Neryl Diphosphate Research Articles

Efficient Biosynthesis of (+)-α-Pinene and de Novo Synthesis of (+)-cis-Verbenol in Escherichia coli.

Bark beetles, major pests that bore into forest stems, cause significant economic damage to forests globally. (+)-α-Pinene is the precursor to (+)-cis-verbenol, a crucial component of the aggregation pheromones produced by bark beetles. This paper describes the de novo synthesis of (+)-cis-verbenol in Escherichia coli. Initially, the truncation position of (+)-α-pinene synthase (PtPS30 from Pinus taeda) and monoterpene precursor (geranyl diphosphate/neryl diphosphate) synthases were evaluated. Neryl diphosphate synthase from Solanum lycopersicum (SlNPPS1) and truncated (+)-α-pinene synthase (PtPS30-39) were selected as promising candidates. Subsequently, the titer of (+)-α-pinene was significantly increased 8.9-fold by using the fusion tag CM29, which enhanced the solubility of PtPS30-39. In addition, by optimizing expression elements (ribosomal binding sites, linkers, and up elements) and overexpressing CM29*PtPS30-39, a yield of 134.12 mg/L (+)-α-pinene was achieved. Finally, the first de novo synthesis of enantiopure (+)-cis-verbenol was achieved by introducing a cytochrome P450 mutant from Pseudomonas putida (P450camF89W,Y98F,L246A), resulting in a yield of 11.13 mg/L. This study lays the groundwork for developing verbenol-based trapping technology for controlling bark beetles.

Efficient synthesis of limonene production in Yarrowia lipolytica by combinatorial engineering strategies

BackgroundLimonene has a variety of applications in the foods, cosmetics, pharmaceuticals, biomaterials, and biofuels industries. In order to meet the growing demand for sustainable production of limonene at industry scale, it is essential to find an alternative production system to traditional plant extraction. A promising and eco-friendly alternative is the use of microbes as cell factories for the synthesis of limonene.ResultsIn this study, the oleaginous yeast Yarrowia lipolytica has been engineered to produce d- and l-limonene. Four target genes, l- or d-LS (limonene synthase), HMG (HMG-CoA reductase), ERG20 (geranyl diphosphate synthase), and NDPS1 (neryl diphosphate) were expressed individually or fused together to find the optimal combination for higher limonene production. The strain expressing HMGR and the fusion protein ERG20-LS was the best limonene producer and, therefore, selected for further improvement. By increasing the expression of target genes and optimizing initial OD, 29.4 mg/L of l-limonene and 24.8 mg/L of d-limonene were obtained. We also studied whether peroxisomal compartmentalization of the synthesis pathway was beneficial for limonene production. The introduction of d-LS and ERG20 within the peroxisome improved limonene titers over cytosolic expression. Then, the entire MVA pathway was targeted to the peroxisome to improve precursor supply, which increased d-limonene production to 47.8 mg/L. Finally, through the optimization of fermentation conditions, d-limonene production titer reached 69.3 mg/L.ConclusionsIn this work, Y. lipolytica was successfully engineered to produce limonene. Our results showed that higher production of limonene was achieved when the synthesis pathway was targeted to the peroxisome, which indicates that this organelle can favor the bioproduction of terpenes in yeasts. This study opens new avenues for the efficient synthesis of valuable monoterpenes in Y. lipolytica.

Nudix hydrolase WvNUDX24 is involved in borneol biosynthesis in Wurfbainia villosa.

