Platform For Sustainable Production Research Articles

Enhancement of Sesquiterpenoid Production by Methyl Jasmonate in Atractylodes chinensis Adventitious Root Culture and its Transcriptional Regulation.

The adventitious root (AR) culture of Atractylodes chinensis is an efficient platform for sustainable production of its sesquiterpenoid compounds (atractylon and β-eudesmol). However, their limited accumulation levels need an effective elicitation approach, and the present study solved this problem using methyl jasmonate (MeJA) as an elicitor. The effects of its treatment concentration and duration on metabolite production were investigated. The ARs treated with 100 µM MeJA for seven days increased atractylon and β-eudesmol by 3.64- and 1.90-fold, respectively, compared with the control. This study further performed transcriptome analysis to explore the transcriptional regulation mechanism of the MeJA elicitation. A total of 124,464 unigenes were identified in A. chinensis ARs, of which 3,568 genes were upregulated and 3,864 genes were downregulated under the MeJA treatment. The MeJA treatment activated the endogenous JA biosynthesis and signaling pathways and sesquiterpenoid biosynthesis. The MeJA treatment more significantly activated the MEP pathway than the MVA pathway. In addition, 14 genes encoding terpene synthase were identified to be significantly upregulated. A total of 2,700 transcription factors (TFs) were identified in A. chinensis ARs, of which Tify, MYB, and MADS were significantly enriched under the MeJA treatment. We predicted a new antagonistic interaction between MYC2 and CPP TFs, which was significantly regulated by the MeJA treatment. The results of real-time quantitative PCR and enzyme activity assays proved the reliability of the transcriptome data. This study will help improve the in vitro production system of A. chinensis sesquiterpenoids and understand the transcriptional regulation mechanism of MeJA elicitation.

Tailoring regulatory components for metabolic engineering in cyanobacteria.

The looming climate crisis has prompted an ever-growing interest in cyanobacteria due to their potential as sustainable production platforms for the synthesis of energy carriers and value-added chemicals from CO2 and sunlight. Nonetheless, cyanobacteria are yet to compete with heterotrophic systems in terms of space-time yields and consequently production costs. One major drawback leading to the low production performance observed in cyanobacteria is the limited ability to utilize the full capacity of the photosynthetic apparatus and its associated systems, i.e. CO2 fixation and the directly connected metabolism. In this review, novel insights into various levels of metabolic regulation of cyanobacteria are discussed, including the potential of targeting these regulatory mechanisms to create a chassis with a phenotype favorable for photoautotrophic production. Compared to conventional metabolic engineering approaches, minor perturbations of regulatory mechanisms can have wide-ranging effects.

Characterization of cinnamate 4-hydroxylase (CYP73A) and p-coumaroyl 3′-hydroxylase (CYP98A) from Leucojum aestivum, a source of Amaryllidaceae alkaloids

Biosynthesis of Amaryllidaceae alkaloids (AA) starts with the condensation of tyramine with 3,4-dihydroxybenzaldehyde. The latter derives from the phenylpropanoid pathway that involves modifications of trans-cinnamic acid, p-coumaric acid, caffeic acid, and possibly 4-hydroxybenzaldehyde, all potentially catalyzed by hydroxylase enzymes. Leveraging bioinformatics, molecular biology techniques, and cell biology tools, this research identifies and characterizes key enzymes from the phenylpropanoid pathway in Leucojum aestivum. Notably, we focused our work on trans-cinnamate 4-hydroxylase (LaeC4H) and p-coumaroyl shikimate/quinate 3ʹ-hydroxylase (LaeC3′H), two key cytochrome P450 enzymes, and on the ascorbate peroxidase/4-coumarate 3-hydroxylase (LaeAPX/C3H). Although LaeAPX/C3H consumed p-coumaric acid, it did not result in the production of caffeic acid. Yeasts expressing LaeC4H converted trans-cinnamate to p-coumaric acid, whereas LaeC3′H catalyzed specifically the 3-hydroxylation of p-coumaroyl shikimate, rather than of free p-coumaric acid or 4-hydroxybenzaldehyde. In vivo assays conducted in planta in this study provided further evidence for the contribution of these enzymes to the phenylpropanoid pathway. Both enzymes demonstrated typical endoplasmic reticulum membrane localization in Nicotiana benthamiana adding spatial context to their functions. Tissue-specific gene expression analysis revealed roots as hotspots for phenylpropanoid-related transcripts and bulbs as hubs for AA biosynthetic genes, aligning with the highest AAs concentration. This investigation adds valuable insights into the phenylpropanoid pathway within Amaryllidaceae, laying the foundation for the development of sustainable production platforms for AAs and other bioactive compounds with diverse applications.

