Bioinformatics-aided identification, characterization and applications of mushroom linalool synthases

Enzymes empower chemical industries and are the keystone for metabolic engineering. For example, linalool synthases are indispensable for the biosynthesis of linalool, an important fragrance used in 60–80% cosmetic and personal care products. However, plant linalool synthases have low activities while expressed in microbes. Aided by bioinformatics analysis, four linalool/nerolidol synthases (LNSs) from various Agaricomycetes were accurately predicted and validated experimentally. Furthermore, we discovered a linalool synthase (Ap.LS) with exceptionally high levels of selectivity and activity from Agrocybe pediades, ideal for linalool bioproduction. It effectively converted glucose into enantiopure (R)-linalool in Escherichia coli, 44-fold and 287-fold more efficient than its bacterial and plant counterparts, respectively. Phylogenetic analysis indicated the divergent evolution paths for plant, bacterial and fungal linalool synthases. More critically, structural comparison provided catalytic insights into Ap.LS superior specificity and activity, and mutational experiments validated the key residues responsible for the specificity.

Efficient Biosynthesis of R-(-)-linalool through Adjusting Expression Strategy and Increasing GPP Supply in Escherichia coli

Background: R-(-)-linalool is a versatile acyclic monoterpene alcohol with applications in the flavor and fragrance, pharmaceutical, and agrochemical industries. However, plant extraction furnishes only limited and unstable R-(-)-linalool yields that do not satisfy market demand. Therefore, a sustainable yet efficient and productive method of R-(-)-linalool synthesis is urgently needed. Results: To induce the R-(-)-linalool biosynthesis pathway in E. coli , we expressed heterologous (3R)-linalool synthase (LIS) from Lavandula angustifolia (laLIS). We then enhanced R-(-)-linalool production in the cells using a suitable LIS from Streptomyces clavuligerus (bLIS). The bLIS expression was markedly elevated by using optimized ribosomal binding sites (RBSs) and protein fusion tags. R-(-)-linalool output rose from 4.8 mg L -1 to 33.4 mg L -1 . To increase the geranyl diphosphate (GPP) content in E. coli , we tested various alterations in geranyl diphosphate synthases (GPPSs) and their mutants. The final E. coli strain harboring GPPS from Abies grandis ( Ag GPPS) accumulated ≤ 100.1 mg L -1 R-(-)-linalool after 72 h shake-flask fermentation. This yield gain constitutes a 60.7-fold improvement in R-(-)-linalool biosynthesis over the parent strain. Fed-batch cultivation of the engineered strain in a 1.3-L fermenter yielded 1,027.3 mg L -1 R-(-)-linalool. Conclusions: In this study, an efficient R-(-)-linalool production pathway was induced in E. coli via the heterologous MVA pathway, AgGPPS, and (3R)-linalool synthase (bLIS). By overexpressing the key enzyme in the engineered E. coli strain, R-(-)-linalool production reached 100.1 mg L -1 and 1,027.3 mg L -1 under shake flask- and fed-batch fermentation conditions, respectively. The latter is the highest reported R-(-)-linalool yield to date using an engineered E. coli strain. The strategies of key enzyme overexpression and mutation could lay theoretical and empirical foundations for engineering terpenoid pathways and optimizing other metabolic pathways.

De novo assembly and comparative transcriptome analysis revealed the genes that were potentially involved in defensive terpenoids emission in the wheat against the wheat aphid Sitobion avenae (Fabricius)

Background Terpenoid volatiles play an important role directly or indirectly in the plant defense mechanisms against herbivores, including the gramineous crops such as transgenic rice and corn. The conventional varieties of wheat are important gramineous cereal crops that lack aphid-resistant genes. Therefore, it is necessary to seek the aphid-resistant genes by screening for potential terpenoid synthase genes in the wheat germplasm resources. Results The result showed that aphid-damaged Octoploid Tirtitrigia emitted a higher amount of S-linalool, ent-kaurene, (+)-delta-cadinene, (3S,6E)-nerolidol as compared to the intact plant. In addition, (E)-β-caryophyllene, β‐Myrcene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) were new volatile terpenoids emitted by the damaged plant. Further olfactory responses tests showed that S-linalool significantly repelled Sitobion avenae (Fabricius). After de novo assembly and quantitative assessment, a total of 182348 (74.8%) unigenes were annotated by alignment with the public protein databases. Of these unigenes, 2389 differentially expressed genes were identified between the intact and damaged ears of Octoploid Trititrigia. The expression profile of 10 randomly selected TPSs was confirmed with RT-qPCR. Candidate genes involved in terpenes biosynthesis were identified by showing the significant transcript changes between the intact and damaged plant ears of Octoploid Trititrigia. The transcript abundances of terpenes biosynthetic pathway-related genes were also positively correlated with the production of volatile terpenoids in the ears. The unigenes of S-linalool synthase gene was mapped to the cloned cDNA WT008_M07 (AK333728) and WT013_P07 (AK335856) of the Chinese spring wheat cultivar. The predicted protein complete ORF sequence (TaLIS1/2) when compared with the S-linalool synthase gene of other species, contained an aspartate-rich region DDxxD motif. Its function was characterized as coordinating the divalent metal ions involved in substrate binding. Furthermore, the phylogenetic tree results indicated that the TaLIS1 and TuNES1 are highly homologous. Conclusions This assembled transcriptome of S. avenae-damaged Octoploid Trititrigia and the intact ears could provide more molecular resources for the future functional characterization analysis of genomics in volatile terpenoids involved in direct or indirect defenses. Our study describes the metabolic regulation mechanism of the volatile terpenoids in the gramineous crops, which provides support for both breeding and genetic modification of the wheat varieties resistant to wheat aphid.

