Engineering peroxisomal biosynthetic pathways for maximization of triterpene production in Yarrowia lipolytica.

Constructing efficient cell factories for product synthesis is frequently hampered by competing pathways and/or insufficient precursor supply. This is particularly evident in the case of triterpenoid biosynthesis in Yarrowia lipolytica, where squalene biosynthesis is tightly coupled to cytosolic biosynthesis of sterols essential for cell viability. Here, we addressed this problem by reconstructing the complete squalene biosynthetic pathway, starting from acetyl-CoA, in the peroxisome, thus harnessing peroxisomal acetyl-CoA pool and sequestering squalene synthesis in this organelle from competing cytosolic reactions. This strategy led to increasing the squalene levels by 1,300-fold relatively to native cytosolic synthesis. Subsequent enhancement of the peroxisomal acetyl-CoA supply by two independent approaches, 1) converting cellular lipid pool to peroxisomal acetyl-CoA and 2) establishing an orthogonal acetyl-CoA shortcut from CO2-derived acetate in the peroxisome, further significantly improved local squalene accumulation. Using these approaches, we constructed squalene-producing strains capable of yielding 32.8 g/L from glucose, and 31.6 g/L from acetate by employing a cofeeding strategy, in bioreactor fermentations. Our findings provide a feasible strategy for protecting intermediate metabolites that can be claimed by multiple reactions by engineering peroxisomes in Y. lipolytica as microfactories for the production of such intermediates and in particular acetyl-CoA-derived metabolites.

Engineering a universal and efficient platform for terpenoid synthesis in yeast.

Engineering microbes for the production of valuable natural products is often hindered by the regulation of native competing metabolic networks in host. This is particularly evident in the case of terpenoid synthesis in yeast, where the canonical terpenoid precursors are tightly coupled to the biosynthesis of sterols essential for yeast viability. One way to circumvent this limitation is by engineering product pathways less connected to the host native metabolism. Here, we introduce a two-step isopentenol utilization pathway (IUP) in Saccharomyces cerevisiae to augment the native mevalonate pathway by providing a shortcut to the synthesis of the common terpenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). As such, the IUP was capable of elevating the IPP/DMAPP pool by 147-fold compared with the native pathway. We further demonstrate that cofeeding isoprenol and prenol enhances geranyl diphosphate (GPP) content for monoterpene biosynthesis. More importantly, we established a synthetic three-step route for efficient synthesis of di-and tetraterpene precursor geranylgeranyl diphosphate (GGPP), circumventing the competition with farnesyl diphosphate (FPP) for sterol biosynthesis and elevating the GGPP level by 374-fold. We combine these IUP-supported precursor-forming platforms with downstream terpene synthases to harness their potential and improve the production of industrially relevant terpenoids by several fold. Our exploration provides a universal and effective platform for supporting terpenoid synthesis in yeast.

Vacuolar MATE/DTX protein-mediated cucurbitacin C transport is co-regulated with bitterness biosynthesis in cucumber.

Membrane-localized transporters constitute important components for specialized metabolism in plants. However, due to the vast array of specialized metabolites produced by plants, and the large families of transporter genes, knowledge about the intracellular and intercellular transport of plant metabolites is still in its infancy. Cucurbitacins are bitter and defensive triterpenoids produced mainly in the cucurbits. Using a comparative genomics and multi-omics approach, a MATE gene (CsMATE1), physically clustered with cucurbitacin C (CuC) biosynthetic genes, was identified and functionally shown to sequester CuC in cucumber leaf mesophyll cells. Notably, the CuC transport process is strictly co-regulated with CuC biosynthesis. CsMATE1 clustering with bitterness biosynthesis genes may provide benefits and a basis for this feedback regulation on CuC sequestration and biosynthesis. Identification of transport systems for plant-specialized metabolites can accelerate the metabolic engineering of high-value-added compounds by simplifying their purification process.

Is bitter actually better? Targeting a soyasaponin acetyltransferase affects soybean seed germination.

This Highlight features a recent study showing that the soybean BAHD-type soyasaponin acetyltransferase GmSSAcT1 is responsible for three or four sequential acetylation steps in type-A soyasaponin biosynthesis and that loss of GmSSAcT1 function strongly inhibits soybean seed germination.

Targeting pathway expression to subcellular organelles improves astaxanthin synthesis in Yarrowia lipolytica.

