EP2167533A2

EP2167533A2 - DNA SEQUENCE ENCODING A SPECIFIC L-ARABINOSE TRANSPORTER, A cDNA MOLECULE, A PLASMID COMPRISING THE SAID DNA SEQUENCE, HOST CELL TRANSFORMED WITH SUCH PLASMID AND APPLICATION THEREOF

Info

Publication number: EP2167533A2
Application number: EP08766990A
Authority: EP
Inventors: César SIMÕES DA FONSECA; Rita BÄRBEL HAHN-HÄGERDAL; Isabel Maria Spencer Vieira Martins
Original assignee: Universidade Nova de Lisboa
Current assignee: Universidade Nova de Lisboa
Priority date: 2007-07-06
Filing date: 2008-07-04
Publication date: 2010-03-31
Also published as: WO2009008756A2; WO2009008756A4; WO2009008756A3; PT103780A

Abstract

The present invention refers to host cells genetically modified microorganisms by introducing a nucleic acid sequence encoding a specific L-arabinose transporter from yeast. The host cells are preferably those of an eukaryotic microorganism such as yeasts or filamentous fungi. The invention is useful for the complete use of sugar obtained from plant biomass and other ligno-cellulose materials of biotechnological importance in the production of biofuels, bulk and platform chemicals including ethanol, butanol, lactate, 1,4-diacids (succinate, fumaric, malic), glycerol, sorbitol, mannitol, xylitol/arabinitol, L-ascorbic acid, xylitol, hydrogen gas, 2,5-furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glutaric acid, glutamic acid, itaconic acid, levulinic acid, and 3-hydroxybutyrolactone, fatty acids, fatty-derived molecules, isoprenoids, isoprenoid-derived molecules, alkanes, isopentanol, isoamylacetate, using genetically-modified microorganisms to consume and ferment L-arabinose in hexose and pentose mixtures resulting from renewable raw materials.

Description

"DNA SEQUENCE ENCODING A SPECIFIC L-ARABINOSE TRANSPORTER,

A CDNA MOLECULE, A PLASMID COMPRISING THE SAID DNA SEQUENCE, HOST CELL TRANSFORMED WITH SUCH PLASMID AND

APPLICATION THEREOF"

OBJECT OF THE INVENTION

The present invention refers to a DNA sequence containing the codifying region of a new gene encoding a specific L- arabinose transporter with high uptake capacity, which once inserted in a yeast, preferably Saccharomyces cerevisiae, modifies it in terms of its capacity to make a highly- effective use of L-arabinose.

The objective of the present invention is to provide the biorefinary, biofuels and bioethanol production industry with a genetically-modified yeast capable of consuming L- arabinose more rapidly and with larger specificity in D- glucose and D-xylose mixtures as well as of fermenting L- arabinose with higher productivity.

STATE OF THE ART

The vast dependence on oil and other fossil fuels, together with the great concern towards the reduction of greenhouse gas emissions and consequent climatic changes, have drawn attention to renewable energies and, in particular, the biofuel production integrated in the wide scope of biorefining. The biorefinaries have the objective of using microorganisms in biomass conversion into value-added compounds . The main products of these biorefinaries are biofuels, bulk and platform chemicals including ethanol, butanol, lactate, 1,4-diacids (succinate, fumaric, malic), glycerol, sorbitol, mannitol, xylitol/arabinitol, L- ascorbic acid, xylitol, hydrogen gas, 2,5-furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glutaric acid, glutamic acid, itaconic acid, levulinic acid, and 3-hydroxybutyrolactone, fatty acids, fatty- derived molecules, isoprenoids, isoprenoid-derived molecules, alkanes, isopentanol, isoamylacetate. Nowadays, cereals or other (starch- or sucrose-based) hexose-rich agricultural substrates are used for the industrial production of ethanol by Saccharomyces cerevisiae. However, plant biomass is mainly composed of lignocel^'lulose materials. They represent the main forest product and a considerable portion of agricultural wastes. The development of processes aiming for the bioconversion of this biomass into value-added products, namely into biofuels and platform chemicals, is potentially important and strongly encouraged.