Borneol, camphor, and bornyl acetate are highly promising monoterpenoids widely used in medicine, flavor, food, and chemical applications. Bornyl diphosphate (BPP) serves as a common precursor for the biosynthesis of these monoterpenoids. Although bornyl diphosphate synthase (BPPS) that catalyzes the cyclization of geranyl diphosphate (GPP) to BPP has been identified in multiple plants, the enzyme responsible for the hydrolysis of BPP to produce borneol has not been reported. Here, we conducted in vitro and in vivo functional characterization to identify the Nudix hydrolase WvNUDX24 from W. villosa, which specifically catalyzes the hydrolysis of BPP to generate bornyl phosphate (BP), and then BP forms borneol under the action of phosphatase. Subcellular localization experiments indicated that the hydrolysis of BPP likely occurs in the cytoplasm. Furthermore, site-directed mutagenesis experiments revealed that four critical residues (R84, S96, P98, and G99) for the hydrolysis activity of WvNUDX24. Additionally, the functional identification of phosphatidic acid phosphatase (PAP) demonstrated that WvPAP5 and WvPAP10 were able to hydrolyze geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate (FPP) to generate geranylgeranyl phosphate (GGP) and farnesyl phosphate (FP), respectively, but could not hydrolyze BPP, GPP, and neryl diphosphate (NPP) to produce corresponding monophosphate products. These findings highlight the essential role of WvNUDX24 in the first step of BPP hydrolysis to produce borneol and provide genetic elements for the production of BPP-related terpenoids through plant metabolic engineering and synthetic biology.

Structural-Functional Correlations between Unique N-terminal Region and C-terminal Conserved Motif in Short-chain cis-Prenyltransferase from Tomato.

Neryl diphosphate (C10) synthase (NDPS1), a homodimeric soluble cis-prenyltransferase from tomato, contains four disulfide bonds, including two inter-subunit S-S bonds in the N-terminal region. Mutagenesis studies demonstrated that the S-S bond formation affects not only the stability of the dimer but also the catalytic efficiency of NDPS1. Structural polymorphs in the crystal structures of NDPS1 complexed with its substrate and substrate analog were identified by employing massive data collections and hierarchical clustering analysis. Heterogeneity of the C-terminal region, including the conserved RXG motifs, was observed in addition to the polymorphs of the binding mode of the ligands. One of the RXG motifs covers the active site with an elongated random coil when the ligands are well-ordered. Conversely, the other RXG motif was located away from the active site with a helical structure. The heterogeneous C-terminal regions suggest alternating structural transitions of the RXG motifs that result in closed and open states of the active sites. Site-directed mutagenesis studies demonstrated that the conserved glycine residue cannot be replaced. We propose that the putative structural transitions of the order/disorder of N-terminal regions and the closed/open states of C-terminal regions may cooperate and be important for the catalytic mechanism of NDPS1.

Mutational Analysis of (+)-Limonene Synthase.

The monoterpene limonene is produced by the enzyme limonene synthase in one of the simplest terpene cyclization reactions. The enzyme can use linalyl diphosphate (LPP) and neryl diphosphate (NPP) as substrates in addition to the naturally occurring substrate geranyl diphosphate (GPP), but the relationship among the three alternative substrates is not well understood. We explored the (+)-limonene synthase ((+)-LS) reaction using site-directed mutagenesis with the three different substrates (GPP, NPP, and LPP) to tease out details of the mechanism. In total, 23 amino acid positions in the active site of (+)-LS were targeted for mutation. In all cases, substitution with Ala resulted in a significant loss of enzyme activity using GPP or NPP as the substrate, but the mutations fell into two groups depending on the effect of using LPP as a substrate: group 1 mutations resulted in the loss of activity with all three substrates (GPP, NPP, and LPP); group 2 mutations resulted in loss of activity with GPP and NPP, but retained near-WT activity with LPP as a substrate. Importantly, mutations resulting in loss of activity with LPP but retention of activity with GPP and NPP were never observed. These data, in combination with the substrate order of reactivity for the WT enzyme (LPP > NPP > GPP), are consistent with a role for LPP as an intermediate in the (+)-LS reaction using either GPP or NPP as a substrate.

Metabolic Engineering for Efficient Production of Z,Z-Farnesol in E. coli.

