Precursor Feeding Research Articles

An improved Nicotiana benthamiana bioproduction chassis provides novel insights into nicotine biosynthesis.

The model plant Nicotiana benthamiana is an increasingly attractive organism for the production of high-value, biologically active molecules. However, N. benthamiana accumulates high levels of pyridine alkaloids, in particular nicotine, which complicates the downstream purification processes. Here, we report a new assembly of theN. benthamiana genome as well as the generation of low-nicotine lines by CRISPR/Cas9-based inactivation of berberine bridge enzyme-like proteins (BBLs). Triple as well as quintuple mutants accumulated three to four times less nicotine than the respective control lines. The availability of lines without functional BBLs allowed us to probe their catalytic role in nicotine biosynthesis, which has remained obscure. Notably, chiral analysis revealed that the enantiomeric purity of nicotine was fully lost in the quintuple mutants. In addition, precursor feeding experiments showed that these mutants cannot facilitate the specific loss of C6 hydrogen that characterizes natural nicotine biosynthesis. Our work delivers an improved N. benthamiana chassis for bioproduction and uncovers the crucial role of BBLs in the stereoselectivity of nicotine biosynthesis.

Developments in biotechnological tools and techniques for production of reserpine.

In the quest for novel medications, researchers have kept on studying nature to unearth beneficial plant species with medicinal qualities that may cure various diseases and disorders. These medicinal plants produce different bioactive secondary metabolites with immense therapeutic importance. One such valuable secondary metabolite, reserpine (C33H40N2O9), has been used for centuries to cure various ailments like hypertension, cardiovascular diseases, neurological diseases, breast cancer, and human promyelocytic leukaemia. Rauvolfia spp. (family Apocynaceae) is an essential reservoir of this reserpine. The current review thoroughly covers different non-conventional or in vitro-mediated biotechnological methods adopted for pilot-scale as well as large-scale production of reserpine from Rauvolfia spp., including techniques like multiple shoot culture, callus culture, cell suspension culture, precursor feeding, elicitation, synthetic seed production, scale-up via bioreactor, and hairy root culture. This review further analyses the unexplored and cutting-edge biotechnological tools and techniques to alleviate reserpine production. KEY POINTS: • Reserpine, a vital indole alkaloid from Rauvolfia spp., has been used for centuries to cure several ailments. • Overview of biosynthetic pathways and biotechnological applications for enhanced production of reserpine. • Probes the research gaps and proposes novel alternative techniques to meet the pharmaceutical industry's need for reserpine while reducing the over-exploitation of natural resources.

A Type I/Type III PKS Hybrid Generates Cinnamomycin A-D.

3,5-Dihydroxybenzoic acid (3,5-DHBA), biosynthesized by type III PKS and tailoring enzymes, is an unconventional starter unit for bacterial type I PKS. Genome mining of 3,5-DHBA-specific biosynthetic gene clusters could lead to discovering new type I/type III PKS hybrids. Herein, we report the discovery and characterization of atypical compounds, namely cinnamomycin A-D, exhibiting selective antiproliferative activity. The biosynthetic pathway of cinnamomycins was proposed based on genetic manipulation, enzymatic reaction, and precursor feeding.

Insights into chlorogenic acids' efficient biosynthesis through Carthamus tinctorius cell suspension cultures and their potential mechanism as α-glucosidase inhibitors

