Metabolic Engineering of Arabidopsis Plant to Produce Triterpenoid Hydrocarbons for Biofuel

Wenxu Zhou1,2, Jing Li1,2, Steven M. Smith2, Dongke Zhang1* 1Centre for Energy (M473)

2ARC Centre of Excellence in Plant Energy Biology The University of Western Australia

35 Stirling Highway, Crawley, WA 6009 * Dongke.Zhang@uwa.edu.au

Abstract— The microalga Botryococcus braunii (B. braunii) can produce extracellular oil in the form of triterpenoid hydrocarbons up to 40 precent of their dry mass. These hydrocarbons with carbon chain lengths ranging from C30 to C40 including botryococcene, squalene and methylated squalenes, are ideal for the production of high quality liquid transport fuels and petrochemical alternatives. However, commercial-scale production of B. braunii is hampered by its slow growth rate. Other fast growing microalgae and terrestrial plants, unlike B. braunii, do not accumulate triterpene hydrocarbons, because squalene, a key metabolite of the plant triterpene pathway, is rapidly converted to downstream products. With the aim of modifying plants to accumulate substantial amounts of squalene-like triterpenes for biofuel, we used a metabolic engineering approach, to divert the plant triterpene pathway for hydrocarbon production. A B. braunii triterpene methyltransferase 3 (BbTMT-3) gene was cloned and transferred into Arabidopsis thaliana. The function of BbTMT-3 was found to add methyl groups to squalene, which prevented squalene from being further metabolized. Further chemical analysis indicated that the transgenic plants contained 30µµµµg/g fresh weight of monomethylsqualene and 50µµµµg/g of dimethylsqualene, and these new hydrocarbon molecules were not present in untransformed plants. The accumulation of the triterpenoid hydrocarbons indicated that the intended transformation was successful, and demonstrated the feasibility of new biofuel production via metabolic engineering. Further research is now aimed to engineer the upstream genes to direct greater carbon flux into the triterpene pathway in order to increase the hydrocarbon content.

Keywords: Arabidopsis thaliana; biofuel; Botryococcus braunii; metabolic engineering; triterpenoid methyltransferase

I. INTRODUCTION

Renewable energy carried by fuels generated from biomass is becoming an essential part of energy infrastructure, because of the rapid depletion of fossil fuel and global warming concerns [1, 2]. Natural products with characteristics of traditional petroleum or diesel are desirable, since they can be used to replace part of the fossil fuel based transportation fuels [3]. In this context, Bio-alcohol (ethanol, butanol) from the fermentation pathway, and bio-diesel from the fatty acid biosynthetic pathway have been intensively studied. Recently, an isoprenoid (terpenoid) pathway has been proposed as an alternative for biofuel production [4, 5]

Terpenoids are a class of natural products made from the isoprenoid metabolic pathways located in the cytosol and chloroplast [6]. Up to 50,000 different terpenoids are found in nature, and their natural synthesis pathways provide enormous opportunities for metabolic engineering to modify resource allocation, and to produce many novel products as candidate molecules for biofuels [7].

Squalene is a liquid triterpenoid hydrocarbon (C30H50) synthesized from six 5-carbon isoprene building blocks, and is widely distributed in all kingdoms, as the precursor for hopanoids in prokaryotes and for sterols in eukaryotes. Its potential as a feedstock for biofuel production is demonstrated by the accumulation of extracellular lipids by the green alga Botryococcous braunii RACE B. Major components of B. braunii lipid are squalene-like hydrocarbons collectively called botryococcenes, including squalene and methylated squalenes [8, 9]. The lipids can accumulate up to 40% of dry weight. Botryococenes are considered to be one of the best sources for fossil fuel replacement due to their high energy density and suitability as feedstocks for existing petroleum refinery systems to produce high quality, sulphur-free transport fuel such as octane, kerosene (aviation fuels), diesel, and petrochemicals [10] (Figure 1).

Figure 1. Typical structures of extracellular lipids of B. braunii

and the products of hydrogenation and cracking of the lipids.

Botryococcenes have not yet been produced commercially, because of the extremely slow growth rate of B. braunii, and they are so far only identified in this single algal species. Although many researchers have been trying to find fast growing strains and optimizing culture conditions, the results are, up till now, far from satisfactory.

Squalene is the starting molecule for all triterpenoids and has been identified in almost all plant species, but usually in very small amounts, far short of biofuel application potentials. Over expression of squalene synthase (SQS) in Arabidopsis resulted in accumulation sterols, showing that squalene is rapidly metabolised [11] . Thus squalene has to be diverted from its native pathway for meaningful accumulation. As indicated in Botryococcous lipids, squalene can be methylated by triterpenoid methyltransferases (BbTMT) [12], so escaping conversion to sterols.

