydes, acids, lactones and derivatives thereof.

Acetyl-CoA Acetyl-CoA ERG10 Acetoacetyl-CoA Acetyl-CoA ERG13 3-Hydroxy-3-methylglutaryl-CoA HMG1, 2 Mevalonic acid O

Mevalonolactone

OH

HO

OH OH O

ERG12

O

Mevalonate-5-phosphate ERG8 Mevalonate-5-pyrophosphate ERG19 Dimethylallyl-PP

IDI1

Dodecane layer and EtOAc dilution HCl treatment and EtOAc extraction Base saponification and dodecane extraction

Isopentenyl pyrophosphate

Single enzymatic transformation Multiple enzymatic steps HCl-catalyzed transformation


ERG20 Geranyl-PP ERG20

Heme Ubiquinones

Sesquiterpene (Amorphadiene)

Farnesyl-PP

OPP

Dolichols Farnesol ERG9

Prenylation BTS1

Nerolidol

OH

HO

Squalene

Geranylgeranyl-PP

Ergosterol

HO

Figure 1 | Key metabolites of the S. cerevisiae mevalonate pathway and major farnesyl-pyrophosphate (farnesyl-PP) side products. Metabolites that may be quantified using the three protocols described here are highlighted in color.

After condensation of IPP and DMAPP to form prenyl phosphates with longer hydrocarbon chains, a vast array of terpenes may be synthesized from these precursors by a diverse group of terpene synthases (TPSs). Sesquiterpene biosynthesis and diversity. Sesquiterpenes, a subclass within the isoprenoid family, are C15 compounds synthesized from FPP, which is formed by the condensation of two IPPs and one DMAPP (Fig. 1). STSs catalyze the metal-dependent cleavage of the diphosphate group from FPP and subsequent atomic rearrangement, resulting in one or more unique cyclic or branched olefins with the formula C15H24. The catalytic mechanism of the STS can be divided into two phases: the first is a metal-dependent binding of substrate and cleavage of the C-O bond to generate a farnesyl cation; the second is a carbocationic cascade of events such as ring closures and hydride or methyl-group shifts to 1982 | VOL.9 NO.8 | 2014 | nature protocols

Isolation of STS genes. The majority of sesquiterpenes produced in S. cerevisiae have been synthesized using STSs of plant origin. The gene responsible for the conversion of FPP into the desired sesquiterpene product is often unknown and is typically isolated from plant cDNA by PCR-based techniques, or mined from a genomic DNA sequence, if available. A description of approaches for isolation of TPSs from cDNA or genomic sequences is beyond the scope of this article, but it has been described previously19–22. The fact that TPSs share a high degree of protein sequence identity facilitates categorization of a candidate enzyme as a TPS on the basis of sequence data alone, but it carries the downside that prediction of substrate or product specificity is difficult. However, STSs can normally be distinguished from monoterpene and diterpene synthases on the basis of their shorter length, the former being cytosolic enzymes and the remainder being plastidic in the vast majority of cases23. An alignment of the full-length protein sequence from a TPS of unknown function with sequences from known STSs and diterpene synthases is a convenient way to check for the presence of an N-terminal extension corresponding to a plastid transit peptide in the unknown enzyme. The absence of a transit peptide indicates that the enzyme is most likely to be an STS. Often, several candidate STSs are identified in the gene discovery phase, and some prioritization can be useful before embarking on characterization. For example, candidates may be excluded from initial screening if they share over 85% protein sequence identity with STSs that are known to make nontarget terpenes that are found in the plant under investigation. When the target sesquiterpene is known to accumulate to high levels in the native plant, expression of the correct gene in an engineered strain of S. cerevisiae is likely to yield a quantity of terpene sufficient for identification4,5. When genes are isolated through genome mining rather than cDNA approaches, it should be noted that a relatively high proportion of candidate TPS genes in some plants appear to encode nonfunctional (or very poorly functional) TPSs, probably a result of genetic duplication and drift5,24. After identification of candidate sesquiterpenes olefins (characteristic ions: M+, 204; [M-CH3]+, 189; and common fragmentation ions, 91, 105 and 119), full mass spectra are typically searched against the NIST mass spectral database or a more specialized terpene mass spectral library, as described above12,25,26. If an authentic standard for the target terpene is not available, then candidate genes may be eliminated on the basis of verification that they make nontarget terpenes (chromatographic RT and mass spectrum match against a standard), followed by purification of remaining terpenes for structural elucidation. To achieve desired

protocol levels of the target sesquiterpene product (in or above the mg per liter range), it may be necessary to optimize the host strain and/or expression level of the STS gene.


Optimization and expression of heterologous STS genes in S. cerevisiae. After a candidate STS gene has been chosen for further study and expression, the researcher is faced with the challenge of designing an expression cassette that yields an optimal level of enzyme in the yeast cell. We review factors related to transcription, translation and protein stability that we have found to be important when optimizing the expression of STSs in S. cerevisiae. Transcriptional control through promoter choice. A wide variety of promoters in S. cerevisiae has been characterized with respect to both regulation and relative strength, and an extensive review has recently been published27. The first consideration for promoter selection is whether gene expression is to be constitutive or inducible. A commonly used set of constitutive promoters for pathway engineering is derived from the translation elongation factor TEF1 (ref. 28). The native promoter sequence provides high expression during both glucose fermentation and ethanol consumption29, and a collection of mutagenized TEF1 promoters has been constructed that spans a range of expression levels 30. A promoter library for lower expression levels, based on the promoter of the native yeast gene PFY1 (required for actin organization), was designed such that its members share a low level of DNA sequence identity, thus minimizing recombination events in cases when several of them are used together31. Glycolysis promoters have also been extensively used for so-called constitutive expression. However, many of these promoters drive high expression during fermentative growth phase but are downregulated during ethanol consumption29. Regulated gene expression can prove advantageous, particularly in cases when the heterologous gene(s) have a deleterious effect on cell growth, or when production is desired only after the exponential growth phase. The inducible promoters most widely used for S. cerevisiae engineering are those of the native galactose-responsive genes. The GAL promoter system is widely used, and it is a preferred induction system for the expression of STSs in yeast (Table 1). As the GAL1, GAL7 and GAL10 promoters are tightly repressed during growth on glucose, deleterious effects of heterologous gene expression can be avoided during genetic engineering32. Once the target pathway has been sufficiently optimized, glucose repression can be relieved by deletion of the GAL80 repressor gene1,3,33, thereby enabling production in medium containing glucose or ethanol instead of the relatively more expensive galactose. This is possible because all galactoseresponsive promoters are regulated by the Gal4p-activator/ Gal80p-repressor transcription factor complex. Optimization of translational efficiency. The relationship between coding sequence and expression level is not completely understood, but there are general considerations that should be taken into account when designing a coding sequence for heterologous expression34. As 18 of the 20 amino acids are encoded by multiple codons, there are an enormous number of codon permutations for even a small peptide. It is generally accepted that codons with more abundant cognate tRNAs are translated more

efficiently because their abundance ensures translational accuracy and minimizes ribosome pausing35. Indeed, modifying codons in heterologous genes to match usage in the host organism can lead to large increases in expression36, and it is common practice to begin sequence expression-optimization by choosing the codons that are most frequently used in the host of interest, which can be found in codon-usage tables37. Codon composition is associated not just with translational efficiency but also with transcriptional efficiency38. Codon usage in the most abundant transcripts in S. cerevisiae is significantly different from the average across the transcriptome, with highly expressed genes having a higher GC content39. Evidence suggests that the transcription rate is higher for GC-rich genes with more stable mRNA secondary structures, as these structures in the nascent mRNA prevent RNA polymerase backtracking40. At the translational level, however, there is a bias against strong mRNA secondary structures immediately surrounding the start codon41. This is supported by experimental work indicating that the most significant determinant of translational initiation is RNA secondary structure42,43. Strong mRNA secondary structures can form between a GC-rich multiple cloning site or 5′ untranslated region (UTR) and the 5′ end of the coding sequence, preventing ribosomes from efficiently scanning the mRNA and recognizing the intended start codon41. If highly stable mRNA secondary structures are predicted (by programs such as Mfold44) for this region (~100 nt containing the start codon as the midpoint), it is recommended to adjust codon usage or to clone the gene into an alternative site. Taking this information together, we routinely optimize genes for yeast with codons favored by highly expressed genes, resulting in GC content a few percent higher than the 40% average in yeast, while minimizing any mRNA secondary structures surrounding the start codon. In addition, to optimize the translation start site, we normally insert the sequence AAAA or AACA immediately upstream of the start codon41,45. Plant cDNAs expressed in yeast are not likely to contain sequences that would be recognized as polyadenylation sites or introns in yeast. However, if the sequence is codon-optimized for expression (or if the gene is bacterial), then it is worth checking the revised sequence for cryptic sites before the gene is synthesized. Tools are available for identifying polyadenylation sites46, as well as intron splicing sites47. In addition to the effect on total expression level, there are cases in which codon usage can affect protein folding and increase the ratio of insoluble to soluble protein4. This could be explained by the presence of rare codons in plant gene sequences that may coordinate co-translational protein folding48,49. Codon harmonization may be a useful approach if a high proportion of the protein is found to be insoluble, particularly where proteins contain multiple domains50. Designing protein stability. The two major considerations for ensuring the stability of heterologous proteins are the N-end rule and the potential for an N-terminal localization sequence. The N-end rule states that heterologous protein stability depends on the N-terminal amino acid (after the methionine residue is cleaved), and was initially demonstrated through heterologous expression of β-galactosidase variants in yeast, where protein half-lives ranged from 20 h to 3 min depending on the N-terminal amino acid51. Further work has identified an N-recognin protein—in nature protocols | VOL.9 NO.8 | 2014 | 1983

protocol Table 1 | Summary of yeast metabolic engineering approaches for sesquiterpene production.