Metabolic engineering approaches for the production of high-value chemicals in microorganisms mostly use the cytosol as general reaction vessel. However, sequestration of enzymes and substrates, and metabolic cross-talk frequently prevent efficient synthesis of target compounds in the cytosol. Organelle compartmentalization in eukaryotic cells suggests ways for overcoming these challenges. Here we have explored this strategy by expressing the astaxanthin biosynthesis pathway in sub-organelles of the oleaginous yeast Yarrowia lipolytica. We first showed that fusion of the two enzymes converting ß-carotene to astaxanthin, ß-carotene ketolase and hydroxylase, performs better than the expression of individual enzymes. We next evaluated the pathway when expressed in compartments of lipid body, endoplasmic reticulum or peroxisome, individually and in combination. Targeting the astaxanthin pathway to subcellular organelles not only accelerated the conversion of ß-carotene to astaxanthin, but also significantly decreased accumulation of the ketocarotenoid intermediates. Anchoring enzymes simultaneously to all three organelles yielded the largest increase of astaxanthin synthesis, and ultimately produced 858 mg/L of astaxanthin in fed-batch fermentation (a 141-fold improvement over the initial strain). Our study is expected to help unlock the full potential of subcellular compartments and advance LB-based compartmentalized isoprenoid biosynthesis in Y. lipolytica.

The role of H2S in low temperature-induced cucurbitacin C increases in cucumber.

KEY MESSAGE: In this study, we first linked the signal molecule H2S with cucurbitacin C, which can cause the bitter taste of cucumber leaves and fruit, and specifically discuss its molecular mechanism. Cucurbitacin C (CuC), a triterpenoid secondary metabolite, enhances the resistance of cucumber plants to pathogenic bacteria and insect herbivores, but results in bitter-tasting fruits. CuC can be induced in some varieties of cucumber on exposure to plant stressors. The gasotransmitter hydrogen sulfide (H2S) participates in multiple physiological processes relating to plant stress resistance. This study focused on the effect of H2S on low temperature-induced CuC synthesis in cucumber. The results showed that treatment of cucumber leaves at 4 °C for 12 h enhanced the content and production rate of H2S and increased the expression of genes encoding enzymes involved in H2S generation, Csa2G034800.1 (CsaLCD), Csa1G574800.1 (CsaDES1), and Csa1G574810.1 (CsaDES2). In addition, treatment at 4 °C or with exogenous H2S upregulated the expression of CuC synthetase-encoding genes and the resulting CuC content in cucumber leaves, whereas pretreatment with hypotaurine (HT, a H2S scavenger) before treatment at 4 °C offset these effects. In vitro, H2S could increase the S-sulfhydration level of His-Csa5G156220 and His-Csa5G157230 (both bHLH transcription factors), as well as their binding activity to the promoter of Csa6G088690, which encodes the key synthetase for CuC generation. H2S pretreatment enhanced the cucumber leaves resistance to the Phytophthora melonis. Together, these results demonstrated that H2S acts as a positive regulator of CuC synthesis as a result of the modification of proteins by S-sulfhydration, also providing indirect evidence for the role of H2S in improving the resistance of plants to abiotic stresses and biotic stresses by regulating the synthesis of secondary metabolites.

Genetic analysis and identification of a candidate gene associated with in vitro regeneration ability of cucumber.

KEY MESSAGE: Candidate genes associated with in vitro regeneration were identified in cucumber. The ability to regenerate shoots or whole plants from differentiated plant tissues is essential for plant transformation. In cucumber (Cucumis sativus L.), regeneration ability varies considerably across accessions, but the genetic mechanism has not yet been demonstrated. In the present study, 148 recombinant inbred lines and a core collection were examined to identify candidate genes involved in cucumber regeneration. Four QTL for cotyledon regeneration that explained 9.7-16.6% of the phenotypic variation in regeneration were identified on cucumber chromosomes 1, 3, and 6. The loci Fcrms1.1 and Fcrms+1.1 were consistently detected in the same genetic interval on two regeneration media. A genome-wide association study revealed 18 SNPs (- log(p) > 5) significantly associated with cotyledon regeneration. Three candidate genes in this region were identified. RT-PCR analyses revealed that Csa1G642540 was significantly more highly expressed in genotypes with high cotyledon regeneration rates than in those with low regeneration. The Csa1G642540 CDS driven by its native promoter was transformed into cucumber line 9110Gt; molecular analyses showed that the T-DNA had integrated into the genomes of 8.6% of regenerated plantlets. The seeds from T0 plants expressing Csa1G642540 were tested for regeneration from cotyledon explants, and the segregate ratio in regeneration frequency is 3:1. The AT3G44110.1, the homologue gene of Csa1G642540 in Arabidopsis, has been reported as PM H+-ATPase activity regulation, integrating flowering signals and enlarging meristem function. These results demonstrate that Csa1G642540 might play an important role in regeneration in cucumber and could serve as a selectable marker for regeneration from cotyledons.

