Novel routes towards bioplastics from plants: elucidation of the methylperillate biosynthesis pathway from Salvia dorisiana trichomes

Plants produce a large variety of highly functionalized terpenoids. Functional groups such as partially unsaturated rings and carboxyl groups provide handles to use these compounds as feedstock for biobased commodity chemicals. For instance, methylperillate, a monoterpenoid found in Salvia dorisiana, may be used for this purpose, as it carries both an unsaturated ring and a methylated carboxyl group. The biosynthetic pathway of methylperillate in plants is still unclear. In this work, we identified glandular trichomes from S. dorisiana as the location of biosynthesis and storage of methylperillate. mRNA from purified trichomes was used to identify four genes that can encode the pathway from geranyl diphosphate towards methylperillate. This pathway includes a (–)-limonene synthase (SdLS), a limonene 7-hydroxylase (SdL7H, CYP71A76), and a perillyl alcohol dehydrogenase (SdPOHDH). We also identified a terpene acid methyltransferase, perillic acid O-methyltransferase (SdPAOMT), with homology to salicylic acid OMTs. Transient expression in Nicotiana benthamiana of these four genes, in combination with a geranyl diphosphate synthase to boost precursor formation, resulted in production of methylperillate. This demonstrates the potential of these enzymes for metabolic engineering of a feedstock for biobased commodity chemical

Generation of flavors and fragrances through biotransformation and de novo synthesis

Flavors and fragrances are the result of the presence of volatile and non-volatile compounds, appreciated mostly by the sense of smell once they usually have pleasant odors. They are used in perfumes and perfumed products, as well as for the flavoring of foods and beverages. In fact the ability of the microorganisms to produce flavors and fragrances has been described for a long time, but the relationship between the flavor formation and the microbial growth was only recently established. After that, efforts have been put in the analysis and optimization of food fermentations that led to the investigation of microorganisms and their capacity to produce flavors and fragrances, either by de novo synthesis or biotransformation. In this review, we aim to resume the recent achievements in the production of the most relevant flavors by bioconversion/biotransformation or de novo synthesis, its market value, prominent strains used, and their production rates/maximum concentrations.We would like to thank the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469 unit, COMPETE 2020 (POCI-01-0145FEDER-006684), and BiotecNorte operation (NORTE-01-0145FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

From plant to plastic : Metabolic engineering of plant monoterpenes for biobased commodity chemicals

This thesis aimed to investigate how plant monoterpenes can be used to produce biobased plastics. Monoterpenes are volatile compounds, produced by plants to defend themselves against insects and pathogens or to attract pollinators. Many monoterpenes have a characteristic odour, and are used by humans in all kinds of products for their nice smell or taste. For example the monoterpene (+)-limonene has a fresh citrus odour, and is used in cosmetics and sodas. Recently, however, it was demonstrated that the chemical structure of some monoterpenes may also be suitable to serve as a feed stock for the synthesis of commodity chemicals and biomaterials. Plants produce monoterpenes in specialized structures, such as glandular trichomes. Trichomes are gland-like structures on the leaf surface that serve as small biochemical factories. Plants produce and store monoterpenes and other volatile compounds in these trichomes. However, the amount of monoterpenes in plants is often not large enough for bulk applications. Therefore, I set out to investigate which genes plants use to produce monoterpenes, and if I can express these genes in a better production platform, in order to produce larger amounts of monoterpenes. Monoterpenes consist of 10 carbon atoms and are synthesized from the precursor geranyl diphosphate (GPP) in the plastids of the plant cell. After synthesis of the monoterpene backbone, usually several structural modifications, for example oxidation, take place, by other enzymes in the cell. Chapter 1 of this thesis introduces what monoterpenes are, how they are synthesized in plants and how they can be produced by metabolic engineering in heterologous hosts like micro-organisms for human applications. One of the best studied monoterpenes is limonene. Chapter 2 reviews the existing and potential applications of limonene as well as the state of the art in its microbial production. The chapter describes which genes have been used for the biosynthesis of limonene, as well as the strategies that have been employed to enhance the production in micro-organisms. Chapter 3 describes our production of limonene using the micro-organism Saccharomyces cerevisieae (yeast). For this purpose, a mutated yeast strain was used, which produces a small amount of GPP as precursor for limonene biosynthesis. Limonene has a chiral centre, which means it can exist in two enantiomers, (+) or (-), which are mirror images. I showed that it is possible to produce both forms in yeast by introducing limonene synthase genes from different plant species. It turned out that it is not straightforward to harvest limonene from yeast cultures, as it is very volatile and does not mix well with the culture broth. Therefore, a system was developed to trap limonene from the yeast culture headspace during production. Compared to other limonene harvesting systems, this resulted in a better yield. Chapter 4 describes how a natural derivative of limonene, methylperillate, can be converted to plastic. Methylperillate has a suitable structure to be converted into a polymer building block. To demonstrate this, methylperillate was converted to the bulk chemical terephthalic acid, which is the building block of polyethylene terephthalate (PET). Due to the high structural similarity between methylperillate and terephthalic acid, a short chemical synthesis route consisting of two steps could be developed. For the large scale application of methylperillate for biobased commodity chemicals, it would be useful to produce methylperillate in micro-organisms. Methylperillate has the same backbone as limonene, but with a methylated carboxyl group at the C7-position. At the onset of this thesis not much was known about the enzymes involved in the biosynthesis of such methylated carboxyl groups. In Chapter 5, I characterise a biosynthetic pathway to methylperillate. After screening several plant species we found that Salvia dorisiana, a sage species, can produce methylperillate in the glandular trichomes on its leaves. Trichomes were isolated from the leaves and used as the source, using genomics techniques, for the isolation of four genes, which I showed are involved in the biosynthesis of methylperillate. Production of methylperillate was established in the tobacco-like model plant, Nicotiana benthamiana, using these four Salvia genes. In the future these genes could also be used in yeast or other microbes to produce methylperillate in fermenters. In Chapter 6 the research results of this thesis are discussed. A perspective is provided on producing bioplastics from compounds like methylperillate. Questions addressed in this chapter include how much monoterpenes should be produced to realistically use them for the production of biomaterials, and which possible solutions can be foreseen to produce monoterpenes on a larger scale. One future scenario is to focus on the use of monoterpenes for more specific, high-value applications by taking advantage of their natural chirality. All in all, this research is an important first step to use specific molecules from plants as an alternative source for biomaterials. Potentially, this will decrease dependence on fossil oil, and improve sustainability of production processes.</p

