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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7337-7) contains supplementary material, which is available to authorized users.

SummarySubcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria.A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana benthamiana indicated local GDP availability for each compartment but resulted in different product levels. A GDP synthase from Picea abies (PaGDPS1) was shown to boost GDP production. PaGDPS1 was also targeted to plastids, cytosol or mitochondria and PaGDPS1 and GES were coexpressed in all possible combinations.Geraniol and geraniol-derived products were analyzed by GC-MS and LC-MS, respectively. GES product levels were highest for plastid-targeted GES, followed by mitochondrial-and then cytosolic-targeted GES. For each compartment local boosting of GDP biosynthesis increased GES product levels.GDP exchange between compartments is not equal: while no GDP is exchanged from the cytosol to the plastids, 100% of GDP in mitochondria can be exchanged to plastids, while only 7% of GDP from plastids is available for mitochondria. This suggests a direct exchange mechanism for GDP between plastids and mitochondria. Cytosolic PaGDPS1 competes with plastidial GES activity, suggesting an effective drain of isopentenyl diphosphate from the plastids to the cytosol.

Monoterpene olefins such as limonene are plant compounds with applications as flavouring and fragrance agents, as solvents and potentially also in polymer and fuel chemistry. We engineered baker's yeast Saccharomyces cerevisiae to express a (À)-limonene synthase from Perilla frutescens and a (+)-limonene synthase from Citrus limon. Both proteins were expressed either with their native plastid targeting signal or in a truncated form in which the plastidial sorting signal was removed. The yeast host strain for expression was AE9 K197G, which expresses a mutant Erg20 enzyme. This enzyme catalyses the formation of geranyl diphosphate, which is the precursor for monoterpenes. Several methods were tested to capture limonene produced by the yeast. Extraction from the culture medium by pentane, or by the addition of CaCl 2 followed by solid-phase micro-extraction, did not lead to detectable limonene, indicating that limonene is rapidly lost from the culture medium. Volatile terpenes such as limonene may also be trapped in a dodecane phase added to the medium during fermentation. This method resulted in recovery of 0.028 mg/l (+)-limonene and 0.060 mg/l (À)-limonene in strains using the truncated Citrus and Perilla synthases, respectively. Trapping the headspace during culture of the limonene synthaseexpressing strains resulted in higher titres, at 0.12 mg/l (+)-limonene and 0.49 mg/l (À)-limonene. These results show that the volatile properties of the olefins produced require specific methods for efficient recovery of these molecules from biotechnological production systems. Gene Bank Nos were: KM015220 (Perilla limonene synthase; this study); AF317695 (Perilla limonene synthase; Yuba et al., 1996); AF514287.1 (Citrus limonene synthase; Lucker et al., 2002).

BackgroundCollimonas 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.ResultsIn 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.ConclusionsOverall 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2289-3) contains supplementary material, which is available to authorized users.

For tuber-bearing Solanum species, six monogenic recessive synaptic mutants, designated sy-1, sy-2, sy-3, sy-4, ds, and dsc have been reported in the literature. In the present investigation no indication for the existence of the mutant sy-1, affecting megasporogenesis only, was found. The mutant ds was confirmed to display typical desynaptic behaviour in microsporogenesis and shown to similarly affect megasporogenesis. It furthermore proved to be allelic to the mutants sy-3 and dsc. It is proposed that the mutants sy-3, ds, and dsc be uniformly designated ds-1, whereas the remaining mutants sy-2 and sy-4 (possibly identical) may be designated simply as synaptic mutant until their actual identity has been established. The observed F1 segregations generally support monogenic recessive inheritance of ds-1. However, in one cross progeny the expected mutant phenotype was not clearly expressed in contrast with its reciprocal, which might indicate cross-specific influence of the cytoplasm on ds-1 expression. The potential value and limitations of desynaptic (ds-1ds-1) mutants for potato breeding and true potato seed production are discussed.Key words: Solanum, (de)synaptic mutants, microsporogenesis, megasporogenesis, 2n gametes.