Removal of lycopene substrate inhibition enables high carotenoid productivity in Yarrowia lipolytica.

Substrate inhibition of enzymes can be a major obstacle to the production of valuable chemicals in engineered microorganisms. Here, we show substrate inhibition of lycopene cyclase as the main limitation in carotenoid biosynthesis in Yarrowia lipolytica. To overcome this bottleneck, we exploit two independent approaches. Structure-guided protein engineering yields a variant, Y27R, characterized by complete loss of substrate inhibition without reduction of enzymatic activity. Alternatively, establishing a geranylgeranyl pyrophosphate synthase-mediated flux flow restrictor also prevents the onset of substrate inhibition by diverting metabolic flux away from the inhibitory metabolite while maintaining sufficient flux towards product formation. Both approaches result in high levels of near-exclusive ß-carotene production. Ultimately, we construct strains capable of producing 39.5 g/L ß-carotene at a productivity of 0.165 g/L/h in bioreactor fermentations (a 1441-fold improvement over the initial strain). Our findings provide effective approaches for removing substrate inhibition in engineering pathways for efficient synthesis of natural products.

Root-secreted bitter triterpene modulates the rhizosphere microbiota to improve plant fitness.

Underground microbial ecosystems have profound impacts on plant health1-5. Recently, essential roles have been shown for plant specialized metabolites in shaping the rhizosphere microbiome6-9. However, the potential mechanisms underlying the root-to-soil delivery of these metabolites remain to be elucidated10. Cucurbitacins, the characteristic bitter triterpenoids in cucurbit plants (such as melon and watermelon), are synthesized by operon-like gene clusters11. Here we report two Multidrug and Toxic Compound Extrusion (MATE) proteins involved in the transport of their respective cucurbitacins, a process co-regulated with cucurbitacin biosynthesis. We further show that the transport of cucurbitacin B from the roots of melon into the soil modulates the rhizosphere microbiome by selectively enriching for two bacterial genera, Enterobacter and Bacillus, and we demonstrate that this, in turn, leads to robust resistance against the soil-borne wilt fungal pathogen, Fusarium oxysporum. Our study offers insights into how transporters for specialized metabolites manipulate the rhizosphere microbiota and thereby affect crop fitness.

Synthesis of high-titer alka(e)nes in Yarrowia lipolytica is enabled by a discovered mechanism.

Alka(e)nes are ideal fuel components for aviation, long-distance transport, and shipping. They are typically derived from fossil fuels and accounting for 24% of difficult-to-eliminate greenhouse gas emissions. The synthesis of alka(e)nes in Yarrowia lipolytica from CO2-neutral feedstocks represents an attractive alternative. Here we report that the high-titer synthesis of alka(e)nes in Yarrowia lipolytica harboring a fatty acid photodecarboxylase (CvFAP) is enabled by a discovered pathway. We find that acyl-CoAs, rather than free fatty acids (FFAs), are the preferred substrate for CvFAP. This finding allows us to debottleneck the pathway and optimize fermentation conditions so that we are able to redirect 89% of acyl-CoAs from the synthesis of neutral lipids to alka(e)nes and reach titers of 1.47 g/L from glucose. Two other CO2-derived substrates, wheat straw and acetate, are also demonstrated to be effective in producing alka(e)nes. Overall, our technology could advance net-zero emissions by providing CO2-neutral and energy-dense liquid biofuels.

A structural and data-driven approach to engineering a plant cytochrome P450 enzyme.

