Strategies, Achievements, and Potential Challenges of Plant and Microbial Chassis in the Biosynthesis of Plant Secondary Metabolites.

Diverse secondary metabolites in plants, with their rich biological activities, have long been important sources for human medicine, food additives, pesticides, etc. However, the large-scale cultivation of host plants consumes land resources and is susceptible to pest and disease problems. Additionally, the multi-step and demanding nature of chemical synthesis adds to production costs, limiting their widespread application. In vitro cultivation and the metabolic engineering of plants have significantly enhanced the synthesis of secondary metabolites with successful industrial production cases. As synthetic biology advances, more research is focusing on heterologous synthesis using microorganisms. This review provides a comprehensive comparison between these two chassis, evaluating their performance in the synthesis of various types of secondary metabolites from the perspectives of yield and strategies. It also discusses the challenges they face and offers insights into future efforts and directions.

AGC kinases OXI1 and AGC2-2 regulate camalexin secretion and disease resistance by phosphorylating transporter PDR6.

Plant transporters regulating the distribution of secondary metabolites play critical roles in defending against pathogens, insects, and interacting with beneficial microbes. The phosphorylation of these transporters can alter their activity, stability, and intracellular protein trafficking. However, the regulatory mechanism underlying this modification remains elusive. In this study, we discovered two orthologs of mammalian PKA, PKG, and PKC (AGC) kinases, oxidative signal-inducible 1 (OXI1) and its closest homologue, AGC subclass 2 member 2 (AGC2-2; 75% amino acid sequence identity with OXI1), associated with the extracellular secretion of camalexin and Arabidopsis (Arabidopsis thaliana) resistance to Pseudomonas syringae, and Botrytis cinerea. These kinases can undergo in vitro kinase reactions with three pleiotropic drug resistance (PDR) transporters: PDR6, PDR8, and PDR12. Moreover, our investigation confirmed PDR6 interaction with OXI1 and AGC2-2. By performing LC-MS/MS and parallel reaction monitoring, we identified the phosphorylation sites on PDR6 targeted by these kinases. Notably, chitin-induced PDR6 phosphorylation at specific residues, namely S31, S33, S827, and T832. Additional insights emerged by expressing dephosphorylated PDR6 variants in a pdr6 mutant background, revealing that the target residues S31, S33, and S827 promote PDR6 efflux activity, while T832 potentially contributes to PDR6 stability within the plasma membrane. The findings of this study elucidate partial mechanisms involved in the activity regulation of PDR-type transporters, providing valuable insights for their potential application in future plant breeding endeavors.

Enhancing tomato disease resistance through endogenous antifungal proteins and introduced nematode-targeting dsRNA of biocontrol agent Bacillus velezensis HS-3.

BACKGROUND: As a type of biological control agent (BCA), Bacillus velezensis possesses the efficacy of inhibiting pathogenic microorganisms, promoting plant growth, and overcoming continuous cropping obstacles (CCOs). However, there is limited reporting on the optimization of the cultivation conditions for such biocontrol agents and their role as double-stranded RNA (dsRNA) delivery vectors. RESULTS: In this study, a Bacillus velezensis strain HS-3 was isolated from the root zone of tomato plants with in vitro anti-Botrytis cinerea activity. The investigation into active compounds revealed that HS-3 predominantly employs proteins with molecular weights greater than 3 kDa for its antifungal activity. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified various proteases and chitosanase, further suggesting that HS-3 most likely employs these enzymes to degrade fungal cell walls for its antifungal effect. To optimize the production of extracellular proteins, fermentation parameters for HS-3 were systematically optimized, leading to an optimized medium (OP-M). HS-3 cultured in OP-M demonstrated enhanced capacity to assist tomato plants in withstanding CCOs. However, the presence of excessive nematodes in diseased soil resulted in the disease severity index (DSI) remaining high. An RNA interference mechanism was further introduced to HS-3, targeting the nematode tyrosine phosphatase (TP) gene. Ultimately, HS-3 expressing dsRNA of TP in OP-M effectively assisted tomatoes in mitigating CCOs, reducing DSI to 2.2% and 17.8% of the control after 45 and 90 days of growth, respectively. CONCLUSION: The advantages of Bacillus velezensis in crop disease management and the mitigation of CCOs become even more pronounced when utilizing both optimized levels of endogenous enzymes and introduced nematode-targeting dsRNA. © 2024 Society of Chemical Industry.

Chloroplast display of subunit vaccines and their efficacy via oral administration.