Carthamus tinctorius is commonly used as food and medicine because it contains various bioactive components, particularly chlorogenic acids (CGAs). However, few research works focused on the development and application of C. tinctorius-derived CGAs. In this study, the technology system for efficient CGAs biosynthesis through C. tinctorius cells was successfully developed, and the feasibility of C. tinctorius cells-derived CGAs as α-glucosidase inhibitors was evaluated. At first, the inflorescence explant was used to induce C. tinctorius embryogenic callus (induction rate: 95%), in which a total of 10 CGAs, including rare caffeoyl and feruloyl-substituted derivatives, were identified and quantified by liquid chromatography-mass spectrometry. Secondly, an excellent C. tinctorius cell suspension culture was established. To further promote CGAs biosynthesis, parameters of elicitation combined with precursor feeding were optimized through Plackett-Burman’s Design and Central Composite Design. Under the optimal condition [cinnamic acid: 283.05 μM, methyl jasmonate: 80.98 μM and yeast extract: 666.66 mg·L−1], CGAs content could reach up to 103.234 mg·g−1 (DW), which was 2.55 times that of the cells in the initial condition. The CGAs production first time reached the highest level among plant-cultured cells in the available literature. At last, the inhibition activity test results indicated good inhibitory activities of C. tinctorius cells-derived CGAs against α-glucosidase (IC50: 0.15–1.38 g·L−1), especially di- and tri-acyl CGAs. Moreover, molecular docking simulation showed that these CGAs could interact with the active site pocket of α-glucosidase to thereby achieve the inhibitory activity. Therefore, these results provided a solid foundation for the large-scale production of CGAs and revealed the application prospects of CGAs in functional food and pharmaceutical industries as α-glucosidase inhibitor.

Micropropagation and elicited production of triterpenoid saponin glycosides and stigmasterol via precursor and elicitor feeding in Bacopa floribunda (R.Br.) Wettst.-A potential nootropic herb.

Bacopa floribunda (Family: Plantaginaceae) is used in folklore medicines for the management of cognitive dysfunction. It has nootropic, antiaging, anti-inflammatory, anti-cholinesterase, and antioxidant properties. We developed an efficient and reproducible protocol for in vitro propagation of B. floribunda using the nodal explants. We assessed the effects of Murashige and Skoog (MS) medium fortified with various plant growth regulatory substances (PGRs), a precursor, and elicitors and their optimal combinations on regeneration and production of total saponins, triterpenoid saponin glycosides (bacoside A3, bacopaside X, bacopaside II, and bacosaponin C), and stigmasterol content in in vitro grown biomass of B. floribunda. The growth of the shoots and roots was stimulated by MS + 2.0 mg/l BAP + 2.0 mg/l KIN and MS + 0.5 mg/l IAA + 0.5 mg/l IBA + 1.0 mg/l NAA, respectively. After 10 weeks of acclimatization, plantlets of B. floribunda had a survival rate of 95%. The highest total saponin content (35.95 ± 0.022 mg DE/g DW) was noted in the treatment of MS + 2.0 mg/l BAP + 1.5 μM SQ. Similarly, total triterpenoid saponin glycosides and stigmasterol were found maximum in biomass derived from MS + 2.0 mg/l BAP + 1.5 μM SQ and MS + 2.0 mg/l BAP, respectively. At the same treatments, bacoside A3 (1.01 ± 0.195 mg/g DW), bacopaside II (43.62 ± 0.657 mg/g DW), bacopaside X (1.23 ± 0.570 mg/g DW), bacosaponin C (0.19 ± 0.195 mg/g DW), and stigmasterol (7.69 ± 0.102 mg/g DW) were reported. The present findings will help to highlight B. floribunda as a potent memory-enhancing herb, and in future also, it could be a potential substitute to B. monnieri. The current work is the first to describe the micropropagation and the elicited production of bioactive metabolites from the in vitro grown biomass of B. floribunda. In addition, further research is needed on production of bioactives, their pharmacological effects, and the elicited production using callus, cell suspension, and hairy root cultures.

