Molecular Mechanisms of Natural Rubber Biosynthesis.

Natural rubber (NR), principally comprising cis-1,4-polyisoprene, is an industrially important natural hydrocarbon polymer because of its unique physical properties, which render it suitable for manufacturing items such as tires. Presently, industrial NR production depends solely on latex obtained from the Pará rubber tree, Hevea brasiliensis. In latex, NR is enclosed in rubber particles, which are specialized organelles comprising a hydrophobic NR core surrounded by a lipid monolayer and membrane-bound proteins. The similarity of the basic carbon skeleton structure between NR and dolichols and polyprenols, which are found in most organisms, suggests that the NR biosynthetic pathway is related to the polyisoprenoid biosynthetic pathway and that rubber transferase, which is the key enzyme in NR biosynthesis, belongs to the cis-prenyltransferase family. Here, we review recent progress in the elucidation of molecular mechanisms underlying NR biosynthesis through the identification of the enzymes that are responsible for the formation of the NR backbone structure.

Plant terpenoid metabolism co-opts a component of the cell wall biosynthesis machinery.

Glycosylation is one of the most prevalent molecular modifications in nature. Single or multiple sugars can decorate a wide range of acceptors from proteins to lipids, cell wall glycans and small molecules, dramatically affecting their activity. Here, we discovered that by 'hijacking' an enzyme of the cellulose synthesis machinery involved in cell wall assembly, plants evolved cellulose synthase-like enzymes (Csls) and acquired the capacity to glucuronidate specialized metabolites, that is, triterpenoid saponins. Apparently, endoplasmic reticulum-membrane localization of Csls and of other pathway proteins was part of evolving a new glycosyltransferase function, as plant metabolite glycosyltransferases typically act in the cytosol. Discovery of glucuronic acid transferases across several plant orders uncovered the long-pursued enzymatic reaction in the production of a low-calorie sweetener from licorice roots. Our work opens the way for engineering potent saponins through microbial fermentation and plant-based systems.

Farnesyl pyrophosphate compartmentalization in the green microalga Chlamydomonas reinhardtii during heterologous (E)-α-bisabolene production.

BACKGROUND: Eukaryotic algae have recently emerged as hosts for metabolic engineering efforts to generate heterologous isoprenoids. Isoprenoid metabolic architectures, flux, subcellular localization, and transport dynamics have not yet been fully elucidated in algal hosts. RESULTS: In this study, we investigated the accessibility of different isoprenoid precursor pools for C15 sesquiterpenoid generation in the cytoplasm and chloroplast of Chlamydomonas reinhardtii using the Abies grandis bisabolene synthase (AgBS) as a reporter. The abundance of the C15 sesquiterpene precursor farnesyl pyrophosphate (FPP) was not increased in the cytosol by co-expression and fusion of AgBS with different FPP synthases (FPPSs), indicating limited C5 precursor availability in the cytoplasm. However, FPP was shown to be available in the plastid stroma, where bisabolene titers could be improved several-fold by FPPSs. Sesquiterpene production was greatest when AgBS-FPPS fusions were directed to the plastid and could further be improved by increasing the gene dosage. During scale-up cultivation with different carbon sources and light regimes, specific sesquiterpene productivities from the plastid were highest with CO2 as the only carbon source and light:dark illumination cycles. Potential prenyl unit transporters are proposed based on bioinformatic analyses, which may be in part responsible for our observations. CONCLUSIONS: Our findings indicate that the algal chloroplast can be harnessed in addition to the cytosol to exploit the full potential of algae as green cell factories for non-native sesquiterpenoid generation. Identification of a prenyl transporter may be leveraged for further extending this capacity.

Molecular and Chemical Screening for Inherent Disease Resistance Factors of Norway Spruce (Picea abies) Clones Against Conifer Stem Rot Pathogen Heterobasidion parviporum.

