Systematic metabolic engineering for improved synthesis of perillic acid in Candida tropicalis.

Perillic acid has been studied as an anticancer and antimicrobial drug. Production of perillic acid has attracted considerable attention. Meanwhile, Candida tropicalis is an unconventional diploid yeast, most significantly characterized by its ability to metabolize alkanes or fatty acids for growth and proliferation. Therefore, perillic acid's precursor (L-limonene) in C. tropicalis was firstly synthesized by expressing a Mentha spicata L-limonene synthase gene, LS_Ms in this work. Expression of a gene which encoded for a truncated version of tLS_Ms increased the production of L-limonene with a 2.78-fold increase in the titer over C. tropicalis GJR-LS-01. Compartmentalized expression of the gene tLS_Ms inhibited the production of L-limonene in C. tropicalis compared to cytoplasmic expression. Cytoplasmic overexpression of seven precursor synthesis genes significantly enhanced the production of L-limonene in C. tropicalis compared to their compartmentalized expression (mitochondria or peroxisomes), which increased by 31.7-fold in C. tropicalis GJR-tLS-01. The L-limonene titer in C. tropicalis GJR-EW-tLS-04 overexpressing the mutant gene ERG20WW in the cytoplasm was significantly increased, 11.33-fold higher than the control. The titer of L-limonene for 60 g/L glucose was increased by 1.40-fold compared to the control. Finally, a Salvia miltiorrhiza cytochrome P450 enzyme gene CYP7176 and an Arabidopsis thaliana NADPH cytochrome P450 reductase gene CPR were heterologously expressed in C. tropicalis GJR-EW-tLS-04C for the synthesis of perillic acid, which reached a titer of 106.69 mg/L in a 5-L fermenter. This is the first report of de novo synthesis of perillic acid in engineered microorganisms. The results also showed that other chemicals may be efficiently produced in C. tropicalis. KEY POINTS: • Key genes cytoplasmic expression was conducive to L-limonene production in C. tropicalis. • Perillic acid was first synthesized de novo in engineered microorganisms. • The titer of perillic acid reached 106.69 mg/L in a 5-L fermenter.

Weed interference with peppermint (Mentha x piperita L.) and spearmint (Mentha spicata L.) crops under different herbicide treatments: effects on biomass and essential oil yield.

BACKGROUND: 'Minor crops' such as spearmint and peppermint are high added value crops, despite the fact that their production area is comparably small worldwide. The main limiting factor in mint commercial cultivation is weed competition. Thus, field experiments were carried out to evaluate the effects of weed interference on growth, biomass and essential oil yield in peppermint and spearmint under different herbicide treatments. RESULTS: The application of pendimethalin and oxyfluorfen provided better control of annual weeds resulting in higher crop yield. Additionally, when treated with herbicides both crops were more competitive against annual weeds in the second year than in the first year. All pre-emergence herbicides increased biomass yield, since pendimethalin, linuron and oxyfluorfen reduced the density of annual weeds by 71-92%, 63-74% and 86-95%, respectively. Weed interference and herbicide application had no effect on essential oil content; however, a relatively strong impact on essential oil production per cultivated area unit was observed, mainly due to the adverse effect of weed interference on plant growth. CONCLUSION: Considering that pendimethalin and oxyfluorfen were effective against annual weeds in both spearmint and peppermint crops, these herbicides should be included in integrated weed management systems for better weed management in mint crops. © 2017 Society of Chemical Industry.

Spearmint R2R3-MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit (MsGPPS.LSU).

Many aromatic plants, such as spearmint, produce valuable essential oils in specialized structures called peltate glandular trichomes (PGTs). Understanding the regulatory mechanisms behind the production of these important secondary metabolites will help design new approaches to engineer them. Here, we identified a PGT-specific R2R3-MYB gene, MsMYB, from comparative RNA-Seq data of spearmint and functionally characterized it. Analysis of MsMYB-RNAi transgenic lines showed increased levels of monoterpenes, and MsMYB-overexpressing lines exhibited decreased levels of monoterpenes. These results suggest that MsMYB is a novel negative regulator of monoterpene biosynthesis. Ectopic expression of MsMYB, in sweet basil and tobacco, perturbed sesquiterpene- and diterpene-derived metabolite production. In addition, we found that MsMYB binds to cis-elements of MsGPPS.LSU and suppresses its expression. Phylogenetic analysis placed MsMYB in subgroup 7 of R2R3-MYBs whose members govern phenylpropanoid pathway and are regulated by miR858. Analysis of transgenic lines showed that MsMYB is more specific to terpene biosynthesis as it did not affect metabolites derived from phenylpropanoid pathway. Further, our results indicate that MsMYB is probably not regulated by miR858, like other members of subgroup 7.

