High-value Metabolites Research Articles

Enhancing poly(3-hydroxybutyrate) production in halophilic bacteria through improved salt tolerance

Polyhydroxyalkanoates (PHA) have emerged as a promising bio-compound in the industrial application due to their potential to replace conventional petroleum-based plastics with sustainable bioplastics. This study focuses on Halomonas sp. YJPS3-3, a halophilic bacterium, and presents a novel approach to enhance PHA production by exploiting its salt tolerance toward PHA biosynthesis. Through gamma irradiation-induced mutants with enhanced salt tolerance from 15% NaCl to 20% NaCl, mutant halo6 showing a significant 11% increase in PHA yield, was achieved. Moreover, the mutants displayed not only higher PHA content but also remarkable cell morphology with elongation. In addition, this research unravels the genetic determinants behind the elevated PHA content and identifies a corresponding shift in fatty acid composition favoring PHA accumulation. This novel mutant obtained from gamma irradiation with enhanced salt tolerance in halophilic bacteria opens up new avenues not only for the bioplastic industry but also for applications in the production of high-value metabolites.

Emerging Technologies for the Discovery of Novel Diversity in Cyanobacteria and Algae and the Elucidation of Their Valuable Metabolites

Until recently, the study of cyanobacteria and microalgae has been hampered by the need to cultivate these organisms to gain insight into their cytomorphology, life cycle and molecular biology. However, various microbial species characterized by thick sheaths of exopolymeric substances were difficult to isolate in culture due to their associated symbiotic bacteria. Other microbes evaded culture. Such challenges have now been overcome by the development of metagenomic techniques that allow direct DNA sequencing from environmental samples, as well as high resolution microscopy techniques that permit direct imaging of environmental samples. The sampling of understudied taxa from extreme environments and of toxic species has been facilitated by specialized robotic equipment. Single-cell sequencing has allowed for the proper characterization of microalgal species and their response to environmental changes. Various strains of cyanobacteria, microalgae and macroalgae have gained renewed interest for their high-value metabolites. This paper provides an overview of the emerging technologies and explains how they are being used to identify such strains and their products for industrial application. Advances in genetic engineering and CRISPR technology have facilitated the production of strains that are more amenable to culture, metabolite extraction, scale-up and application in biorefinery approaches. Emerging analytical techniques are discussed, with the advent of multiomics and its application in this field.

Current Nuclear Engineering Strategies in the Green Microalga Chlamydomonas reinhardtii.

The green model microalga Chlamydomonas reinhardtii recently emerged as a sustainable production chassis for the efficient biosynthesis of recombinant proteins and high-value metabolites. Its capacity for scalable, rapid and light-driven growth in minimal salt solutions, its simplicity for genetic manipulation and its "Generally Recognized As Safe" (GRAS) status are key features for its application in industrial biotechnology. Although nuclear transformation has typically resulted in limited transgene expression levels, recent developments now allow the design of powerful and innovative bioproduction concepts. In this review, we summarize the main obstacles to genetic engineering in C. reinhardtii and describe all essential aspects in sequence adaption and vector design to enable sufficient transgene expression from the nuclear genome. Several biotechnological examples of successful engineering serve as blueprints for the future establishment of C. reinhardtii as a green cell factory.

Two-phase microalgae cultivation for RAS water remediation and high-value biomass production.

