Programmable transcriptional factors based on the CRISPR architecture are becoming commonly used in plants for endogenous gene regulation. In plants, a potent CRISPR tool for gene induction is the so-called dCasEV2.1 activation system, which has shown remarkable genome-wide specificity combined with a strong activation capacity. To explore the ability of dCasEV2.1 to act as a transactivator for orthogonal synthetic promoters, a collection of DNA parts was created (GB_SynP) for combinatorial synthetic promoter building. The collection includes (i) minimal promoter parts with the TATA box and 5'UTR regions, (ii) proximal parts containing single or multiple copies of the target sequence for the gRNA, thus functioning as regulatory cis boxes, and (iii) sequence-randomized distal parts that ensure the adequate length of the resulting promoter. A total of 35 promoters were assembled using the GB_SynP collection, showing in all cases minimal background and predictable activation levels depending on the proximal parts used. GB_SynP was also employed in a combinatorial expression analysis of an autoluminescence pathway in Nicotiana benthamiana, showing the value of this tool in extracting important biological information such as the determination of the limiting steps in an enzymatic pathway.

Plant-based bioproduction of insect sex pheromones has been proposed as an innovative strategy to increase the sustainability of pest control in agriculture. Here, we describe the engineering of transgenic plants producing (Z)-11-hexadecenol (Z11-16OH) and (Z)-11-hexadecenyl acetate (Z11-16OAc), two main volatile components in many Lepidoptera sex pheromone blends. We assembled multigene DNA constructs encoding the pheromone biosynthetic pathway and stably transformed them into Nicotiana benthamiana plants. The constructs contained the Amyelois transitella AtrΔ11 desaturase gene, the Helicoverpa armigera fatty acyl reductase HarFAR gene, and the Euonymus alatus diacylglycerol acetyltransferase EaDAct gene in different configurations. All the pheromone-producing plants showed dwarf phenotypes, the severity of which correlated with pheromone levels. All but one of the recovered lines produced high levels of Z11-16OH, but very low levels of Z11-16OAc, probably as a result of recurrent truncations at the level of the EaDAct gene. Only one plant line (SxPv1.2) was recovered that harboured an intact pheromone pathway and which produced moderate levels of Z11-16OAc (11.8μgg^-1 FW) and high levels of Z11-16OH (111.4μgg^-1). Z11-16OAc production was accompanied in SxPv1.2 by a partial recovery of the dwarf phenotype. SxPv1.2 was used to estimate the rates of volatile pheromone release, which resulted in 8.48ngg^-1 FW per day for Z11-16OH and 9.44ngg^-1 FW per day for Z11-16OAc. Our results suggest that pheromone release acts as a limiting factor in pheromone biodispenser strategies and establish a roadmap for biotechnological improvements.

Summary: Previous work has demonstrated that plants can be used as production platforms for molecules used in health, medicine, and agriculture. Production has been exemplified in both stable transgenic plants and using transient expression strategies. In particular, species of Nicotiana have been engineered to produce a range of useful molecules, including insect sex pheromones, which are valued for species‐specific control of agricultural pests. To date, most studies have relied on strong constitutive expression of all pathway genes. However, work in microbes has demonstrated that yields can be improved by controlling and balancing gene expression. Synthetic regulatory elements that provide control over the timing and levels of gene expression are therefore useful for maximizing yields from heterologous biosynthetic pathways. In this study, we demonstrate the use of pathway engineering and synthetic genetic elements for controlling the timing and levels of production of Lepidopteran sex pheromones in Nicotiana benthamiana. We demonstrate that copper can be used as a low‐cost molecule for tightly regulated inducible expression. Further, we show how construct architecture influences relative gene expression and, consequently, product yields in multigene constructs. We compare a number of synthetic orthogonal regulatory elements and demonstrate maximal yields from constructs in which expression is mediated by dCas9‐based synthetic transcriptional activators. The approaches demonstrated here provide new insights into the heterologous reconstruction of metabolic pathways in plants.