Biotechnological Approaches to Producing Natural Antioxidants: Anti-Ageing and Skin Longevity Prospects

Plants are the main source of bioactive compounds that can be used for the formulation of cosmetic products. Plant extracts have numerous proven health benefits, among which are anti-ageing and skin-care properties. However, with the increased demand for plant-derived cosmetic products, there is a crucial prerequisite for establishing alternative approaches to conventional methods to ensure sufficient biomass for sustainable production. Plant tissue culture techniques, such as in vitro root cultures, micropropagation, or callogenesis, offer the possibility to produce considerable amounts of bioactive compounds independent of external factors that may influence their production. This production can also be significantly increased with the implementation of other biotechnological approaches such as elicitation, metabolic engineering, precursor and/or nutrient feeding, immobilization, and permeabilization. This work aimed to evaluate the potential of biotechnological tools for producing bioactive compounds, with a focus on bioactive compounds with anti-ageing properties, which can be used for the development of green-label cosmeceutical products. In addition, some examples demonstrating the use of plant tissue culture techniques to produce high-value bioactive ingredients for cosmeceutical applications are also addressed, showing the importance of these tools and approaches for the sustainable production of plant-derived cosmetic products.

Improving ATP availability by sod1 deletion with a strategy of precursor feeding enhanced S-adenosyl-L-methionine accumulation in Saccharomyces cerevisiae

S-adenosyl-L-methionine (SAM), used in diverse pharmaceutical applications, was biosynthesized from L-methionine (L-met) and adenosine triphosphate (ATP). This study aims to increase the accumulation of SAM in Saccharomyces cerevisiae by promoting ATP availability. Strain ΔSOD1 was obtained from the parent strain WT15–33 (CCTCC M 2021915) by deleting gene sod1, which improved the supply of ATP. The SAM content in strain ΔSOD1 exhibited a 22.3% improvement compared to the parent strain, which reached 93.6 mg g−1. The transformation of NADH (reduced nicotinamide adenine dinucleotide) and the relative expression of ATPase essential genes were investigated, respectively. The results showed that the lack of gene sod1 benefited the generation of ATP, which positively regulated the synthesis of SAM. Besides that, the production of SAM was further enhanced by improving substrate assimilation. With the infusion of 1.44 g L−1L-met and 0.60 g L−1 adenosine at 24 h (h) and 0 h following fermentation, the optimum medium could produce 1.54 g L−1 SAM. Based on the regulations mentioned above, the SAM concentration of strain ΔSOD1 enhanced from 7.3 g L−1 to 10.1 g L−1 in a 5-L fermenter in 118 h. This work introduces a novel idea for the biosynthesis of ATP and SAM, and the strain ΔSOD1 has the potential for industrial production.

Control strategies for atmospheric pressure plasma polymerization of fluorinated silane thin films with antiadhesive properties

AbstractFinding proper strategies to control plasma polymerization processes is a crucial aspect to produce thin films with tailored characteristics. In this work, the validity of the Yasuda parameter W/FM (W: discharge power and FM: precursor feed rate) as a controlling parameter for a polymerization process assisted by an atmospheric pressure single electrode plasma jet and the aerosolized fluorinated silane precursor trimethoxy(3,3,3‐trifluoropropyl)silane is demonstrated. The properties of thin films deposited under different W/FM values are discussed using attenuated total reflectance—Fourier transform infrared (ATR‐FTIR) spectroscopy, X‐ray photoelectron spectroscopy (XPS), water contact angle (WCA) measurements, and scanning electron microscopy (SEM). Results suggest the presence of two deposition domains as a function of W/FM (an energy‐deficient domain and a monomer‐deficient domain), each inducing coatings with different chemical and physical properties. Furthermore, coatings deposited under the same W/FM values exhibit similar characteristics regardless of the power and feed rate values adopted. Considering the potential use of the deposited coatings to increase the antiadhesive properties of implantable medical devices, preliminary results on coatings' antiadhesive activity against Pseudomonas aeruginosa and Staphylococcus aureus are presented.