Converting S-limonene synthase to pinene or phellandrene synthases reveals the plasticity of the active site.

S-limonene synthase is a model monoterpene synthase that cyclizes geranyl pyrophosphate (GPP) to form S-limonene. It is a relatively specific enzyme as the majority of its products are composed of limonene. In this study, we converted it to pinene or phellandrene synthases after introducing N345A/L423A/S454A or N345I mutations. Further studies on N345 suggest the polarity of this residue plays a critical role in limonene production by stabilizing the terpinyl cation intermediate. If it is mutated to a non-polar residue, further cyclization or hydride shifts occurs so the carbocation migrates towards the pyrophosphate, leading to the production of pinene or phellandrene. On the other hand, mutant enzymes that still possess a polar residue at this position produce limonene as the major product. N345 is not the only polar residue that may stabilize the terpinyl cation because it is not strictly conserved among limonene synthases across species and there are also several other polar residues in this area. These residues could form a "polar pocket" that may collectively play this stabilizing role. Our study provides important insights into the catalytic mechanism of limonene synthases. Furthermore, it also has wider implications on the evolution of terpene synthases.

Generation and Functional Evaluation of Designer Monoterpene Synthases.

Monoterpene synthases are highly versatile enzymes that catalyze the first committed step in the pathways toward terpenoids, the structurally most diverse class of plant natural products. Recent advancements in our understanding of the reaction mechanism have enabled engineering approaches to develop mutant monoterpene synthases that produce specific monoterpenes. In this chapter, we are describing protocols to introduce targeted mutations, express mutant enzyme catalysts in heterologous hosts, and assess their catalytic properties. Mutant monoterpene synthases have the potential to contribute significantly to synthetic biology efforts aimed at producing larger amounts of commercially attractive monoterpenes.

Metabolic engineering of terpene biosynthesis in plants using a trichome-specific transcription factor MsYABBY5 from spearmint (Mentha spicata).

In many aromatic plants including spearmint (Mentha spicata), the sites of secondary metabolite production are tiny specialized structures called peltate glandular trichomes (PGT). Having high commercial values, these secondary metabolites are exploited largely as flavours, fragrances and pharmaceuticals. But, knowledge about transcription factors (TFs) that regulate secondary metabolism in PGT remains elusive. Understanding the role of TFs in secondary metabolism pathway will aid in metabolic engineering for increased yield of secondary metabolites and also the development of new production techniques for valuable metabolites. Here, we isolated and functionally characterized a novel MsYABBY5 gene that is preferentially expressed in PGT of spearmint. We generated transgenic plants in which MsYABBY5 was either overexpressed or silenced using RNA interference (RNAi). Analysis of the transgenic lines showed that the reduced expression of MsYABBY5 led to increased levels of terpenes and that overexpression decreased terpene levels. Additionally, ectopic expression of MsYABBY5 in Ocimum basilicum and Nicotiana sylvestris decreased secondary metabolite production in them, suggesting that the encoded transcription factor is probably a repressor of secondary metabolism.

Next generation sequencing unravels the biosynthetic ability of spearmint (Mentha spicata) peltate glandular trichomes through comparative transcriptomics.

BACKGROUND: Plant glandular trichomes are chemical factories with specialized metabolic capabilities to produce diverse compounds. Aromatic mint plants produce valuable essential oil in specialised glandular trichomes known as peltate glandular trichomes (PGT). Here, we performed next generation transcriptome sequencing of different tissues of Mentha spicata (spearmint) to identify differentially expressed transcripts specific to PGT. Our results provide a comprehensive overview of PGT's dynamic metabolic activities which will help towards pathway engineering. RESULTS: Spearmint RNAs from 3 different tissues: PGT, leaf and leaf stripped of PGTs (leaf-PGT) were sequenced by Illumina paired end sequencing. The sequences were assembled de novo into 40,587 non-redundant unigenes; spanning a total of 101 Mb. Functions could be assigned to 27,025 (67%) unigenes and among these 3,919 unigenes were differentially expressed in PGT relative to leaf - PGT. Lack of photosynthetic transcripts in PGT transcriptome indicated the high levels of purity of isolated PGT, as mint PGT are non-photosynthetic. A significant number of these unigenes remained unannotated or encoded hypothetical proteins. We found 16 terpene synthases (TPS), 18 cytochrome P450s, 5 lipid transfer proteins and several transcription factors that were preferentially expressed in PGT. Among the 16 TPSs, two were characterized biochemically and found to be sesquiterpene synthases. CONCLUSIONS: The extensive transcriptome data set renders a complete description of genes differentially expressed in spearmint PGT. This will facilitate the metabolic engineering of mint terpene pathway to increase yield and also enable the development of strategies for sustainable production of novel or altered valuable compounds in mint.