The cellulose component of lignocellulose materials is exclusively composed of glucose polymers, whereas the hemicellulose fraction is composed of polymers containing a mixture of hexoses (D-glucose, D-galactose, D-manose) and pentoses (D-xylose, L-arabinose) . D-Xylose is the main pentose present in hemicelluloses, accounting up to .40% of the carbohydrate fraction. L-Arabinose may also be present in significant amounts, mainly in agricultural residues, accounting up to 20% of the carbohydrate fraction. The majority of these pentoses (D-xylose and L-arabinose) can be recovered as fermentable sugars in the hemicellulose hydrolysate. The use of lignocellulose materials for a cost-effective production of biofuels and platform chemicals by S. cerevisiae requires the complete fermentation of pentoses. However, this yeast does not have natural ability to use D-xylose and L-arabinose. There are other yeasts and different groups of microorganisms comprising that capacity, but which are not necessarily adequate for industrial application. S. cerevisiae is clearly the most adequate yeast to this aggressive environment, in many cases maintaining good fermentation capacity.

Different strategies have been applied to produce recombinant strains of S. cerevisiae able to use D-xylose and L-arabinose. The term "use" should be understood in as much as D-xylose or L-arabinose can be metabolized as carbon and energy source, being converted into biomass, ethanol or other useful compounds.

To confer to S. cerevisiae the ability to use D-xylose, two different strategies were applied: the expression of two genes of Pichia stipitis, encoding a xylose reductase (XR) , which reduces D-xylose into xylitol, and a xylitol dehydrogenase (XDH) , which oxidizes xylitol into D- xylulose; and the expression of the gene encoding a xylose isomerase (XI), of Thermus thermophilυs or Piromyces sp. , which converts D-xylose directly into D-xylulose. The latest is already naturally metabolized by 5. cerevisiae, through the pentose phosphate pathway and the glycolytic pathway.

To confer to S. cerevisiae the ability to use L-arabinose, two different strategies were applied. One involving the introduction of the metabolic pathway present in bacteria, corresponding to the expression of the gene encoding a L- arabinose isomerase (AI) of Bacillus subt±l±s, together with the genes encoding a L-ribulose kinase (RK) and a L- ribulose-5-phosphate-4-epimerase (RPE) of Escherichia coli (WO2003095627) . Another approach involving the expression of two heterologous genes in a strain of S. cerevisiae already modified with XR and XDH: a Trichoderma reesei gene, encoding a L-arabitol-4-dehydrogenase (LAD) , which oxidizes L-arabitol into L-xylulose, and the expression of the L-xylulose reductase gene (LXR) of T. reesei or Ambrosiozyma monospora, which reduces L-xylulose into xylitol (WO2002066616 and WO2005026339) . The enzyme XR is unspecific and might convert L-arabinose into L-arabitol, whereas the XDH enzyme converts xylitol, produced by LAD and LXR, into D-xylulose.

A S. cerevisiae strain able to utilize D-xylose and L- arabinose was obtained by combining the heterologous expression of XR/XDH and AI/RK/RPE (WO2006096130) .

Although the best modified S. cerevisiae strains for D- xylose fermentation already originate good ethanol yields, but still with insufficient productivity when compared to those obtained in glucose fermentation, in L-arabinose fermentation there are no 5. cerevisiae strains with good yields and ethanol productivities.

The sugar transporter from the environment into the cell can be ^' an obstacle to the efficient fermentation of pentoses by S. cerevisiae. In this yeast, the L-arabinose is transported by GAL2, an unspecific transporter capable to carry D-galactose, D-glucose, D-xylose and L-arabinose. Howover, the overexpression of the gene GAL2 improved L- arabinose fermentation capacity in the S. cerevisiae strain modified with AI/RK/RPE. However, this strain shows relatively low yields of ethanol yield and productivity.

The ethanol yield and productivity will be improved when S. cerevisiae strains are obtained with an efficient pentose fermentation capacity. Specific pentose transporters are necessary to increase the metabolic flux and the consequent ethanol production, by means of fermentation, or other compounds obtained from the sugar which is present in the hemicellulose hydrolysates from plant biomass handling.

The expression, in a microorganism, preferably in S. cerevisiae, of a specific L-arabinose transporter with high uptake capacity will be a surplus in the efficient use of this pentose.