Preparation of ZrC coating in TRISO fuel particles by precise transportation of solid precursor and its microstructure evolution

Very-high-temperature gas-cooled reactors (VHTRs) with outlet temperatures above 1273 K show higher thermal efficiencies, which require fuel elements to operate at higher temperatures. In such situations, the conventional silicon carbide (SiC) coatings in TRISO particles might not be able to adapt to such high temperatures, and zirconium carbide (ZrC) can function as a potential fission product barrier material. For ZrC coating, controlling the stoichiometric ratio is a key challenge in the preparation process in fluidized bed. In this paper, a novel solid precursor feeding mode was proposed, combining quantitative powder feeding with cold transport. By controlling the powder feeding rate of ZrCl4 and the flow rate of propylene, the C/Zr atomic ratio in ZrC coating was controlled accurately. ZrC coatings with different stoichiometric ratios were successfully prepared via fluidized-bed chemical vapor deposition (FB-CVD). The ZrC coatings were subjected to high temperature heat treatment for different times from 2473 to 2673 K under argon atmosphere and the microstructure and property evolutions after high-temperature heat treatment were investigated. The deposition mechanism of ZrC coating was proposed.

Influence of gamma radiation and phenylalanine on secondary metabolites in callus cultures of milk thistle (Silybum marianum L.)

BackgroundA useful technique for growing large amounts of plant material is in vitro propagation of important medicinal plants. The present investigation deals with the enhancement of secondary metabolite production via elicitation using gamma (γ)-radiation and phenylalanine (Phe) precursor feeding in callus cultures of Silybum marianum L. ResultsSeeds were exposed to two doses of γ-radiation (25 and 50 Gy) and the calli derived from stem explants obtained from seedlings of these radiated seeds were treated with different concentrations of Phe. The biosynthesis of phenols and flavonoids was evaluated. It was found that callus cultures derived from explants of the seeds exposed to 25 Gy γ-radiation and treated with 4 mg/l Phe accumulated the maximum phenolic content (34.27±0.02 mg/g d.wt.), while the highest flavonoid content (9.56±0.12 mg/g d.wt.) was found in callus cultures derived from explants of seeds radiated with 25 Gy γ-radiation and subjected to 1 mg/l Phe. Similarly, HPLC quantification revealed that the production of flavonoids was highly accumulated (1343.06 μg/mg d.wt.) in callus cultures from explants of seeds exposed to 25 Gy γ-radiation and grown at 1 mg/l Phe compared to the other treatments. In addition, a total of 11 important flavonoids have been determined in all callus cultures, except for acacetin-7-O-rutinoside, which was not found in the callus culture of the control. ConclusionsThese findings suggest that γ-radiation combined with Phe can improve the metabolism of S. marianum L. and could be used to produce such valuable metabolites on a commercial scale.

Thiamyxins: Structure and Biosynthesis of Myxobacterial RNA-Virus Inhibitors.

During our search for novel myxobacterial natural products, we discovered the thiamyxins: thiazole- and thiazoline-rich non-ribosomal peptide-polyketide hybrids with potent antiviral activity. We isolated four congeners of this unprecedented natural product family with the non-cyclized thiamyxin D fused to a glycerol unit at the C-terminus. Alongside their structure elucidation, we present a concise biosynthesis model based on biosynthetic gene cluster analysis and isotopically labelled precursor feeding. We report incorporation of a 2-(hydroxymethyl)-4-methylpent-3-enoic acid moiety by a GCN5-related N-acetyltransferase-like decarboxylase domain featuring polyketide synthase. The thiamyxins show potent inhibition of RNA viruses in cell culture models of corona, zika and dengue virus infection. Their potency up to a half maximal inhibitory concentration of 560 nM combined with milder cytotoxic effects on human cell lines indicate the potential for further development of the thiamyxins.

