Conventional coffee production faces sustainability challenges. Coffee cell cultures (CC) are a promising alternative coffee raw material. This study investigated the composition of CC in comparison to conventional green coffee beans (GB) using untargeted metabolomics and lipidomics approaches. The lipid content (g/100 g) of GB was approximately four times higher than that of CC with triglycerides as the predominant lipid class identified followed by diterpenes. Additionally, differences were observed in the diterpene profiles of CC and GB. CC accumulated more thiamine and choline but lower contents of caffeine, trigonelline, and alpha-tocopherol compared to GB. The detected carbohydrates of statistical significance between CC and GB were mostly monosaccharides. This study provides information on the bioactive metabolites in CC, demonstrates the possibility of tailor-made coffee products from CC, and encourages further optimization of the production process of CC to enable development of sustainable, healthy coffee beverages for the future.

Plant cell culture technology can produce valuable compounds for various industries. Interest in plant cells for food has grown due to challenges in conventional agriculture, including climate change and food supply issues. Bioreactor-grown cells offer high product consistency without pesticide risks. Although plant cell cultures can have high nutritional value, their overall flavour remains mild. This study investigated the induction of flavour in plant cell cultures, specifically focusing on arctic bramble, strawberry, and lingonberry cell cultures. Flavour induction was performed with additives and mild heating. Sensory odour profiling was performed by a trained panel, and selected samples were analyzed for volatile compounds using HS-SPME-GC–MS.Across seven experiments, low-dose ethanol treatment at 0.25–0.50% (v/v) enhanced fruity, berry and sweet odour attributes, with the strongest effects observed after short exposure times. Arctic bramble cells showed the most pronounced fruity response whereas strawberry cells were more associated with berry-like odour Mild heating at 40 °C increased fruity and sweet odours in some cultures but also contributed to nutty notes. Ethanol and commercial juices like grape-cranberry juice increased fruity (on average 2 units on a 0–15 scale), berry, and sweet odours. Volatile analysis identified 114 compounds; ethanol-treated samples were associated particularly with ethyl acetate, acetaldehyde, and 6-methyl-5-hepten-2-one, associated with fruity and sweet odours.Low amounts of EtOH can induce desirable flavours in plant cell cultures, making it a cost-effective and safe food additive. Additionally, short incubation in a medium partially replaced by commercial juice shows potential.

Abstract Cultivating dedifferentiated plant cells in bioreactors offers a continuous, scalable, reproducible, and fully controlled platform for producing valuable secondary metabolites. However, performance is highly dependent on cultivation conditions. In this study, we investigated the impact of different bioreactor configurations on the growth and metabolite production of an apple plant cell suspension culture. Stirred-tank bioreactors (STRs) with different vessel geometries (flat and round-bottom) were evaluated at the multi-liter scale (2–5 L) and compared, for the first time with plant cells, with a high-throughput small volume parallel system (240 mL). Using these mini-bioreactors, we studied the effect of impeller type (Rushton, marine, elephant ear) and pO2 cascade controls on cell growth and triterpene production. Results revealed that triterpene content and biomass yield varied significantly across cultivation systems and that plant cells were highly sensitive to shear stress, with variation coefficient reaching 56.2%-63.5%. Configurations with elephant ear-type impellers seemed to reduce shear stress, supporting stable triterpene accumulation (14.15 mg/g cells DW) and higher biomass (11.8 g DW/L). The high-throughput system closely mirrored multi-liter STR performance, demonstrating consistent and reliable prediction at larger scales. In contrast, wave bioreactors and flask cultures yielded distinct results, emphasizing the importance of continuous monitoring and system-specific optimization. Overall, these findings demonstrate that careful selection of bioreactor design and control strategy is critical for maximizing plant cell growth and metabolite production, and that small-scale high-throughput systems can be effectively used as process development tool for plant cell-based biomanufacturing.

In this paper, an analytical method for the analysis of molecular lipids in algae samples is reported. The sample preparation is based on a modified Folch extraction, and the analysis is carried out with ultrahigh performance liquid chromatography combined with mass spectrometry (UPLC-MS). For the characterization of lipids, data dependent acquisition (DDA) analyses are carried out utilizing a high-resolution quadrupole-time-of-flight (Q-ToF) instrument. Throughput of the method is over 100 samples/day. The repeatability is good, and the relative standard deviation of spiked samples is <15%.