Z,Z-farnesol (Z,Z-FOH), the all-cis isomer of farnesol, holds enormous potential for application in cosmetics, daily chemicals, and pharmaceuticals. In this study, we aimed to metabolically engineer Escherichia coli to produce Z,Z-FOH. First, we tested five Z,Z-farnesyl diphosphate (Z,Z-FPP) synthases that catalyze neryl diphosphate to form Z,Z-FPP in E. coli. Furthermore, we screened thirteen phosphatases that could facilitate the dephosphorylation of Z,Z-FPP to produce Z,Z-FOH. Finally, through site-directed mutagenesis of cis-prenyltransferase, the optimal mutant strain was able to produce 572.13 mg/L Z,Z-FOH by batch fermentation in a shake flask. This achievement represents the highest reported titer of Z,Z-FOH in microbes to date. Notably, this is the first report on the de novo biosynthesis of Z,Z-FOH in E. coli. This work represents a promising step toward developing synthetic E. coli cell factories for the de novo biosynthesis of Z,Z-FOH and other cis-configuration terpenoids.

Functional characterization of a bark-specific monoterpene synthase potentially involved in wounding- and methyl jasmonate-induced linalool emission in rubber (Hevea brasiliensis)

Rubber (Hevea brasiliensis) is a latex-producing plant that often encounters mechanical wounding, as well as pathogen and pest attacks through wound sites during and after tapping. Terpenoids play an important role in the ecological interactions of many plant species, and their diversity is mainly generated by enzymes known as terpene synthases (TPS). In this study, one cDNA sequence encoding a putative terpene synthase, HbTPS20, was obtained from the bark tissues of H. brasiliensis. The encoded protein contains 610 amino acids with a putative N-terminal plastid transit peptide of approximately 70 residues. It belongs to the TPS-b subfamily. Further phylogenetic analysis showed that HbTPS20 formed a separate branch that diverged from the progenitor of all other potentially functional terpene synthases of the rubber TPS-b subfamily. The truncated HbTPS20 without the signal peptide coding sequence was successfully expressed in E. coli and in vitro enzymatic assays with geranyl diphosphate (GPP) or neryl diphosphate (NPP) as a substrate defined HbTPS20 as an active linalool synthase (HbLIS) with the ability to produce linalool as the principal product. RT-qPCR analysis showed that the highest transcript levels of HbTPS20 were found in barks, and this gene was expressed at 2.26- and 250-fold greater levels in the bark tissues of wounded and MeJA-treated plants, respectively, than in those of the control plants. This indicates that this gene may be involved in the induced stress responses of rubber.

Functional characterization of a terpene synthase responsible for (E)-β-ocimene biosynthesis identified in Pyrus betuleafolia transcriptome after herbivory.

(E)-β-ocimene, a ubiquitous monoterpene volatile in plants, is emitted from flowers to attract pollinators and/or from vegetative tissues as part of inducible defenses mediated by complex signaling networks when plants are attacked by insect herbivores. Wild pear species Pyrus betuleafolia used worldwide as rootstock generally displays valuable pest-resistant traits and is a promising genetic resource for pear breeding. In the current study, transcriptional changes in this wild pear species infested with a polyphagous herbivore Spodoptera litura and the underlying molecular mechanisms were fully investigated. A total of 3,118 differentially expressed genes (DEGs) were identified in damaged pear leaf samples. Spodoptera litura larvae infestation activated complex phytohormonal signaling networks in which jasmonic acid, ethylene, brassinosteroids, cytokinin, gibberellic acid and auxin pathways were induced, whereas salicylic acid and abscisic acid pathways were suppressed. All DEGs associated with growth-related photosynthesis were significantly downregulated, whereas most DEGs involved in defense-related early signaling events, transcription factors, green leaf volatiles and volatile terpenes were significantly upregulated. The PbeOCS (GWHGAAYT028729), a putative (E)-β-ocimene synthase gene, was newly identified in P. betuleafolia transcriptome. The upregulation of PbeOCS in S. litura-infested pear leaves supports a potential role for PbeOCS in herbivore-induced plant defenses. In enzyme-catalyzed reaction, recombinant PbeOCS utilized only geranyl pyrophosphate but not neryl diphosphate, farnesyl pyrophosphate or geranylgeranyl diphosphate as a substrate, producing (E)-β-ocimene as the major product and a trace amount of (Z)-β-ocimene. Moreover, as a catalytic product of PbeOCS, (E)-β-ocimene showed repellent effects on larvae of S. litura in dual-choice bioassays. What is more, (E)-β-ocimene increased mortalities of larvae in no-choice bioassays. These findings provide an overview of transcriptomic changes in wild pears in response to chewing herbivores and insights into (E)-β-ocimene biosynthesis in pear plants, which will help elucidate the molecular mechanisms underlying pear-insect interactions.

