Integrating structure-based machine learning and co-evolution to investigate specificity in plant sesquiterpene synthases.

Sesquiterpene synthases (STSs) catalyze the formation of a large class of plant volatiles called sesquiterpenes. While thousands of putative STS sequences from diverse plant species are available, only a small number of them have been functionally characterized. Sequence identity-based screening for desired enzymes, often used in biotechnological applications, is difficult to apply here as STS sequence similarity is strongly affected by species. This calls for more sophisticated computational methods for functionality prediction. We investigate the specificity of precursor cation formation in these elusive enzymes. By inspecting multi-product STSs, we demonstrate that STSs have a strong selectivity towards one precursor cation. We use a machine learning approach combining sequence and structure information to accurately predict precursor cation specificity for STSs across all plant species. We combine this with a co-evolutionary analysis on the wealth of uncharacterized putative STS sequences, to pinpoint residues and distant functional contacts influencing cation formation and reaction pathway selection. These structural factors can be used to predict and engineer enzymes with specific functions, as we demonstrate by predicting and characterizing two novel STSs from Citrus bergamia.

Inactivation of the germacrene A synthase genes by CRISPR/Cas9 eliminates the biosynthesis of sesquiterpene lactones in Cichorium intybus L.

Chicory (Cichorium intybus var. sativum) is an industrial crop species cultivated for the production of a fructose polymer inulin, which is used as a low-calorie sweetener and prebiotic. Besides, inulin chicory taproots also accumulate sesquiterpene lactones (STLs). These are bitter tasting compounds, which need to be removed during inulin extraction, resulting in additional costs. In this work, we describe chicory lines where STL accumulation is almost completely eliminated. Genome editing using the CRISPR/Cas9 system was used to inactivate four genes that encode the enzyme that performs the first dedicated step in STL synthesis, germacrene A synthase (CiGAS). Chicory lines were obtained that carried null mutations in all four CiGAS genes. Lines lacking functional CiGAS alleles showed a normal phenotype upon greenhouse cultivation and show nearly complete elimination of the STL synthesis in the roots. It was shown that the reduction in STLs could be attributed to mutations in genetically linked copies of the CiGAS-short gene and not the CiGAS-long gene, which is relevant for breeding the trait into other cultivars. The inactivation of the STL biosynthesis pathway led to increase in phenolic compounds as well as accumulation of squalene in the chicory taproot, presumably due to increased availability of farnesyl pyrophosphate (FFP). These results demonstrate that STLs are not essential for chicory growth and that the inhibition of the STL biosynthesis pathway reduced the STL levels chicory which will facilitate inulin extraction.

Several geranylgeranyl diphosphate synthase isoforms supply metabolic substrates for carotenoid biosynthesis in tomato.

Geranylgeranyl diphosphate (GGPP) produced by GGPP synthase (GGPPS) serves as a precursor for many plastidial isoprenoids, including carotenoids. Phytoene synthase (PSY) converts GGPP into phytoene, the first committed intermediate of the carotenoid pathway. Here we used biochemical, molecular, and genetic tools to characterise the plastidial members of the GGPPS family in tomato (Solanum lycopersicum) and their interaction with PSY isoforms. The three tomato GGPPS isoforms found to localise in plastids (SlG1, 2 and 3) exhibit similar kinetic parameters. Gene expression analyses showed a preferential association of individual GGPPS and PSY isoforms when carotenoid biosynthesis was induced during root mycorrhization, seedling de-etiolation and fruit ripening. SlG2, but not SlG3, physically interacts with PSY proteins. By contrast, CRISPR-Cas9 mutants defective in SlG3 showed a stronger impact on carotenoid levels and derived metabolic, physiological and developmental phenotypes compared with those impaired in SlG2. Double mutants defective in both genes could not be rescued. Our work demonstrates that the bulk of GGPP production in tomato chloroplasts and chromoplasts relies on two cooperating GGPPS paralogues, unlike other plant species such as Arabidopsis thaliana, rice or pepper, which produce their essential plastidial isoprenoids using a single GGPPS isoform.

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.

Metabolic flux ratio analysis by parallel 13C labeling of isoprenoid biosynthesis in Rhodobacter sphaeroides.