Functional manipulation of biosynthetic enzymes such as cytochrome P450s (or P450s) has attracted great interest in metabolic engineering of plant natural products. Cucurbitacins and mogrosides are plant triterpenoids that share the same backbone but display contrasting bioactivities. This structural and functional diversity of the two metabolites can be manipulated by engineering P450s. However, the functional redesign of P450s through directed evolution (DE) or structure-guided protein engineering is time consuming and challenging, often because of a lack of high-throughput screening methods and crystal structures of P450s. In this study, we used an integrated approach combining computational protein design, evolutionary information, and experimental data-driven optimization to alter the substrate specificity of a multifunctional P450 (CYP87D20) from cucumber. After three rounds of iterative design and evaluation of 96 protein variants, CYP87D20, which is involved in the cucurbitacin C biosynthetic pathway, was successfully transformed into a P450 mono-oxygenase that performs a single specific hydroxylation at C11 of cucurbitadienol. This integrated P450-engineering approach can be further applied to create a de novo pathway to produce mogrol, the precursor of the natural sweetener mogroside, or to alter the structural diversity of plant triterpenoids by functionally manipulating other P450s.

The Genome of Medicinal Plant Macleaya cordata Provides New Insights into Benzylisoquinoline Alkaloids Metabolism.

The overuse of antibiotics in animal agriculture and medicine has caused a series of potential threats to public health. Macleaya cordata is a medicinal plant species from the Papaveraceae family, providing a safe resource for the manufacture of antimicrobial feed additive for livestock. The active constituents from M. cordata are known to include benzylisoquinoline alkaloids (BIAs) such as sanguinarine (SAN) and chelerythrine (CHE), but their metabolic pathways have yet to be studied in this non-model plant. The active biosynthesis of SAN and CHE in M. cordata was first examined and confirmed by feeding 13C-labeled tyrosine. To gain further insights, we de novo sequenced the whole genome of M. cordata, the first to be sequenced from the Papaveraceae family. The M. cordata genome covering 378 Mb encodes 22,328 predicted protein-coding genes with 43.5% being transposable elements. As a member of basal eudicot, M. cordata genome lacks the paleohexaploidy event that occurred in almost all eudicots. From the genomics data, a complete set of 16 metabolic genes for SAN and CHE biosynthesis was retrieved, and 14 of their biochemical activities were validated. These genomics and metabolic data show the conserved BIA metabolic pathways in M. cordata and provide the knowledge foundation for future productions of SAN and CHE by crop improvement or microbial pathway reconstruction.

Convergence and divergence of bitterness biosynthesis and regulation in Cucurbitaceae.

Differentiation of secondary metabolite profiles in closely related plant species provides clues for unravelling biosynthetic pathways and regulatory circuits, an area that is still underinvestigated. Cucurbitacins, a group of bitter and highly oxygenated tetracyclic triterpenes, are mainly produced by the plant family Cucurbitaceae. These compounds have similar structures, but differ in their antitumour activities and ecophysiological roles. By comparative analyses of the genomes of cucumber, melon and watermelon, we uncovered conserved syntenic loci encoding metabolic genes for distinct cucurbitacins. Characterization of the cytochrome P450s (CYPs) identified from these loci enabled us to unveil a novel multi-oxidation CYP for the tailoring of the cucurbitacin core skeleton as well as two other CYPs responsible for the key structural variations among cucurbitacins C, B and E. We also discovered a syntenic gene cluster of transcription factors that regulates the tissue-specific biosynthesis of cucurbitacins and may confer the loss of bitterness phenotypes associated with convergent domestication of wild cucurbits. This study illustrates the potential to exploit comparative genomics to identify enzymes and transcription factors that control the biosynthesis of structurally related yet unique natural products.

Plant science. Biosynthesis, regulation, and domestication of bitterness in cucumber.

Cucurbitacins are triterpenoids that confer a bitter taste in cucurbits such as cucumber, melon, watermelon, squash, and pumpkin. These compounds discourage most pests on the plant and have also been shown to have antitumor properties. With genomics and biochemistry, we identified nine cucumber genes in the pathway for biosynthesis of cucurbitacin C and elucidated four catalytic steps. We discovered transcription factors Bl (Bitter leaf) and Bt (Bitter fruit) that regulate this pathway in leaves and fruits, respectively. Traces in genomic signatures indicated that selection imposed on Bt during domestication led to derivation of nonbitter cucurbits from their bitter ancestors.