Plant exosomes exhibit high stability and easy absorption, and have emerged as promising bioactive tools due to their potential health benefits and biomedical applications. Saffron tepals contain abundant metabolites with potential therapeutic properties and were used for exosome extraction by ultracentrifugation and gradient purification. The exosomes showed an average particle size of 151.5 ± 79.6 nm and exhibited a spherical morphology. Five well-conserved miRNAs—miR157, miR166, miR168, miR396, and miR398—were identified in the exosomes, which are involved in the coordination of growth and physiological plant responses with endogenous and environmental abiotic and biotic signals, and their potential targets in mammals are upregulated in specific cancer types and associated with inflammation. Proteome analysis revealed an enrichment of proteasome proteins, ribosomal proteins, and proteins involved in the cytoskeleton, transport across the membrane (ABC transporters), and vesicle trafficking (RAB GTPases, TM9SF and Coatomer subunits). Metabolite analyses showed mainly anthocyanins. The exosomes have selective stimulatory activity on macrophages, increasing the expression of surface molecules (CD80 and CD86), and cytokines (IL-1β, IL-6, and TNF-α), but not the levels of IL-10. Overall, these results indicated that saffron flowers are an effective and abundant source of exosomes as new nanomedicines for human health.

Summary: Fungal antifungal proteins (AFPs) have attracted attention as novel biofungicides. Their exploitation requires safe and cost‐effective producing biofactories. Previously, Penicillium chrysogenum and Penicillium digitatum produced recombinant AFPs with the use of a P. chrysogenum ‐based expression system that consisted of the paf gene promoter, signal peptide (SP)‐pro sequence and terminator. Here, the regulatory elements of the afpA gene encoding the highly produced PeAfpA from Penicillium expansum were developed as an expression system for AFP production through the FungalBraid platform. The afpA cassette was tested to produce PeAfpA and P. digitatum PdAfpB in P. chrysogenum and P. digitatum , and its efficiency was compared to that of the paf cassette. Recombinant PeAfpA production was only achieved using the afpA cassette, being P. chrysogenum a more efficient biofactory than P. digitatum . Conversely, P. chrysogenum only produced PdAfpB under the control of the paf cassette. In P. digitatum , both expression systems allowed PdAfpB production, with the paf cassette resulting in higher protein yields. Interestingly, these results did not correlate with the performance of both promoters in a luciferase reporter system. In conclusion, AFP production is a complex outcome that depends on the regulatory sequences driving afp expression, the fungal biofactory and the AFP sequence.

Agaricus bisporus (white button mushroom), Pleurotus ostreatus (oyster mushroom), and Lentinula edodes (shiitake mushroom) are the most cultivated mushrooms in the Spanish region of Castilla‐La Mancha. The increasing production of these mushrooms has created opportunities for sustainable utilization of their by‐products such as undersized mushrooms, stalks, and stems. This study evaluated the antioxidant, prebiotic, and antimicrobial properties of aqueous, ethanolic, and alkaline extracts derived from these by‐products. Aqueous extracts from oyster mushroom, rich in glucose‐polysaccharides (59.4 ± 0.4% mushroom dw), promoted the growth and lactic acid production of the probiotic bacteria Lactocaseibacillus casei and Lactoplantibacillus plantarum . Shiitake extracts demonstrated antimicrobial activity against Pseudomonas aeruginosa , Escherichia coli, and Salmonella enterica (minimal inhibitory concentrations: 15, 7.5, and 7.5 mg/mL, respectively) and stimulated the growth of lactic acid bacteria at low concentrations (1.875 mg/mL) but inhibiting them at higher concentrations. Extracts from white button mushrooms exhibited the strongest antioxidant activity, particularly ethanolic extracts rich in phenolic compounds (0.015 mg gallic acid/mg extract). These results highlight the potential of extracts of mushroom by‐products as organic sources of antioxidants, antimicrobials, and prebiotics, creating new avenues for food product development. Reusing these by‐products could encourage sustainability and assist the mushroom sector in implementing zero‐waste and circular economy methods.