The overall goal of this study was to provide solutions to innovative microalgae-based technology for wastewater remediation in a cold-water recirculating marine aquaculture system (RAS). This is based on the novel concept of integrated aquaculture systems in which fish nutrient-rich rearing water will be used for microalgae cultivation. The produced biomass can be used as fish feed, while the cleaned water can be reused, to create a highly eco-sustainable circular economy. Here, we tested three microalgae species Nannochloropis granulata (Ng), Phaeodactylum tricornutum (Pt), and Chlorella sp (Csp) for their ability to remove nitrogen and phosphate from the RAS wastewater and simultaneously produce high-value biomass, i.e., containing amino acids (AA), carotenoids, and polyunsaturated fatty acids (PUFAs). A high yield and value of biomass were achieved for all species in a two-phase cultivation strategy: i) a first phase using a medium optimized for best growth (f/2 14x, control); ii) a second "stress" phase using the RAS wastewater to enhance the production of high-value metabolites. Ng and Pt performed best in terms of biomass yield (i.e., 5-6 g of dry weight, DW.L-1) and efficient cleaning of the RAS wastewater from nitrite, nitrate, and phosphate (i.e., 100% removal). Csp produced about 3 g L-1 of DW and reduced efficiently only nitrate, and phosphate (i.e., about 76% and 100% removal, respectively). The biomass of all strains was rich in protein (30-40 % of DW) containing all the essential AA except Methionine. The biomass of all three species was also rich in PUFAs. Finally, all tested species are excellent sources of antioxidant carotenoids, including fucoxanthin (Pt), lutein (Ng and Csp) and β-carotene (Csp). All tested species in our novel two-phase cultivation strategy thus showed great potential to treat marine RAS wastewater and provide sustainable alternatives to animal and plant proteins with extra added values.

Comparative life cycle assessment of marine microalgae, Nannochloropsis sp. and fishmeal for sustainable protein ingredients in aquaculture feeds

Fishmeal from small marine pelagic fishes reduces their availability for marine wildlife forage and artisanal fishing catches that support food security in lower income coastal nations. Fishmeal is primarily used in feeds for aquaculture, the world’s fastest-growing food sector. Replacing fishmeal in aquafeeds with more environmentally responsible alternative ingredients can help feed aquaculture transition to more sustainable production methods. Protein from defatted marine microalga, Nannochloropsis sp., produced alongside polyunsaturated fatty acids (PUFAs) for the nutraceutical market lacks a comprehensive open-access analysis of environmental impacts of producing these products from biorefineries. This study compared life cycle impacts (global warming potential, water use, land use, marine eutrophication potential, freshwater eutrophication potential, and biotic resource use) of protein from fishmeal produced in a small pelagic fish biorefinery to protein from defatted Nannochloropsis meal. We conducted an attributional life cycle assessment using primary data provided by Cellana LLC to model biomass cultivation and harvesting at the Kona Demonstration Facility (Hawaii, USA) and literature data to model the downstream processing of biomass into a high-protein fishmeal replacement for the aquafeed market and concentrated PUFAs for the nutraceutical market. Material and energy inputs from a Nannochloropsis biorefinery included 2 harvesting scenarios (wet and dry biomass) and 2 scenarios for oil extraction and processing (i.e., oil fractionation and concentration of PUFAs): solvents or supercritical carbon dioxide. Results for aquafeed protein from defatted Nannochloropsis were that cultivation processes had the largest overall effect for all scenarios; urea and pure liquid carbon dioxide were environmental hot spots; and the processing scenario involving dry biomass followed by oil extraction and oil processing with solvent had significantly lower environmental impacts than protein from fishmeal from a small pelagic fish biorefinery for global warming potential, water use, marine eutrophication potential, freshwater eutrophication potential, and biotic resource use, but not for land use. These results suggest that aquafeed from marine microalgae can be an environmentally sustainable replacement for fishmeal if high-value metabolites are coproduced in a biorefinery.

Focus and Insights into the Synthetic Biology-Mediated Chassis of Economically Important Fungi for the Production of High-Value Metabolites.

Substantial progress has been achieved and knowledge gaps addressed in synthetic biology-mediated engineering of biological organisms to produce high-value metabolites. Bio-based products from fungi are extensively explored in the present era, attributed to their emerging importance in the industrial sector, healthcare, and food applications. The edible group of fungi and multiple fungal strains defines attractive biological resources for high-value metabolites comprising food additives, pigments, dyes, industrial chemicals, and antibiotics, including other compounds. In this direction, synthetic biology-mediated genetic chassis of fungal strains to enhance/add value to novel chemical entities of biological origin is opening new avenues in fungal biotechnology. While substantial success has been achieved in the genetic manipulation of economically viable fungi (including Saccharomyces cerevisiae) in the production of metabolites of socio-economic relevance, knowledge gaps/obstacles in fungal biology and engineering need to be remedied for complete exploitation of valuable fungal strains. Herein, the thematic article discusses the novel attributes of bio-based products from fungi and the creation of high-value engineered fungal strains to promote yield, bio-functionality, and value-addition of the metabolites of socio-economic value. Efforts have been made to discuss the existing limitations in fungal chassis and how the advances in synthetic biology provide a plausible solution.