Sesquiterpene Origin of TPS

Plasmid/ promoter used for STS expression

Host strain

Additional pathway modifications (chromosomally integrated unless indicated otherwise); notes Titer attained

Reference

Amorphadiene

A. annua

pRS425 (2 µ)/GAL1

BY4742

EPY300: MATα PGAL1-tHMGR PGAL1upc2-1 erg9::PMET3-ERG9 PGAL1tHMGR PGAL1-ERG20

153 mg per liter

2

Artemisinic acid

A. annua

pRS425 (2 µ)/GAL10

BY4742

MATα PGAL1-tHMGR PGAL1-upc2-1 erg9::PMET3-ERG9 PGAL1-tHMGR PGAL1-ERG20

32 mg per liter in shake flasks, 115 mg per liter in a 1-liter bioreactor

2

Amorphadiene

A. annua

pAM426 (2 µ)/GAL1 (Leu2d marker)

CEN.PK2

MATa erg9∆::kanr_PMET3-ERG9, leu2-3,112::HIS_PGAL1MVD1PGAL10-ERG8, his3∆1:: HIS_PGAL1-ERG12_PGAL10-ERG10, ade1∆::PGAL1-tHMG1_PGAL10-IDI1_ ADE1, ura3-52::PGAL1-tHMG1_ PGAL10-ERG13_URA3, trp1-289:: PGAL1-tHMG1_PGAL10-ERG20_TRP1

1,250 mg per liter in shake flasks; 40 grams per liter in a fermenter

1

Bisabolene

Abies grandis (grand fir)

pRS425Leu2d (2 µ)/GAL1

BY4742

MATα PGAL1-tHMGR PGAL1-upc2-1 900 mg per liter erg9::PMET3-ERG9 PGAL1-tHMGR PGAL1-ERG20; Titers for strains expressing bisabolene synthases from other species were markedly lower

4

Cubol

Citrus paradisi (grapefruit)

pESC-URA (2 µ)/GAL1

CEN.PK113-5D

MATa erg9::PMET3-ERG9; tHMG1 was 10 mg per liter expressed from the PGAL10 promoter on the pESC-URA plasmid

59

Epi-cedrol

A. annua (sweet wormwood)

pRS426 (2 µ)/GAL1

JBY575

MATa upc2-1 PGAL1-tHMG1::LEU2; The ‘a’ mating type produced more epi-cedrol than the equivalent α-strain (The a-type pheromone contains a farnesyl group.)

0.37 mg per liter

60

Patchulol

Pogostemon cablin (patchouli)


CEN.PK113-5D

erg9::PMET3-ERG9; valencene and cubol synthases from C. paradisi were also expressed in this strain

16.9 mg per liter in a 5-liter bioreactor. 20 mg per liter farnesol was detected

61

Patchulol

P. cablin


CEN.PK113-5D

erg9::PMET3-ERG9; highest titers were achieved when the patchoulol synthase was expressed as a fusion to FPPS

23 mg per liter in shake flasks, 41 mg per liter in a 1.1-liter bioreactor

62

Santalene

Clausena lansium (wampee)

pSP-GM2 (2 µ)/TEF1

CEN.PK113-5D

lpp1∆::loxP dpp1∆::loxP PERG9∆::loxP-PHXT1gdh1∆::loxP PTEF1-ERG20 PPGK1-GDH2

22 mg per liter in a 1-liter chemostat

63

Santalene

C. lansium

pICK01 (2 µ)/TEF1

CEN.PK113-5D

lpp1 ∆::loxP dpp1 ∆::loxP PERG9 ∆::loxP- PHXT1

92 mg per liter in fed-batch shake flasks

64

Valencene

Callitropsis nootkatensis (cypress)

pYES3 (2 µ)/GAL1

WAT11

This strain was not engineered for sesquiterpene (FPP) production. An alternative valencene synthase from citrus yielded detectable levels of valencene

1.3 mg per liter

65

1984 | VOL.9 NO.8 | 2014 | nature protocols

protocol


yeast, Ubr1p—that recognizes the N-terminal amino acids Arg, Lys, His, Leu, Phe, Trp, Tyr, and Ile. In addition, Ubr1p can target Met, Ala, Val, Ser, Thr and Cys when they are acetylated. The amino acids that are generally immune from degradation in yeast, mammals and bacteria are Gly and Pro52. Encoding the heterologous protein to begin with Met-Gly may be the optimal choice, not only in terms of the N-end rule but also optimization of the translation initiation sequence and neutrality of the amino acid41. A further consideration for designing the N terminus of a heterologous protein is whether it contains an undesired subcellular targeting sequence. For example, the incidence of mitochondrial targeting sequences in bacterial genes is estimated at around 5% (ref. 53). The PSORT programs have been developed to predict subcellular localization signals54, and although the overall accuracy of the program is not perfect (a set of ~1,000 yeast genes was predicted with 63% accuracy using PSORTII) each prediction is accompanied with a probability estimating accuracy. Insertion of heterologous genes. The typical approaches for the expression of heterologous genes in yeast are integration into the genome or maintenance on a plasmid. Chromosomal integration may be the best approach when low or moderate expression levels are sufficient, and several groups have developed engineering platforms for efficient integration3,55,56. The main alternative to chromosomal integration is to use a plasmid for gene expression, and two classes of vectors are typically used for pathway engineering. Yeast episomal plasmids (YEps) or ‘high-copy’ plasmids contain a 2 µ origin of replication, and they are typically maintained at 10–100 copies per cell34. The second vector class, yeast centromere plasmid (YCps), or ‘low-copy’ plasmids, contain an origin of replication and a centromere sequence, and they are maintained at one or two copies per cell, normally segregating as chromosomes34. For engineered strains intended for large-scale production, 2 µ–based plasmids have several disadvantages. Cells that are selected to maintain high numbers of 2 µ plasmids exhibit low viability and slower growth57. This phenotype is partly owing to the fact that only a fraction of a cell population can maintain an elevated plasmid copy number58. However, as STS kinetics

are typically slow, a high level of enzyme is usually required to consume the FPP produced in engineered strains and to avoid toxicity associated with accumulation of prenyl phosphate intermediates. A survey of engineering approaches for sesquiterpene production in yeast shows a pattern of chromosomal integration for insertion of MEV pathway genes and the use of 2 µ plasmids for STS expression (Table 1). Host engineering for a high-flux MEV pathway. Several publications have reported genetic modifications that improve the production of sesquiterpenes in S. cerevisiae (Table 1). These engineering strategies achieve a wide range of product titers, but the approaches taken have several features in common. In general, the strategies for increased production aim to increase flux through the MEV pathway to the sesquiterpene precursor FPP, and/or reduce flux of FPP to routes other than the target sesquiterpene. In order to further improve production levels for any given sesquiterpene, it is usually necessary to balance MEV pathway flux with the STS, dictated by limitations in enzyme kinetics, solubility or expression level. Expression of an unoptimized or kinetically slow STS in an engineered strain often results in the accumulation of FPP and related prenyl phosphate precursors to toxic levels. Accumulation of FPP to high levels usually results in de-phosphorylation to farnesol and its isomer nerolidol, both of which can be sequestered in a dodecane layer added to yeast cultures to facilitate detection. Ergosterol, a major component of the yeast cell membrane, and its precursor squalene are also derived from FPP and they may be isolated by saponification followed by solvent extraction. Increases in FPP-derived metabolites farnesol, nerolidol, squalene and ergosterol are potential indicators of a pathway imbalance. MEV is the only metabolite of the MEV pathway that is neither conjugated to a CoA nor phosphorylated. This six-carbon metabolite accumulates without apparent toxicity and it is predominantly found in the medium when produced in excess. An increase in MEV levels may indicate an increase in flux into the upper MEV pathway coupled with a bottleneck in conversion of IPP/DMAPP to sesquiterpene product.