Metabolic engineering of Ashbya gossypii for limonene production from xylose

BackgroundLimonene is a cyclic monoterpene that has applications in the food, cosmetic, and pharmaceutical industries. The industrial production of limonene and its derivatives through plant extraction presents important drawbacks such as seasonal and climate issues, feedstock limitations, low efficiency and environmental concerns. Consequently, the implementation of efficient and eco-friendly bioprocesses for the production of limonene and other terpenes constitutes an attractive goal for microbial biotechnology. In this context, novel biocatalysts with the ability to produce limonene from alternative carbon sources will help to meet the industrial demands of limonene.ResultsEngineered strains of the industrial fungus Ashbya gossypii have been developed to produce limonene from xylose. The limonene synthase (LS) from Citrus limon was initially overexpressed together with the native HMG1 gene (coding for HMG-CoA reductase) to establish a limonene-producing platform from a xylose-utilizing A. gossypii strain. In addition, several strategies were designed to increase the production of limonene. Hence, the effect of mutant alleles of ERG20 (erg20F95W and erg20F126W) were evaluated together with a synthetic orthogonal pathway using a heterologous neryl diphosphate synthase. The lethality of the A. gossypii double mutant erg20F95W−F126W highlights the indispensability of farnesyl diphosphate for the synthesis of essential sterols. In addition, the utilization of the orthogonal pathway, bypassing the Erg20 activity through neryl diphosphate, triggered a substantial increase in limonene titer (33.6 mg/L), without critically altering the fitness of the engineered strain. Finally, the overexpression of the native ERG12 gene further enhanced limonene production, which reached 336.4 mg/L after 96 h in flask cultures using xylose as the carbon source.ConclusionsThe microbial production of limonene can be carried out using engineered strains of A. gossypii from xylose-based carbon sources. The utilization of a synthetic orthogonal pathway together with the overexpression of ERG12 is a highly beneficial strategy for the production of limonene in A. gossypii. The strains presented in this work constitute a proof of principle for the production of limonene and other terpenes from agro-industrial wastes such as xylose-rich hydrolysates in A. gossypii.

Characterization of a Novel Insect-Induced Sesquiterpene Synthase GbTPS1 Based on the Transcriptome of Gossypium barbadense Feeding by Cotton Bollworm.