Closing the Gap between Bio-Based and Petroleum-Based Plastic through Bioengineering.

Bioplastics, which are plastic materials produced from renewable bio-based feedstocks, have been investigated for their potential as an attractive alternative to petroleum-based plastics. Despite the harmful effects of plastic accumulation in the environment, bioplastic production is still underdeveloped. Recent advances in strain development, genome sequencing, and editing technologies have accelerated research efforts toward bioplastic production and helped to advance its goal of replacing conventional plastics. In this review, we highlight bioengineering approaches, new advancements, and related challenges in the bioproduction and biodegradation of plastics. We cover different types of polymers, including polylactic acid (PLA) and polyhydroxyalkanoates (PHAs and PHBs) produced by bacterial, microalgal, and plant species naturally as well as through genetic engineering. Moreover, we provide detailed information on pathways that produce PHAs and PHBs in bacteria. Lastly, we present the prospect of using large-scale genome engineering to enhance strains and develop microalgae as a sustainable production platform.

Transcriptional deregulation of stress-growth balance in Nicotiana benthamiana biofactories producing insect sex pheromones.

Plant biofactories are a promising platform for sustainable production of high-value compounds, among which are insect sex pheromones, a green alternative to conventional insecticides in agriculture. Recently, we have constructed transgenic Nicotiana benthamiana plants ("Sexy Plants", SxP) that successfully produce a blend of moth (Lepidoptera) sex pheromone compounds (Z)-11-hexadecen-1-ol and (Z)-11-hexadecenyl acetate. However, efficient biosynthesis of sex pheromones resulted in growth and developmental penalty, diminishing the potential for commercial use of SxP in biomanufacturing. To gain insight into the underlying molecular responses, we analysed the whole-genome transcriptome and evaluated it in relation to growth and pheromone production in low- and high-producing transgenic plants of v1.0 and v1.2 SxP lines. In our study, high-producing SxPv1.2 plants accumulated the highest amounts of pheromones but still maintained better growth compared to v1.0 high producers. For an in-depth biological interpretation of the transcriptomic data, we have prepared a comprehensive functional N. benthamiana genome annotation as well as gene translations to Arabidopsis thaliana, enabling functional information transfer by using Arabidopsis knowledge networks. Differential gene expression analysis, contrasting pheromone producers to wild-type plants, revealed that while only a few genes were differentially regulated in low-producing plants, high-producing plants exhibited vast transcriptional reprogramming. They showed signs of stress-like response, manifested as downregulation of photosynthesis-related genes and significant differences in expression of hormonal signalling and secondary metabolism-related genes, the latter presumably leading to previously reported volatilome changes. Further network analyses confirmed stress-like response with activation of jasmonic acid and downregulation of gibberellic acid signalling, illuminating the possibility that the observed growth penalty was not solely a consequence of a higher metabolic burden imposed upon constitutive expression of a heterologous biosynthetic pathway, but rather the result of signalling pathway perturbation. Our work presents an example of comprehensive transcriptomic analyses of disadvantageous stress signalling in N. benthamiana biofactory that could be applied to other bioproduction systems.