A green and efficient technology for sequential extraction of lipid and paramylon from Euglena gracilis

Euglena gracilis is rich in nutrients and high-value metabolites. However, at present, most studies only extract the paramylon of E. gracilis, which is a waste of resources. In this study, a method of sequential extraction of lipid and paramylon from E. gracilis was studied, and the extraction conditions were optimized. Firstly, by optimizing the solid-liquid ratio, extraction time, extraction temperature and volume ratio of n-hexane to ethanol, the lipid extraction efficacy was increased from 73.0 % to 96.0 %. Next, paramylon was extracted from the E. gracilis residue. After optimizing the enzyme type, enzyme concentration, ratio of material to liquid and enzymolysis time, the protein removal rate was 91.3 %, the yield of paramylon was 58.3 % and the purity reached 89.3 %. Compared with the traditional method, this sequential extraction method was greener and more effective for the extraction of paramylon and lipids from E. gracilis, which can promote the comprehensive utilization of algae resources.

A Culturomics-Based Bacterial Synthetic Community for Improving Resilience towards Arsenic and Heavy Metals in the Nutraceutical Plant Mesembryanthemum crystallinum.

Plant-growth-promoting bacteria (PGPB) help plants thrive in polluted environments and increase crops yield using fewer inputs. Therefore, the design of tailored biofertilizers is of the utmost importance. The purpose of this work was to test two different bacterial synthetic communities (SynComs) from the microbiome of Mesembryanthemum crystallinum, a moderate halophyte with cosmetic, pharmaceutical, and nutraceutical applications. The SynComs were composed of specific metal-resistant plant-growth-promoting rhizobacteria and endophytes. In addition, the possibility of modulating the accumulation of nutraceutical substances by the synergetic effect of metal stress and inoculation with selected bacteria was tested. One of the SynComs was isolated on standard tryptone soy agar (TSA), whereas the other was isolated following a culturomics approach. For that, a culture medium based on M. crystallinum biomass, called Mesem Agar (MA), was elaborated. Bacteria of three compartments (rhizosphere soil, root endophytes, and shoot endophytes) were isolated on standard TSA and MA media, stablishing two independent collections. All bacteria were tested for PGP properties, secreted enzymatic activities, and resistance towards As, Cd, Cu, and Zn. The three best bacteria from each collection were selected in order to produce two different consortiums (denominated TSA- and MA-SynComs, respectively), whose effect on plant growth and physiology, metal accumulation, and metabolomics was evaluated. Both SynComs, particularly MA, improved plant growth and physiological parameters under stress by a mixture of As, Cd, Cu, and Zn. Regarding metal accumulation, the concentrations of all metals/metalloids in plant tissues were below the threshold for plant metal toxicity, indicating that this plant is able to thrive in polluted soils when assisted by metal/metalloid-resistant SynComs and could be safely used for pharmaceutical purposes. Initial metabolomics analyses depict changes in plant metabolome upon exposure to metal stress and inoculation, suggesting the possibility of modulating the concentration of high-value metabolites. In addition, the usefulness of both SynComs was tested in a crop plant, namely Medicago sativa (alfalfa). The results demonstrate the effectiveness of these biofertilizers in alfalfa, improving plant growth, physiology, and metal accumulation.

Microalgae as next generation plant growth additives: Functions, applications, challenges and circular bioeconomy based solutions.