When attacked by insect herbivores, plants initiate sophisticated defenses mediated by complex signaling networks and usually release a blend of functional volatiles such as terpenes against infestation. The extra-long staple cotton Gossypium barbadense cultivated worldwide as natural textile fiber crop is frequently exposed to a variety of herbivores, such as cotton bollworm Helicoverpa armigera. However, little is known about insect-induced transcriptional changes and molecular mechanisms underlying subsequent defense responses in G. barbadense. In the current study, transcriptome changes in G. barbadense infested with chewing H. armigera larvae were investigated, and we identified 5,629 differentially expressed genes (DEGs) in the infested cotton leaves compared with non-infested controls. H. armigera feeding triggered complex signaling networks in which almost all (88 out of 90) DEGs associated with the jasmonic acid (JA) pathway were upregulated, highlighting a central role for JA in the defense responses of G. barbadense against target insects. All DEGs involved in growth-related photosynthesis were downregulated, whereas most DEGs associated with defense-related transcript factors and volatile secondary metabolism were upregulated. It was noteworthy that a terpene synthase gene in the transcriptome data, GbTPS1, was strongly expressed in H. armigera-infested G. barbadense leaves. The upregulation of GbTPS1 in qPCR analysis also suggested an important role for GbTPS1 in herbivore-induced cotton defense. In vitro assays showed that recombinant GbTPS1 catalyzed farnesyl pyrophosphate and neryl diphosphate to produce three sesquiterpenes (selinene, α-gurjunene, and β-elemene) and one monoterpene (limonene), respectively. Moreover, these catalytic products of GbTPS1 were significantly elevated in G. barbadense leaves after H. armigera infestation, and elemene and limonene had repellent effects on H. armigera larvae in a dual-choice bioassay and increased larval mortality in a no-choice bioassay. These findings provide a valuable insight into understanding the transcriptional changes reprogramming herbivore-induced sesquiterpene biosynthesis in G. barbadense infested by H. armigera, which help elucidate the molecular mechanisms underlying plant defense against insect pests.

Structure-based engineering of a short-chain cis-prenyltransferase to biosynthesize nonnatural all-cis-polyisoprenoids: molecular mechanisms for primer substrate recognition and ultimate product chain-length determination.

Most cis-prenyltransferases (cPTs) use all-trans-oligoprenyl diphosphate, such as (E,E)-farnesyl diphosphate (FPP, C15 ), but scarcely accept dimethylallyl diphosphate (DMAPP, C5 ), as an allylic diphosphate primer in consecutive cis-condensations of isopentenyl diphosphate. Consequently, naturally occurring cis-1,4-polyisoprenoids contain a few trans-isoprene units at their ω-end. However, some Solanum plants have distinct cPTs that primarily use DMAPP as a primer to synthesize all-cis-oligoprenyl diphosphates, such as neryl diphosphate (NPP, C10 ). However, the mechanism underlying the allylic substrate preference of cPTs remains unclear. In this study, we determined the crystal structure of NDPS1, an NPP synthase from tomato, and investigated critical residues for primer substrate preference through structural comparisons of cPTs. Highly conserved Gly and Trp in the primer substrate-binding region of cPTs were discovered to be substituted for Ile/Leu and Phe, respectively, in DMAPP-preferring cPTs. An I106G mutant of NDPS1 exhibited a low preference for DMAPP, but a higher preference for FPP. However, an I106G/F276W mutant preferred not only DMAPP but also all-trans-oligoprenyl diphosphates, with 15-fold higher catalytic efficiency than WT. Surprisingly, the mutant synthesized longer polyisoprenoids (~C50 ). Furthermore, one of the helix domains that constitute the hydrophobic cleft for accommodating elongating prenyl chains was also demonstrated to be critical in primer substrate preference. An NDPS1 I106G/F276W mutant with a chimeric helix domain swapped with that of a medium-chain cPT synthesizing C50-60 polyisoprenoids showed over 94-fold increase in catalytic efficiency for all primer substrates tested, resulting in longer products (~C70 ). These NDPS1 mutants could be used in the enzymatic synthesis of nonnatural all-cis-polyisoprenoids.