Hairy root cultures offer a sustainable alternative to field-grown plants for the production of bioactive compounds, but their translation to industrial-scale processes is limited by the challenges of high-volume cultivation and downstream processing. In the current study, a large-scale production pipeline for basil (Ocimum basilicum) hairy roots, a rich source of specialized bioactive metabolites, is described. The production of hairy root biomass was optimized by developing a robust and scalable cultivation protocol, enabling consistent and reproducible growth in bioreactor systems with working volumes of up to 1000 L. The entire production pipeline from initial Petri dish cultures to industrial-scale bioreactors was established by adopting a systematic, stepwise methodology, ensuring process reliability and scalability. A customized bioreactor with controlled aeration maintained biomass integrity and metabolic stability during long-term axenic cultivation, yielding 96.4 kg of biomass fresh weight within 84 days. Complementary solvent-based extraction and pulsed electric field processing allowed the recovery of metabolites with a broad range of polarities. Untargeted metabolomics revealed a chemically diverse profile comprising primary metabolites, phenolic acids, flavonoids, terpenoids and triterpenoids with relevance for cosmetic applications. These results demonstrate the feasibility of industrial-scale basil hairy root cultures and support their use as a source of plant-derived cosmetic ingredients.

Constituents of microalgae and sample preparation for UPLC-ELSD and GC-MS analyses are described. Bound fatty acids from acylglycerols, alkylacylglycerols, galactosyldiacylglycerols, glycerophospholipids, and sterol esters are derivatized by using transesterification with sodium methoxide to form fatty acid methyl esters. Compounds containing free hydroxyl groups, either present originally or formed during previous step, like free fatty acids, sterols, α-tocopherol, phytol, and nonesterified alkoxyglycerols, are trimethylsilylated. The compounds in algal lipid extract are subsequently derivatized by these two steps.

The SaCas9 from Staphylococcus aureus has been shown to be more effective than the commonly used SpCas9 from Streptococcus pyogenes for the generation of mutations in plants and has boosted CRISPR/Cas systems. CRISPRa/Cas systems on the other hand have been focused on the use of SpCas9. Building on foundational work, we developed a modular CRISPRa/Cas ortholog system based on a single plasmid infiltration for the use and comparison of both SaCas9 and SpCas9 for enhanced gene expression. Utilizing the multi-kingdom Golden Gate cloning platform, we integrated various elements to create a widely adaptable system. For the first time in the current study, we demonstrate that the dSaCas9 effector induces stronger activation of reporter gene expression by targeted promoters than dSpCas9 in plants. As both effectors are sharing a common target site for the tested promoter, they could be directly compared with each other showing that the dSaCas9-effector exhibited a stronger effect in the expression of the reporter genes. This allows fine-tuning the expression of genes by using different effectors. In addition, we could show on the example of the pNOS promoter and a truncated version of the pNOS promoter, that a lower basic expression can lead to an increased relative induction.

Crocins are water-soluble apocarotenoids from saffron (Crocus sativus L.) with potent antioxidant and neuroprotective properties, yet their supply is limited by low yields and labor-intensive cultivation. We previously engineered Nicotiana tabacum L. to express carotenoid cleavage dioxygenase 2 (CCD2) to establish an alternative production platform. Transgenic lines accumulated crocins at variable levels, including a subset of high-producing genotypes. However, these lines displayed chlorosis, impaired growth, and reduced viability in soil, highlighting a trade-off between crocin biosynthesis and photosynthetic function. To overcome this limitation, selected best performer lines were subsequently used to establish callus cultures, which were grown under light and dark conditions, elicited using nanoparticles and finally analyzed for crocin accumulation, carotenoid precursor profiles, and expression of key biosynthetic genes. Integration of metabolite and transcript data revealed complex relationships between carotenoid metabolism and crocin production, indicating that crocin accumulation might likely depend not only on gene expression but also on precursor availability, post-translational and traditional effects and metabolic flux. To develop a scalable production platform, the highest-producing line was transferred to suspension cultures and, for the first time, successfully cultivated in a bioreactor system. This approach yielded crocin-enriched extracts dominated by glycosylated crocin species and demonstrates the potential of engineered tobacco cell cultures for sustainable large-scale crocin production. Finally, hydroalcoholic extracts from the bioreactor culture showed no cytotoxicity in the human SK-N-BE neuroblastoma cell line and significantly protected cells from hydrogen peroxide-induced oxidative stress. Overall, these results demonstrate that engineered N. tabacum callus cultures are a promising platform for scalable crocin production and might represent a potential source of bioactive apocarotenoids for pharmaceutical applications.