Sustainable agriculture practices involve the application of environment-friendly plant growth promoters and additives that do not negatively impact the health of the ecosystem. Stringent regulatory frameworks restricting the use of synthetic agrochemicals and the increase in demand for organically grown crops have paved the way for the development of novel bio-based plant growth promoters. In this context, microalgae biomass and derived agrochemicals offer novel sources of plant growth promotors that enhance crop productivity and impart disease resistance. These beneficial effects could be attributed to the presence of wide range of biomolecules such as soluble amino acid (AA), micronutrients, polysaccharides, phytohormones and other signaling molecules in microalgae biomass. In addition, their phototrophic nature, high photosynthetic efficiency, and wide environmental adaptability make them an attractive source of biostimulants, biofertilizers and biopesticides. The present review aims to describe the various plant growth promoting metabolites produced by microalgae and their effects on plant growth and productivity. Further, the effects elicited by microalgae biostimulants with respect to different modes of applications such as seed treatments, foliar spray and soil/root drenching is reviewed in detail. In addition, the ability of microalgae metabolites to impart tolerance against various abiotic and biotic stressors along with the mechanism of action is discussed in this paper. Although the use of microalgae based biofertilizers and biostimulants is gaining popularity, the high nutrient and water requirements and energy intensive downstream processes makes microalgae based technology commercially unsustainable. Addressing this challenge, we propose a circular economy model of microalgae mediated bioremediation coupled with biorefinery approaches of generating high value metabolites along with biofertilizer applications. We discuss and review new trends in enhancing the sustainability of microalgae biomass production by co-cultivation of algae with hydroponics and utilization of agriculture effluents.

Combined application of exogenous phytohormones and blue light illumination to the marine diatom Phaeodactylum tricornutum

Diatoms constitute a diverse group of photosynthetic microorganisms with great potential for biotechnological applications. Compounds which they can synthesise, such as ω3-fatty acids or carotenoid pigments, can find uses in the pharmacology, nutraceutical or cosmetic sectors. However, a major challenge to date has been to stimulate the synthesis of valuable metabolites without hampering cell growth.In this study, the model microalgal species Phaeodactylum tricornutum (marine diatom) was subjected to blue light illumination and phytohormonal supplementation (gibberellic acid, GA3, and abscisic acid, ABA) as single factors or in combination. P. tricornutum was grown under a batch culture regime for 30 days and treatments were tested at two different stages (day 0 and day 20).The highest yield of dry biomass was obtained when combining blue light and GA3 from day 0 (25 % higher than control) while no relevant variation occurred when applying blue light from day 20. When applied from day 0, blue light led to a reduction in the fucoxanthin and PUFA content of the biomass. Single dose supplementations of the phytohormones seemed less dependent on the time of application. When applied without blue light, GA3 (2 mg/l) increased the fucoxanthin content and yield by 25 and 30 %, respectively. However, the co-supplementation of GA3 and ABA (2 mg/l) was more effective than single applications, significantly boosting fucoxanthin yield up to 1.3 mg/l and EPA proportions by 17 %, compared to the control set. These increments were also associated with enhanced antioxidant activity (60 % increment).These results highlight the importance of optimising the timing of application of treatments to avoid deleterious effects on pigment and PUFA accumulation in cells. Switching to blue light illumination and applying GA3 and ABA later in the growth of the diatom P. tricornutum constitutes a promising strategy to improve its yields in high-value metabolites prior to biomass harvest.

Highly efficient co-production of fucoxanthin and eicosapentaenoic acid by heterotrophic cultivation of a newly isolated microalga Nitzschia sp. FZU62