Dynamic control of ERG20 expression to improve production of monoterpenes by engineering Saccharomyces cerevisiae

Monoterpenes are widely used in cosmetics, food, medicine, agriculture and other fields. With the development of synthetic biology, it is considered as a potential way to create microbial cell factories to produce monoterpenes. Engineering Saccharomyces cerevisiae to produce monoterpenes has been a research hotspot in synthetic biology. In S. cerevisiae, the production of geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) is catalyzed by a bifunctional enzyme farnesyl pyrophosphate synthetase(encoded by ERG20 gene) which is inclined to synthesize FPP essential for yeast growth. Therefore, reasonable control of FPP synthesis is the basis for efficient monoterpene synthesis in yeast cell factories. In order to achieve dynamic control from GPP to FPP biosynthesis in S. cerevisiae, we obtained a novel chassis strain HP001-pERG1-ERG20 by replacing the ERG20 promoter of the chassis strain HP001 with the promoter of cyclosqualene cyclase(ERG1) gene. Further, we reconstructed the metabolic pathway by using GPP and neryl diphosphate(NPP), cis-GPP as substrates in HP001-pERG1-ERG20. The yield of GPP-derived linalool increased by 42.5% to 7.6 mg·L~(-1), and that of NPP-derived nerol increased by 1 436.4% to 8.3 mg·L~(-1). This study provides a basis for the production of monoterpenes by microbial fermentation.

Engineering of a Plant Isoprenyl Diphosphate Synthase for Development of Irregular Coupling Activity.

We performed mutagenesis on a regular isoprenyl diphosphate synthase (IDS), neryl diphosphate synthase from Solanum lycopersicum (SlNPPS), that has a structurally related analogue performing non‐head‐to‐tail coupling of two dimethylallyl diphosphate (DMAPP) units, lavandulyl diphosphate synthase from Lavandula x intermedia (LiLPPS). Wild‐type SlNPPS catalyses regular coupling of isopentenyl diphosphate (IPP) and DMAPP in cis‐orientation resulting in the formation of neryl diphosphate. However, if the enzyme is fed with DMAPP only, it is able to catalyse the coupling of two DMAPP units and synthesizes two irregular monoterpene diphosphates; their structures were elucidated by the NMR analysis of their dephosphorylation products. One of the alcohols is lavandulol. The second compound is the trans‐isomer of planococcol, the first example of an irregular cyclobutane monoterpene with this stereochemical configuration. The irregular activity of SlNPPS constitutes 0.4 % of its regular activity and is revealed only if the enzyme is supplied with DMAPP in the absence of IPP. The exchange of asparagine 88 for histidine considerably enhanced the non‐head‐to‐tail coupling. While still only observed in the absence of IPP, irregular activity of the mutant reaches 13.1 % of its regular activity. The obtained results prove that regular IDS are promising starting points for protein engineering aiming at the development of irregular activities and leading to novel monoterpene structures.

Combining Metabolic and Monoterpene Synthase Engineering for de Novo Production of Monoterpene Alcohols in Escherichia coli

The monoterpene alcohols acyclic nerol and bicyclic borneol are widely applied in the food, cosmetic, and pharmaceutical industries. The emerging synthetic biology enables microbial production to be a promising alternative for supplying monoterpene alcohols in an efficient and sustainable approach. In this study, we combined metabolic and plant monoterpene synthase engineering to improve the de novo production of nerol and borneol in prene-overproducing Escherichia coli. We engineered the growth-orthogonal neryl diphosphate (NPP) as the universal precursor of monoterpene alcohol biosynthesis and coexpressed nerol synthase (GmNES) from Glycine max to generate nerol or coexpressed the truncated bornyl diphosphate synthase (LdtBPPS) from Lippia dulcis for borneol production. Further, through site-directed mutation of LdtBPPS based on the structural simulation, we screened multiple variants that markedly elevated the production of acyclic nerol or bicyclic borneol, of which the LdtBPPSS488T mutant outperformed the wild-type LdtBPPS on borneol synthesis and the LdtBPPSF612A variant was superior to GmNES on nerol production. Subsequently, we overexpressed the endogenous Nudix hydrolase NudJ to facilitate the dephosphorylation of precursors and boosted the production of nerol and borneol from glucose. Finally, after the optimization of the fermentation process, the engineered strain ENO2 produced 966.55 mg/L nerol, and strain ENB57 generated 87.20 mg/L borneol in a shake flask, achieving the highest reported titers of nerol and borneol in microbes to date. This work shows a combinatorial engineering strategy for microbial production of natural terpene alcohols.