As a safe and natural "capsule," plants have several advantages over mammals and microorganisms for the production of oral vaccines. In this study, we innovatively utilized the transmembrane region of the pea Translocase of chloroplast 34 (TOC34) protein to display two subunit vaccines, capsid protein VP2 of Porcine parvovirus (PPV) and the heat-labile enterotoxin B (LTB) of Escherichia coli, on the surface of chloroplasts. Unlike microbial display techniques, chloroplast display circumvents antigen degradation in the stomach while retaining the size characteristic of microorganisms. Additionally, a co-expressed peptide adjuvant, antimicrobial peptide protegin-1 (PG1), was used to enhance the strength of oral immunization. Immunohistochemistry and trypsin digestion of chloroplast surface proteins confirmed the successful localization of both antigens on the chloroplast surface. In stable transgenic tobacco plants, the expression level of VP2-TOC34 ranged from 0.21 to 6.83 µg/g FW, while LTB-TOC34 ranged from 2.42 to 10.04 µg/g FW. By contrasting the digestive characteristics of plant materials with different particle sizes, it was observed that plant materials with diameters around 1 mm exhibited more prominent advantages in terms of chloroplast release and antigen exposure compared to both larger and smaller particles. Oral immunization resulted in significantly increased levels of specific IgG and secretory IgA in the mice compared to the control, with similar effects observed between the groups receiving oral immunization alone and those receiving a combination of initial injection and subsequent oral immunization. Challenge experiments further demonstrated the effective protection against infection in mice using this approach. These findings highlight the potential of chloroplast display technology for the development of effective oral vaccines.

Identification and Characterization of Two Novel Extracellular ß-Glucanases from Chaetomium globosum against Fusarium sporotrichioides.

Chaetomium globosum can inhibit the growth of fusarium by means of their extracellular proteins. Two novel ß-glucanases, designated Cgglu17A and Cgglu16B, were separated from the supernatant of C. globosum W7 and verified to have the ability to hydrolyze cell walls of Fusarium sporotrichioides MLS-19. Cgglu17A (397 amino acids) was classified as glycoside hydrolase family 17 while Cgglu16B belongs to the family16 (284 amino acids). Recombinant protein Cgglu17A was successfully expressed in Escherichia coli, and the enzymes were purified by affinity chromatography. Maximum activity of Cgglu17A appeared at the pH 5.5 and temperature 50 °C, but Cgglu16B shows the maximum activity at the pH 5.0 and temperature 50 °C. Most of heavy metal ions had inhibition effect on the two enzymes, but Cgglu17A and Cgglu16B were respectively activated by Ba2+ and Mn2+. Cgglu17A exhibited high substrate specificity, almost only catalyzing the cleavage of ß-1,3-glycosidic bond, in various polysaccharose, to liberate glucose. However, Cgglu16B showed high catalytic activities to both ß-1,3-glycosidic and ß-1,3-1,4-glycosidic bonds. Cgglu17A was an exo-glucanase, but Cgglu16B was an endo-glucanase based on hydrolytic properties assay. Both of two enzymes showed potential antifungal activity, and the synergistic effect was observed in the germination experiment of pathogenic fungus. In conclusion, Cgglu17A (exo-1,3-ß-glucanase) and Cgglu16B (endo-1,3(4)-ß-glucanase) were confirmed to play a key role in the process of C. globosum controlling fusarium and have potential application value on industry and agriculture for the first time.

Identification and functional characterization of a PDR transporter in Tripterygium wilfordii Hook.f. that mediates the efflux of triptolide.

KEY MESSAGE: TwPDR1, a PDR transporter from Tripterygium wilfordii Hook.f., was proved to efflux triptolide and its stability could be enhanced by A1033T mutation. Triptolide, an abietane-type diterpene in Tripterygium wilfordii Hook.f., possesses many pharmacological activities. However, triptolide is in short supply and very expensive because it is present at low amounts in natural plants and lack alternative production methods. Transporter engineering, which increases the extracellular secretion of secondary metabolites in in vitro culture systems, is an effective strategy in metabolic engineering but is rarely reported. In this study, TwPDR1, a pleiotropic drug resistance-type ATP binding cassette transporter, was identified as the best efflux pump candidate for diterpenoids through bioinformatics analysis. TwPDR1 was located in the plasma membrane, highly expressed in adventitious roots, and induced by methyl jasmonate. The triptolide efflux function of TwPDR1 was confirmed by transient expression in tobacco BY-2 cells and by downregulation via RNA interference in the native host. However, the overexpression of TwPDR1 had a limited effect on the secretion of triptolide. As shown by previous studies, a single amino acid mutation might increase the abundance of TwPDR1 by increasing protein stability. We identified the A1033 residue in TwPDR1 by sequence alignment and confirmed that A1033T mutation could increase the expression of TwPDR1 and result in the higher release ratio of triptolide (78.8%) of the mutants than that of control (60.1%). The identification and functional characterization of TwPDR1 will not only provide candidate gene material for the metabolic engineering of triptolide but also guide other transporter engineering researches in the future.