Elicitor and precursor-induced approaches to enhance the in vitro production of L-DOPA from cell cultures of Mucuna pruriens

Elicitation and precursor feeding are two important strategies in the in vitro techniques to enhance metabolite production to meet the demand of mankind. The secondary metabolites produced by the plants are extensively used in pharmaceutical, food and agro-chemical industries. One such metabolite is 3,4 dihydroxyphenylalanine ( L -DOPA) produced from Mucuna pruriens (L.) DC. is used since ancient times to treat Parkinson’s disease. Though all parts produce L -DOPA, the seed has the highest quantity. To overcome the extensive usage of the natural sources whose growth and metabolite production is highly dependent on edaphic and ecological factors, in vitro techniques like establishing cell culture for continuous production of metabolites, precursor feeding and elicitation of cell cultures to enhance the metabolite production has been reported in the present study. Callus was developed from the in vitro leaf explant and cell suspension culture was established in the liquid Murashige and Skoog’s medium fortified with 0.5 mg/L picloram . Amino acid precursors like tyrosine , phenylalanine and chemical elicitors like methyl jasmonate , salicylic acid, sodium nitroprusside and silver nitrate were exposed to cell cultures for different periods (3, 6 and 9 days respectively). The precursors showed a better response in enhancing both the biomass and L -DOPA when compared to the elicitors. 500 and 1000 mg/L tyrosine showed a 1.6- and an 8.1-fold increase in biomass and L -DOPA production respectively when supplemented with MS media . However, though all the elicitors enhanced the L -DOPA production by 1.1–3.3-folds they did not show much significant increase in biomass. Precursor feeding approaches enhanced the metabolite considerably more than the elicitor treatment. Based on the productivity (Biomass × L -DOPA conc.) precursors like Tyrosine>Phenylalanine and elicitors like Sodium nitroprusside>Silver nitrate>Methyl jasmonate>Salicylic acid showed better response. • First report to elicit L -DOPA from Mucuna pruriens cell cultures using Phenylalanine. • A highest of 8.1-fold increase of L -DOPA was achieved using Tyrosine as a precursor. • Among the elicitors, a 3.3-fold increase of L -DOPA was seen using sodium nitroprusside. • On the basis of productivity, Tyrosine and sodium nitroprusside showed best results.

Highly Efficient Deposition of Centimeter-Scale MoS2 Monolayer Film on Dragontrail Glass with Large Single-Crystalline Domains.

Highly efficient growth of a centimeter-scale MoS2 monolayer film by oxide scale sublimation chemical vapor deposition (OSSCVD) in a time as short as 60 s is reported. Benefiting from the superior catalytic ability of Dragontrail glass (DT-glass) substrate and the controlled large vapor supersaturation of the molybdenum source, the ultrafast deposition of MoS2 is realized with maintaining large-sized single-crystalline domains over 20µm at maximum in the film. It is comparable to those reported for MoS2 grown in tens of minutes and even hours. Similar to the face-to-face precursor feed route, the gas-controlled OSSCVD with a showerhead configuration facilitates a homogeneous and controllable source supply. It enables high-quality monolayer MoS2 film deposition on 2 × 2 cm2 DT-glass with centimeter-scale uniformity confirmed by microscopic, spectroscopic, and electrical characterizations. Back-gate MoS2 field-effect transistors fabricated on polycrystalline continuous film exhibit the maximum field-effect mobility of 5.1 cm2 V-1 s-1 and a peak Ion /Ioff ratio of 5 × 108 . They reach 40 cm2 V-1 s-1 and 1.2 × 109 , respectively, on single-crystalline domains. These results are even greater than those for MoS2 grown using 1-2 orders of magnitude longer deposition time and higher temperatures. This study highlights the opportunities for low-cost high-throughput production of large-area high-quality monolayer MoS2 .

A p-Coumaroyl-CoA Biosensor for Dynamic Regulation of Naringenin Biosynthesis in Saccharomyces cerevisiae.

In vivo biosensors that can convert metabolite concentrations into measurable output signals are valuable tools for high-throughput screening and dynamic pathway control in the field of metabolic engineering. Here, we present a novel biosensor in Saccharomyces cerevisiae that is responsive to p-coumaroyl-CoA, a central precursor of many flavonoids. The sensor is based on the transcriptional repressor CouR from Rhodopseudomonas palustris and was applied in combination with a previously developed malonyl-CoA biosensor for dual regulation of p-coumaroyl-CoA synthesis within the naringenin production pathway. Using this approach, we obtained a naringenin titer of 47.3 mg/L upon external precursor feeding, representing a 15-fold increase over the nonregulated system.