Among the yeasts capable of naturally utilize L-arabinose, Candida arabinofermentans PYCC 5603^T, Pichia guilliermondii PYCC 3012 and Ambrosiozyma monospora PYCC 4390^T stood out due to its high specific growth rate. It was demonstrated that the first two comprised two types of transport systems for L-arabinose: a facilitated diffusion, with high uptake capacity and high specificity (unable to transport D-xylose and D-glucose) ; a L-arabinose/proton simporter, with higher affinity for L-arabinose but with much lower uptake capacity and specificity. C. arabinofermentans PYCC 5603^T was considered the best yeast to isolate the gene, encoding the high-capacity and specific L-arabinose transporter (ARTl), to be expressed in S. cerevisiae.

In spite of the already demonstrated breakthroughs, the recombinant yeasts so far developed do not demonstrate enough efficiency in ethanol production from L-arabinose. There is the need of improving the state of the art in order to use lignocellulose materials in biorefinaries, namely for production at an industrial scale of biofuels, bulk and platform chemicals including ethanol, butanol, lactate, 1,4-diacids (succinate, fumaric, malic), glycerol, sorbitol, mannitol, xylitol/arabinitol, L-ascorbic acid, xylitol, hydrogen gas, 2,5-furan dicarboxylic acid, 3- hydroxy propionic acid, aspartic acid, glutaric acid, glutamic acid, itaconic acid, levulinic acid, and 3- hydroxybutyrolactone, fatty acids, fatty-derived molecules, isoprenoids, isoprenoid-derived molecules, alkanes, isopentanol, isoamylacetate.

SUMMARY OF THE INVENTION

The present invention provides a means for allowing the process for the use of lignocellulose materials to become more efficient and highly cost effective, namely in production of biofuels, bulk and platform chemicals including ethanol, butanol, lactate, 1,4-diacids (succinate, fumaric, malic) , glycerol, sorbitol, mannitol, xylitol/arabinitol, L-ascorbic acid, xylitol, hydrogen gas, 2,5-furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glutaric acid, glutamic acid, itaconic acid, levulinic acid, and 3-hydroxybutyrolactone, fatty acids, fatty-derived molecules, isoprenoids, isoprenoid-derived molecules, alkanes, isopentanol, isoamylacetate.

The solution to this problem is based on the fact that the present inventors have identified and isolated a gene encoding a C. arabinofermentans transporter with a surprisingly uptake capacity and specificity for L- arabinose, when compared to sugar transporters that occur naturally in fermenting yeasts. The amino acid sequence and the corresponding coding genes are not known for a specific and high capacity L-arabinose transporter. One of these genes is now disclosed. Since this transporter is specific for L-arabinose, not using D-xylose or D-glucose as substrates, when the corresponding gene is inserted into a host cell, this gene turns the cell into a potentially more efficient cell to consume and ferment the L-arabinose which is present in the hexose and pentose mixture resulting from lignocellulose plant biomass material, which is one of the raw materials of industrial interest, namely for production of biofuels, bulk and platform chemicals including ethanol, butanol, lactate, 1,4-diacids (succinate, fumaric, malic), glycerol, sorbitol, mannitol, xylitol/arabinitol, L- ascorbic acid, xylitol, hydrogen gas, 2,5-furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glutaric acid, glutamic acid, itaconic acid, levulinic acid, and 3-hydroxybutyrolactone, fatty acids, fatty- derived molecules, isoprenoids, isoprenoid-derived molecules, alkanes, isopentanol, isoamylacetate.

So being, a first aspect of the invention refers to an isolated DNA fragment encoding a transporter with high uptake capacity and specificity for L-arabinose, comprising:

a) a nucleotide sequence SEQ ID No. 1_/ or b⁾ a functionally-equivalent variant of the nucleotide sequence SEQ ID No. 1, or complementary strings thereof.

In a second aspect, the invention refers to a πDNA molecule comprising: a) a nucleotide sequence SEQ ID No. 1; or b) a functionally-equivalent variant of the nucleotide sequence SEQ ID No. 1, or complementary strings thereof.

In a third aspect, the invention refers to plasmids comprising a DNA fragment according to claim 1.

In a fourth aspect, the invention refers to a host cell characterized in that it is transformed with the DNA fragment according to claim 1, in order to allow the host cell to express the said L-arabinose transporter.