A droplet-based microfluidic platform enables high-throughput combinatorial optimization of cyanobacterial cultivation

Cyanobacteria are fast-growing, genetically accessible, photoautotrophs. Therefore, they have attracted interest as sustainable production platforms. However, the lack of techniques to systematically optimize cultivation parameters in a high-throughput manner is holding back progress towards industrialization. To overcome this bottleneck, here we introduce a droplet-based microfluidic platform capable of one- (1D) and two-dimension (2D) screening of key parameters in cyanobacterial cultivation. We successfully grew three different unicellular, biotechnologically relevant, cyanobacteria: Synechocystis sp. PCC 6803, Synechococcus elongatus UTEX 2973 and Synechococcus sp. UTEX 3154. This was followed by a highly-resolved 1D screening of nitrate, phosphate, carbonate, and salt concentrations. The 1D screening results suggested that nitrate and/or phosphate may be limiting nutrients in standard cultivation media. Finally, we use 2D screening to determine the optimal N:P ratio of BG-11. Application of the improved medium composition in a high-density cultivation setup led to an increase in biomass yield of up to 15.7%. This study demonstrates that droplet-based microfluidics can decrease the volume required for cyanobacterial cultivation and screening up to a thousand times while significantly increasing the multiplexing capacity. Going forward, microfluidics have the potential to play a significant role in the industrial exploitation of cyanobacteria.

Engineering type I methanotrophic bacteria as novel platform for sustainable production of 3-hydroxybutyrate and biodegradable polyhydroxybutyrate from methane and xylose

Methylotuvimicrobium alcaliphilum20Z recombinant strain co-utilizing methane and xylose from anthropogenic activities and lignocellulose biomassis a promising cell factory platform. In this study, the production of (R)-3-hydroxybutyrate and poly (3-hydroxybutyrate) inM. alcaliphilum20Z was demonstrated. The production of (R)-3-hydroxybutyrate was optimized by introducing additional thioesterase, and a tunable genetic module. The final recombinant strain produced the highest titer of 334.52 ± 2 mg/L (R)-3-hydroxybutyrate (yield of 1,853 ± 429 mg/g dry cell weight). The poly (3-hydroxybutyrate) yielded 1.29 ± 0.08% (w/w) from methane and xylose in one-stage cultivation. Moreover, the study demonstrated the importance of pathway reversibility as an effective design strategy for balancing the driving force and intermediate accumulation. This is the first demonstration of the production ofbiodegradablepoly (3-hydroxybutyrate) from methane in type I methanotrophs, which is a key step toward sustainable biomanufacturing and carbon–neutral society.

Gardenia carotenoid cleavage dioxygenase 4a is an efficient tool for biotechnological production of crocins in green and non-green plant tissues.

Crocins are beneficial antioxidants and potential chemotherapeutics that give raise, together with picrocrocin, to the colour and taste of saffron, the most expensive spice, respectively. Crocins are formed from crocetin dialdehyde that is produced in Crocus sativus from zeaxanthin by the carotenoid cleavage dioxygenase 2L (CsCCD2L), while GjCCD4a from Gardenia jasminoides, another major source of crocins, converted different carotenoids, including zeaxanthin, into crocetin dialdehyde in bacterio. To establish a biotechnological platform for sustainable production of crocins, we investigated the enzymatic activity of GjCCD4a, in comparison with CsCCD2L, in citrus callus engineered by Agrobacterium-mediated supertransformation of multi genes and in transiently transformed Nicotiana benthamiana leaves. We demonstrate that co-expression of GjCCD4a with phytoene synthase and β-carotene hydroxylase genes is an optimal combination for heterologous production of crocetin, crocins and picrocrocin in citrus callus. By profiling apocarotenoids and using in vitro assays, we show that GjCCD4a cleaved β-carotene, in planta, and produced crocetin dialdehyde via C30 β-apocarotenoid intermediate. GjCCD4a also cleaved C27 β-apocarotenoids, providing a new route for C17 -dialdehyde biosynthesis. Callus lines overexpressing GjCCD4a contained higher number of plastoglobuli in chromoplast-like plastids and increased contents in phytoene, C17:0 fatty acid (FA), and C18:1 cis-9 and C22:0 FA esters. GjCCD4a showed a wider substrate specificity and higher efficiency in Nicotiana leaves, leading to the accumulation of up to 1.6 mg/g dry weight crocins. In summary, we established a system for investigating CCD enzymatic activity in planta and an efficient biotechnological platform for crocins production in green and non-green crop tissues/organs.