Biotechnological production of limonene in microorganisms

This mini review describes novel, biotechnology-based, ways of producing the monoterpene limonene. Limonene is applied in relatively highly priced products, such as fragrances, and also has applications with lower value but large production volume, such as biomaterials. Limonene is currently produced as a side product from the citrus juice industry, but the availability and quality are fluctuating and may be insufficient for novel bulk applications. Therefore, complementary microbial production of limonene would be interesting. Since limonene can be derivatized to high-value compounds, microbial platforms also have a great potential beyond just producing limonene. In this review, we discuss the ins and outs of microbial limonene production in comparison with plant-based and chemical production. Achievements and specific challenges for microbial production of limonene are discussed, especially in the light of bulk applications such as biomaterials.</p

Use of high-resolution mass spectrometry for veterinary drug multi-residue analysis

National and international food and feed safety authorities are shifting from routine-to risk-based monitoring. Risk-based monitoring requires flexibility in the scope of analytes, matrices, and sampling. Also, risk-based monitoring implies a desire for retrospective analysis using different scope(s) to follow trends, identify new food safety threats, and monitor the effectiveness of policy interventions. The current availability of sensitive and accurate high-resolution mass spectrometry (HRMS) fits within this approach. This writing reviews the applicability of HRMS techniques for food control laboratories in the analysis of veterinary medicinal products and hormones in food, using HRMS and legislative background. Different HRMS measurement and data evaluation strategies are identified and discussed. Among them, routine screening and confirmation, suspect screening, semi-untargeted analysis (common mass pattern search), metabolite and degradation product identification, profiling for deviating samples, physiological markers or treatments, and identification of unknowns can be found. The food safety competent authorities could shift from methods with predefined scope to real risk-based monitoring by implementing HRMS for routine food and feed analysis

Novel routes towards bioplastics from plants: elucidation of the methylperillate biosynthesis pathway from Salvia dorisiana trichomes.

Plants produce a large variety of highly functionalized terpenoids. Functional groups such as partially unsaturated rings and carboxyl groups provide handles to use these compounds as feedstock for biobased commodity chemicals. For instance, methylperillate, a monoterpenoid found in Salvia dorisiana, may be used for this purpose, as it carries both an unsaturated ring and a methylated carboxyl group. The biosynthetic pathway of methylperillate in plants is still unclear. In this work, we identified glandular trichomes from S. dorisiana as the location of biosynthesis and storage of methylperillate. mRNA from purified trichomes was used to identify four genes that can encode the pathway from geranyl diphosphate towards methylperillate. This pathway includes a (-)-limonene synthase (SdLS), a limonene 7-hydroxylase (SdL7H, CYP71A76), and a perillyl alcohol dehydrogenase (SdPOHDH). We also identified a terpene acid methyltransferase, perillic acid O-methyltransferase (SdPAOMT), with homology to salicylic acid OMTs. Transient expression in Nicotiana benthamiana of these four genes, in combination with a geranyl diphosphate synthase to boost precursor formation, resulted in production of methylperillate. This demonstrates the potential of these enzymes for metabolic engineering of a feedstock for biobased commodity chemicals