Metabolic engineering for increased isoprenoid production often benefits from the simultaneous expression of the two naturally available isoprenoid metabolic routes, namely the 2-methyl-D-erythritol 4-phosphate (MEP) pathway and the mevalonate (MVA) pathway. Quantification of the contribution of these pathways to the overall isoprenoid production can help to obtain a better understanding of the metabolism within a microbial cell factory. Such type of investigation can benefit from 13C metabolic flux ratio studies. Here, we designed a method based on parallel labeling experiments (PLEs), using [1-13C]- and [4-13C]glucose as tracers to quantify the metabolic flux ratios in the glycolytic and isoprenoid pathways. By just analyzing a reporter isoprenoid molecule and employing only four equations, we could describe the metabolism involved from substrate catabolism to product formation. These equations infer 13C atom incorporation into the universal isoprenoid building blocks, isopentenyl-pyrophosphate (IPP) and dimethylallyl-pyrophosphate (DMAPP). Therefore, this renders the method applicable to the study of any of isoprenoid of interest. As proof of principle, we applied it to study amorpha-4,11-diene biosynthesis in the bacterium Rhodobacter sphaeroides. We confirmed that in this species the Entner-Doudoroff pathway is the major pathway for glucose catabolism, while the Embden-Meyerhof-Parnas pathway contributes to a lesser extent. Additionally, we demonstrated that co-expression of the MEP and MVA pathways caused a mutual enhancement of their metabolic flux capacity. Surprisingly, we also observed that the isoprenoid flux ratio remains constant under exponential growth conditions, independently from the expression level of the MVA pathway. Apart from proposing and applying a tool for studying isoprenoid biosynthesis within a microbial cell factory, our work reveals important insights from the co-expression of MEP and MVA pathways, including the existence of a yet unclear interaction between them.

A MYB Triad Controls Primary and Phenylpropanoid Metabolites for Pollen Coat Patterning.

The pollen wall is a complex, durable structure essential for plant reproduction. A substantial portion of phenylpropanoids (e.g. flavonols) produced by pollen grain tapetal cells are deposited in the pollen wall. Transcriptional regulation of pollen wall formation has been studied extensively, and a specific regulatory mechanism for Arabidopsis (Arabidopsis thaliana) pollen flavonol biosynthesis has been postulated. Here, metabolome and transcriptome analyses of anthers from mutant and overexpression genotypes revealed that Arabidopsis MYB99, a putative ortholog of the petunia (Petunia hybrida) floral scent regulator ODORANT1 (ODO1), controls the exclusive production of tapetum diglycosylated flavonols and hydroxycinnamic acid amides. We discovered that MYB99 acts in a regulatory triad with MYB21 and MYB24, orthologs of emission of benzenoids I and II, which together with ODO1 coregulate petunia scent biosynthesis genes. Furthermore, promoter-activation assays showed that MYB99 directs precursor supply from the Calvin cycle and oxidative pentose-phosphate pathway in primary metabolism to phenylpropanoid biosynthesis by controlling TRANSKETOLASE2 expression. We provide a model depicting the relationship between the Arabidopsis MYB triad and structural genes from primary and phenylpropanoid metabolism and compare this mechanism with petunia scent control. The discovery of orthologous protein triads producing related secondary metabolites suggests that analogous regulatory modules exist in other plants and act to regulate various branches of the intricate phenylpropanoid pathway.

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.

The santalene synthase from Cinnamomum camphora: Reconstruction of a sesquiterpene synthase from a monoterpene synthase.

Plant terpene synthases (TPSs) can mediate formation of a large variety of terpenes, and their diversification contributes to the specific chemical profiles of different plant species and chemotypes. Plant genomes often encode a number of related terpene synthases, which can produce very different terpenes. The relationship between TPS sequence and resulting terpene product is not completely understood. In this work we describe two TPSs from the Camphor tree Cinnamomum camphora (L.) Presl. One of these, CiCaMS, acts as a monoterpene synthase (monoTPS), and mediates the production of myrcene, while the other, CiCaSSy, acts as a sesquiterpene synthase (sesquiTPS), and catalyses the production of α-santalene, ß-santalene and trans-α-bergamotene. Interestingly, these enzymes share 97% DNA sequence identity and differ only in 22 amino acid residues out of 553. To understand which residues are essential for the catalysis of monoterpenes resp. sesquiterpenes, a number of hybrid synthases were prepared, and supplemented by a set of single-residue variants. These were tested for their ability to produce monoterpenes and sesquiterpenes by in vivo production of sesquiterpenes in E. coli, and by in vitro enzyme assays. This analysis pinpointed three residues in the sequence which could mediate the change in product specificity from a monoterpene synthase to a sesquiterpene synthase. Another set of three residues defined the sesquiterpene product profile, including the ratios between sesquiterpene products.