Effects of food matrix and probiotics on the bioavailability of curcumin in different nanoformulations.

BACKGROUND: Nanoparticles can improve the bioavailability of bioactive compounds. Concomitant intake of food can affect pharmacokinetic profiles by altering dissolution, absorption, metabolism, and elimination behavior. Studies on the effects of food and its supplements on the bioavailability of bioactives in nanoformulations are few. In this study, the effects of typical food (milk, sugar, high-fat diet, and regular kibble) and a widely consumed probiotic [Bifidobacterium lactis Bb-12® (Bb-12)] on the bioavailability of curcumin in four formulations [simply suspended curcumin (Cur-SS) and curcumin in nanoemulsions (Cur-NEs), in single-walled carbon nanotubes (Cur-SWNTs), and in nanostructured lipid carriers (Cur-NLCs)] were investigated. RESULTS: Fasting treatment and sugar co-ingestion can significantly enhance the bioavailability of curcumin in Cur-NEs and Cur-SWNTs, respectively. Compared with the fasting treatment, co-ingestion with regular kibble reduced the absorption of curcumin in Cur-NEs and Cur-SWNTs. Ingesting milk along with Cur-NE is also not recommended. The mechanisms behind these phenomena were briefly discussed. This study revealed for the first time that the intestinal colonization of Bb-12 reduces the bioavailability of curcumin and this reduction can be attenuated by nanoformulations SWNTs and NLCs, but not NEs. The reason for this difference was the protective effects of the former two nanoformulations against curcumin degradation by Bb-12 according to in vitro experiments. CONCLUSION: Dietary status (including supplementary probiotics) can dramatically influence the bioavailability of curcumin in nanoformulations. © 2021 Society of Chemical Industry.

A class I TGA transcription factor from Tripterygium wilfordii Hook.f. modulates the biosynthesis of secondary metabolites in both native and heterologous hosts.

Class I TGA transcription factors (TFs) are known to participate in plant resistance responses, however, their regulatory functions in the biosynthesis of secondary metabolites were rarely revealed. In this study, a class I TGA TF, TwTGA1, from Tripterygium wilfordii Hook.f. was cloned and characterized. Overexpression of TwTGA1 in T. wilfordii Hook.f. cells increased the production of triptolide and two sesquiterpene pyridine alkaloids, which was further enhanced by methyl jasmonate (MeJA) treatment. RNA interference of TwTGA1 showed no significant effects on the production of these metabolites, indicating the existence of other TGA partner(s) with overlapping functions. Heterologous expression of TwTGA1 in tobacco By-2 cells promoted the biosynthesis of pyridine alkaloids. Under the elicitation of MeJA, the contents of nonpyrrolidine alkaloids further increased but not for nicotine. TwTGA1 could induce the expression of Putrescine N-methyltransferase (PMT) and N-methylputrescine oxidase 1 (MPO1) through binding to their promoters. Finally, transient expression of TwTGA1 in leaves of Catharanthus roseus changed both the profiles of vinca alkaloids (increased contents of serpentine and catharanthine, but decreased that of vinblastine) and the expressions of biosynthesis-related genes. The metabolic and transcriptional data indicated a relationship between jasmonic acid signaling pathway and the functions of TwTGA1.

Targeting Pyruvate Kinase M2 and Hexokinase II, Pachymic Acid Impairs Glucose Metabolism and Induces Mitochondrial Apoptosis.