Root and stem rot of conifer trees caused by Heterobasidion annosum species complex leads to huge economic losses in Europe, yet not much is known about the molecular and chemical basis for host resistance. To identify inherent chemical or molecular markers in clones found to be either resistant or susceptible, we sampled needle tissues of all the clones before pathogen inoculation. We conducted a short-term resistance screening by using the pathogen H. parviporum to inoculate 70 Norway spruce clones. Based on lesion size, subsets of highly susceptible and resistant clones were further analyzed. Terpene detection and RNA sequencing were performed to explore inherent variations in genotypes differing in resistance to pathogenic challenge at chemical and transcriptional levels. A negative correlation emerged between resistance and growth. Terpene profiles of resistant clones showed higher content of monoterpenes and sesquiterpenes, with concomitant increased transcript abundance of genes involved in the terpenoid pathway. A set of upregulated genes relevant to flavonoid biosynthesis was observed in resistant genotypes, whereas higher transcripts of lignin biosynthetic genes were prevalent in susceptible clones. Genes involved in flavonoid and lignin biosynthesis as well as terpene content may have a role in facilitating resistance of Norway spruce against H. parviporum. Our results provide strong support on the feasibility of sampling needle tissues before pathogen inoculation, and the approach could be of value for large-scale screening of novel biomarkers for durable resistance. The additional insights could form a basis for further research on resistance screening in this pathosystem.

The transition of Rhodobacter sphaeroides into a microbial cell factory.

Microbial cell factories are the workhorses of industrial biotechnology and improving their performances can significantly optimize industrial bioprocesses. Microbial strain engineering is often employed for increasing the competitiveness of bio-based product synthesis over more classical petroleum-based synthesis. Recently, efforts for strain optimization have been standardized within the iterative concept of "design-build-test-learn" (DBTL). This approach has been successfully employed for the improvement of traditional cell factories like Escherichia coli and Saccharomyces cerevisiae. Within the past decade, several new-to-industry microorganisms have been investigated as novel cell factories, including the versatile α-proteobacterium Rhodobacter sphaeroides. Despite its history as a laboratory strain for fundamental studies, there is a growing interest in this bacterium for its ability to synthesize relevant compounds for the bioeconomy, such as isoprenoids, poly-ß-hydroxybutyrate, and hydrogen. In this study, we reflect on the reasons for establishing R. sphaeroides as a cell factory from the perspective of the DBTL concept. Moreover, we discuss current and future opportunities for extending the use of this microorganism for the bio-based economy. We believe that applying the DBTL pipeline for R. sphaeroides will further strengthen its relevance as a microbial cell factory. Moreover, the proposed use of strain engineering via the DBTL approach may be extended to other microorganisms that have not been critically investigated yet for industrial applications.

De novo transcriptome assembly and protein profiling of copper-induced lignocellulolytic fungus Ganoderma lucidum MDU-7 reveals genes involved in lignocellulose degradation and terpenoid biosynthetic pathways.

Ganoderma lucidum is an important medicinal fungus that possesses exceedingly high lignocellulose degrading ability. Evidently, Cu2+ has decisive roles on the mycelial growth and enzyme production. To reveal the effect of Cu2+ on G. lucidum transcriptome, predominantly associated with lignocellulolytic progression, we conducted comparative NGS based de novo transcriptome assembly using Illumina Hi SeqTM sequencing platform. We obtained 26,083,372 and 35,713,076 high-quality reads from induced and uninduced cultures. For wood degrading activity, 194 transcript coding for oxidoreductases and 402 transcripts for CAZymes were predicted. Further, secretome studies revealed high score GO terms related to oxidoreductases, glycosyl hydrolases, and chitinases from Cu-induced mycelia. The increased Cu2+ concentrations showed higher secretion of lignocellulases such as laccases, cellulases, and xylanases along with increased production of phenolics and antioxidants. Several differences in the transcriptomic and proteomic signatures for lignocellulolytic enzymes provide vital clues about Cu2+ mediated gene regulation and metabolic pathways in basidiomycetous fungi.

Conversion of poplar biomass into high-energy density tricyclic sesquiterpene jet fuel blendstocks.