Nicotiana benthamiana α-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease.

α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall-associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activity-based probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-d-galactopyranoside substrate (Km = 0.17 mm) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management.

Transcription Factor-Mediated Control of Anthocyanin Biosynthesis in Vegetative Tissues.

Plants accumulate secondary metabolites to adapt to environmental conditions. These compounds, here exemplified by the purple-colored anthocyanins, are accumulated upon high temperatures, UV-light, drought, and nutrient deficiencies, and may contribute to tolerance to these stresses. Producing compounds is often part of a more broad response of the plant to changes in the environment. Here we investigate how a transcription-factor-mediated program for controlling anthocyanin biosynthesis also has effects on formation of specialized cell structures and changes in the plant root architecture. A systems biology approach was developed in tomato (Solanum lycopersicum) for coordinated induction of biosynthesis of anthocyanins, in a tissue- and development-independent manner. A transcription factor couple from Antirrhinum that is known to control anthocyanin biosynthesis was introduced in tomato under control of a dexamethasone-inducible promoter. By application of dexamethasone, anthocyanin formation was induced within 24 h in vegetative tissues and in undifferentiated cells. Profiles of metabolites and gene expression were analyzed in several tomato tissues. Changes in concentration of anthocyanins and other phenolic compounds were observed in all tested tissues, accompanied by induction of the biosynthetic pathways leading from Glc to anthocyanins. A number of pathways that are not known to be involved in anthocyanin biosynthesis were observed to be regulated. Anthocyanin-producing plants displayed profound physiological and architectural changes, depending on the tissue, including root branching, root epithelial cell morphology, seed germination, and leaf conductance. The inducible anthocyanin-production system reveals a range of phenomena that accompanies anthocyanin biosynthesis in tomato, including adaptions of the plants architecture and physiology.

Engineering storage capacity for volatile sesquiterpenes in Nicotiana benthamiana leaves.

Plants store volatile compounds in specialized organs. The properties of these storage organs prevent precarious evaporation and protect neighbouring tissues from cytotoxicity. Metabolic engineering of plants is often carried out in tissues such as leaf mesophyll cells, which are abundant and easily accessible by engineering tools. However, these tissues are not suitable for the storage of volatile and hydrophobic compound such as sesquiterpenes and engineered volatiles are often lost into the headspace. In this study, we show that the seeds of Arabidopsis thaliana, which naturally contain lipid bodies, accumulate sesquiterpenes upon engineered expression. Subsequently, storage of volatile sesquiterpenes was achieved in Nicotiana benthamiana leaf tissue, by introducing oleosin-coated lipid bodies through metabolic engineering. Hereto, different combinations of genes encoding diacylglycerol acyltransferases (DGATs), transcription factors (WRINKL1) and oleosins (OLE1), from the oil seed-producing species castor bean (Ricinus communis) and Arabidopsis, were assessed for their suitability to promote lipid body formation. Co-expression of α-bisabolol synthase with Arabidopsis DGAT1 and WRINKL1 and OLE1 from castor bean promoted storage of α-bisabolol in N. benthamiana mesophyll tissue more than 17-fold. A clear correlation was found between neutral lipids and storage of sesquiterpenes, using synthases for α-bisabolol, (E)-ß-caryophyllene and α-barbatene. The co-localization of neutral lipids and α-bisabolol was shown using microscopy. This work demonstrates that lipid bodies can be used as intracellular storage compartment for hydrophobic sesquiterpenes, also in the vegetative parts of plants, creating the possibility to improve yields of metabolic engineering strategies in plants.

Engineering de novo anthocyanin production in Saccharomyces cerevisiae.