Microalgae can accumulate various compounds simultaneously, thus co-production of high-value metabolites to improve the economic value of microalgae production is feasible. In this study, a microalga strain Nitzschia sp. FZU62 capable of co-producing fucoxanthin and eicosapentaenoic acid (EPA) was isolated and identified. Further, heterotrophic conditions were optimized to improve the co-production efficiency in flasks. Results showed that the optimal conditions for flask culture were cultivation for 84 h with an inoculum size of 0.47 g/L and a temperature of 19 °C. Besides, three fed-batch culture strategies with distinct nutrient compositions were explored to improve the heterotrophic performance of strain FZU62. Results indicated that the highest biomass, fucoxanthin, and EPA productivities were obtained by Fed-batch III, in which concentrated medium was pulse-fed to supply 60 % initial nutrients once glucose concentration dropped to about 2 g/L. Moreover, the heterotrophic performance was verified in a 5 L fermenter, resulting in excellent fucoxanthin and EPA productions of 63.03 and 398.60 mg/L, and productivities of 3.41 and 22.52 mg/L/d, respectively. These results, especially the EPA production and productivity, are superior to many literature reports. The present study provides new insights into the highly efficient co-production of fucoxanthin and EPA by marine microalgae and has guiding significance for commercial production in heterotrophic mode.

Microencapsulation of Yarrowia lipolytica: cell viability and application in vitro ruminant diets.

Microencapsulation is an alternative to increase the survival capacity of microorganisms, including Yarrowia lipolytica, a widely studied yeast that produces high-value metabolites, such as lipids, aromatic compounds, biomass, lipases, and organic acids. Thus, the present study sought to investigate the effectiveness of different wall materials and the influence of the addition of salts on the microencapsulation of Y. lipolytica, evaluating yield, relationship with cell stability, ability to survive during storage, and in vitro application of ruminant diets. The spray drying process was performed via atomization, testing 11 different compositions using maltodextrin (MD), modified starch (MS) and whey protein concentrate (WPC), Y. lipolytica (Y. lipo) cells, tripolyphosphate (TPP), and sodium erythorbate (SE). The data show a reduction in the water activity value in all treatments. The highest encapsulation yield was found in treatments using MD + TPP + Y. lipo (84.0%) and WPC + TPP + Y. lipo (81.6%). Microencapsulated particles showed a survival rate ranging from 71.61 to 99.83% after 24h. The treatments WPC + Y. lipo, WPC + SE + Y. lipo, WPC + TPP + Y. lipo, and MD + SE + Y. lipo remained stable for up to 105days under storage conditions. The treatment WPC + SE + Y. lipo (microencapsulated yeast) was applied in the diet of ruminants due to the greater stability of cell survival. The comparison between the WPC + SE + Y. lipo treatment, wall materials, and the non-microencapsulated yeast showed that the microencapsulated yeast obtained a higher soluble fraction, degradability potential, and release of nutrients.

Isolating Fistulifera pelliculosa from the northern Bohai Sea and analyzing biochemical composition, antibacterial and nutrient removal potential

The microalgae located near the estuary of the Liaohe River along the coast of Panjin have long been in an area with large fluctuations in salinity, temperature, and nutrients, and have high-quality alternatives for high-value metabolites. Three strains of microalgae were screened and the biomass of microalgae could be optimized 0.313–0.790 g L−1 in 10 L bioreactor. The determination results of bioactive substances in these three microalgae showed that, the amount of fucoxanthin in the growth phase II (14 days) was maximum, at 5.354, 6.284 and 14.837 mg g−1 respectively. The diatoxanthin of Dut-wj-J1 in growth phase III (21 days) could reach 5.158 mg g−1. Dut-wj-J4 had the highest lipid production efficiency (9.45 mg L−1 d−1) followed by Dut-wj-J2 (8.49 mg L−1 d−1) and Dut-wj-J1 (8.18 mg L−1 d−1) respectively. These bioactive substances have inhibition zones of 7–13 mm against all four strains of bacteria ie., Acetobacter, Rhodococcus erythropolis, Escherichia coli and Bacillus subtilis Cohn respectively. In addition, these microalgae can play a potential role in nutrient enrichment in eutrophic seawater. The NO3− degradation rates of these three algae in the first 14 days were 75.0 %, 45.8 % and 100 % respectively, as well as the PO4− degradation rates in the first 7 days were 94.8 %, 100 % and 80.9 % respectively. This work manifests the plasticity of algae isolated from the Bohai Sea and provides useful insights for further joint production of bioactive substances.