Pachymic acid (PA), a triterpenoid from Poria cocos, has various pharmacological effects, including anti-inflammatory, anti-cancer, anti-aging, and insulin-like properties. PA has gained considerable research attention, but the mechanism of its anti-cancer effects remains unclear. In this study, pyruvate kinase M2 (PKM2) was discovered as a PA target via the drug affinity responsive target stability. Molecular docking and enzyme assay revealed that PA is a competing activator of PKM2, and mimics the natural activator, fructose-1,6-bisphosphate. PKM2 activation should augment the flux of glycolysis. However, decreased glucose uptake and lactate production after PA treatment was observed in SK-BR-3 breast carcinoma cells, indicating a blockage or downregulation of glycolysis. The potential of previously reported triterpenoids in blocking hexokinase II (HK2) activity inspired us to investigate the inhibition effect of PA on HK2 activity. Molecular docking and enzyme assay confirmed that PA was an inhibitor of HK2, with an IC50 of 5.01 µM. The possible consequences of glycometabolic regulation by PA, such as dissociation of HK2 from the mitochondria, release of mitochondrial cytochrome (Cyt) c, depletion of ATP, and generation of reactive oxygen species, were further validated. Furthermore, the details of the possible linkage of targeting PKM2 and HK2 with previously reported actions of PA were discussed. The results of our study provided valuable information on the anti-cancer mechanisms of PA.

Efficiency and Safety Assurance of Six Fungicides Applied on Postharvest Cabbages Stored in a Natural Environment.

Postharvest disease is a major factor in the limited shelf life of many fruits and vegetables, and it is often managed using fungicidal spraying or soaking. In this study, we first tested the efficiency of six common fungicides on postharvest head cabbage ( Brassica oleracea var. capitata) against Botrytis cinerea. Afterward, the elimination abilities of these six fungicides on different layers of cabbage heads were examined, and the effects of the household processes on residue removal were evaluated. Results showed that very low contents of residues reached the inner layers and that peeling the three outmost leaves of cabbage could eliminate most of the investigated fungicides. All six fungicides disappeared during washing, stir-frying, or boiling, among which cyprodinil was the easiest to be eliminated. Furthermore, the combined processes reduced the residues below the limits of quantification for all six investigated fungicides, even after 2 days of spraying.

Cadmium-induced serotonergic neuron and reproduction damages conferred lethality in the nematode Caenorhabditis elegans.

Cadmium is a ubiquitous environmental toxicant. The use of Caenorhabditis elegans as a model for monitoring cadmium exposure has revealed several conserved signaling pathways. However, little is known about the killing process during lethality assay. In the present study, we investigated the effects serotonergic neuronal and reproductive damages on cadmium exposure in C. elegans. We found that sterile hermaphrodites, males and worms that passed reproduction span presented high cadmium resistance compared to those of young adults. The results demonstrated that reproduction process other than reproduction capacity conferred cadmium sensitivity. Cadmium exposure resulted in high ratio bagging phenotype, which was a severe reproductive deficit with embryos hatched internally that could cause worms to die early. The mechanism of bagging formation was ascribed to cadmium-induced egg laying deficiency that led embryos to retain and hatch in uterus. The addition of serotonin and imipramine promoted egg laying and thereby increased cadmium resistance. The results demonstrated that vulval muscles responsible for egg laying were still functional, while the serotonergic hermaphrodite specific neurons might be dysfunctional under cadmium exposure. Cadmium exposure resulted in shrinkage of serotonergic neuronal body and reduced expressions of tryptophan hydroxylase, the key enzyme for serotonin synthesis. The protection of serotonergic neuron through transient thermal preconditioning improved survival rate. In conclusion, our study demonstrated that damages of serotonergic neurons and reproduction conferred to cadmium-induced lethality.

A MDR transporter contributes to the different extracellular production of sesquiterpene pyridine alkaloids between adventitious root and hairy root liquid cultures of Tripterygium wilfordii Hook.f.

KEY MESSAGE: TwMDR1 transports sesquiterpene pyridine alkaloids, wilforine and wilforgine, into the hairy roots of T. wilfordii Hook.f. resulting in low secretion ratio of alkaloids. Hairy roots (HRs) exhibit high growth rate and biochemical and genetic stability. However, varying secondary metabolites in HR liquid cultures mainly remain in root tissues, and this condition may affect cell growth and cause inconvenience in downstream extraction. Studies pay less attention to adventitious root (AR) liquid cultures though release ratio of some metabolites in AR liquid cultures is significantly higher than that of HR. In Tripterygium wilfordii Hook.f., release ratio of wilforine in AR liquid cultures reached 92.75 and 13.32% in HR on day 15 of culture. To explore potential roles of transporters in this phenomenon, we cloned and functionally identified a multidrug resistance (MDR) transporter, TwMDR1, which shows high expression levels in HRs and is correlated to transmembrane transportation of alkaloids. Nicotiana tabacum cells with overexpressed TwMDR1 efficiently transported wilforine and wilforgine in an inward direction. To further prove the feasibility of genetically engineered TwMDR1 and improve alkaloid production, we performed a transient RNAi experiment on TwMDR1 in T. wilfordii Hook.f. suspension cells. Results indicated that release ratios of wilforine and wilforgine increased by 1.94- and 1.64-folds compared with that of the control group, respectively. This study provides bases for future studies that aim at increasing secretion ratios of alkaloids in root liquid cultures in vitro.