BACKGROUND: Anthocyanins are polyphenolic pigments which provide pink to blue colours in fruits and flowers. There is an increasing demand for anthocyanins, as food colorants and as health-promoting substances. Plant production of anthocyanins is often seasonal and cannot always meet demand due to low productivity and the complexity of the plant extracts. Therefore, a system of on-demand supply is useful. While a number of other (simpler) plant polyphenols have been successfully produced in the yeast Saccharomyces cerevisiae, production of anthocyanins has not yet been reported. RESULTS: Saccharomyces cerevisiae was engineered to produce pelargonidin 3-O-glucoside starting from glucose. Specific anthocyanin biosynthetic genes from Arabidopsis thaliana and Gerbera hybrida were introduced in a S. cerevisiae strain producing naringenin, the flavonoid precursor of anthocyanins. Upon culturing, pelargonidin and its 3-O-glucoside were detected inside the yeast cells, albeit at low concentrations. A number of related intermediates and side-products were much more abundant and were secreted into the culture medium. To optimize titers of pelargonidin 3-O-glucoside further, biosynthetic genes were stably integrated into the yeast genome, and formation of a major side-product, phloretic acid, was prevented by engineering the yeast chassis. Further engineering, by removing two glucosidases which are known to degrade pelargonidin 3-O-glucoside, did not result in higher yields of glycosylated pelargonidin. In aerated, pH controlled batch reactors, intracellular pelargonidin accumulation reached 0.01 µmol/gCDW, while kaempferol and dihydrokaempferol were effectively exported to reach extracellular concentration of 20 µM [5 mg/L] and 150 µM [44 mg/L], respectively. CONCLUSION: The results reported in this study demonstrate the proof-of-concept that S. cerevisiae is capable of de novo production of the anthocyanin pelargonidin 3-O-glucoside. Furthermore, while current conversion efficiencies are low, a number of clear bottlenecks have already been identified which, when overcome, have huge potential to enhance anthocyanin production efficiency. These results bode very well for the development of fermentation-based production systems for specific and individual anthocyanin molecules. Such systems have both great scientific value for identifying and characterising anthocyanin decorating enzymes as well as significant commercial potential for the production of, on-demand, pure bioactive compounds to be used in the food, health and even pharma industries.

Identification of major loci and genomic regions controlling acid and volatile content in tomato fruit: implications for flavor improvement.

Plant metabolites are important to world food security due to their roles in crop yield and nutritional quality. Here we report the metabolic profile of 300 tomato accessions (Solanum lycopersicum and related wild species) by quantifying 60 primary and secondary metabolites, including volatile organic compounds, over a period of 2 yr. Metabolite content and genetic inheritance of metabolites varied broadly, both within and between different genetic groups. Using genotype information gained from 10 000 single nucleotide polymorphism markers, we performed a metabolite genome-wide association mapping (GWAS) study. We identified 79 associations influencing 13 primary and 19 secondary metabolites with large effects at high resolution. Four genome regions were detected, highlighting clusters of associations controlling the variation of several metabolites. Local linkage disequilibrium analysis and allele mining identified possible candidate genes which may modulate the content of metabolites that are of significant importance for human diet and fruit consumption. We precisely characterized two associations involved in fruit acidity and phenylpropanoid volatile production. Taken together, this study reveals complex and distinct metabolite regulation in tomato subspecies and demonstrates that GWAS is a powerful tool for gene-metabolite annotation and identification, pathways elucidation, and further crop improvement.

A Single Arabidopsis Gene Encodes Two Differentially Targeted Geranylgeranyl Diphosphate Synthase Isoforms.

A wide diversity of isoprenoids is produced in different plant compartments. Most groups of isoprenoids synthesized in plastids, and some produced elsewhere in the plant cell derive from geranylgeranyl diphosphate (GGPP) synthesized by GGPP synthase (GGPPS) enzymes. In Arabidopsis (Arabidopsis thaliana), five genes appear to encode GGPPS isoforms localized in plastids (two), the endoplasmic reticulum (two), and mitochondria (one). However, the loss of function of the plastid-targeted GGPPS11 isoform (referred to as G11) is sufficient to cause lethality. Here, we show that the absence of a strong transcription initiation site in the G11 gene results in the production of transcripts of different lengths. The longer transcripts encode an isoform with a functional plastid import sequence that produces GGPP for the major groups of photosynthesis-related plastidial isoprenoids. However, shorter transcripts are also produced that lack the first translation initiation codon and rely on a second in-frame ATG codon to produce an enzymatically active isoform lacking this N-terminal domain. This short enzyme localizes in the cytosol and is essential for embryo development. Our results confirm that the production of differentially targeted enzyme isoforms from the same gene is a central mechanism to control the biosynthesis of isoprenoid precursors in different plant cell compartments.

Processing black mulberry into jam: effects on antioxidant potential and in vitro bioaccessibility.