Exotic Hydrogel Matrix as an Efficient Platform for Sustainable Production of Biomass and Lipid from Chlorella sorokiniana

Concerning the climate crisis, energy disaster, and greenhouse effects, microalgae have paved the way for consideration as a biofuel feed material. The advent of polymeric materials with unique architecture at nanoscale, in combination with microalgae, has given direction for the bioeconomic yield of highly valued compounds, essentially lipid. Herein, we discuss the paramount significance of exotic hydrogel matrix (HM) with efficient violet light absorption, far-red emission, CO2-adsorbing capability and catalyst-free condition that could increase the photosynthesis activity, alleviating the microalgal growth for the effective augmentation of lipid, protein, and chlorophyll. The intrinsic morphological and structural features of HM were revealed by a suite of characterizations that confirm its hollow tubular architecture. Fluorescence intensity measurement confirmed the electron transfer from HM to Chlorella sorokiniana, accelerating the photosynthetic rate for the improved production of lipids (98%), proteins (60%), and chlorophyll a (121%), compared to untreated C. sorokiniana control cells. Moreover, by visualizing the Nile red (NR) fluorescence response from C. sorokiniana/HM cells, a high lipid content was observed with a larger cell size (14.6 μm) compared to control cells (8.7 μm). Fatty acid methyl esters (FAMEs), obtained from C. sorokiniana/HM, were noted with a large-scale volume of C16:C18 fatty acids (>80%). We, therefore, envisage that HM plays a significant role in enhancing the generation of lipids and proteins from C. sorokiniana. These outcomes assure a qualitative transit in the bioenergy domain.

Current Status and Future Strategies to Increase Secondary Metabolite Production from Cyanobacteria.

Cyanobacteria, given their ability to produce various secondary metabolites utilizing solar energy and carbon dioxide, are a potential platform for sustainable production of biochemicals. Until now, conventional metabolic engineering approaches have been applied to various cyanobacterial species for enhanced production of industrially valued compounds, including secondary metabolites and non-natural biochemicals. However, the shortage of understanding of cyanobacterial metabolic and regulatory networks for atmospheric carbon fixation to biochemical production and the lack of available engineering tools limit the potential of cyanobacteria for industrial applications. Recently, to overcome the limitations, synthetic biology tools and systems biology approaches such as genome-scale modeling based on diverse omics data have been applied to cyanobacteria. This review covers the synthetic and systems biology approaches for advanced metabolic engineering of cyanobacteria.

A marine photosynthetic microbial cell factory as a platform for spider silk production

Photosynthetic microorganisms such as cyanobacteria, purple bacteria and microalgae have attracted great interest as promising platforms for economical and sustainable production of bioenergy, biochemicals, and biopolymers. Here, we demonstrate heterotrophic production of spider dragline silk proteins, major ampullate spidroins (MaSp), in a marine photosynthetic purple bacterium, Rhodovulum sulfidophilum, under both photoheterotrophic and photoautotrophic growth conditions. Spider silk is a biodegradable and biocompatible material with remarkable mechanical properties. R. sulfidophilum grow by utilizing abundant and renewable nonfood bioresources such as seawater, sunlight, and gaseous CO2 and N2, thus making this photosynthetic microbial cell factory a promising green and sustainable production platform for proteins and biopolymers, including spider silks.

An environmentally sustainable biomimetic production of cyclic disulfide-rich peptides

An environmentally sustainable production platform for a variety of correctly folded cyclic disulfide-rich peptides with enhanced yields.

A novel strategy to enhance terpenoids production using cambial meristematic cells of Tripterygium wilfordii Hook. f.