BACKGROUND: In an effort to ensure future energy security, reduce greenhouse gas emissions and create domestic jobs, the US has invested in technologies to develop sustainable biofuels and bioproducts from renewable carbon sources such as lignocellulosic biomass. Bio-derived jet fuel is of particular interest as aviation is less amenable to electrification compared to other modes of transportation and synthetic biology provides the ability to tailor fuel properties to enhance performance. Specific energy and energy density are important properties in determining the attractiveness of potential bio-derived jet fuels. For example, increased energy content can give the industry options such as longer range, higher load or reduced takeoff weight. Energy-dense sesquiterpenes have been identified as potential next-generation jet fuels that can be renewably produced from lignocellulosic biomass. RESULTS: We developed a biomass deconstruction and conversion process that enabled the production of two tricyclic sesquiterpenes, epi-isozizaene and prespatane, from the woody biomass poplar using the versatile basidiomycete Rhodosporidium toruloides. We demonstrated terpene production at both bench and bioreactor scales, with prespatane titers reaching 1173.6 mg/L when grown in poplar hydrolysate in a 2 L bioreactor. Additionally, we examined the theoretical fuel properties of prespatane and epi-isozizaene in their hydrogenated states as blending options for jet fuel, and compared them to aviation fuel, Jet A. CONCLUSION: Our findings indicate that prespatane and epi-isozizaene in their hydrogenated states would be attractive blending options in Jet A or other lower density renewable jet fuels as they would improve viscosity and increase their energy density. Saturated epi-isozizaene and saturated prespatane have energy densities that are 16.6 and 18.8% higher than Jet A, respectively. These results highlight the potential of R. toruloides as a production host for the sustainable and scalable production of bio-derived jet fuel blends, and this is the first report of prespatane as an alternative jet fuel.

Differential Gene Profiling of the Heartwood Formation Process in Taiwania cryptomerioides Hayata Xylem Tissues.

Taiwania (Taiwania cryptomerioides) is an important tree species in Taiwan because of the excellent properties of its wood and fascinating color qualities of its heartwood (HW), as well as the bioactive compounds therein. However, limited information is available as to the HW formation of this species. The objective of this research is to analyze the differentially expressed genes (DEGs) during the HW formation process from specific Taiwania xylem tissues, and to obtain genes that might be closely associated with this process. The results indicated that our analyses have captured DEGs representative to the HW formation process of Taiwania. DEGs related to the terpenoid biosynthesis pathway were all up-regulated in the transition zone (TZ) to support the biosynthesis and accumulation of terpenoids. Many DEGs related to lignin biosynthesis, and two DEGs related to pinoresinol reductase (PrR)/pinoresinol lariciresinol reductase (PLR), were up-regulated in TZ. These DEGs together are likely involved in providing the precursors for the subsequent lignan biosynthesis. Several transcription factor-, nuclease-, and protease-encoding DEGs were also highly expressed in TZ, and these DEGs might be involved in the regulation of secondary metabolite biosynthesis and the autolysis of the cellular components of ray parenchyma cells in TZ. These results provide further insights into the process of HW formation in Taiwania.

Functional analysis of isoprenoid precursors biosynthesis by quantitative metabolomics and isotopologue profiling.

INTRODUCTION: Isoprenoids are amongst the most abundant and diverse biological molecules and are involved in a broad range of biological functions. Functional understanding of their biosynthesis is thus key in many fundamental and applicative fields, including systems biology, medicine and biotechnology. However, available methods do not yet allow accurate quantification and tracing of stable isotopes incorporation for all the isoprenoids precursors. OBJECTIVES: We developed and validated a complete methodology for quantitative metabolomics and isotopologue profiling of isoprenoid precursors in the yeast Saccharomyces cerevisiae. METHODS: This workflow covers all the experimental and computational steps from sample collection and preparation to data acquisition and processing. It also includes a novel quantification method based on liquid chromatography coupled to high-resolution mass spectrometry. Method validation followed the Metabolomics Standards Initiative guidelines. RESULTS: This workflow ensures accurate absolute quantification (RSD < 20%) of all mevalonate and prenyl pyrophosphates intermediates with a high sensitivity over a large linear range (from 0.1 to 50 pmol). In addition, we demonstrate that this workflow brings crucial information to design more efficient phytoene producers. Results indicate stable turnover rates of prenyl pyrophosphate intermediates in the constructed strains and provide quantitative information on the change of the biosynthetic flux of phytoene precursors. CONCLUSION: This methodology fills one of the last technical gaps for functional studies of isoprenoids biosynthesis and should be applicable to other eukaryotic and prokaryotic (micro)organisms after adaptation of some organism-dependent steps. This methodology also opens the way to 13C-metabolic flux analysis of isoprenoid biosynthesis.

Yarrowia lipolytica: more than an oleaginous workhorse.

Microbial production of fuels and chemicals offers a means by which sustainable product manufacture can be achieved. In this regard, Yarrowia lipolytica is a unique microorganism suitable for a diverse array of biotechnological applications. As a robust oleaginous yeast, it has been well studied for production of fuels and chemicals derived from fatty acids. However, thanks in part to newfound genetic tools and metabolic understanding, Y. lipolytica has been explored for high-level production of a variety of non-lipid products. This mini-review will discuss some of the recent research surrounding the ability of Y. lipolytica to support bio-based chemical production outside the realm of fatty acid metabolism including polyketides, terpenes, carotenoids, pentose phosphate-derived products, polymers, and nanoparticles.