[Molecular identification of Mentha haplocalyx and Mentha spicata with specific primers multi-PCR system].

OBJECTIVE: To screen specific SNPs loci of Mentha haplocalyx and Mentha spicata,and then specific primers were designed to identify the two species and their mixture rapidly. METHODS: PsbA-trnH sequences of Mentha haplocalyx and Mentha spicata were obtained by PCR product sequencing and downloading from GenBank. SNPs in the psbA-trnH sequences of Mentha haplocalyx and Mentha spicata were found by ClustulW program and Bioedit software. Primers for authentication of the two species were designed according to the SNP loci, and PCR reaction system was optimized to identify the original plants. RESULTS: Multi-PCR reaction system was constructed. The 181 bp identification band for Mentha haplocalyx or(and) 288 bp identification band for Mentha spicata could be produced by a single PCR reaction,which showed good identification ability to the two species. CONCLUSION: The multi-PCR reaction system can be applied to identify Mentha haplocalyx and Mentha spicata as well as their mixture.

Yield, content, and composition of peppermint and spearmints as a function of harvesting time and drying.

Peppermint ( Mentha × piperita L.) and spearmints ('Scotch' spearmint, M. × gracilis Sole, and 'Native' spearmint, Mentha spicata L.) are widely grown essential oil crops in more northern latitudes; however, there is limited information on how harvest time and drying influence peppermint and spearmint yield, oil composition, and bioactivity, when grown south of the 41st parallel. In this 2-year study, the effects of harvest time and drying on the yield, oil composition, and bioactivity of peppermint ('Black Mitcham' and 'B90-9'), 'Scotch' spearmint, and 'Native' spearmint were evaluated. Peppermint oil from the dried material had higher menthol and eucalyptol concentrations. Menthone in both peppermint cultivars decreased from harvest 1 (late June) to harvest 5 (late August) or 6 (early September), whereas menthol increased. (-)-Carvone in spearmints accumulated early, before flowering, allowing for early harvest. Oil yields from the dried spearmint biomass reached the maximum at harvest 3 (mid-July). The essential oil compositions of the four mint genotypes were similar to that of 11 commercially available oils, suggesting that these genotypes can be grown in the hot, humid environment of the southeastern United States. The antioxidant activities (ORAC(oil) values) of the essential oils were 4372, 1713, 1107, and 471 µmol of TE L(-1) for 'Scotch' spearmint, 'Native' spearmint, peppermint, and Japanese cornmint ( Mentha canadensis ), respectively. The oils of the four mint genotypes did not affect ruminal fermentation in vivo, and did not exhibit antimicrobial, antileishmanial, or antimalarial activity at levels that would warrant bioassay-directed fractionation in a drug-discovery screening program. Specifically, the oils did not show greater than 50% growth inhibition against Leishmania donovani , Plasmodium falciparum clones D6 and W2, Candida albicans , Escherichia coli , Pseudomonas aeruginosa , Cryptococcus neoformans , Mycobacterium intracellulare , or Aspergillus fumigates at 50 µg mL(-1).

Protection against LPS-induced cartilage inflammation and degradation provided by a biological extract of Mentha spicata.