Linum lewisii Adventitious and Hairy-Roots Cultures as Lignan Plant Factories

Plants synthesize specific secondary metabolites for survival, reproduction, environmental resilience, and defense. Among them, lignans are a class of polyphenols with several bioactive properties: chemopreventive, anti-inflammatory, antiviral, and antioxidant. These compounds are often extracted from field-grown plants with very low yields. To overcome these constraints, in vitro tissue cultures provide a tool to optimize large-scale production. Moreover, the use of elicitation to increase secondary metabolite production is gaining importance. The aim of this work was to develop adventitious (ARL) and hairy roots (HRL) from Linum lewisi, a species able to synthesize arylnaphthalene lignans such as justicidin B. The ARL and HRL were obtained for the first time and characterized for their phenol content, antioxidant activity, and the production of justicidin B after treatments with several elicitors and precursor feeding. Through NMR spectroscopy, other four lignans were highlighted and identified in the roots extracts. A pilot-scale bioreactor was adopted to assess the suitability of the developed root cultures for future large-scale production. The ARL and HRL cultures showed a justicidin B production higher than other Linum species cultures described up to now (75.8 mg/L and 82.2 g/L), and the production more than doubled after elicitation with MeJA.

Exploring the core microbiota in scented rice (Oryza sativa L.) rhizosphere through metagenomics approach

Rice is a major food crop cultivated around the globe. Specially scented rice varieties are of commercial importance but they are low-yielding. The rhizospheric microflora plays a significant role in improving yield and aroma. However, the core microbiome of the scented rice rhizosphere is comparatively less explored. Here, we analyzed the core microbiome associated with the rhizosphere of the scented (Ambemohar-157 and Dehradun basmati) in comparison with non-scented rice (Kolam and Arize 6444 Gold) cultivated at two different geoclimatic zones of India (Maharashtra and Uttarakhand) using the metagenomics approach. The alpha and beta diversity analysis showed that the microbial communities associated with scented and non-scented varieties significantly changes with respect to richness, diversity, and evenness. The taxonomic profiling revealed the variation in composition, diversity, and abundance of the microbiome in terms of phyla and genera associated with scented rice varieties over non-scented. The cluster analysis distinguishes the microbial communities based on their geographical positions. The core microbiome analysis revealed that scented rice rhizosphere shelters distinct and unique microbiota. 28.6 % of genera were exclusively present only in the scented rice rhizosphere. The putative functional gene annotation revealed the high abundance of genes related to the biosynthesis of 2-acetyl-1-pyrroline (2AP) precursors in scented rice. The precursor feeding analysis revealed proline as a preferred substrate by 2AP synthesizing bacteria. The 2AP precursor proline and proline metabolism genes showed a positive correlation. The scented rice-specific rhizobacteria pointed out in this study can be used as bio-inoculants for enhancing aroma, yield, and sustainable rice cultivation.

Biotechnological production of β-carotene using plant in vitro cultures.

β-carotene is biologically active compound widely distributed in plants. The use of plant in vitro cultures and genetic engineering is a promising strategy for its sustainable production. β-carotene is an orange carotenoid often found in leaves as well as in fruits, flowers, and roots. A member of the tetraterpene family, this 40-carbon isoprenoid has a conjugated double-bond structure, which is responsible for some of its most remarkable properties. In plants, β-carotene functions as an antenna pigment and antioxidant, providing protection against photooxidative damage caused by strong UV-B light. In humans, β-carotene acts as a precursor of vitamin A, prevents skin damage by solar radiation, and protects against several types of cancer such as oral, colon and prostate. Due to its wide spectrum of applications, the global market for β-carotene is expanding, and the demand can no longer be met by extraction from plant raw materials. Considerable research has been dedicated to finding more efficient production alternatives based on biotechnological systems. This review provides a detailed overview of the strategies used to increase the production of β-carotene in plant in vitro cultures, with particular focus on culture conditions, precursor feeding and elicitation, and the application of metabolic engineering.