In a last aspect, the invention refers to a process for using plant biomass or other lignocellulose materials in the production of biofuels and platform chemicals, comprising the use of L-arabinose from an environment including a L-arabinose source with a host cell transformed according to claims 4 to 6, wherein the host cell uses L- arabinose, generating value-added compounds, such as biofuels, bulk and platform chemicals including ethanol, butanol, lactate, 1,4-diacids (succinate, fumaric, malic), glycerol, sorbitol, mannitol, xylitol/arabinitol, L- ascorbic acid, xylitol, hydrogen gas, 2,5-furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glutaric acid, glutamic acid, itaconic acid, levulinic acid, and 3~hydroxybutyrolactone, fatty acids, fatty- derived molecules, isoprenoids, isoprenoid-derived molecules, alkanes, isopentanol, isoamylacetate.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure J. : Gel electrophoresis of denaturant polyacrylamide (10% T) with 20 μg total protein from plasma or mitochondrial membranes isolated from C. arabinofermentans cells cultivated in 0.5% L-arabinose (Ara) or 0.5% D- glucose (GIu) . The gel was stained with Coomassie Blue. M - Sigma Marker (Wide Range), MW - molecular weight; pm - plasma membranes; mm - mitochondrial membranes.

Figure 2 : Amino acid sequence of the N-terminus region of the ARTl protein and degenerate primers designed from this region.

Figure 3: Analysis by Northern blot of the expression of ARTl gene. The total RNA was isolated from C. arabinofermentans PYCC 5603^T cultures in mineral medium containing 0.5% L-arabinose (L-Ara) or 0.5% D-glucose (D- GIu) as the sole carbon and energy source. Each sample contains 10 μg total RNA, separated in 1.2% denaturing agarose gel and subsequently transferred to a nylon membrane (Hybond-N) . A 810 pb fragment, amplified from C. arabinofermentans PYCC 5603^T genomic DNA with ATCA05_FOR ( 5 ' -ACAATGGTCAATTGAATGGGGTAT-S ' ) and ATCA06_REV ( 5 ' GCAGCAGCT GACATACCTTTTGAT-3 ') primers, was used as specific probe for the ARTl gene. The probe was labelled with [α- ³²P]-ATP (Amersham Biosciences) using Prime-a-Gene Labelling System (Promega) . The hybridization and washing were carried out as described by Griffioen et al. (1996).

Figure 4: a) Nucleotide sequence of ARTl gene (SEQ ID No. 1), from the first (ATG) to the last codon (TAA); b) Amino acid sequence of the ARTl protein.

DETAILED DESCRIPTION OF THE INVENTION

According to a preferred embodiment of tho present invention, a process was developed to express a specific L- arabinose transporter in Saccharomyces cerevisiae. This process includes the introduction of heterologous DNA in yeasts which become integral within a gene for the transport of L-arabinose, with high specificity and uptake capacity.

A process was followed for the isolation, cloning and expression of the gene of the present invention. However, alternative processes can be used by ones skilled in the art.

Identification of the specific L-arabinose transporter for SDS-PAGE

The specific C. arabinofermentans L-arabinose transporter was identified by comparing the relative abundance of proteins present in plasma membranes isolated from C. arabinofermentans cells cultivated under induction and repression conditions. To this end, plasma and mitochondrial membranes were isolated from cells cultivated in defined mineral medium (Verduyn et al., 1992) alternatively containing 0.5% L-arabinose or 0.5% of D- glucose as sole carbon and energy source. The cells were harvested during the exponential growth phase (DO₆₄₀ =0.8- 2.0) and washed twice with cold distilled water and once with buffer A (0.1 M glycine, 0.3 M KCl, pH 7.0). Ten to fifteen grams of cells were subsequently resuspended in 15 mL Buffer A containing 0.1 rtiM PMSF. Membranes were isolated as described by van Leeuwen et al. (1991) . Using membrane protein samples (20 μg) an electrophoresis was carried out in denaturing polyacrylamide qel in the presence of tricine (Tricine SDS-PAGE; Schagger, 1994) . The acrylamido and bisacrylamide concentrations used in the gel were 10%T and 3%C (%T= total concentration of acrylamide+bisacrylamide and %C= bisacrylamide percentage to the total) . The plasma membrane samples presented a band pattern clearly different from the one presented by the corresponding samples of mitochondrial membranes (Figure 1), indicating an efficient separation of the two types of membrane proteins. One has consequently concluded that the differences observed among the band patterns of plasma membrane samples corresponding to the different carbon sources are not the result of contamination by mitochondrial proteins.