Improve the production of D-limonene by regulating the mevalonate pathway of Saccharomyces cerevisiae during alcoholic beverage fermentation.

D-Limonene, a cyclized monoterpene, possesses citrus-like olfactory property and multi-physiological functions, which can be used as a bioactive compound and flavor to improve the overall quality of alcoholic beverages. In our previous study, we established an orthogonal pathway of D-limonene synthesis by introducing neryl diphosphate synthase 1 (tNDPS1) and D-limonene synthase (tLS) in Saccharomyces cerevisiae. To further increase D-limonene formation, the metabolic flux of the mevalonate (MVA) pathway was enhanced by overexpressing the key genes tHMGR1, ERG12, IDI1, and IDI1WWW, respectively, or co-overexpressing. The results showed that strengthening the MVA pathway significantly improved D-limonene production, while the best strain yielded 62.31mg/L D-limonene by co-expressing tHMGR1, ERG12, and IDI1WWW genes in alcoholic beverages. Furthermore, we also studied the effect of enhancing the MVA pathway on the growth and fermentation of engineered yeasts during alcoholic beverage fermentation. Besides, to further resolve the problem of yeast growth inhibition, we separately investigated transporter proteins of the high-yielding D-limonene yeasts and the parental strain under the stress of different D-limonene concentration, suggesting that the transporters of Aus1p, Pdr18p, Pdr5p, Pdr3p, Pdr11p, Pdr15p, Tpo1p, and Ste6p might play a more critical role in alleviating cytotoxicity and improving the tolerance to D-limonene. Finally, we verified the functions of three transporter proteins, finding that the transporter of Aus1p failed to transport D-limonene, and the others (Pdr5p and Pdr15p) could improve the tolerance of yeast to D-limonene. This study provided a valuable platform for other monoterpenes' biosynthesis in yeast during alcoholic beverage fermentation.

Origin and functional differentiation of (E)-β-ocimene synthases reflect the expansion of monoterpenes in angiosperms.

The acquisition of new metabolic activities is a major force driving evolution. We explored, from the perspectives of gene family expansion and the evolutionary adaptability of proteins, how new functions have arisen in which terpene synthases diverged. Monoterpenoids are diverse natural compounds that can be divided into cyclic and acyclic skeleton forms according to their chemical structure. We demonstrate, through phylogenetic reconstructions and genome synteny analyses, that the (E)-β-ocimene synthases, which are acyclic monoterpene synthases (mTPSs), appear to have arisen several times in independent lineages during plant evolution. Bioinformatics analyses and classical mutation experiments identified four sites (I388, F420, S446, and F485) playing important roles in the neofunctionalization of mTPSs. Incubation of neryl diphosphate with Salvia officinalis 1,8-cineole synthase (SCS) and mutated proteins show that these four sites obstruct the isomerization of geranyl diphosphate. Quantum mechanical/molecular mechanical molecular dynamics simulations of models of SCS, SCSY420F/I446S, and SCSN338I/Y420F/I446S/L485F with (3R)-linalyl diphosphate suggest that mutations changed the configuration of the intermediate to obtain new activities. These results provide new perspectives on the evolution of mTPSs, explain the convergent evolution of (E)-β-ocimene synthases at the molecular level, and identify key residues to control the specificity of engineered mTPSs.