In situ glucosylceramide synthesis and its pharmacological inhibition analysed in cells by 13 C5 -sphingosine precursor feeding and mass spectrometry.

Glycosphingolipids (GSLs) fulfil diverse functions in cells. Abnormalities in their metabolism are associated with specific pathologies and, consequently, the pharmacological modulation of GSLs is considered a therapeutic avenue. The accurate measurement of in situ metabolism of GSLs and the modulatory impact of drugs is warranted. Employing synthesised sphingosine and sphinganine containing 13 C atoms, we developed a method to monitor the de novo synthesis of glucosylceramide, the precursor of complex GSLs, by the enzyme glucosylceramide synthase (GCS). We show that feeding cells with isotope-labelled precursor combined with liquid chromatography-mass spectrometry (MS)/MS analysis allows accurate determination of the IC50 values of therapeutically considered inhibitors (iminosugars and ceramide mimics) of GCS in cultured cells. Acquired data were comparable to those obtained with an earlier method using artificial fluorescently labelled ceramide to feed cells.

Cell type matters: competence for alkaloid metabolism differs in two seed-derived cell strains of Catharanthus roseus

Since the discovery of the anticancer drugs vinblastine and vincristine, Catharanthus roseus has been intensively studied for biosynthesis of several terpene indole alkaloids (TIAs). Due to their low abundance in plant tissues at a simultaneously high demand, modes of production alternative to conventional extraction are mandatory. Plant cell fermentation might become one of these alternatives, yet decades of research have shown limited success to certain product classes, leading to the question: how to preserve the intrinsic ability to produce TIAs (metabolic competence) in cell culture? We used the strategy to use the developmental potency of mature embryos to generate such strains. Two cell strains (C1and C4) from seed embryos of Catharanthus roseus were found to differ not only morphologically, but also in their metabolic competence. This differential competence became manifest not only under phytohormone elicitation, but also upon feeding with alkaloid pathway precursors. The more active strain C4 formed larger cell aggregates and was endowed with longer mitochondria. These cellular features were accompanied by higher alkaloid accumulation in response to methyl jasmonate (MeJA) elicitation. The levels of catharanthine could be increased significantly, while the concurrent vindoline branch of the pathway was blocked, such that no bisindole alkaloids were detectable. By feeding vindoline to MeJA-elicited C4 cells, vincristine became detectable; however, only to marginal amounts. In conclusion, these results show that cultured cells are not “de-differentiated”, but can differ in metabolic competence. In addition to elicitation and precursor feeding, the cellular properties of the “biomatter” are highly relevant for the success of plant cell fermentation.

Advanced Atomic Layer Deposition: Ultrathin and Continuous Metal Thin Film Growth and Work Function Control Using the Discrete Feeding Method.

The ultrathin and continuous ruthenium (Ru) film was deposited through an improved atomic layer deposition (ALD) process with a discrete feeding method (DFM), called DF-ALD, employing a cut-in purge step during the precursor feeding. The excess precursor molecules can be physically adsorbed onto the chemisorbed precursors on the substrate during precursor feeding, which screens the reactive sites on the surface. Using DF-ALD, surface coverage of precursors was enhanced because the cut-in purge removes the physisorbed precursors securing the reactive sites beneath them; thus, nucleation density was greatly increased. Therefore, the grain size decreased, which changed the microstructure and increased oxygen impurity concentration. However, a more metallic Ru thin film was formed due to thermodynamic stability and improved physical density. Consequently, DF-ALD enables the deposition of the ultrathin (3 nm) and continuous Ru film with a low resistivity of ∼60 μΩ cm and a high effective work function of ∼4.8 eV.