MATERIALS REAGENTS Yeast growth and expression of STS • Yeast complete supplemental mixture (CSM) amino acid dropout formula to match auxotrophic marker(s) used (Sunrise Science Products, cat. no. 1005-010) • Difco yeast nitrogen base (YNB) without amino acids, with ammonium sulfate (Becton-Dickinson, Wickerham formula, cat. no. 233520) • Glucose (Sigma-Aldrich, cat. no. G8270) • Galactose (Sigma-Aldrich, cat. no. G0750) • Dodecane (Sigma-Aldrich, cat. no. 29787) Extraction, preparation and detection of sesquiterpene, farnesol and nerolidol • Ethyl acetate (EtOAc; Sigma-Aldrich, cat. no. W241407) ! CAUTION Use EtOAc in a chemical hood and wear protective gloves and suitable protective clothing. • (−)-Trans-caryophyllene (Sigma-Aldrich, cat. no. 22075) Extraction, preparation and detection of MEV • Hydrochloric acid (HCl) solution, 2 M (Sigma-Aldrich, cat. no. 653799) ! CAUTION This solution is poisonous and corrosive; HCl liquid and fumes

cause severe burns to all body tissues. Inhalation may cause lung damage. All handling of HCl should be performed in a chemical hood while wearing protective goggles and gloves and suitable protective clothing. • (–)-Trans-caryophyllene (Sigma-Aldrich, cat. no. 22075) • Ethyl acetate (Sigma-Aldrich, cat. no. W241407) ! CAUTION See notes above. • (±)-Mevalonolactone (Sigma-Aldrich, cat. no. M4667). Use it if absolute quantification is required Extraction, preparation and detection of ergosterol and squalene • Dodecane (Sigma-Aldrich, cat. no. D221104) ! CAUTION Dodecane is flammable. Do not expose it to heat or flame. • (–)-Trans-caryophyllene (Sigma-Aldrich, cat. no. 22075) • Ethyl acetate (Sigma-Aldrich, cat. no. W241407) ! CAUTION Ethyl acetate is flammable. See notes above. • Potassium hydroxide (KOH), 90% flakes, (Sigma-Aldrich, cat. no. 484016) ! CAUTION KOH is caustic. Heat is liberated when KOH and water are mixed, which can result in splattering or dangerous mists. Use sodium hydroxide in a chemical hood and wear protective goggles and gloves and suitable protective clothing. • Cholesterol, Sigma grade, ≥99% (Sigma-Aldrich, cat. no. C8667) nature protocols | VOL.9 NO.8 | 2014 | 1985


protocol • Ethanol, ≥99.5% (Sigma-Aldrich, cat. no. 459844) ! CAUTION Ethanol is flammable. Do not expose it to heat or flame. • Ergosterol, ≥95.0% (Sigma-Aldrich, cat. no. 45480) • Squalene, ≥98%, liquid (Sigma-Aldrich, cat. no. S3626) • Trans, trans-farnesol, 96% (Sigma-Aldrich, cat. no. 277541) • Trans-nerolidol, ≥85% (Sigma-Aldrich, cat. no. 18143 Fluka) EQUIPMENT Yeast growth and expression of STS • 1.7-ml, snap-cap microcentrifuge tubes, polypropylene, nonsterile (Corning Costar, cat. no. 3620) • Bottle-top vacuum filter (0.22-µm pore size), polyethersulfone membrane filter, 45-mm neck diameter (Corning, cat. no. 431153) • Glass or Pyrex round medium storage bottles with GL45 screw cap, sterile, 500 ml (Corning, cat. no. 1395-500) • Eppendorf benchtop microcentrifuge (Eppendorf, with 48 ×1.5/2 ml QL-AT rotor, 120 V, 50/60 Hz, cat. no. 022620603) • Glass culture tubes, sterile, 55 ml (25 × 150 mm, VWR, cat. no. 89000-512) • Nonbaffled Erlenmeyer flask, sterile, 250 ml (Corning, cat. no. 4980-250) The flask is used for 50-ml cell culture, inoculated with precultured cells • Benchtop spectrophotometer for UV/visible absorbance measurements (Cole-Parmer UV/visible spectrophotometer, 115 VAC, 60 Hz, cat. no. EW-83059-10) • Plastic cuvettes (Sigma-Aldrich, polystyrene, semi-micro without stopper, cat. no. C5416) • Magnetic stir bar (Sigma-Aldrich, BRAND magnetic stirring bar, polytetrafluoroethylene (PTFE), cylindrical, 45-mm length, 8-mm bar diameter, flattened sides, cat. no. Z328820) • Stir plate (Corning, hot plate and stirrer with digital display, AC input 120 V, plate 5 × 7 inches (length × width), cat. no. 6795-420D) • Large water bath set to 50 °C (NeuTec Lab Supplies, analog 2-liter water bath, cat. no. WA02A11B) • Glass GC autosampler vials (Sigma-Aldrich, 2-ml screw-top vials, large opening, cat. no. 29118-U) • PCR tube strips (Corning, Thermowell Gold PCR eight-well tube strips, 0.2 ml, no caps, clear, cat. no. 3741) • Benchtop mini centrifuge (Sigma, Spectrafuge mini centrifuge, cat. no. S7816) • Storage freezer held at −20 °C for sample storage (Fisher Scientific, Isotemp freezer, cat. no. 11-670-202) Extraction, preparation and detection of sesquiterpene, farnesol and nerolidol • Glass screw-cap GC autosampler vials (National Scientific, 2 ml, Clear Glass I-D, 12 × 32 mm, flat base, Target DP screw thread vial, cat. no. C4000-1W) • Caps with PTFE septa for screw-cap vials (Agilent Technologies, cat. no. 5182-0717) • Benchtop mini centrifuge (Sigma, Spectrafuge mini centrifuge, cat. no. S7816) • Glass inserts for GC-autosampler vials (Supelco, inserts for 1.5-ml large opening vials size 0.10 ml, vial insert, glass conical with bottom spring, 6 mm × 29 mm (outer diameter (o.d.) × height), cat. no. 860066) • Glass or Pyrex round medium storage bottles with GL45 screw cap, sterile, 500 ml (Corning, cat. no. 1395-500) • GC ‘chiral’ column, CycloSil-B (Agilent, 30-m length, 0.25-mm inner diameter (i.d.), 0.25-µm film thickness, cat. no. 112-6632) • GC-MS instrument (Agilent Technologies, Agilent GC system 6890 series, Agilent mass selective detector 5973 network) • GC autoinjection system (CTC Analytics, Combi-PAL) • MSD Productivity ChemStation software package (Agilent Technologies) for data acquisition and data processing Extraction, preparation and detection of MEV • Glass screw-cap GC-autosampler vials (National Scientific, 2 ml, clear glass I-D , 12 × 32 mm, flat base, Target DP screw thread vial, cat. no. C4000-1W) • Caps with PTFE septa for screw-cap vials (Agilent Technologies, cat. no. 5182-0717) • Glass inserts for GC-autosampler vials (Supelco, inserts for 1.5 ml large opening vials size 0.10 ml, vial insert, glass conical with bottom spring, 6 mm × 29 mm (o.d. × height), cat. no. 860066) • Analog vortex mixer (Fisher Scientific, cat. no. 02-215-365) • Foam rack vortex mixer accessory to hold microcentrifuge tubes and GC vials (Fisher Scientific, cat. no. 02-215-386) • Falcon 15-ml conical centrifuge tubes (Corning, cat. no. 430766) 1986 | VOL.9 NO.8 | 2014 | nature protocols