De novo assembly and comparative transcriptome analysis reveal genes potentially involved in changes of terpenoids in wheat germplasm resource, that defend against the wheat aphid Sitobion avenae (Fabricius)

Background Terpenoid volatiles play an important role in direct and indirect plant defense responses against herbivores, including in gramineous crops such as transgenic rice and corn. The conventional varieties of wheat, an important gramineous cereal crop, lack aphid-resistant genes. It is therefore, necessary to seek aphid-resistant genes by screening for potential terpenoid synthase genes in wheat germplasm resources. Results The result showed that aphid-damaged Octoploid Tirtitrigia emitted a higher amount of S-linalool, ent-kaurene, (+)-delta-cadinene, (3S,6E)- nerolidol compared to intact plant. In addition, (E)-β-caryophyllene, β‐Myrcene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) were new volatile terpenoids emitted by the damaged plant. Further olfactory responses tests showed that S-linalool significantly repelled Sitobion avenae (Fabricius). Using the Illumina sequencing platform, approximately 203.09 million high quality paired-end reads were obtained. After de novo assembly and quantitative assessment, a total of 182,348(74.8%) unigenes were annotated by alignment with public protein databases. Of these unigenes, 2,389 differentially expressed genes were identified between intact and damaged ears of Octoploid Trititrigia. The expression profile of 10 randomly selected TPSs was confirmed with RT-qPCR. Candidate genes involved in terpenes biosynthesis were identified showing significant transcript changes between intact and damaged plant ears of Octoploid Trititrigia. Also transcript abundances of terpenes biosynthetic pathway-related genes were positively correlated with the production of volatile terpenoids in ears. The unigenes of S-linalool synthase gene was mapped to the cloned cDNA WT008_M07 (AK333728) and WT013_P07 (AK335856) of the Chinese spring wheat cultivar. The predicted protein complete ORF sequence (TaLIS1/2) when compared with the S-linalool synthase gene of other species, contained an aspartate-rich region DDxxD motif. Its function was characterized as coordinating the divalent metal ions involved in substrate binding. Furthermore, phylogenetic tree results showed that the TaLIS1 and TuNES1 are highly homologous. Conclusions This assembled transcriptome of S. avenae-damaged Octoploid Trititrigia and intact ears can provide more molecular resources for future functional characterization analysis of genomics in volatile terpenoids involved in direct or indirect defenses. Our study describes the metabolic regulation mechanism of volatile terpenoids in gramineous crops, which provides support for both breeding and genetic modification of wheat varieties resistant to wheat aphids.

An unbiased approach elucidates variation in (S)-(+)-linalool, a context-specific mediator of a tri-trophic interaction in wild tobacco

Plant volatile organic compounds (VOCs) mediate many interactions, and the function of common VOCs is especially likely to depend on ecological context. We used a genetic mapping population of wild tobacco, Nicotiana attenuata, originating from a cross of 2 natural accessions from Arizona and Utah, separated by the Grand Canyon, to dissect genetic variation controlling VOCs. Herbivory-induced leaf terpenoid emissions varied substantially, while green leaf volatile emissions were similar. In a field experiment, only emissions of linalool, a common VOC, correlated significantly with predation of the herbivore Manduca sexta by native predators. Using quantitative trait locus mapping and genome mining, we identified an (S)-(+)-linalool synthase (NaLIS). Genome resequencing, gene cloning, and activity assays revealed that the presence/absence of a 766-bp sequence in NaLIS underlies the variation of linalool emissions in 26 natural accessions. We manipulated linalool emissions and composition by ectopically expressing linalool synthases for both enantiomers, (S)-(+)- and (R)-(−)-linalool, reported to oppositely affect M. sexta oviposition, in the Arizona and Utah accessions. We used these lines to test ovipositing moths in increasingly complex environments. The enantiomers had opposite effects on oviposition preference, but the magnitude of the effect depended strongly both on plant genetic background, and complexity of the bioassay environment. Our study reveals that the emission of linalool, a common VOC, differs by orders-of-magnitude among geographically interspersed conspecific plants due to allelic variation in a linalool synthase, and that the response of a specialist herbivore to linalool depends on enantiomer, plant genotype, and environmental complexity.