BACKGROUND: A variety of mint [Mentha spicata] has been bred which over-expresses Rosmarinic acid (RA) by approximately 20-fold. RA has demonstrated significant anti-inflammatory activity in vitro and in small rodents; thus it was hypothesized that this plant would demonstrate significant anti-inflammatory activity in vitro. The objectives of this study were: a) to develop an in vitro extraction procedure which mimics digestion and hepatic metabolism, b) to compare anti-inflammatory properties of High-Rosmarinic-Acid Mentha spicata (HRAM) with wild-type control M. spicata (CM), and c) to quantify the relative contributions of RA and three of its hepatic metabolites [ferulic acid (FA), caffeic acid (CA), coumaric acid (CO)] to anti-inflammatory activity of HRAM. METHODS: HRAM and CM were incubated in simulated gastric and intestinal fluid, liver microsomes (from male rat) and NADPH. Concentrations of RA, CA, CO, and FA in simulated digest of HRAM (HRAMsim) and CM (CMsim) were determined (HPLC) and compared with concentrations in aqueous extracts of HRAM and CM. Cartilage explants (porcine) were cultured with LPS (0 or 3 microg/mL) and test article [HRAMsim (0, 8, 40, 80, 240, or 400 microg/mL), or CMsim (0, 1, 5 or 10 mg/mL), or RA (0.640 microg/mL), or CA (0.384 microg/mL), or CO (0.057 microg/mL) or FA (0.038 microg/mL)] for 96 h. Media samples were analyzed for prostaglandin E2 (PGE2), interleukin 1beta (IL-1), glycosaminoglycan (GAG), nitric oxide (NO) and cell viability (differential live-dead cell staining). RESULTS: RA concentration of HRAMsim and CMsim was 49.3 and 0.4 microg/mL, respectively. CA, FA and CO were identified in HRAMsim but not in aqueous extract of HRAM. HRAMsim (> or = 8 microg/mL) inhibited LPS-induced PGE2 and NO; HRAMsim (> or = 80 microg/mL) inhibited LPS-induced GAG release. RA inhibited LPS-induced GAG release. No anti-inflammatory or chondroprotective effects of RA metabolites on cartilage explants were identified. CONCLUSIONS: Our biological extraction procedure produces a substance which is similar in composition to post-hepatic products. HRAMsim is an effective inhibitor of LPS-induced inflammation in cartilage explants, and effects are primarily independent of RA. Further research is needed to identify bioactive phytochemical(s) in HRAMsim.

Metabolic engineering of monoterpene biosynthesis: two-step production of (+)-trans-isopiperitenol by tobacco.

Monoterpenoid biosynthesis in tobacco was modified by introducing two subsequent enzymatic activities targeted to different cell compartments. A limonene-3-hydroxylase (lim3h) cDNA was isolated from Mentha spicata L. 'Crispa'. This cDNA was used to re-transform a transgenic Nicotiana tabacum'Petit Havana' SR1 (tobacco) line expressing three Citrus limon L. Burm. f. (lemon) monoterpene synthases producing (+)-limonene, gamma-terpinene and (-)-beta-pinene as their main products. The targeting sequences of these synthases indicate that they are probably localized in the plastids, whereas the sequence information of the P450 hydroxylase indicates targeting to the endoplasmatic reticulum. Despite the different location of the enzymes, the introduced P450 hydroxylase proved to be functional in the transgenic plants as it hydroxylated (+)-limonene, resulting in the emission of (+)-trans-isopiperitenol. Some further modifications of the (+)-trans-isopiperitenol were also detected, resulting in the additional emission of 1,3,8-p-menthatriene, 1,5,8-p-menthatriene, p-cymene and isopiperitenone.

Molecular evaluation of a spearmint mutant altered in the expression of limonene hydroxylases that direct essential oil monoterpene biosynthesis.

Gamma irradiation of Scotch spearmint created a mutant line, 643-10-74, which has an altered essential oil reminiscent of peppermint because the monoterpene metabolites in the oil glands of the mutant are predominantly oxygenated at the C3 position of the p-menthane ring instead of the C6 position normally found in spearmint. The limonene hydroxylase genes responsible for directing the regiochemistry of oxygenation were cloned from Scotch spearmint and mutant 643 and expressed in Escherichia coli. The limonene bydroxylase from the wild-type parent hydroxylated the C6 position while the enzyme from the mutant oxygenated the C3 position. Comparison of the amino acid sequences with other limonene hydroxylases showed that the mutant enzyme was more closely related to the peppermint limonene-3-hydroxylases than to the spearmint limonene-6-hydroxylases. Because of the sequence differences between the Scotch spearmint and mutant 643 limonene hydroxylases, it is most likely that the mutation did not occur within the structural gene for limonene hydroxylase but rather at a regulatory site within the genome that controls the expression of one or the other regiospecific variants.