• Eppendorf benchtop microcentrifuge (Eppendorf, with 48 × 1.5/2 ml QL-AT rotor, 120 V, 50/60 Hz, cat. no. 022620603) • Benchtop centrifuge (Beckman Coulter, Allegra 25R centrifuge, cat. no. 369434) • Rotor for benchtop centrifuge; holds up to 6 × 250 ml tubes (Beckman Coulter, TA-10-250 fixed-angle rotor, Beckman Coulter, cat. no. 368293) • Adapters for TA-10-250 fixed-angle rotor, One-Place polypropylene adapter for 50-ml conical-bottom tube (Beckman Coulter, cat. no. 356966) • Glass or Pyrex round medium storage bottles with GL45 screw cap, sterile, 500 ml (Corning, cat. no. 1395-500) • GC ‘chiral’ column, CycloSil-B (Agilent, 30-m length, 0.25-mm i.d., 0.25-µm film thickness, part. no. 112-6632) • GC-MS instrument (Agilent Technologies, Agilent GC system 6890 series, Agilent mass selective detector 5973 network) • GC autoinjection system (CTC Analytics, Combi-PAL) • MSD Productivity ChemStation software package (Agilent Technologies) for data acquisition and data processing Extraction, preparation and detection of ergosterol and squalene • Glass screw-cap GC-autosampler vials (National Scientific, 2 ml, Clear Glass I-D, 12 × 32 mm, flat base, Target DP screw thread vial, cat. no. C4000-1W) • Caps with PTFE septa for screw-cap vials (Agilent Technologies, cat. no. 5182-0717) • Glass inserts for GC-autosampler vials (Supelco, inserts for 1.5-ml large opening vials, size 0.10 ml, vial insert, glass conical with bottom spring, 6 mm × 29 mm (o.d. × height), cat. no. 860066) • Analog vortex mixer (Fisher Scientific, cat. no. 02-215-365) • Eppendorf benchtop microcentrifuge (Eppendorf, with 48 × 1.5/2 ml QL-AT rotor, 120 V, 50/60 Hz, cat. no. 022620603) • Benchtop centrifuge (Beckman Coulter, Allegra 25R Centrifuge, cat. no. 369434) • GC column DB-5MS (Agilent, 30-m length, 0.25-mm i.d., 0.25-µm film thickness, part no. 122-5532) • GC-MS instrument (Agilent Technologies, Agilent GC system 6890 Series, Agilent mass selective detector 5973 network) • GC autoinjection system (CTC Analytics, Combi-PAL) • MSD Productivity ChemStation software package (Agilent Technologies) for data acquisition and data processing • Falcon 15-ml conical centrifuge tubes (Corning, cat. no. 14-432-22) • Snap-cap microcentrifuge tubes, polypropylene, nonsterile, 1.7 ml (Corning Costar, cat. no. 3620) • Floating microtube racks (foam works best to fit glass screw cap vials, VWR, cat. no. 82024-508) • Large (1 liter) glass beaker (VWR, cat. no. 89000-212) • Professional hot plate (VWR, cat. no. 97042-738) • Screw-cap microcentrifuge tubes, 2.0 ml (VWR, cat. no. 89004-326) REAGENT SETUP Yeast host engineering and culturing Several different strains of S. cerevisiae, combined with various engineering approaches for improvement of pathway flux, have been used for the production of sesquiterpenes (see INTRODUCTION section; Fig. 2 and Table 1). Before culturing and modifying the selected yeast strain, it is important to confirm that the genotype is compatible with the genome/plasmid-based marker(s) to be used for selection of the STS and any other introduced genes. Here we assume that the STS gene is under control of a GAL promoter and therefore requires galactose for induction. We use the example of amorphadiene as a representative sesquiterpene in the procedure below. Media component stocks (general) Media consist of three components: a carbon source, yeast complete supplemental mixture lacking the amino acid(s) used for selection of transgenes (CSM) and YNB without amino acids (see Reagents). Each of these components is prepared as a 10× stock, as described in the following items. Glucose (20% (wt/vol)), sterile Add 100 g of glucose to a large 500-ml medium storage bottle. Add 400 ml of deionized water and mix it with a stir bar on a heated stir plate until glucose is fully in solution. To aid solvation, it may be necessary to microwave briefly or warm it in a 50 °C water bath for ~20 min. After the glucose is fully dissolved, sterilize it by filtration into a sterile 500-ml glass medium bottle using a bottle-top 0.22-µm filter, under vacuum. This can be stored at 20–25 °C for at least 60 d. Galactose (20% (wt/vol)), sterile Add 100 g of galactose to a large 500-ml media storage bottle. Add 400 ml of deionized water to the bottle and prepare

protocol a

d

Isolation of STS genes

Protocols Strain assessment for production and pathway efficiency

cDNA isolation 3′AAAAA(A)n

mRNA 5′ cDNA 3′ Or

5′ TTTTT(T)n

mined genomic sequences 1. Sampling Overlay

Metabolite:

b

Optimization of STS expression

Sesquiterpene Farnesol Nerolidol

Culture

Culture

Squalene Ergosterol

Mevalonate

HCl Kozak sequence Promoter choice

Intron removal

2. Preparation Terminator


Dilute in EtOAc

Genome or plasmid expression

5′ end secondary structure removal

KOH/ EtOH

Extract in EtOAc

Extract in dodecane

3. Detection and quantitation

Coding sequence - Codon optimization - N-end rule

Culture extract

Identify with standard

Quantify 7000 6000

c

5000 4000

Host modifications

3000 2000 1000 0

e

0

20

40

60

80

100

120

Final engineered strain

Product Acetyl-CoA Increased titers

Figure 2 | Overview of processes involved in engineering S. cerevisiae for sesquiterpene production. (a–c) Information related to isolation of sesquiterpene synthase (STS) genes (a); optimization of STS expression (b); and S. cerevisiae host modifications for increased mevalonate pathway flux (c) can be found within the introductory sections. (d) Illustration of the protocols for detection and quantification of sesquiterpenes and key related metabolites. Iteration of steps b–d are typically used during the process of engineering yeast to maximize sesquiterpene titers (e).

the solution as per directions for glucose. This solution can be stored at 20–25 °C for at least 60 d. YNB solution (10×), sterile Add 37.5 g of YNB powder to a large 500-ml medium storage bottle, and then add 362.5 ml of deionized water to the bottle. Mix with a stir bar on a stir plate until the YNB is fully dissolved, and then sterilize it by filtration into a sterile 500-ml glass medium bottle using a bottle-top 0.22-µm filter, under vacuum. This can be stored at 4 °C for at least 60 d. CSM amino acid dropout solution (10×), sterile The amount of CSM mixture required to make a 10× solution varies with the particular amino acid(s) dropped out. For example, to make 10× CSM–Leu, add 3.45 g of CSM–Leu to deionized water to a final volume of 500 ml, and mix with a stir bar on a stir plate until dissolved. Sterilize the solution by filtration into a sterile 500-ml glass medium bottle using a bottle-top 0.22-µm filter, under vacuum. This can be stored at 20–25 °C for at least 60 d. Pre-production selection medium (2% (wt/vol) glucose) To make 500 ml of the pre-production medium, combine 50 ml of 20% (wt/vol) sterile glucose solution, 50 ml of sterile 10× YNB solution and 50 ml of 10× CSM amino acid dropout solution in a sterile 500-ml glass medium bottle. Add sterile deionized water to a final volume of 500 ml. This can be stored at 20–25 °C for at least 7 d.

Production medium (1.8% galactose and 0.2% glucose, wt/vol) Production medium contains a small amount of glucose to minimize lag phase during metabolic adaption to galactose. To make 500 ml of production medium, combine 45 ml of 20% (wt/vol) sterile galactose solution, 5 ml of 20% (wt/vol) of sterile glucose solution, 50 ml of sterile 10× YNB solution and 50 ml of 10× CSM amino acid dropout solution in a sterile 500-ml glass medium bottle. Add sterile deionized water to a final volume of 500 ml. This can be stored at 20–25 °C for at least 7 d. Ethyl acetate spiked with 15 g/ml trans-caryophyllene To provide an internal standard for GC-MS quantification, transfer 500 ml of ethyl acetate into a glass medium bottle, add 8.31 µl of trans-caryophyllene (neat) and mix thoroughly.  CRITICAL Store the spiked ethyl acetate in a cool, dark storage area. Trans-caryophyllene may oxidize with prolonged exposure to light and air to form caryophyllene oxide. Caryophyllene standard stock solution Prepare 40 ml of a solution of 100 µg/ml trans-caryophyllene in ethyl acetate, mix it well and store it as 1-ml aliquots in glass screw-cap GC vials at −20 °C (aliquots are stable for at least 12 months). Dilutions of this stock solution are used to generate a standard curve for GC-MS quantification of sesquiterpenes. Mevalonolactone standard stock solution Prepare a 1 mg/ml solution of mevalonolactone in ethyl acetate spiked with caryophyllene, mix it well and nature protocols | VOL.9 NO.8 | 2014 | 1987