The most evident difference among the two plasma membrane samples is indicated by the arrow in Figure 1. It corresponds to a protein of about 40 kDa molecular weight that is present mainly in plasma membranes of cells cultivated in 0.5% L-arabinose. Since the molecular weight of this protein is within the expected range for a sugar transporter, it has been considered that the band would correspond to the C. arabinofermentans L-arabinose transporter.

Cloning of cDNA encoding the specific L-arabinose transporter

The identified membrane protein, as herein described, was isolated from a preparative gel loaded with 250 μg of total membrane protein of C. arabinofermentans cultivated in 0.5% L-arabinose. After electrophoresis, the proteins were transferred to a PVDF membrane (sequi-blot of BIO-RAD) . The electrophoresis and the transfer were carried out according to the manufacturer's instructions. The gel and the membrane containing the proteⁱn were used for sequencing of the N-terminus of the protein {Protein Core Facility, Columbia University, USA) . The 15-amino acid sequence obtained is shown in Figure 2. From this sequence degenerate primers were drawn (Figure 2) . These primers were used to amplify the complete ARTl cDNA by RACE technique {Rapid Amplification of cDNA Ends) from total RNA of cells cultivated in 0.5% L-arabinose. To this end, First Choice RLM-RACE kit (Ambion) was used, according to the manufacturer's instructions. RNA was extracted as described by Griffioen et al. (1996) and purified using RNA cleanup protocol (RNeasy kit, Quiagen) . RNA was used as template for the 3'-RACE protocol, in combination with the ATCA01D_FOR and ATCA02D_FOR (5 ' -TTYGGIAAYMGICARATGCC-3 ' and 5' -AARTTYTAYAAYCCITAYATG-3', primers respectively; I=inosina, Y=C/T, R=A/G, M=A/C) . The primers design was based on the amino acid sequence of the protein N-terminus. The amplified fragments were cloned into the pMOSBlue vector (Amersham Biosciences) and sequenced using an ALFexpress™ II DNA Analyzer (Amersham Biosciences) , and 5' -Cy5-labelled vector-specific primers (Thermo Sequenase Primer Cycle Sequencing kit) . The protein encoded by this molecule presented the characteristic properties of a sugar transporter. An analysis by Northern blot was followed, which demonstrated that the respective mRNA was very abundant in cells cultivated in 0.5% L-arabinose, but it was not detectable in cells cultivated in 0.5% glucose (Figure 3) .

The cDNA end 5¹ was obtained by 5'-RACE, using the ATCAO3_REV (5' CTGAACCAATAATCCAAAATCCAC-3 ' ) primer. The obtained fragment was cloned and sequenced as described in the previous paragraph, being demonstrated that tb^ cDNA encodes a further amino acid (the initiation methionine) and a not-translated sequence '5' of 29 amino acids. The new gene was designated ARTl (ARabinose Transporter 1⁾ . The respective nucleotide sequence (SEQ ID No. 1) is shown in Figure 4.

Expression in S. cerevisiae

Several plasmids were constructed containing the gene ARTl that allows the cDNA expression in S. cerevisiae.