Production of Metabolites in Microalgae Under Alkali Halophilic Growth Medium Using a Dissolved Inorganic Carbon Source

The production of metabolites in microalgae is influenced by extreme cultivation conditions. Bicarbonate is an inorganic carbon source for phototrophic microalgae culturing. In this study, the effect of dissolved inorganic carbon (DIC) (0.4–13 g L−1) on the accumulation of metabolites in Dunaliella tertiolecta biomass is presented. The highest levels of primary metabolites [lipid (239.6 ± 24.3 mg g−1), protein (336.2 ± 47.5 mg g−1)], secondary metabolites [total phenolic (12.8 ± 2.0 mg g−1), total flavonoid (14.4 ± 2.3 mg g−1), total ascorbate (4.7 ± 1.1 mg g−1)], and pigments [chlorophyll (27.2 ± 3.1 mg g−1), carotenoid (2.0 ± 0.1 mg g−1)] were observed when cells were grown with 5.7 g L−1 of DIC (NaHCO3). The highest biomass concentrations (1.5 ± 0.1 g L−1) were obtained for cells grown in a mixture of DIC (4.3 + 1.1 g L−1, NaHCO3 + Na2CO3). This study recommends the optimal levels of bicarbonate carbon of 5.7 g L−1 for maximising the generation of metabolites in the biomass. It also demonstrates that exogenous excessive DIC in the growth medium would be an effective stressor to produce high-value metabolites in Dunaliella or alkali-halophilic strains.Graphical

Natural promoters and promoter engineering strategies for metabolic regulation in Saccharomyces cerevisiae.

Sharomyces cerevisiae is currently one of the most important foreign gene expression systems. S. cerevisiae is an excellent host for high-value metabolite cell factories due to its advantages of simplicity, safety, and nontoxicity. A promoter, as one of the basic elements of gene transcription, plays an important role in regulating gene expression and optimizing metabolic pathways. Promoters control the direction and intensity of transcription, and the application of promoters with different intensities and performances will largely determine the effect of gene expression and ultimately affect the experimental results. Due to its significant role, there have been many studies on promoters for decades. While some studies have explored and analyzed new promoters with different functions, more studies have focused on artificially modifying promoters to meet their own scientific needs. Thus, this article reviews current research on promoter engineering techniques and related natural promoters in S. cerevisiae. First, we introduce the basic structure of promoters and the classification of natural promoters. Then, the classification of various promoter strategies is reviewed. Finally, by grouping related articles together using various strategies, this review anticipates the future development direction of promoter engineering.

Cellular and metabolic characteristics of peach anther-derived callus

Callus is an important material for horticulture research, especially for biotechnological applications, and can be used for the production of high-value metabolites. In this paper, anthers of peach (Prunus persica L.) varieties ‘Zaojiubao’, ‘Yanhong’, and ‘Xueyulu’ were used to induce callus lines, and the tissue morphology, subcellular structure, and metabolite profiles were characterized. Peach anther-derived calli were successfully induced in MS+1.0 mg/l ZT+1.0 mg/l 2,4-D + 0.5 mg/l IAA medium, and were stably subcultured in MS+1.0 mg/l BA+0.2 mg/l IBA+0.1 mg/l GA3 medium. SSR analysis confirmed the callus lines originated from somatic cells. Furthermore, microscopic observation of callus cells revealed some interesting cytological features, such as abundant mitochondria, which may be associated with vigorous growth and strong resistance to senescence, and suggests their potential for protoplast-related experiments such as transient expression. The metabolites in anther-derived calli were comparatively analyzed. Naringenin-7-O-glucoside (prunin), which has medicinal value as an anti-inflammatory and antiviral compound, was highly enriched in the callus lines, and cyanidin-3-O-glucoside caused the red coloration of peach anther calli. Additionally, chlorogenic acid, and amino acid derivatives such as aspartic acid and leucine, as well as other organic acids, lipids, and alkaloids, were detected in the callus lines. This study showed a series of peach anther-derived callus lines have great potential for cell biology and transformation research, especially for the production of high value components, which can be used for medicinal purposes, health products, and food additives.