protocol store it as 1-ml aliquots in glass screw-cap GC vials at −20 °C (aliquots are stable for at least 6 months). This solution is 10× the highest concentration of mevalonolactone recommended for a set of standards (100 µg/ml), which should be freshly prepared for each GC-MS run. Cholesterol internal standard stock solution Prepare a 10-ml solution of 1 mg/ml cholesterol in ethanol for dilution 100-fold in the KOH solution below. The stock solution can be stored at −20 °C for at least 6 months. KOH (20% (wt/vol)) in 50% ethanol containing 10 g/ml cholesterol Mix 5 ml of ethanol (≥99.5%) and 100 µl of the cholesterol internal standard stock solution in a 15-ml Falcon tube. Weigh out 2 g of KOH. Add KOH to the 5-ml alcoholic cholesterol solution and mix it well. ! CAUTION KOH is very caustic; all handling of KOH should be performed in a chemical hood while wearing protective goggles, gloves and suitable protective clothing. Be sure that the cap is screwed on tightly before mixing. Slowly add deionized water to a final volume of 10 ml. This solution should be freshly prepared for each experiment. Squalene standard stock solution (100×) Prepare a 10 mg/ml solution of squalene in ethyl acetate, mix it well and store it as 1-ml aliquots in glass screw-cap GC vials at −20 °C; aliquots can be stored for at least 6 months. Ergosterol standard stock solution (10×) In a 15-ml Falcon tube, prepare a 10-ml solution of 1 mg/ml ergosterol in ethanol. If exactly 10 mg cannot be weighed, adjust the volume of ethanol in order to achieve a 1 mg/ml final concentration. The stock solution can be stored at −20 °C for at least 6 months. EQUIPMENT SETUP Glassware sterilization for yeast growth and expression of STS Sterilization (121 °C, 30 min) of 500-ml medium bottles, glass culture tubes and 250-ml Erlenmeyer growth flasks must be performed before the start of the procedure. GC-MS metabolite profiling of the sesquiterpene of interest (e.g., amorphadiene), farnesol and nerolidol: injection parameters 1 µl of sample is injected (splitless) using He as the carrier gas. The inlet and MS transfer line are held at 250 °C throughout the run. Chromatography parameters for sesquiterpene, farnesol and nerolidol The preferred column for amorphadiene separation is the Agilent CycloSil-B. The carrier gas should be held at a constant flow rate of 1.0 ml/min. After each sample injection, the oven temperature is held at 100 °C for 0.75 min, followed by a ramp of 40 °C/min to a final temperature of 250 °C, holding at 250 °C for 3 min. The total run time is 5.5 min. Mass spectrometer parameters for sesquiterpene, farnesol and nerolidol The MS solvent delay should be set at 3.5 min, and the electron multiplier voltage (EMV) mode is set to a gain factor of 1. The MS instrument should be set to selected ion mode (SIM) for acquisition, monitoring m/z ions 189 (targeting amorphadiene and caryophyllene), along with 81, 93 and 136 (predominantly targeting farnesol and nerolidol). Ions should be selected

from the full mass spectra of target metabolites in order to maximize response and minimize background signal. For example, the 189 ion represents the [M-CH3]+ fragment from amorphadiene, and it was chosen as a quantification target owing to its relative abundance following fragmentation of amorphadiene coupled with low background levels in yeast cell extracts. Ions can be selected for quantification of other terpenes based on their full mass spectra, and by following a rule of thumb that selecting abundant ions with high masses should generate a high signal-to-noise ratio. Other fragmentation ions that are often abundant in sesquiterpene mass spectra include 91, 93, 105 and 119. GC-MS metabolite profiling of MEV: Injection parameters 1 µl of sample is injected (splitless), by using He as the carrier gas. The inlet and MS transfer line are held at 250 °C throughout the run. Chromatography parameters for MEV The preferred column for MEV separation is the Agilent CycloSil-B. The carrier gas should be held at a constant flow rate of 1.0 ml/min. After each sample injection, the oven temperature is held at 90 °C for 1 min, followed by a ramp of 30 °C/min to a final temperature of 250 °C, holding at 250 °C for 2 min. The total run time is 8.33 min. Mass spectrometer parameters for MEV The MS solvent delay should be set at 4.5 min, and the EMV mode is set to a gain factor of 1. The MS instrument should be set to SIM for acquisition, monitoring m/z ions 58 and 71 (mevalonolactone ions), along with 189 and 204 (caryophyllene internal standard ions). As for selection of ions for amorphadiene, the mevalonolactone and caryophyllene ions should be selected on the basis of their abundance in the full mass spectra of target molecules and rarity as background ions. GC-MS metabolite profiling of ergosterol and squalene: injection parameters 1 µl of sample is injected (splitless), by using He as the carrier gas. The inlet is held at 250 °C, and the MS transfer line is held at 280 °C throughout the run. Chromatography parameters for ergosterol and squalene The selected column for ergosterol and squalene separation is the Agilent DB-5MS. The DB-5MS + DG contains a guard column at the front end and also works well for this application. The carrier gas should be held at a constant flow rate of 1.0 ml/min. After each sample injection, the oven temperature is held at 80 °C for 1 min, followed by a ramp of 20 °C/min to 280 °C, a hold for 15 min at 280 °C and a ramp to 300 °C at a rate of 20 °C/min with a final hold at 300 °C for 2 min. The total run time is 29 min. Mass spectrometer parameters for ergosterol and squalene The MS solvent delay should be set at 10 min, and the EMV mode is set to a gain factor of 1. The mass spectrometer should be set to SIM acquisition mode, monitoring m/z ions 218, 386 and 396. The temperatures of the quadrupole and the ion source should be set to 200 and 300 °C, respectively. As for selection of ions for amorphadiene, the ergosterol and squalene ions were selected based on their abundance in the full mass spectra of target molecules and rarity as background ions.

PROCEDURE Preparation of pre-production and production cultures ● TIMING 3 h hands-on time (days 1–2) 1| Transfer 5 ml of sterile pre-production selection medium to a sterile glass culture tube, and inoculate it with a single yeast colony picked from a selective agar plate (after transformation with a plasmid harboring the STS gene of interest). We highly recommend including an empty-vector control strain and picking at least three individual colonies from each strain to be tested; each colony would be inoculated into a separate culture tube. 2| Incubate the colony at 30 °C, with shaking at 200 r.p.m., for 24 h. 3| Check the OD600 of the pre-production culture before proceeding to Step 3. Ideally, the culture will be between an OD600 of 1 and 3. Check the OD of the culture by removing 50 µl and diluting it 1/20 with water in a cuvette. Determine the absorbance at 600 nm, setting the baseline with a water blank.


protocol 4| Transfer 50 ml of sterile production medium to a sterile 250-ml Erlenmeyer flask. Add 5 ml of dodecane to the sterile flask (10% of the total volume). The dodecane acts to trap the sesquiterpene product and minimizes the loss due to evaporation. A flask containing blank medium and a flask inoculated with a yeast strain lacking the STS gene may be included as controls for comparison of GC-MS chromatograms. 5| Inoculate the 50 ml of production medium with the pre-production culture to a calculated starting OD600 of 0.05 (e.g., by adding 1.25 ml of a pre-production culture with an OD600 of 2.0). 6| Incubate the culture at 30 °C, with shaking at 200 r.p.m., for 144 h (6 d). At each 24-h interval, sample the culture for measurement of amorphadiene, farnesol, nerolidol, MEV, squalene and ergosterol (see the following sampling procedures for each metabolite).