BackgroundTripterygium wilfordii Hook. f. (T. wilfordii) is an important medicinal plant with anti-inflammatory, immunosuppressive and anti-tumor activities. The main bioactive ingredients are diterpenoids and triterpenoids, such as triptolide, triptophenolide and celastrol. However, the production of terpenoids from original plants, hairy roots and dedifferentiated cells (DDCs) are not satisfactory for clinical applications. To find a new way to further improve the production of terpenoids, we established a new culture system of cambial meristematic cells (CMCs) with stem cell-like properties, which had strong vigor and high efficiency to produce large amounts of terpenoids of T. wilfordii.ResultsCMCs of T. wilfordii were isolated and cultured for the first time. CMCs were characterized consistent with stem cell identities based on their physiological and molecular analysis, including morphology of CMCs, hypersensitivity to zeocin, thin cell wall and orthogonal partial least square-discriminant analysis, combination of transcriptional data analysis. After induction with methyl jasmonate (MJ), the maximal production of triptolide, celastrol and triptophenolide in CMCs was 312%, 400% and 327% higher than that of control group, respectively. As for medium, MJ-induced CMCs secreted 231% triptolide and 130% triptophenolide at the maximum level into medium higher than that of control group. Maximal celastrol production of induced CMCs medium was 48% lower than that of control group. Long-term induction significantly enhanced the production of terpenoids both in cells and medium. The reason for increasing the yield of terpenoids was that expression levels of 1-deoxy-d-xylulose-5-phosphate synthase (DXS), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) and hydroxymethylglutaryl-CoA synthase (HMGS) were upregulated in CMCs after induction.ConclusionsFor the first time, CMCs of T. wilfordii were isolated, cultured, characterized and applied. Considering the significant enrichment of terpenoids in CMCs of T. wilfordii, CMCs could provide an efficient and controllable platform for sustainable production of terpenoids, which can be a better choice than DDCs.

Single mutation at a highly conserved region of chloramphenicol acetyltransferase enables isobutyl acetate production directly from cellulose by Clostridium thermocellum at elevated temperatures

BackgroundEsters are versatile chemicals and potential drop-in biofuels. To develop a sustainable production platform, microbial ester biosynthesis using alcohol acetyltransferases (AATs) has been studied for decades. Volatility of esters endows high-temperature fermentation with advantageous downstream product separation. However, due to the limited thermostability of AATs known, the ester biosynthesis has largely relied on use of mesophilic microbes. Therefore, developing thermostable AATs is important for ester production directly from lignocellulosic biomass by the thermophilic consolidated bioprocessing (CBP) microbes, e.g., Clostridium thermocellum.ResultsIn this study, we engineered a thermostable chloramphenicol acetyltransferase from Staphylococcus aureus (CATSa) for enhanced isobutyl acetate production at elevated temperatures. We first analyzed the broad alcohol substrate range of CATSa. Then, we targeted a highly conserved region in the binding pocket of CATSa for mutagenesis. The mutagenesis revealed that F97W significantly increased conversion of isobutanol to isobutyl acetate. Using CATSa F97W, we demonstrated direct conversion of cellulose into isobutyl acetate by an engineered C. thermocellum at elevated temperatures.ConclusionsThis study highlights that CAT is a potential thermostable AAT that can be harnessed to develop the thermophilic CBP microbial platform for biosynthesis of designer bioesters directly from lignocellulosic biomass.