Exploring the genomic traits of fungus-feeding bacterial genus Collimonas

Background Collimonas is a genus belonging to the class of Betaproteobacteria and consists mostly of soil bacteria with the ability to exploit living fungi as food source (mycophagy). Collimonas strains differ in a range of activities, including swimming motility, quorum sensing, extracellular protease activity, siderophore production, and antimicrobial activities. Results In order to reveal ecological traits possibly related to Collimonas lifestyle and secondary metabolites production, we performed a comparative genomics analysis based on whole-genome sequencing of six strains representing 3 recognized species. The analysis revealed that the core genome represents 43.1 to 52.7 % of the genomes of the six individual strains. These include genes coding for extracellular enzymes (chitinase, peptidase, phospholipase), iron acquisition and type II secretion systems. In the variable genome, differences were found in genes coding for secondary metabolites (e.g. tripropeptin A and volatile terpenes), several unknown orphan polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), nonribosomal peptide synthetase (NRPS) gene clusters, a new lipopeptide and type III and type VI secretion systems. Potential roles of the latter genes in the interaction with other organisms were investigated. Mutation of a gene involved in tripropeptin A biosynthesis strongly reduced the antibacterial activity against Staphylococcus aureus, while disruption of a gene involved in the biosynthesis of the new lipopeptide had a large effect on the antifungal/oomycetal activities. Conclusions Overall our results indicated that Collimonas genomes harbour many genes encoding for novel enzymes and secondary metabolites (including terpenes) important for interactions with other organisms and revealed genomic plasticity, which reflect the behaviour, antimicrobial activity and lifestylesof Collimonas spp

Additional file 1: Figure S1. of Exploring the genomic traits of fungus-feeding bacterial genus Collimonas

Phylogenetic tree based on 16S rDNA depicting the relationships of sequenced strains of Collimonas. Underneath the genes are the module and domain organization of PKS or NRPS genes. The domains are as follows: C, condensation; A, adenylation; T, thiolation; E, Epimerization; TE, thioesterification; CAL, Co-enzyme A ligase domain; KS, Ketosynthase domain; AT, Acyltransferase domain; ER, Enoylreductase domain; KR, Ketoreductase domain; DH, Dehydratase domain; and ACP, Acyl-carrier protein domain. Underneath the domains are the amino acids that are incorporated into the peptide moiety. The number associated with the amino acid refers to the position of the amino acid in the peptide chain. Figure S2. Synteny of the six Collimonas genomes. Pairwise alignments of genomes were generated using Mauve (A) C. fungivorans (B) C. pratensis (C) C. arenae and (D) The three species together. Colored outlined blocks surround the regions of the genomic sequence that aligned to another genome. The colored bars inside the blocks are related to the level of sequence similarities. The analysis showed that the highest number of rearrangements was evident between all the three species. Figure S3. (A) Conserved gene clusters for type II (T2SSa/T2SSb), III (T3SSa/T3SSb) and VI (T6SS) secretion systems identified in Collimonas strains. Quorum sensing assays of the Collimonas strains. Quorum sensing activity of Collimonas strains Ter331, Ter6, Ter91, Ter291, Ter10 and Ter282 with indicator strain (B) C. violaceum CV026 and (C) A. tumefaciens NT1 (outer colonies). A purple (C. violaceum CV026) or blue (A. tumefaciens NT1) pigment produced by the indicator strains is indicative of quorum sensing activity of the tested strains. Figure S4. Organization of the orphan gene clusters and predicted amino acid compositions. Figure S5. Amino acid alignment of Collimonas terpene synthases CPter91_2617 and CPter291_2730 with previously characterised bacterial terpene synthases. The Collimonas terpene synthases were aligned with the Streptomyces exfoliatus pentalenene synthase (Se_pentalenene), S. coelicolor geosmin synthase (Sc_geosmin, 336 amino acids of the N-terminus), Streptosporangium roseum epi-cubenol synthase (Sr_epicubenol), S. avermitilis avermitilol synthase (Sa_avermitilol), S. clavuligerus 1,8-cineole synthase (Scl_cineole) and Pseudomonas fluorescens 2-methylenebornane synthase (Pf_methylenebornane). The characteristic terpene synthase divalent metal-binding motifs, namely the acidic amino acid-rich motif and the NSE triad, are boxed. Figure S6. GC-MS chromatograms of Ter91 terpene cyclase incubated with different substrates, and mass spectra of major products. The GC-MS chromatograms of Ter291 were identical to Ter91 (data not shown). Empty vector chromatograms shows products from a control enzyme extract (pACYC-duet-1). (A) Ter91 terpene synthase with GPP. TIC 100 % = 1.19E4. Major monoterpene product at RT 6.79, identified as Beta-pinene by authentic standard. (B) Ter91 terpene synthase with GGPP. TIC 100 % = 2.12E4. Major diterpene product at RT 20.24, identified as 13-epimanool comparison of mass spectra to the NIST8 library. (PDF 570 kb