Procedure for sampling and sample storage ● TIMING 1–3 h of hands-on time each day for 6–30 samples (days 3–10) 7| OD measurement. Check the OD600 of the culture as in Step 3. 8| Sampling the dodecane layer for detection of amorphadiene. Remove 50 µl of the dodecane layer and transfer it to a PCR tube.  CRITICAL STEP Sampling of the dodecane layer may be difficult, as it is likely to be in the form of droplets rather than a contiguous layer; tilting the Erlenmeyer flask to one side is usually helpful. Try to minimize the medium that is aspirated along with the dodecane layer, as the bulk of sesquiterpenes are dissolved in this phase. 9| Repeat Step 8, so that a total of 100 µl of the dodecane/medium mixture is sampled for each flask. 10| Spin PCR tubes for 1 min in the benchtop mini centrifuge so that the medium and dodecane layers are clearly separated.  CRITICAL STEP It is important to ensure that the sample separates into two separate layers and to sample only the dodecane (top).  PAUSE POINT At this point, samples can be stored at −20 °C for up to 3 months before analysis. Further preparation of amorphadiene is continued at Step 14. 11| Sampling culture for the detection of MEV. By taking care to not sample the dodecane layer, pipette 200 µl of whole culture (medium and cells) from each flask into a glass GC vial.  PAUSE POINT At this point, the sample can be stored at −80 °C for up to 3 months before analysis. Further preparation of MEV is continued at Step 22. 12| Sampling culture for the detection of ergosterol and squalene. Collect 2 ml of yeast cell culture in a 2.0-ml microcentrifuge tube. 13| Centrifuge the culture for 15 s at 15,000g (room temperature, 20–25 °C) in a benchtop microcentrifuge to pellet the cells. Aspirate and discard the supernatant.  PAUSE POINT At this point, the sample can be stored at −80 °C for up to 6 months before analysis. Further preparation of ergosterol and squalene is continued at Step 36. Preparation of samples for amorphadiene analysis ● TIMING 5–30 min of hands-on time  CRITICAL The samples used here are those resulting from Step 10 of the PROCEDURE. 14| If samples were frozen, thaw the samples to room temperature and then spin them for 1 min in a benchtop mini centrifuge to clearly separate the medium and the dodecane layer. 15| Transfer 10 µl of the dodecane (top layer) from each sample into 990 µl of caryophyllene-spiked ethyl acetate (dilution of 1:100) in a glass GC vial. ! CAUTION Perform this step in a chemical hood while wearing protective goggles and gloves and suitable protective clothing.  CRITICAL STEP To avoid errors in terpene quantification due to evaporation of ethyl acetate, cap the vial promptly after diluting each sample. Preparation of a standard curve using caryophyllene equivalents ● TIMING 20–30 min of hands-on time 16| Perform serial dilutions of the caryophyllene 100 µg/ml stock solution (see Reagent Setup) in GC vials. Serially dilute 600 µl of this stock 50:50 in ethyl acetate five times to give a set of standards at 50, 25, 12.5, 6.25 and 3.125 µg/ml. To use as a blank, add an aliquot of ethyl acetate to a glass GC vial. nature protocols | VOL.9 NO.8 | 2014 | 1989

protocol GC-MS separation and detection of amorphadiene and caryophyllene standards ● TIMING ~8 min per sample 17| Transfer diluted dodecane samples (from Step 15) and caryophyllene standards (from Step 16) to the GC-MS autosampler for sesquiterpene detection and quantification (see Equipment Setup). Quantification and data analysis: quantification of sesquiterpene (amorphadiene) using a caryophyllene standard curve ● TIMING 1–3 h of hands-on time 18| The chromatographic peak areas for caryophyllene and amorphadiene are determined using the integration tools of the MSD Productivity ChemStation software. It is important to use the same integration parameters for an entire set of samples, and to visually confirm that the baselines drawn under the peaks are equivalent. RTs for authentic standards of caryophyllene and amorphadiene are ~3.95 and 4.10 min, respectively. RTs will vary according to factors such as column age, liner and inlet seal cleanliness, and column length (if it has been trimmed at some point). ? TROUBLESHOOTING


19| Plot the caryophyllene peak areas against their respective concentrations and generate a calibration curve by linear regression analysis.  CRITICAL STEP The calibration curve is expected to be linear within a range of 0.5–50 µg/ml caryophyllene with an r2 value of >0.980. If this is not the case, use only concentrations that lie within the linear range. 20| For quantification of sesquiterpene levels, integrate the peak at the expected RT from each sample using the MSD Productivity ChemStation software (~4.10 min for amorphadiene; for other sesquiterpenes, use an authentic standard to verify RT). Normalize the amorphadiene peak area for each sample to adjust for variations in injection volume and GC-MS response by using the peak corresponding to the internal standard caryophyllene (i.e., multiply the amorphadiene peak area for Sample 1 by the average caryophyllene peak area for all samples (excluding standards), divided by the caryophyllene peak area for Sample 1). Apply the linear equation resulting from the calibration curve to each adjusted amorphadiene peak area to calculate the sesquiterpene concentration in the diluted dodecane sample. ? TROUBLESHOOTING 21| Apply the dilution factor to calculate the concentration of amorphadiene in the neat dodecane phase and then divide this by 10 to determine the amount of amorphadiene produced per milliliter of culture (as the dodecane layer constitutes a second phase (10%, vol/vol) in the culture). Normalize this production value by dividing by the OD600 for each culture to calculate the specific production value (µg/ml/OD600). Note that concentrations of farnesol and nerolidol from wild-type strains may fall below the limit of detection when using these extraction and detection protocols. Therefore, do not anticipate detection in samples prepared from an unengineered host (Fig. 3). Preparation of samples for MEV analysis ● TIMING 40 min–2 h hands-on time (for 6–30 samples)  CRITICAL The samples used here are those resulting from Step 11 of the PROCEDURE. 22| If samples were frozen, allow them to thaw at room temperature for 5 min. 23| Add 50 µl of 2 M HCl to each glass GC vial. ! CAUTION Only use screw-cap vials, not snap-cap microcentrifuge tubes. Snap-cap tubes can release HCl during vigorous shaking (Step 24). 24| Vortex the vials at top speed for 15 min using a foam rack vortex mixer accessory. ! CAUTION Close the caps tightly to prevent HCl leakage.

a

1

25| Add 250 µl of ethyl acetate spiked with caryophyllene to each vial.

2

3 Caryophyllene

5

Farnesol

4

Figure 3 | GC-MS chromatograms (representing ions 189 and 204) relevant to quantification of the sesquiterpene amorphadiene and FPP-derived products. (a) Chromatograms for standards of (1) caryophyllene, (2) (E,Z)-farnesol, (3) (E,E)-farnesol, (4) (Z)-nerolidol, (5) (E)-nerolidol, and (6) amorphadiene. (b) Dodecane fractions from cultures of BY4742, a wild-type yeast strain; EPY300, corresponding to BY4742 engineered for high FPP production but lacking a sesquiterpene synthase; and EPY224, corresponding to EPY300 containing a high-cop-number plasmid harboring ADS from A. annua. 1990 | VOL.9 NO.8 | 2014 | nature protocols

Nerolidol

6

b

Amorphadiene

6

5

BY4742 3 1 3.5

5 3.7

3.9

4.1

EPY300

3 4.3 4.5 4.7 Retention time (min)

EPY224 4.9

5.1

5.3

5.5

protocol 26| Vortex the vials at top speed for 5 min using a foam rack vortex mixer accessory. ! CAUTION Close the caps tightly to prevent HCl leakage. 27| Centrifuge the vials at 3,000g for 3 min at room temperature. Glass screw-cap vials cannot normally be centrifuged in benchtop microcentrifuges. To circumvent this, carefully place up to three glass screw-cap vials into a 15-ml Falcon tube and centrifuge them in a benchtop centrifuge.  CRITICAL STEP When removing the Falcon tubes, take care to not disturb the separated phases, as this may contaminate your sample. Remove the GC vials from the Falcon tubes with tweezers. 28| Remove 100 µl of the ethyl acetate (top phase) and place it in a new GC vial containing a glass insert adapter (for smaller volumes) for MEV detection and quantification.  PAUSE POINT At this point, samples can be stored at −20 °C for up to 3 months before analysis.


Preparation of a standard curve using mevalonolactone ● TIMING 20–30 min of hands-on time 29| Dilute the 1 mg/ml mevalonolactone stock (see Reagent Setup) to 100 µg/ml by adding 100 µl of the stock to 900 µl of ethyl acetate spiked with the internal standard caryophyllene in a GC vial. 30| Perform serial dilutions of the 100 µg/ml mevalonolactone solution in GC vials. Serially dilute 600 µl of this stock 50:50 in ethyl acetate five times to give a set of standards at 50, 25, 12.5, 6.25 and 3.125 µg/ml. To use as a blank, add an aliquot of ethyl acetate to a glass GC vial. GC-MS separation and detection of mevalonolactone ● TIMING ~11 min per sample 31| Transfer prepared samples (from Step 28) and mevalonolactone standards (from Step 30) to the GC-MS autosampler for sesquiterpene detection and quantification (see Equipment Setup). Quantification and data analysis: quantification of mevalonolactone concentration using a mevalonolactone standard curve ● TIMING 1–3 h of hands-on time 32| Determine the chromatographic peak areas for mevalonolactone and the internal standard caryophyllene using the integration tools of the MSD Productivity ChemStation software. It is important to use the same integration parameters for an entire set of samples, and to visually confirm that the baselines drawn under the peaks are equivalent. RTs for authentic standards of mevalonolactone and caryophyllene are ~5.90 and 5.07 min, respectively. RTs will vary according to factors such as column age, liner and inlet seal cleanliness, and column length (if it has been trimmed at some point). 33| Plot the mevalonolactone peak areas against their respective concentrations and generate a calibration curve by linear regression analysis. The calibration curve is expected to be linear within a range of 0.5–50 µg/ml mevalonolactone with an r2 value >0.980. If this is not the case, use only concentrations that lie within the linear range. Poor chromatography (peak shape) at the lower end of this range can often be improved by trimming the front end of the GC column and/or replacing the liner and inlet seal. 34| To quantify mevalonolactone levels, integrate each peak at the expected RT using the MSD Productivity ChemStation software. Normalize the mevalonolactone peak area for each sample to adjust for variations in injection volume and GC-MS response by using the peak corresponding to the internal standard caryophyllene (i.e., multiply the mevalonolactone peak area for Sample 1 by the average caryophyllene peak area for all samples, divided by the caryophyllene peak area for Sample 1). Apply the linear equation resulting from the calibration curve to each adjusted mevalonolactone peak area to calculate the mevalonolactone concentration in each sample. ? TROUBLESHOOTING 35| Apply the dilution factor (i.e., multiply by 1.25, adjusting for the addition of HCl to the cell culture) to calculate the amount of mevalonolactone produced per ml of culture. Normalize this production value by dividing by the OD600 for each culture to calculate the specific production value (µg/ml/OD600).  CRITICAL STEP Concentrations of MEV in wild-type strains may fall below the limit of detection when these extraction and preparation protocols are applied. Therefore, do not anticipate detection in samples prepared from an unengineered host (Fig. 4). Preparation of samples for squalene and ergosterol analysis ● TIMING 1–2 h of hands-on time  CRITICAL The samples used here are those resulting from Step 13 of the PROCEDURE. nature protocols | VOL.9 NO.8 | 2014 | 1991