BACKGROUND: Black mulberries (Morus nigra) were processed into jam on an industrialised scale, including the major steps of: selection of frozen black mulberries, adding glucose-fructose syrup and water, cooking, adding citric acid and apple pectin, removing seeds, and pasteurisation. Qualitative and quantitative determinations of antioxidants in black mulberry samples were performed using spectrophotometric methods, as well as HPLC- and LC-QTOF-MS-based measurements. These analyses included the determination of total polyphenolic content, % polymeric colour, total and individual anthocyanin contents, antioxidant capacity, and in vitro bioaccessibility in processing samples. RESULTS: Jam processing led to a significant reduction in total phenolics (88%), total flavonoids (89%), anthocyanins (97%), and antioxidant capacity (88-93%) (P < 0.05). Individual anthocyanin contents, determined using HPLC analysis, also showed a significant decrease (∼99% loss). In contrast, % recovery of bioaccessible total phenolics, anthocyanins, and antioxidant capacity (ABTS assay) increased after jam processing (16%, 12%, and 37%, respectively). CONCLUSION: Fruit processing resulted in losses of polyphenols, anthocyanins, and antioxidant capacity of black mulberry jam. Optimisation of food processing could help to protect the phenolic compounds in fruits which might be helpful for the food industry to minimise the antioxidant loss and improve the final product quality. © 2016 Society of Chemical Industry.

Non-smoky glycosyltransferase1 prevents the release of smoky aroma from tomato fruit.

Phenylpropanoid volatiles are responsible for the key tomato fruit (Solanum lycopersicum) aroma attribute termed "smoky." Release of these volatiles from their glycosylated precursors, rather than their biosynthesis, is the major determinant of smoky aroma in cultivated tomato. using a combinatorial omics approach, we identified the non-smoky glycosyltransferase1 (NSGT1) gene. Expression of NSGT1 is induced during fruit ripening, and the encoded enzyme converts the cleavable diglycosides of the smoky-related phenylpropanoid volatiles into noncleavable triglycosides, thereby preventing their deglycosylation and release from tomato fruit upon tissue disruption. In an nsgt1/nsgt1 background, further glycosylation of phenylpropanoid volatile diglycosides does not occur, thereby enabling their cleavage and the release of corresponding volatiles. Using reverse genetics approaches, the NSGT1-mediated glycosylation was shown to be the molecular mechanism underlying the major quantitative trait locus for smoky aroma. Sensory trials with transgenic fruits, in which the inactive nsgt1 was complemented with the functional NSGT1, showed a significant and perceivable reduction in smoky aroma. NSGT1 may be used in a precision breeding strategy toward development of tomato fruits with distinct flavor phenotypes.

A Review on the Effect of Drying on Antioxidant Potential of Fruits and Vegetables.

The role of antioxidants in human nutrition has gained increased interest, especially due to their associated health beneficial effects for a number of chronic diseases, including cardiovascular diseases and certain types of cancer. Fruits and vegetables are perishable and difficult to preserve as fresh products. Dried fruits and vegetables can be easily stored, transported at relatively low cost, have reduced packing costs, and their low water content delays microbial spoilage. Air-, freeze-, microwave- and sun-drying are among the most thoroughly studied drying methods. This review provides an overview of recent findings on the effects of different drying techniques on major antioxidants of fruits and vegetables. In particular, changes in ascorbic acid, carotenoids, flavonoids, phenolic acids, total phenolics, and antioxidant activity are discussed in detail.

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.

Fruit Antioxidants during Vinegar Processing: Changes in Content and in Vitro Bio-Accessibility.

BACKGROUND: Vinegars based on fruit juices could conserve part of the health-associated compounds present in the fruits. However, in general very limited knowledge exists on the consequences of vinegar-making on different antioxidant compounds from fruit. In this study vinegars derived from apple and grape are studied. METHODS: A number of steps, starting from the fermentation of the fruit juices to the formation of the final vinegars, were studied from an industrial vinegar process. The effect of each of the vinegar processing steps on content of antioxidants, phenolic compounds and flavonoids was studied, by spectroscopic methods and by high-performance liquid chromatography (HPLC). RESULTS: The major observation was that spectrophotometric methods indicate a strong loss of antioxidant phenolic compounds during the transition from fruit wine to fruit vinegar. A targeted HPLC analysis indicates that metabolites such as gallic acid are lost in later stages of the vinegar process. CONCLUSION: The major conclusion of this work is that major changes occur in phenolic compounds during vinegar making. An untargeted metabolite analysis should be used to reveal these changes in more detail. In addition, the effect of vinegar processing on bio-accessibility of phenolic compounds was investigated by mimicking the digestive tract in an in vitro set up. This study is meant to provide insight into the potential of vinegar as a source of health-related compounds from fruit.