Plants represent commercially relevant production systems for recombinant proteins and chemical compounds. Effective genetic engineering depends on precise control of heterologous gene expression, which remains challenging due to complex transcriptional and post-transcriptional regulation, endogenous gene silencing mechanisms, and notably because of limited number of tools for allowing robust, fine-tuned control of expression levels across different systems/organisms. Some of the most common issues associated with plant expression systems are addressed with our plant-optimized version of a previously developed fungal universal synthetic expression system (SES). Plant SES demonstrates several favorable characteristics for robust heterologous gene expression, including highly constitutive function with apparently reduced sensitivity to endogenous silencing in transient assays, without requiring p19 co‑expression under the tested conditions. These together provide simple, predictable tuning of gene expression levels, and the potential for very high expression levels of the target genes. In all these features, SES shows higher and more stable transcript levels than Cauliflower Mosaic Virus (CaMV) 35 S promoter-based constructs in our experimental setups. The functionality of plant SES was tested by expressing mCherry and three commercially relevant proteins: fungal glucose oxidase (GOX), protein A and human vascular endothelial growth factor 165 (VEGF16) from diverse organisms, supporting high-level accumulation of recombinant proteins. In addition, plant SES retains full functionality in both plant and fungal hosts, which makes this expression system a useful tool for a multitude of genetic engineering applications in other eukaryotic organisms. Plant synthetic expression system (SES) enables tunable transient expression of genes with high recombinant protein yields in Nicotiana benthamiana with the capability of inter-kingdom functionality.

IntroductionPlant cell culture (PCC) technology is currently being developed to produce plant foods partially decoupled from traditional agriculture practices. By now, the safety of the ingredients produced by PCC technology for food or nutritional purposes has to be tested.Materials and methodsIn this study, the oral safety and toxicity of two novel PCCs, scurvy grass (SG) (Cochlearia danica) and rowan (RW) (Sorbus aucuparia), and to characterize the macro- and micronutrient quality, including proteomic profiles, to identify potential allergens.ResultsNutritional composition analysis showed that both SG and RW PCCs profiles are comparable to other berry cell lines with a good amount of protein, dietary fibre and vitamins. Potential allergens were identified via proteomics based on structural similarity. The acute and subacute toxicity profiles of the PCC samples were evaluated based on OECD guidelines. For both PCCs, no deaths, behavioral changes, nor metabolic effects were observed at 2000 mg/kg. In the 28-day repeated oral exposure subacute toxicity study, no mortality or significant adverse clinical, hematological, or metabolic effects were observed for either SG or RW.DiscussionThese findings indicate that the no-observed-adverse-effect level (NOAEL) for both PCCs exceeds 2000 mg/kg. Overall, our findings indicate that the consumption of these PCCs could be considered safe and non-toxic, although further assessments on potential allergens and phytohormone accumulation are necessary to fully ensure consumer safety. This study highlights the oral safety of PCCs for consideration as a novel food ingredient and serve as a basis for evaluating toxicological impacts of PCCs.

Saffron apocarotenoids, including crocins, picrocrocin and safranal, are valuable metabolites with pharmaceutical and cosmetic potential. However, their natural plant sources are difficult to cultivate, which limits large-scale production. The identification of carotenoid cleavage dioxygenases (CCDs), which catalyse the first and most critical step in their biosynthesis, has enabled the production of these apocarotenoids in heterologous plant systems. In this study, we aimed to generate plant cell suspensions expressing Crocus sativus CCD2 and Gardenia jasminoides CCD4a, along with a bacterial phytoene synthase to enhance carotenoid biosynthesis and CsUGT93P1, which improves crocin stability. Transgenic cell suspensions were established from Nicotiana benthamiana plants and Nicotiana tabacum cv. BY-2 cells. In BY-2 cells expressing GjCCD4a, crocin accumulation reached 770 μg/g DW, which further increased upon methyl jasmonate elicitation. Remarkably, the BY-2 transgenic cells exhibited an 18,000-fold increase in β-cyclocitral content compared to wild-type cells. The best-performing N. benthamiana and BY-2 lines were successfully cultivated in wave bioreactors, demonstrating their potential for saffron apocarotenoid production. In the BY-2 bioreactor, apart from saffron apocarotenoids, phytoene and notably high amounts of lycopene were produced, adding value to the platform and indicating a remodelling of the carotenoid pathway. This study establishes the viability and lays the foundation for the scalable production of saffron apocarotenoids and carotenoids in plant cell suspensions.

With global demand for coffee on the rise, coffee cultivation faces multiple challenges, e.g. ensuring supply and reducing their environmental impact. To address these, alternatives to traditional coffee (TC) are proposed such as coffee substitutes and cell-cultured coffees (CC). To assess similarities and differences between traditional and alternative coffees, a three-step analysis is introduced, encompassing precursor analysis in unroasted, aroma in roasted, and sensory in brew. Unroasted CC has higher monosaccharide, lower amino acids and lipids content, and different organic acid profile. Chlorogenic acids, caffeine and trigonelline, were low to absent. Roasted CC revealed lower aroma compound intensity in S- and N-containing compounds, Strecker aldehydes and guaiacols. Nevertheless, furfurals, higher homologous aldehydes, and hydrocarbons were abundant. This provides a protocol for mapping out the performance of alternatives to TC and guiding their optimization.