In this work, we made an Arabidopsis thaliana transgenic plant, in which BbTMT-3 gene was overexpressed. The genetic modified plants grew normally and were able to produce both monomethylatd and dimethylated squalenes, which were absent from the reference plants. Our results indicated that squalene-type hydrocarbons had been successfully produced via metabolic engineering approaches. This finding provides a solid base for further metabolic engineering to allow either plants or microalgae to be modified for high quality biofuel production.

II. EXPERIMENTALS

A. Reagents and instruments

All reagents were purchased from Sigma-Aldrich Pty. Ltd. unless otherwise indicated. The GC/MSD system utilized for triterpenoid analysis included an Agilent GC 6890N gas chromatograph fitted with a 7683B Automatic Liquid Sampler and a 5975B Inert MSD quadrupole MS

detector. The capillary column on the gas chromatograph (0.25 mm (i.d.), 0.25 µm film thickness, 30 m Varian FactorFour VF-5ms) was fitted with a 10 m integrated guard column (Agilent). The injection volume was 1 µL, and inlet temperature was kept constant at 300˚C. The carrier gas (helium) flowed at a constant rate of 1 mL min-1. The GC oven temperature was set at 100˚C initially for 1 min, increased to 320˚C at a rate of 37˚C min-1 and then held for 2 min. The transfer line temperature was set at 280˚C, MS source and quadrupole temperature at 230˚C and 150˚C, respectively. Ionisation was achieved by electron impact at 70 eV. The mass calibrant perfluorotributylamine was used to pre-tune the MSD. Data analysis was performed through the Agilent GC/MSD Productivity Chemstation software.

B. Sequence analysis

The gene encoding TMT and SMT was identified by

BLAST searches of the NCBI protein database using BbTMT-3 as the query sequence. A phylogenetic tree and sequence alignment were generated using Geneious 6.03 (http://www.geneious.com/) (Biomatters, Auckland, New Zealand) with default parameters. Accession numbers of amino acids sequences of TMTs and SMTs from green algae are as follow: Botryococcus braunii, B. braunii TMT-3, (H2E7T7); B. braunii TMT-1, (H2E7T5); B. braunii TMT-2, (H2E7T6); B. braunii SMT-2, (H2E7T9); B. braunii SMT-1, (H2E7T8); B. braunii SMT-3, (H2E7U0); Chlamydomonas reinhardtii, C. reinhardtii SMT, (XP_001690775); Volvox carteri, V. carteri SMT, (XP_002948023); Coccomyxa subellipsoidea, C. subellipsoidea SMT, (EIE25361).

C. . Expression of TBTMT-3 in Arabidopsis thaliana

The coding region deduced from the BbTMT-3 (JN828962.1) was cloned into the pGreen expression vector with Kanamycine as selection marker. The vector was transferred into Agrobacteria (Agrobacterium tumefacien) via heat shock. Transformed agrobacteria cells were inoculated in LB medium and incubated at 30ºC for two days until cell density reached an OD of 1.0 at 600 nm. The Agrobacteria were resuspended in the transformation buffer, and the vector was transferred into 4 weeks Arabidopsis plant via floral dipping (T0 plants). Transformed plants propagated from the seed of T0 plants was selected by their resistance to Kanamycine, and homozygote lines were further selected via kanamycin screening. A blank vector transferring line was also selected as reference.

D. . Product analysis

A sample of the fresh plant tissue from 4 week plants (50mg) was collected into a 2 ml microcentrifuge tube and snap frozen by liquid nitrogen. After adding internal standard (Squalane 10ul 0.05µg/ml in toluene), the plant tissue was grinded in fine powder using a Retsch ball mill (Haan, Germany) at 30Hz for 1 min. The plant tissues were saponified using 10% methanolic KOH. The nonsaponifiable fraction (NSF) was extracted with n-hexane to remove fatty acid from subsequent analysis. To remove the interferences

of fatty alcohols, they were converted to their trimethylsilane esters using N,O-Bis (Trimethylsilyl) trifluoroacetamide (BSTFA) and Trimethylchlorosilane (TMCS) (99:1) with pyridine as catalyst. Squalene and methylated squalenes were identified by their retention time and unique mass spectra

III. RESULTS AND DISCUSSION

A. Characterization of Bb TMT.

The triterpene pathway of B. braunii has attracted significant attention, but the full sequence of genome is not yet available to the public. The first gene coded for triterpene methyltrasferase identified from B. braunii was patented in 2010 [12]. Enzyme from this gene was able to catalyse the conversion of squalene to methylated squalenes. Blasting NCBI database using amino acid sequence deduced from the patented gene, resulted in 6 genes derived from B. braunii sharing very high identities. These 6 genes were classified into two group as sterol methyltransferases (SMTs) and triterpene methyltransferases (TMTs) [13]. A phylogenetic tree (Figure 1) was constructed with amino acid sequences of B. braunii TMTs (BbTMT) and SMTs from other green algae.