Biocatalysis for terpene-based polymers.

Accelerated generation of bio-based materials is vital to replace current synthetic polymers obtained from petroleum with more sustainable options. However, many building blocks available from renewable resources mainly contain unreactive carbon-carbon bonds, which obstructs their efficient polymerization. Herein, we highlight the potential of applying biocatalysis to afford tailored functionalization of the inert carbocyclic core of multicyclic terpenes toward advanced materials. As a showcase, we unlock the inherent monomer reactivity of norcamphor, a bicyclic ketone used as a monoterpene model system in this study, to afford polyesters with unprecedented backbones. The efficiencies of the chemical and enzymatic Baeyer-Villiger transformation in generating key lactone intermediates are compared. The concepts discussed herein are widely applicable for the valorization of terpenes and other cyclic building blocks using chemoenzymatic strategies.

Isoprenoid biosynthesis in dandelion latex is enhanced by the overexpression of three key enzymes involved in the mevalonate pathway.

BACKGROUND: Latex from the dandelion species Taraxacum brevicorniculatum contains many high-value isoprenoid end products, e.g. triterpenes and polyisoprenes such as natural rubber. The isopentenyl pyrophosphate units required as precursors for these isoprenoids are provided by the mevalonate (MVA) pathway. The key enzyme in this pathway is 3-hydroxy-methyl-glutaryl-CoA reductase (HMGR) and its activity has been thoroughly characterized in many plant species including dandelion. However, two enzymes acting upstream of HMGR have not been characterized in dandelion latex: ATP citrate lyase (ACL), which provides the acetyl-CoA utilized in the MVA pathway, and acetoacetyl-CoA thiolase (AACT), which catalyzes the first step in the pathway to produce acetoacetyl-CoA. Here we isolated ACL and AACT genes from T. brevicorniculatum latex and characterized their expression profiles. We also overexpressed the well-characterized HMGR, ACL and AACT genes from Arabidopsis thaliana in T. brevicorniculatum to determine their impact on isoprenoid end products in the latex. RESULTS: The spatial and temporal expression profiles of T. brevicorniculatum ACL and AACT revealed their pivotal role in the synthesis of precursors necessary for isoprenoid biosynthesis in latex. The overexpression of A. thaliana ACL and AACT and HMGR in T. brevicorniculatum latex resulted in the accumulation of all three enzymes, increased the corresponding enzymatic activities and ultimately increased sterol levels by ~5-fold and pentacyclic triterpene and cis-1,4-isoprene levels by ~2-fold. Remarkably high levels of the triterpene precursor squalene were also detected in the triple-transgenic lines (up to 32 mg/g root dry weight) leading to the formation of numerous lipid droplets which were observed in root cross-sections. CONCLUSIONS: We could show the effective expression of up to three transgenes in T. brevicorniculatum latex which led to increased enzymatic activity and resulted in high level squalene accumulation in the dandelion roots up to an industrially relevant amount. Our data provide insight into the regulation of the MVA pathway in dandelion latex and can be used as a basis for metabolic engineering to enhance the production of isoprenoid end products in this specialized tissue.

Head-to-Head Prenyl Synthases in Pathogenic Bacteria.

Many organisms contain head-to-head isoprenoid synthases; we investigated three such types of enzymes from the pathogens Neisseria meningitidis, Neisseria gonorrhoeae, and Enterococcus hirae. The E. hirae enzyme was found to produce dehydrosqualene, and we solved an inhibitor-bound structure that revealed a fold similar to that of CrtM from Staphylococcus aureus. In contrast, the homologous proteins from Neisseria spp. carried out only the first half of the reaction, yielding presqualene diphosphate (PSPP). Based on product analyses, bioinformatics, and mutagenesis, we concluded that the Neisseria proteins were HpnDs (PSPP synthases). The differences in chemical reactivity to CrtM were due, at least in part, to the presence of a PSPP-stabilizing arginine in the HpnDs, decreasing the rate of dehydrosqualene biosynthesis. These results show that not only S. aureus but also other bacterial pathogens contain head-to-head prenyl synthases, although their biological functions remain to be elucidated.

Novel Insights into the Organization of Laticifer Cells: A Cell Comprising a Unified Whole System.