Engineering Saccharomyces cerevisiae for production of the valuable monoterpene d-limonene during Chinese Baijiu fermentation.

d-Limonene, a cyclic monoterpene, possessescitrus-like olfactory property and multi-physiologicalfunctions. In this study, the d-limonene synthase (tLS) from Citrus limon was codon-optimized and heterologously expressed in Saccharomyces cerevisiae. The metabolic flux of canonical pathway based on overexpressing endogenous geranyl diphosphate synthase gene (ERG20) and its variant ERG20F96W-N127W was strengthened for improvement d-limonene production in Chinese Baijiu. To further elevate production, we established an orthogonal pathway by introducing neryl diphosphate synthase 1 (tNDPS1) from Solanum lycopersicum. The results showed that expressing ERG20 and ERG20F96W-N127W could enhance d-limonene synthesis, while expressing heterologous NPP synthase gene significantly increase d-limonene formation. Furthermore, we constructed a tLS-tNDPS1 fusion protein, and the best strain yielded 9.8mg/L d-limonene after optimizing the amino acid linker and fusion order, a 40% improvement over the free enzymes during Chinese Baijiu fermentation. Finally, under the optimized fermentation conditions, a maximum d-limonene content of 23.7mg/L in strain AY12α-L9 was achieved, which was the highest reported production in Chinese Baijiu. In addition, we also investigated that the effect of d-limonene concentration on yeast growth and fermentation. This study provided a meaningful insight into the platform for other valuable monoterpenes biosynthesis in Chinese Baijiu fermentation.

Metabolic engineering of Escherichia coli for the production of neryl acetate

Neryl acetate, a monoterpenoid derivate originated from neryl diphosphate (NPP), has found extensive applications in food, agriculture, and cosmetic fields. In this study, neryl acetate was biosynthesized in recombinant Escherichia coli for the first time. First, the alcohol acetyltransferase, ATF1, was heterologously expressed in a nerol-producing E. coli strain. The resultant engineered strain, LZ006, accumulated 0.250 ± 0.014 mg/L of neryl acetate. Further overexpression of tHMG1, which improved the metabolic flux of MVA pathway, enhanced the yield to 8.064 ± 0.724 mg/L in LZ007, representing a 32.2-fold increase compared to that of LZ006. Finally, supplementation with 2 g/L pyruvate in the culture further increased neryl acetate production to 11.712 ± 0.653 mg/L, representing a 46.8-fold increase over that of the LZ006 strain. This study provides a novel method for the biosynthesis of neryl acetate and the strategies described here could be adapted to biosynthesize many other NPP-derived bioproducts.

Advanced Strategies for Production of Natural Products in Yeast.

Natural products account for more than 50% of all small-molecule pharmaceutical agents currently in clinical use. However, low availability often becomes problematic when a bioactive natural product is promising to become a pharmaceutical or leading compound. Advances in synthetic biology and metabolic engineering provide a feasible solution for sustainable supply of these compounds. In this review, we have summarized current progress in engineering yeast cell factories for production of natural products, including terpenoids, alkaloids, and phenylpropanoids. We then discuss advanced strategies in metabolic engineering at three different dimensions, including point, line, and plane (corresponding to the individual enzymes and cofactors, metabolic pathways, and the global cellular network). In particular, we comprehensively discuss how to engineer cofactor biosynthesis for enhancing the biosynthesis efficiency, other than the enzyme activity. Finally, current challenges and perspective are also discussed for future engineering direction.

Orthogonal monoterpenoid biosynthesis in yeast constructed on an isomeric substrate

Synthetic biology efforts for the production of valuable chemicals are frequently hindered by the structure and regulation of the native metabolic pathways of the chassis. This is particularly evident in the case of monoterpenoid production in Saccharomyces cerevisiae, where the canonical terpene precursor geranyl diphosphate is tightly coupled to the biosynthesis of isoprenoid compounds essential for yeast viability. Here, we establish a synthetic orthogonal monoterpenoid pathway based on an alternative precursor, neryl diphosphate. We identify structural determinants of isomeric substrate selectivity in monoterpene synthases and engineer five different enzymes to accept the alternative substrate with improved efficiency and specificity. We combine the engineered enzymes with dynamic regulation of metabolic flux to harness the potential of the orthogonal substrate and improve the production of industrially-relevant monoterpenes by several-fold compared to the canonical pathway. This approach highlights the introduction of synthetic metabolism as an effective strategy for high-value compound production.