Figure 2 The phylogenetic tree of TMTs and SMTs from

green algae All three BbSMTs were clustered together as their own

clade close to algal SMTs, but BbTMTs were located much further from the SMTs, which was in agreement with the earlier report [13]. Amino acid sequence analysis of BbTMT indicted these TMTs had three signature binding sits for AdoMet (S-Adenosyl-l-Methionine), signified for AdoMet dependent methyltransferas [14] (Figure 2). In addition to the AdoMet binding, TMTs also possessed two regions specified for SMTs (Figure 2). The region 1 (SMT-1) was linked with the sterol substrate binding [15], from which TMTs and SMTs cannot be distinguished.

Figure 3 Partial sequence alignment showing the conserved regions of TMTs and SMTs from green algae However, the SMT specific region two (SMT-2) differed

between BbTMTs and BbSMTs. For SMT the sequence was “IEATCHAP”, which was absolutely conserved among all plant SMTs [16]; whereas the sequence in TMTs was “MDS/ATCHAP”. The distinct sequence variations might have contributed to the TMT and SMT substrate specificities to use squalene or sterols, respectively. Reference [13] has reported that although there were some differences among three BbTMTs in terms of substrate selectivity, they were all able to methylate squalene. In order to achieve our goal that was to divert plant squalene from to be metabolised, BbTMT-3 gene was chosen for the transgenic experiment.

B. Arabidopsis plant overexpressed with BbTMT-3 gene.

The BbTMT-3 gene (JN828964), corresponding to the sequence published in 2010 patent, was synthesized, and cloned into a pGreen vector, and transferred into Arabidopsis plant via agrobacteria mediated transformation method. The BbTMT gene was under control of the Cauliflower mosaic virus (CaMV) 35S promoter to be consecutively expressed in the plant. Since, we have established a GC/MS method for squalene and methylated squalene analyses; the successfully transformed plants were easily selected by the GC/MS assay (Figure 4 ), which was much more specific than PCR assay to identify the gene transfer events.

In order to increase the sensitivity of GC/MS assay, the

leaf samples were put through saponification and silyation to reduce the interference by the fatty acids and long chain fatty alcohols. Using the aforementioned method, the detection limits for squalene and its methylated products were about 1µg/g fresh weight. Squalene and methylated squalenes were identified by the similarity of their mass spectra to the standards spectra in commercial GC/MS database and published data [13] [12].

Three transgenic lines (Bbt-1, 2 and 3) were selected by

their ability to produce the expected products. The plants had no visible phenotype compared to both wild type plants and empty vector control plants (data not show). We plan to check more detailed physiology properties of these transgenic plants in the future.

Figure 4. GC trace of squalene (1) and methylated squalenes (2,3) from

leaves of TMT transformed Arabidopsis plants. Inset: the Mass spectra of the 3 identified triterpenes

We have determined the methyl squalenes contents of 4

week old leaves from the three transgenic lines. Among the three transgenic lines, Bbt-3 produced highest amounts of methylated squalenes with on average 50µg/g fresh weight of monomethylsqualene and 30µg/g of dimethylsqualene.. We noticed that the total triterpene hydrocarbons (including squalene) from the best transgenic line was about 150 µg/g fresh weigh, which was far less compared with the hydrocarbon produced by B. braunii. The reason for the low production rate was likely the low carbon flux to squalene biosynthesis. Further engineering of the upstream genes to direct carbon to squalene production would allow plants to make more triterpene hydrocarbons comparable to algae, and the plant will have much higher growth rate.

CONCLUSIONS

We applied Arabidopsis for proof of concept that we could engineer a plant to produce triterpene hydrocarbons. Although the hydrocarbon yield from our transgenic plants was lower than the native algal system, our research pioneered the application of higher plants for new generation biofuel production that potentially add value to crop products. Knowledge gained from our experiments using Arabidopsis can be easily applied to other organisms, such as fast growing microalgae, which can be cultured on a massive scale without competing with agriculture.

ACKNOWLEDGMENT

The authors gratefully acknowledge the financial and other support provided by the Australian Research Council under the ARC Linkage Projects Scheme (LP100200135), BHP Billiton Iron Ore Pty Ltd, ENN Group and Ansac Pty Ltd.

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