A new vector was built, using the YEplacl95 plasmid (multi^¬ copy) (Gietz et al., 1988), the HXT7 truncated promoter of S. cerevisiae, the ARTl gene and the PGKl terminator of 5. cerevisiae. A DNA fragment comprising the nucleotides -392 to -1 of the HXT7 promoter was amplified by PCR using the HXT7prom_F0R (S'-AACCTGCAGCTCGTAGGAACAATTTCGG-S') and HXT7prom_REV ( 5 ' -GGACGGGACATATGCTGATTAAAATTAAAAAAACTT-S ' ) primers and the YEpkHXT7 plasmid (Krampe et al., 1998) as template. These primers contain, in the 5' end, Pstl and Ndel restriction sites, respectively. The codifying region of the ARTl gene was amplified using the ART1__FOR2 (5' ATAGCAGATCTCATATGGTTTTCGGTAACAGGCAAAT-S ' ) and ART1_REV2 ( 5 ' -ATAGCAGATCTTCTAGATTAACTATCTAAAGACCGAACG-S ' ) primers and genomic DNA from C. arabinofermentans PYCC 5603^T as template. In the 5' end, these primers contain Ndel and Xbal restriction sites, respectively. The fragment containing the HXT7 promotor was digested with Pstl and Ndel, the fragment containing the ARTl gene was digested with Ndel and Xbal and the YEplacl95 plasmid was digested with Pstl and Xbal. These three DNA elements were linked, forming the pHXT7p-ARTl plasmid. Subsequently, a 0,3 kb fragment containing the terminator region of the PGKl gene was amplified from the pMA91 plasmid, with the PGKT_FOR2

( 5 ' --ACCGTGTCTAGATAAATTGAATTGAATTΠAAATCGATAG-3 ' ) and

PGKT_REV ( 5 ' -TAATTAGAGCTCTCGAAAGCTTTAACGAACGCAGAA- 3 ' ) primers that, in the 5' end, contain Xbal and Sad restriction sites, respectively. The fragment containing the terminator region of the PGKl gene was subsequently- digested with these enzymes and linked among the Xbal and Sad restriction sites of the pHXT7p-ART2 plasmid, thus resulting in the pHXT7p-ARTl-PGKlt plasmid.

Homology with other transporters

The ARTl gene is translated into a protein of 521 amino acids (Artlp; Fig. 4). The analysis of similarities between amino acid sequences in available database on the Internet allowed verifying that it is a membrane protein (with transmembrane domains) and with the features inherent to sugar transporters. This protein has homology with sugar transporters from other yeasts and filamentous fungi. For the closest related protein sequences, there is no register of functional study (they are registered in the databases as possible sugar transporters or as proteins with unknown function) . The protein further a homology region with a bacterial L-arabinose transporter family, AraJ (a gene encoding a Escherichia coli L-arabinose permease) .

Claims

1. A fragment of isolated DNA encoding a specific and high-capacity L-arabinose transporter, comprising: a) a nucleotide sequence SEQ ID No. 1; or b) a functionally-equivalent variant of the nucleotide sequence SEQ ID No. 1, or complementary strings thereof.

2. A cDNA molecule comprising: a) a nucleotide sequence SEQ ID No. 1; or b) a functionally-equivalent variant of the nucleotide sequence SEQ ID No. 1, or complementary strings thereof.

3. A plasmid comprising a DNA fragment according to claim 1.

4. A host cell characterized in that it is transformed with the DNA fragment according to claim I₇ such that it allows the host cell to express the corresponding mRNA.

5. Host cell according to claim characterized in that it is a yeast.

6. Host cell according to claim characterized in that the yeast is Saccharσmyces cerevisiae.

7. Process for the use of plant biomass or other lignoce.1 lulose compounds, in the production of value- added compounds, such as biofuels, bulk and platform chemicals including ethanol, butanol, lactate, 1,4- diacids (succinate, fumaric, malic) , glycerol, sorbitol, mannitol, xylitol/arabinitol, L-ascorbic acid, xylitol, hydrogen gas, 2,5-furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glutaric acid, glutamic acid, itaconic acid, levulinic acid, and 3-hydroxybutyrolactone, fatty acids, fatty- derived molecules, isoprenoids, isoprenoid-derived molecules, alkanes, isopentanol, isoamylacetate, comprising the utilization of L-arabinose from an agent comprising a L-arabinose source with a host cell transformed according to claims 4 to 6, wherein the host cell uses L-arabinose, generating value-added compounds, such as biofuels, bulk and platform chemicals including ethanol, butanol, lactate, 1,4- diacids (succinate, fumaric, malic) , glycerol, sorbitol, mannitol, xylitol/arabinitol, L-ascorbic acid, xylitol, hydrogen gas, 2,5-furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glutaric acid, glutamic acid, itaconic acid, levulinic acid, and 3-hydroxybutyrolactone, fatty acids, fatty- derived molecules, isoprenoids, isoprenoid-derived molecules, alkanes, isopentanol, isoamylacetate.