Iron oxide nanoparticles: a novel elicitor to enhance anticancer flavonoid production and gene expression in Dracocephalum kotschyi hairy‐root cultures

Dracocephalum kotschyi Boiss. is a valuable source of rosmarinic acid (RA) and methoxylated hydroxyflavones (such as xanthomicrol and cirsimaritin) with antioxidative and antiplatelet effects and with antiproliferative potential against various cancer cells. The extensive application of nanotechnology in hairy root cultures is a new sustainable production platform for producing these active constituents. In the present study, hairy roots derived from 4-week-old leaves and Agrobacterium rhizogenes strain ATCC15834 were used to investigate the impact of various concentrations of iron oxide nanoparticles (Fe NPs) in two elicitation time exposures (24 and 48 h) on growth, antioxidant enzyme activity, total phenolic and flavonoid content (TPC and TFC), and some polyphenols. Gene expression levels of phenylalanine ammonia-lyase (pal) and rosmarinic acid synthase (ras) were also analyzed. Iron nanoparticles enhanced biomass accumulation in hairy roots. The treatment time and Fe NP dosage largely improved the activity of antioxidant enzymes, TPC and TFC. The highest RA (1194 μg g-1 FW) content (9.7-fold), compared to controls, was detected with 24 h of exposure to 75 mg L-1 Fe NP, which was consistent with the expression of pal and ras genes under the influence of elicitation. The xanthomicrol, cirsimaritin, and isokaempferide content was increased 11.87, 3.85, and 2.27-fold, respectively. Stimulation of D. kotschyi hairy roots by Fe NPs led to a significant increase in the induction and production of important pharmaceutical compounds such as rosmarinic acid and xanthomicrol. © 2019 Society of Chemical Industry.

Robust and biocompatible catalysts for efficient hydrogen-driven microbial electrosynthesis

CO2 reduction by combined electro- and bio-catalytic reactions is a promising technology platform for sustainable production of chemicals from CO2 and electricity. While heterogeneous electrocatalysts can reduce CO2 to a variety of organic compounds at relatively high reaction rates, these catalysts have limitations achieving high selectivity for any single product beyond CO. Conversely, microbial CO2 reduction pathways proceed at high selectivity; however, the rates at bio-cathodes using direct electron supply via electricity are commonly limiting. Here we demonstrate the use of non-precious metal cathodes that produce hydrogen in situ to support microbial CO2 reduction to C1 and C2 compounds. CoP, MoS2 and NiMo cathodes perform durable hydrogen evolution under biologically relevant conditions, and the integrated system achieves coulombic efficiencies close to 100% without accumulating hydrogen. Moreover, the one-reactor hybrid platform is successfully used for efficient acetate production from electricity and CO2 by microbes previously reported to be inactive in bioelectrochemical systems.

Engineered Microorganisms for the Production of Food Additives Approved by the European Union-A Systematic Analysis.

In the 1950s, the idea of a single harmonized list of food additives for the European Union arose. Already in 1962, the E-classification system, a robust food safety system intended to protect consumers from possible food-related risks, was introduced. Initially, it was restricted to colorants, but at later stages also preservatives, antioxidants, emulsifiers, stabilizers, thickeners, gelling agents, sweeteners, and flavorings were included. Currently, the list of substances authorized by the European Food Safety Authority (EFSA) (referred to as “E numbers”) comprises 316 natural or artificial substances including small organic molecules, metals, salts, but also more complex compounds such as plant extracts and polymers. Low overall concentrations of such compounds in natural producers due to inherent regulation mechanisms or production processes based on non-regenerative carbon sources led to an increasing interest in establishing more reliable and sustainable production platforms. In this context, microorganisms have received significant attention as alternative sources providing access to these compounds. Scientific advancements in the fields of molecular biology and genetic engineering opened the door toward using engineered microorganisms for overproduction of metabolites of their carbon metabolism such as carboxylic acids and amino acids. In addition, entire pathways, e.g., of plant origin, were functionally introduced into microorganisms, which holds the promise to get access to an even broader range of accessible products. The aim of this review article is to give a systematic overview on current efforts during construction and application of microbial cell factories for the production of food additives listed in the EU “E numbers” catalog. The review is focused on metabolic engineering strategies of industrially relevant production hosts also discussing current bottlenecks in the underlying metabolic pathways and how they can be addressed in the future.

Neochloris oleoabundans is worth its salt: Transcriptomic analysis under salt and nitrogen stress.