Laticifer cells are specialized plant cells that synthesize and accumulate latex. Studies on laticifers have lagged behind in recent years, and data regarding the functional role of laticifers and their fitness benefit still remain elusive. Laticifer differentiation and its impact on plant growth and development also remain to be investigated. Here, cellular, molecular, and genetic tools were developed to examine the distribution, differentiation, ontogeny, and other characteristic features, as well as the potential developmental role of laticifer cells in the latex-bearing plant Euphorbia lathyris. The organization of the laticiferous system within the E. lathyris plant body is reported, emerging as a single elongated and branched coenocytic cell, constituting the largest cell type existing in plants. We also report the ontogeny and organization of laticifer cells in the embryo and the identification of a laticifer-associated gene expression pattern. Moreover, the identification of laticifer- and latex-deficient mutants (pil mutants) allowed for the identification of distinct loci regulating laticifer differentiation, growth, and metabolic activity. Additionally, pil mutants revealed that laticifer cells appear nonessential for plant growth and development, thus pointing toward their importance, instead, for specific ecophysiological adaptations of latex-bearing plants in natural environments.

Application of a stir bar sorptive extraction method for the determination of volatile compounds in different grape varieties.

BACKGROUND: Stir bar sorptive extraction (SBSE) has been employed for the analysis of aroma compounds directly from Spanish and Italian grape extracts. The extraction conditions have been optimized using a statistical approach, obtaining results that improve the current state of the art. RESULTS: The proposed analytical conditions were: sample volume 40 mL, extraction time 80 min, 25% (w/v) NaCl and stirring speed 750 rpm using 20 mm × 0.5 mm stir bars. Performance characteristics of the SBSE procedure were further studied and showed correlation coefficients, detection and quantification limits, linear ranges, recoveries and precision values adequate for analysing these compounds in grapes. Twenty-four grape varieties (aromatic and non-aromatic) were analysed. Statistically significant differences (P < 0.05) according to geographical origin were found, being derived mainly from the terpenoid content. By principal component analysis it was possible to note that the terpenoids were more effective in the separation of the non-aromatic samples, showing negative and positive scores for Italian and Spanish grapes, respectively. CONCLUSION: For non-aromatic grape varieties, the factor 'geographic location' was the most significant, thus corroborating the high influence of this parameter on the volatile composition of grapes and therefore on the genuineness of wines. © 2016 Society of Chemical Industry.

Proteomic analysis of lettuce seed germination and thermoinhibition by sampling of individual seeds at germination and removal of storage proteins by polyethylene glycol fractionation.

Germination and thermoinhibition in lettuce (Lactuca sativa 'Jianyexianfeng No. 1') seeds were investigated by a proteomic comparison among dry seeds, germinated seeds at 15°C, at 15°C after imbibition at 25°C for 48 h, or at 25°C in KNO3 (all sampled individually at germination), and ungerminated seeds at 25°C, a thermoinhibitory temperature. Before two-dimensional gel electrophoresis analysis, storage proteins (greater than 50% of total extractable protein) were removed by polyethylene glycol precipitation, which significantly improved the detection of less abundant proteins on two-dimensional gels. A total of 108 protein spots were identified to change more than 2-fold (P<0.05) in abundance in at least one germination treatment. Nineteen proteins increasing and one protein decreasing in abundance during germination had higher abundance in germinated 15°C, 15°C after imbibition at 25°C for 48 h, and 25°C in KNO3 seeds than in ungerminated 25°C seeds. Gene expression of 12 of those proteins correlated well with the protein accumulation. Methionine metabolism, ethylene production, lipid mobilization, cell elongation, and detoxification of aldehydes were revealed to be potentially related to lettuce seed germination and thermoinhibition. Accumulation of three proteins and expression of five genes participating in the mevalonate (MVA) pathway of isoprenoid biosynthesis correlated positively with seed germinability. Inhibition of this pathway by lovastatin delayed seed germination and increased the sensitivity of germination to abscisic acid. MVA pathway-derived products, cytokinins, partially reversed the lovastatin inhibition of germination and released seed thermoinhibition at 25°C. We conclude that the MVA pathway for isoprenoid biosynthesis is involved in lettuce seed germination and thermoinhibition.

Functional convergence of hopanoids and sterols in membrane ordering.