Chemotaxonomy is the link between the state of the art in analytical chemistry and the systematic classification and phylogenetic analysis of biota. Although the characteristic secondary metabolites from diverse biotic sources have been used in pharmacology and biological systematics since the dawn of mankind, only comparatively recently established reproducible methods have allowed the precise identification and distinction of structurally similar compounds. Reliable, rapid screening methods like TLC (Thin Layer Chromatography) can be used to investigate sufficiently large numbers of samples for chemotaxonomic purposes. Using distribution patterns of mutually exclusive naphthoquinones, it is demonstrated in this review how a simple set of chemical data from a representative sample of closely related species in the sundew family (Droseraceae, Nepenthales) provides taxonomically and phylogenetically informative signal within the investigated group and beyond.

The so-called “craft beer revolution” has increased the demand for new styles of beers, often with new ingredients like flavour extracts. In recent years, synthetic biology has realized the production of a plethora of plant secondary metabolites in microbial hosts, which could provide an alternative source for these compounds. In this study, we selected a in situ flavour production approach for grape flavour addition. We used an O -methyl anthranilate (OmANT) producing laboratory Saccharomyces cerevisiae strain in co-fermentations with an industrial beer yeast strain WLP644. The laboratory strain provided an ease of genetic manipulation and the desirable properties of the WLP644 strain were not modified in this approach. In shake flasks, a 10:90 ratio of the yeasts produced grape flavoured beer with the yeast produced flavour compound in a range normally used for flavoured beverages. Hopped and unhopped beers were analysed by VTT’s trained sensory panel and with olfactory GC–MS. OmANT was successfully detected from the beers as a floral odour and flavour. Moreover, no off-flavours were detected and aroma profiles outside the grape flavour were rather similar. These results indicate that the co-fermentation principle is a suitable approach to change the flavour profiles of beers with a simple yeast strain drop-in approach.

Raspberry ketone has generated interest in recent years both as a flavor agent and as a health promoting supplement. Raspberry ketone can be synthesized chemically, but the value of a natural nonsynthetic product is among the most valuable flavor compounds on the market. Coumaroyl-coenzyme A (CoA) is the direct precursor for raspberry ketone but also an essential precursor for flavonoid and lignin biosynthesis in plants and therefore highly regulated. The synthetic fusion of 4-coumaric acid ligase (4CL) and benzalacetone synthase (BAS) enables the channeling of coumaroyl-CoA from the ligase to the synthase, proving to be a powerful tool in the production of raspberry ketone in both N. benthamiana and S. cerevisiae. To the best of our knowledge, the key pathway genes for raspberry ketone formation are transiently expressed in N. benthamiana for the first time in this study, producing over 30 μg/g of the compound. Our raspberry ketone producing yeast strains yielded up to 60 mg/L, which is the highest ever reported in yeast.

Veratrum (Melanthiaceae; Liliales) is a genus of perennial herbs known for the production of unique bioactive steroidal alkaloids. However, the biosynthesis of these compounds is incompletely understood because many of the downstream enzymatic steps have yet to be resolved. RNA-Seq is a powerful method that can be used to identify candidate genes involved in metabolic pathways by comparing the transcriptomes of metabolically active tissues to controls lacking the pathway of interest. The root and leaf transcriptomes of wild Veratrum maackii and Veratrum nigrum plants were sequenced and 437,820 clean reads were assembled into 203,912 unigenes, 47.67% of which were annotated. We identified 235 differentially expressed unigenes potentially involved in the synthesis of steroidal alkaloids. Twenty unigenes, including new candidate cytochrome P450 monooxygenases and transcription factors, were selected for validation by quantitative real-time PCR. Most candidate genes were expressed at higher levels in roots than leaves but showed a consistent profile across both species. Among the 20 unigenes putatively involved in the synthesis of steroidal alkaloids, 14 were already known. We identified three new CYP450 candidates (CYP76A2, CYP76B6 and CYP76AH1) and three new transcription factor candidates (ERF1A, bHLH13 and bHLH66). We propose that ERF1A, CYP90G1-1 and CYP76AH1 are specifically involved in the key steps of steroidal alkaloid biosynthesis in V. maackii roots. Our data represent the first cross-species analysis of steroidal alkaloid biosynthesis in the genus Veratrum and indicate that the metabolic properties of V. maackii and V. nigrum are broadly conserved despite their distinct alkaloid profiles.