Neochloris oleoabundans is an oleaginous microalgal species that can be cultivated in fresh water as well as salt water. Using salt water gives the opportunity to reduce production costs and the fresh water footprint for large scale cultivation. Production of triacylglycerols (TAG) usually includes a biomass growth phase in nitrogen-replete conditions followed by a TAG accumulation phase under nitrogen-deplete conditions. This is the first report that provides insight in the saline resistance mechanism of a fresh water oleaginous microalgae. To better understand the osmoregulatory mechanism of N. oleoabundans during growth and TAG accumulating conditions, the transcriptome was sequenced under four different conditions: fresh water nitrogen-replete and -deplete conditions, and salt water (525 mM dissolved salts, 448mM extra NaCl) nitrogen-replete and -deplete conditions. In this study, several pathways are identified to be responsible for salt water adaptation of N. oleoabundans under both nitrogen-replete and -deplete conditions. Proline and the ascorbate-glutathione cycle seem to be of importance for successful osmoregulation in N. oleoabundans. Genes involved in Proline biosynthesis were found to be upregulated in salt water. This was supported by Nuclear magnetic resonance (NMR) spectroscopy, which indicated an increase in proline content in the salt water nitrogen-replete condition. Additionally, the lipid accumulation pathway was studied to gain insight in the gene regulation in the first 24 hours after nitrogen was depleted. Oil accumulation is increased under nitrogen-deplete conditions in a comparable way in both fresh and salt water. The mechanism behind the biosynthesis of compatible osmolytes can be used to improve N. oleoabundans and other industrially relevant microalgal strains to create a more robust and sustainable production platform for microalgae derived products in the future.

Genetically Transformed Root-Based Culture Technology in Medicinal Plant Cosmos bipinnatus

Background: Cosmos bipinnatus is an important medicinal plant with antioxidative, antigenotoxic, anti-inflammatory, and anti-proliferative effects on several cancer cell lines. It has great potential for development as a promising cancer chemo-preventive agent. Hairy root-based culture technology is a new sustainable production platform for producing specific pharmaceutical secondary metabolites. Objectives: The current study developed and introduced a reliable transformation system for C. bipinnatus by optimization of aspects important in transformation frequency using Agrobacterium rhizogenes. Methods: Five bacterial strains, including ATCC 15834, ATCC 31798, A7, MAFF-02-10266, and MSU440, 2 explant types (leaf and stem), and 2 co-cultivation media (full MS and ½ MS) were examined. Genomic DNA was extracted using a modified CTAB protocol from putative transgenic root lines and the control root. Transgenic hairy root lines were approved by means of Polymerase Chain Reaction (PCR) using specific rolB gene primers. Results: The highest ratio of genetically transformed root induction was found from leaf explants using A. rhisogenes strains ATCC15834 and MSU440 (72% to 73%). When ½ MS medium was used as a co-cultivation medium, a significant increase in transformation frequency (84%) was observed. Conclusions: The MSU440 Agrobacterium strain and ½ MS co-cultivation medium could significantly improve genetic transformation efficiency for establishment of hairy root-based cultures for C. bipinnatus.

Effects of hydrostatic pressure and supercritical carbon dioxide on the viability of Botryococcus braunii algae cells

In bio-based industries, Botryococcus braunii is identified as a potential resource for production of hydrocarbons having a wide range of applications in chemical and biopolymer industries. For a sustainable production platform, the algae cultivation should be integrated with downstream processes. Ideally the algae are not harvested, but the product is isolated while cultivation and growth is continued especially if the doubling time is slow. Consequently, hydrocarbons can be extracted while keeping the algae viable. In this study, the effects of pressure on the viability of B. braunii cells were tested hydrostatically and under supercritical CO2 conditions. Viability was determined by light microscopy, methylene blue uptake and by re-cultivation of the algae after treatments to follow the growth. It was concluded that supercritical CO2 was lethal to the algae, whereas hydrostatic pressure treatments up to 150 bar have not affected cell viability and recultivation was successful.