Liquid-ordered phases are one of two biochemically active membrane states, which until now were thought to be a unique consequence of the interactions between eukaryotic membrane lipids. The formation of a liquid-ordered phase depends crucially on the ordering properties of sterols. However, it is not known whether this capacity exists in organisms that lack sterols, such as bacteria. We show that diplopterol, the simplest bacterial hopanoid, has similar properties and that hopanoids are bacterial "sterol surrogates" with the ability to order saturated lipids and to form a liquid-ordered phase in model membranes. These observations suggest that the evolution of an ordered biochemically active liquid membrane could have evolved before the oxygenation of Earth's surface and the emergence of sterols.

Use of nonionic surfactants for improvement of terpene production in Saccharomyces cerevisiae.

To facilitate enzyme and pathway engineering, a selection was developed for improved sesquiterpene titers in Saccharomyces cerevisiae. α-Bisabolene, a candidate advanced biofuel, was found to protect yeast against the disruptive action of nonionic surfactants such as Tween 20 (T20). An experiment employing competition between two strains of yeast, one of which makes twice as much bisabolene as the other, demonstrated that growth in the presence of T20 provided sufficient selective pressure to enrich the high-titer strain to form 97% of the population. Following this, various methods were used to mutagenize the bisabolene synthase (BIS) coding sequence, coupled with selection by subculturing in the presence of T20. Mutagenesis targeting the BIS active site did not yield an improvement in bisabolene titers, although mutants were found which made a mixture of α-bisabolene and ß-farnesene, another candidate biofuel. Based on evidence that the 3' end of the BIS mRNA may be unstable in yeast, we randomly recoded the last 20 amino acids of the enzyme and, following selection in T20, found a variant which increased specific production of bisabolene by more than 30%. Since T20 could enrich a mixed population, efficiently removing strains that produced little or no bisabolene, we investigated whether it could also be applied to sustain high product titers in a monoculture for an extended period. Cultures grown in the presence of T20 for 14 days produced bisabolene at titers up to 4-fold higher than cultures grown with an overlay of dodecane, used to sequester the terpene product, and 20-fold higher than cultures grown without dodecane.

Terpene and dextran renewable resources for the synthesis of amphiphilic biopolymers.

The present work shows the synthesis of amphiphilic polymers based on the hydrophilic dextran and the hydrophobic terpenes as renewable resources. The first step concerns the synthesis of functional terpene molecules by thiol-ene addition chemistry involving amino or carboxylic acid thiols and dihydromyrcenol terpene. The terpene-modified polysaccharides were subsequently synthesized by coupling the functional terpenes with dextran. A reductive amination step produced terpene end-modified dextran with 94% of functionalization, while the esterification step produced three terpene-grafted dextrans with a number of terpene units per dextran of 1, 5, and 10. The amphiphilic renewable grafted polymers were tested as emulsifiers for the stabilization of liquid miniemulsion of terpene droplets dispersed in an aqueous phase. The average hydrodynamic diameter of the stable droplets was observed at about 330 nm.

Gene expression plasticity facilitates different host feeding in Ips sexdentatus (Coleoptera: Curculionidae: Scolytinae).

Host shift is ecologically advantageous and a crucial driver for herbivore insect speciation. Insects on the non-native host obtain enemy-free space and confront reduced competition, but they must adapt to survive. Such signatures of adaptations can often be detected at the gene expression level. It is astonishing how bark beetles cope with distinct chemical environments while feeding on various conifers. Hence, we aim to disentangle the six-toothed bark beetle (Ips sexdentatus) response against two different conifer defences upon host shift (Scots pine to Norway spruce). We conducted bioassay and metabolomic analysis followed by RNA-seq experiments to comprehend the beetle's ability to surpass two different terpene-based conifer defence systems. Beetle growth rate and fecundity were increased when reared exclusively on spruce logs (alternative host) compared to pine logs (native host). Comparative gene expression analysis identified differentially expressed genes (DEGs) related to digestion, detoxification, transporter activity, growth, signalling, and stress response in the spruce-feeding beetle gut. Transporter genes were highly abundant during spruce feeding, suggesting they could play a role in pumping a wide variety of endogenous and xenobiotic compounds or allelochemicals out. Trehalose transporter (TRET) is also up-regulated in the spruce-fed beetle gut to maintain homeostasis and stress tolerance. RT-qPCR and enzymatic assays further corroborated some of our findings. Taken together, the transcriptional plasticity of key physiological genes plays a crucial role after the host shift and provides vital clues for the adaptive potential of bark beetles on different conifer hosts.