protocol Figure 4 | GC-MS chromatograms (representing ions 58, 71, 189 and 204) relevant to quantification of mevalonolactone and the internal standard, caryophyllene. (a) Chromatograms for standards of (1) caryophyllene and (2) mevalonolactone. (b) Extractions from cultures of BY4742, a wild-type yeast strain; EPY300, corresponding to BY4742 engineered for high FPP production but lacking a sesquiterpene synthase; and EPY224, corresponding to EPY300 containing a high-copy-number plasmid harboring ADS from A. annua. Amorphadiene (3) is also detected, but is not quantified using this method.


36| Set up a 1-liter glass beaker approximately half-filled with water, cover the top loosely with aluminum foil and set it to boil on a hot plate. Boiling the water takes ~10 min. Make sure that the water is at a rolling boil before adding samples. 37| If the samples were frozen, allow them to thaw at room temperature. Centrifuge the tubes for 15 s at 15,000g at room temperature in a benchtop microcentrifuge to pellet the cells. Aspirate and discard the medium and add 1 ml of deionized water to wash the yeast cells. Centrifuge again as before, and remove all of the supernatant. 38| Resuspend the cell pellet in 0.4 ml of the alcoholic KOH solution containing 10 µg/ml cholesterol (freshly prepared, see Reagent Setup), and transfer it to a 2.0-ml Eppendorf screw-cap tube. ! CAUTION KOH is corrosive. All handling of KOH should be performed in a chemical hood while wearing protective goggles and gloves and suitable protective clothing. 39| Transfer the tightly closed tubes to a floating Eppendorf tube holder and place them into the boiling water, covering the beaker loosely with aluminum foil again to enable rapid boiling. Once the water is boiling again, set a timer for 5 min. ! CAUTION Use only screw-cap tubes for this step, and ensure that the caps are secured tightly. Snap-cap Eppendorf tubes are likely to pop open during the boiling process, even if wrapped in Parafilm.  CRITICAL STEP Make sure that all of the samples are partially submerged in the boiling water. 40| Remove the tubes from the boiling water bath and allow them to cool for 5 min. ! CAUTION The hot plate, water and beaker are hot. Do not touch them with bare hands; remove the samples with tongs and turn off the hot plate. 41| Add 0.4 ml of dodecane to each tube and vortex vigorously for 5 min. ! CAUTION Be sure that you are using screw-cap tubes and that the caps are secured tightly. 42| Separate the two phases by centrifugation at 3,000g for 5 min at room temperature. 43| Transfer 0.1 ml of the dodecane layer (top layer) to a glass GC vial containing a glass insert adapter (for smaller volumes) for GC-MS analysis.  PAUSE POINT At this point, the sample can be stored at −20 °C for up to 3 months before analysis. Preparation of standard curves for ergosterol, squalene and cholesterol ● TIMING 30–45 min of hands-on time 44| Dilute the 10 mg/ml (100×) squalene standard stock solution (see Reagent Setup) to a final concentration of 100 µg/ml by adding 10 µl of the squalene stock into 990 µl of dodecane in a GC vial and mixing it thoroughly by vortexing. 45| Perform serial dilutions of the squalene 100 µg/ml solution into GC vials. Serially dilute 600 µl of this stock 50:50 in dodecane five times to give a set of standards at 50, 25, 12.5, 6.25 and 3.125 µg/ml. To use as a blank, add an aliquot of dodecane to a glass GC vial. 46| Dilute the 1 mg/ml (10×) ergosterol stock solution (see Reagent Setup) to a final concentration of 100 µg/ml by adding 100 µl of the ergosterol stock to 900 µl of dodecane in a GC vial and mixing it thoroughly by vortexing. As in Step 45, perform serial dilutions to achieve a range of standards from 50 to 3.125 µg/ml. 1992 | VOL.9 NO.8 | 2014 | nature protocols

protocol


Figure 5 | GC-MS chromatograms (representing ions 218, 386 and 396) relevant to quantification of intracellular ergosterol and squalene. (a) Chromatograms for standards of cholesterol (1), squalene (2) and ergosterol (3). (b) Cell extracts from BY4742, a wild-type yeast strain; EPY300, corresponding to BY4742 engineered for high FPP production but lacking a sesquiterpene synthase; and EPY224, corresponding to EPY300 containing a high-copy-number plasmid harboring ADS from A. annua.

a

1 2

3

Cholesterol Ergosterol Squalene

b

47| (Optional) Prepare a standard curve for cholesterol as well. This can be 2 used to estimate the ergosterol extrac10 12 14 tion efficiency by quantification of the cholesterol internal standard in each sample (see Step 50). Repeat Step 46 with the 1 mg/ml cholesterol stock solution (see Reagent Setup).

BY4742 EPY300 1 16

18

3 20

EPY224 22

24

26

28

Retention time (min)

GC-MS separation and detection ● TIMING ~33 min per sample 48| Transfer the prepared samples (from Step 43) and standards for ergosterol, squalene and cholesterol standard curves (Steps 45 and 46) to the GC-MS autosampler for detection and quantification (see Equipment Setup). RTs for authentic standards are ~14.80, 21.13 and 19.41 min for squalene, ergosterol and cholesterol, respectively. RTs will vary according to factors such as column age, liner and inlet seal cleanliness, and column length (if it has been trimmed at some point). Data analysis for ergosterol, squalene and cholesterol ● TIMING 1–3 h of hands-on time 49| Plot the cholesterol peak areas against their respective concentrations and generate a calibration curve by linear regression analysis. The calibration curve is expected to be linear within a range of 0.5–50 µg/ml cholesterol with an r2 value of >0.980. If this is not the case, use only concentrations that lie within the linear range. Poor chromatography (peak shape) at the lower end of this range can often be improved by trimming the front end of the GC column and/or by replacing the liner and inlet seal. 50| For quantification of cholesterol levels, integrate each peak at the expected RT using the MSD Productivity ChemStation software. Apply the linear equation resulting from the cholesterol calibration curve to each cholesterol peak area to calculate the concentration in each sample. ? TROUBLESHOOTING 51| Repeat Steps 48 and 49 for ergosterol (RT 21.13 min), and again for squalene (RT 14.80 min), and then adjust for the fact that 2 ml of culture was extracted into 0.4 ml of dodecane (i.e., multiply the amount of ergosterol and squalene calculated in each dodecane sample by 0.2). 52| (Optional) To adjust for variations in extraction efficiency, injection volume and GC-MS response, use the quantity of cholesterol detected in each sample to normalize the calculated squalene and ergosterol concentrations. The amount of cholesterol expected in each dodecane sample is 10 µg/ml (assuming 100% extraction efficiency), as all samples are prepared using KOH solution spiked with 10 µg/ml cholesterol.  CRITICAL STEP Concentrations of ergosterol and squalene in wild-type strains fall within the limit of detection when applying these extraction protocols (Fig. 5). Therefore, if ergosterol or squalene is not detected, the user should consult the TROUBLESHOOTING section. ? TROUBLESHOOTING ? TROUBLESHOOTING Troubleshooting advice can be found in Table 2.

nature protocols | VOL.9 NO.8 | 2014 | 1993

protocol


Table 2 | Troubleshooting table. Step

Problem

Possible reason

Solution

18

No detectable sesquiterpene, but detectable caryophyllene

Insufficient enzyme expression or enzyme activity of STS is very low; yeast strain is not optimal