Streptomyces kanasensis sp. nov., an Antiviral Glycoprotein Producing Actinomycete Isolated from Forest Soil Around Kanas Lake of China.

A filamentous actinomycete, designated strain ZX01(T), was isolated from forest soil around Kanas Lake of China. A polyphasic taxonomic study was carried out to establish the status of strain ZX01(T). Chemical and morphological properties of the isolate were similar to those of species of the genus Streptomyces. Analysis of the almost complete 16S rRNA gene sequence placed strain ZX01(T) in the genus Streptomyces where it formed a distinct phyletic line with recognized species of this genus. The strain exhibited the highest sequence similarities to Streptomyces lavendofoliae NBRC 12882(T) (99.1%), S. luridus NBRC 12793(T) (99.0%), S. lavendulocolor NBRC 12881(T) (99.0%), S. gobitricini NBRC 15419(T) (99.0%), and S. roseolilacinus NBRC 12815(T) (98.9%). Low DNA-DNA relatedness values of 54.0, 50.0, 60.0, 66.7, and 50.4%, respectively, were found between strain ZX01(T) and corresponding strains above. A number of phenotypic properties also enabled the isolate to be differentiated from related species of the genus Streptomyces. Therefore, it is proposed that strain ZX01(T) should be classified as the type strain of a novel species in the genus Streptomyces, Streptomyces kanasensis sp. nov. The type strain is ZX01(T) (= CGMCC 4893(T) =JCM 30232(T)).

Effects of plant stress signal molecules on the production of wilforgine in an endophytic actinomycete isolated from Tripterygium wilfordii Hook.f.

The endophytic actinomycete F4-20 was isolated from Tripterygium wilfordii Hook.f. and was confirmed to produce wilforgine, a secondary metabolite discovered in its host. F4-20 showed a close phylogenetic relationship to Streptomyces species. To seek elicitors that may enhance the production of wilforgine in F4-20, four plant stress molecules were applied to the in vitro liquid cultures. Results showed that methyl jasmonate (MeJA), salicylic acid (SA), and hydrogen peroxide (H2O2) inhibited bacterial growth, whereas glutathione (GSH) treatment significantly increased bacterial growth. The wilforgine contents in the mycelia of F4-20 were reduced by MeJA and GSH but were induced by SA and H2O2. When added in the end of the culture period (7 day), 1 mM SA and 5 mM H2O2 resulted in 69.35 ± 1.71 and 71.80 ± 3.35 µg/g DW of wilforgine production, 1.55 and 1.60 fold to that of control (44.83 ± 1.35 µg/g DW), respectively. Though this improved production was about 6.5 times lower than that of the natural root (454.00 µg/g dry root bark), it provided an alternative method for the production of valuable plant secondary metabolites.

Establishment of Tripterygium wilfordii Hook. f. Hairy root culture and optimization of its culture conditions for the production of triptolide and wilforine.

In order to solve the shortage of natural Tripterygium wilfordii Hook. f. plant resource for the production of the important secondary metabolites triptolide and wilforine, hairy roots were induced from its root calli by Agrobacterium rhizogenes. Induced hairy roots not only could be maintained and grown well in hormone-free half-strength Murashige and Skoog medium but also could produce sufficient amounts of both triptolide and wilforine. Although hairy roots produced approximately 15% less triptolide than adventitious roots and 10% less wilforine than naturally grown roots, they could grow fast and could be a suitable system for producing both secondary metabolites compared with other tissues. Addition of 50 micrometer methyl jasmonate (MeJA) could slightly affect hairy root growth, but dramatically stimulated the production of both triptolide and wilforine, whereas 50 micrometer salicylic acid had no apparent effect on hairy root growth with slightly stimulatory effects on the production of both secondary metabolites. Addition of precursor nicotinic acid, isoleucine, or aspartic acid at the concentration of 500 micrometer had varying effects on hairy root growth, but none of them had stimulatory effects on triptolide production, and only the former two had slightly beneficial effects on wilforine production. The majority of triptolide produced was secreted into the medium, whereas most of the produced wilforine was retained inside of hairy roots. Our studies provide a promising way to produce triptolide and wilforine in T. wilfordii hairy root cultures combined with MeJA treatment.