Express alternative STS with strong activity in the same strain to deconvolute strain/STS issues. Evaluate STS protein levels. Optimize strain or STS as needed (before Step 1; see Experimental design)

No detectable sesquiterpene, but detectable caryophyllene

Dodecane sample was overdiluted

Run a neat dodecane sample, or run a 1:10 dilution and check for the target terpene (spike the neat sample with caryophyllene standard)

No detectable sesquiterpene in the neat dodecane sample

Sampling the medium layer instead of the dodecane

Take care to sample only the dodecane layer at Steps 8 and 10

Caryophyllene is not detected or an additional peak is observed in the EtOAc-caryophyllene sample

Caryophyllene or caryophyllene-spiked EtOAc has been exposed to light for an extended period, resulting in caryophyllene oxididation. A full scan will reveal the presence of caryophyllene oxide

Use new caryophyllene, and a fresh caryophyllene-spiked EtOAc solution. The solution should be stable for >1 year if stored in the dark in a sealed bottle as instructed in the Reagent Setup section

Poor chromatography (peak shape) at the lower end of concentration range of the standards

Column contamination

This can often be improved by trimming the front end of the GC column and/or replacing the liner and inlet seal

Integrated peak area of the target terpene is not within the linear range of the standard curve

Amount of terpene produced is higher or lower than expected and outside of the range of standards

Either dilute samples to the appropriate range (if peak area was too high) or reduce the concentration of standards at Steps 15 and 16

Amount of terpene produced is lower than expected and outside of the range of standards

Overestimated production levels of sesquiterpene

Reduce the concentration of standards at Step 16, and run the samples and standards again

The sesquiterpene peak areas are greater than the highest value within the calibration curve

Underestimated production levels of sesquiterpene

The sesquiterpene sample should be diluted accordingly at Step 15 and re-analyzed by GC-MS

Inconsistent internal standard levels: random fluctuations in caryophyllene peak area within a GC-MS run

If the caryophyllene peak area fluctuates substantially (>30%) in a random pattern, this may be due to inconsistent sample injection or poor mixing of dodecane and EtOAc

Check the consistency of sample injection volume by injecting the same EtOAc-caryophyllene sample 20 times. If it is inconsistent, change the injection syringe and service the autosampler if needed. Make sure to mix dodecane/ EtOAc well

Inconsistent internal standard levels: the caryophyllene peak area steadily increases or decreases over the length of a run

Drifting of the caryophyllene peak area in a consistent manner is usually a result of instrument response

Initially, change the septum, liner and inlet seal. Check for air leaks. If the problem is not fixed, check the MS electron multiplier voltage; if >2,400, clean the ion source

34

The mevalonolactone peak areas are greater than the highest value within the calibration curve

Underestimated mevalonate production levels

Dilute the sample and re-analyze by GC-MS

50,52

No detectable ergosterol or squalene; internal standard is detected

Insufficient cell density

Check medium components and preparation; wait at least 72 h after induction to sample. Sample a larger volume of culture at Step 12

20

No detectable ergosterol or Poor extraction owing to bad reagents squalene; internal standard is also or insufficient boiling not detectable


Fresh KOH-EtOH solution should be used each time at Step 38; make sure that water is at a rolling boil before adding samples at Step 36


protocol ● TIMING Steps 1–6 (days 1–2), yeast pre-production and production culture preparation: 3 h of hands-on time Steps 7–13 (days 3–10), sampling and sample storage: sample once every 24 h for time course or sample once at a final time point at 144 h (1–3 h hands-on time each day for 6–30 samples) Steps 14 and 15, extraction, preparation, detection and quantification of amorphadiene, farnesol and nerolidol (after the desired sampling is completed): 5–30 min hands-on time (for preparation) Step 16, preparation of a standard curve using caryophyllene equivalents: 30 min of hands-on time Step 17, GC-MS separation and detection of standards: 8 min per sample Steps 18–21, quantification and data analysis using a caryophyllene standard curve: 1–3 h of hands-on time Steps 22–28, extraction, preparation, detection and quantification of MEV (after the desired sampling is completed): 40 min to 2 h hands-on time (for preparation, for 6–30 samples) Steps 29 and 30, preparation of a standard curve using mevalonolactone: 20–30 min of hands-on time Step 31, GC-MS separation and detection of mevalonolactone: 11 min per sample Steps 32–35, quantification and data analysis using a mevalonolactone standard curve: 1–3 h of hands-on time Steps 36–43, extraction, preparation, detection and quantification of ergosterol and squalene (after the desired sampling is completed): 1–2 h hands-on time (for preparation, for 6–30 samples) Steps 44–47, preparation of a standard curve for ergosterol, squalene and cholesterol: 30–45 min of hands-on time Step 48, GC-MS separation and detection, and data analysis: ~33 min per sample, including cool-down time between samples Steps 49–52, data analysis for ergosterol, squalene and cholesterol: 1–3 h of hands-on time ANTICIPATED RESULTS The data shown here (Figs. 3–5) illustrate the metabolic changes observed over the course of a MEV pathway engineering project in yeast. Overall, the wild-type strain S. cerevisiae BY4742 was engineered for the production of the sesquiterpene precursor FPP through engineering of the MEV pathway, resulting in strain EPY300 (PGAL1-tHMGR PGAL1-upc2-1 erg9::PMET3-ERG9 PGAL1-tHMGR PGAL1-ERG20). When amorphadiene synthase (ADS) from A. annua was introduced to this strain on a high-copy-number plasmid under control of the GAL1 promoter (generating EPY224), an amorphadiene production titer of ~153 mg per liter was achieved2. Therefore, to serve as an example of typical results that might be encountered in response to pathway modifications, we show chromatograms for sesquiterpene and key associated metabolites from three strains: BY4742 (wt), EPY300 and EPY224. Increasing flux through the MEV pathway in strain EPY300 yields significant levels of farnesol and nerolidol, compared with negligible amounts in BY4742, indicating an unbalanced pathway owing to the accumulation of FPP (Fig. 3). Squalene and MEV levels are also considerably higher in EPY300 compared with BY4742, indicating high MEV pathway flux and excess FPP (Figs. 4 and 5). Introduction of the ADS-encoding gene (strain EPY224) results in accumulation of amorphadiene in the dodecane overlay, and concomitant reduction of farnesol, nerolidol and squalene (Figs. 3 and 5). In addition to facilitating the monitoring of sesquiterpene product titers, these rapid and robust methods for the quantification of key metabolites provide indicators of strain fitness and pathway imbalances over the course of an iterative engineering project, helping to guide future modifications.

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Detection and quantification of residues and metabolites of medicinal products in environmental compartments, food commodities and workplaces. A review.

Methods for extraction and quantification of receptors.

Identification, characterization, synthesis and HPLC quantification of new process-related impurities and degradation products in retigabine.

Carotenoid Extraction and Quantification from Capsicum annuum.

Detection, quantification, and total synthesis of novel 3-hydroxykynurenine glucoside-derived metabolites present in human lenses.

ShatterProof: operational detection and quantification of chromothripsis.

Quantification of fibrin degradation products in glioma and meningioma patients.

Quantification of mutation-derived bias for alternate mating functionalities of the Saccharomyces cerevisiae Ste2p pheromone receptor.

Quantification of three lidocaine metabolites and their conjugates.

Acquisition and quantification of ion images with a camera-based detection system and classical quantification algorithms.

Slit scanning of Saccharomyces cerevisiae cells: quantification of asymmetric cell division and cell cycle progression in asynchronous culture.

Human norovirus detection and production, quantification, and storage of virus-like particles.

Extraction and Quantification of Cyclic Di-GMP from P. aeruginosa.

Quantification of Bacterial Fatty Acids by Extraction and Methylation.

Detection and quantification of Vibrio parahaemolyticus in shellfish from Italian production areas.

Simultaneous quantification of 20 synthetic cannabinoids and 21 metabolites, and semi-quantification of 12 alkyl hydroxy metabolites in human urine by liquid chromatography-tandem mass spectrometry.

Metabolic engineering of Saccharomyces cerevisiae for caffeine and theobromine production.

Automated detection and quantification of microaneurysms in fluorescein angiograms.

Detection and quantification of RNA 2'-O-methylation and pseudouridylation.

Detection and quantification of extracellular microRNAs in murine biofluids.

Selective detection and quantification of carbon nanotubes in soil.

Detection and quantification of microRNA in cerebral microdialysate.

Detection and quantification of 4(5)-methylimidazole in cooked meat.

MS Analysis.