Improvement in the Sequential Extraction of Phycobiliproteins from Arthrospira platensis Using Green Technologies.

Arthrospira platensis (commercially known as Spirulina) is an excellent source of phycobiliproteins, especially C-phycocyanin. Phycobiliproteins are significant bioactive compounds with useful biological applications. The extraction process plays a significant role in downstream microalga production and utilisation. The important pigments found in A. platensis include chlorophyll and carotenoids as nonpolar pigments and phycobiliproteins as polar pigments. Supercritical fluid extraction (SFE) as a green extraction technology for the high-value metabolites of microalgae has potential for trends in food and human health. The nonpolar bioactive compounds, chlorophyll and carotenoids of A. platensis, were primarily separated using supercritical carbon dioxide (SC-CO2) solvent-free fluid extraction pressure; the temperature and ethanol as cosolvent conditions were compared. The residue from the A. platensis cells was subjected to phycobiliprotein extraction. The phosphate and water extraction of A. platensis SFE residue were compared to evaluate phycobiliprotein extraction. The SFE results exhibited higher pressure (350 bar) and temperature extraction (50 °C) with ethanol-free extraction and increased nonpolar pigment. Phycobiliprotein yield was obtained from A. platensis SFE residue by ethanol-free buffer extraction as a suitable process with antioxidant properties. The C-phycocyanin was isolated and enhanced to 0.7 purity as food grade. This developed method can be used as a guideline and applied as a sustainable process for important pigment extraction from Arthrospira microalgae.

Plant-based expression platforms to produce high-value metabolites and proteins.

Plant-based heterologous expression systems can be leveraged to produce high-value therapeutics, industrially important proteins, metabolites, and bioproducts. The production can be scaled up, free from pathogen contamination, and offer post-translational modifications to synthesize complex proteins. With advancements in molecular techniques, transgenics, CRISPR/Cas9 system, plant cell, tissue, and organ culture, significant progress has been made to increase the expression of recombinant proteins and important metabolites in plants. Methods are also available to stabilize RNA transcripts, optimize protein translation, engineer proteins for their stability, and target proteins to subcellular locations best suited for their accumulation. This mini-review focuses on recent advancements to enhance the production of high-value metabolites and proteins necessary for therapeutic applications using plants as bio-factories.

Advances in plastid transformation for metabolic engineering in higher plants.

The plastid (chloroplast) genome of higher plants is an appealing target for metabolic engineering via genetic transformation. Although the bacterial-type plastid genome is small compared with the nuclear genome, it can accommodate large quantities of foreign genes that precisely integrate through homologous recombination. Engineering complex metabolic pathways in plants often requires simultaneous and concerted expression of multiple transgenes, the possibility of stacking several transgenes in synthetic operons makes the transplastomic approach amazing. The potential for extraordinarily high-level transgene expression, absence of epigenetic gene silencing and transgene containment due to the exclusion of plastids from pollen transmission in most angiosperm species further add to the attractiveness of plastid transformation technology. This minireview describes recent advances in expanding the toolboxes for plastid genome engineering, and highlights selected high-value metabolites produced using transplastomic plants, including artemisinin, astaxanthin and paclitaxel.

Architect: A tool for aiding the reconstruction of high-quality metabolic models through improved enzyme annotation.

Constraint-based modeling is a powerful framework for studying cellular metabolism, with applications ranging from predicting growth rates and optimizing production of high value metabolites to identifying enzymes in pathogens that may be targeted for therapeutic interventions. Results from modeling experiments can be affected at least in part by the quality of the metabolic models used. Reconstructing a metabolic network manually can produce a high-quality metabolic model but is a time-consuming task. At the same time, current methods for automating the process typically transfer metabolic function based on sequence similarity, a process known to produce many false positives. We created Architect, a pipeline for automatic metabolic model reconstruction from protein sequences. First, it performs enzyme annotation through an ensemble approach, whereby a likelihood score is computed for an EC prediction based on predictions from existing tools; for this step, our method shows both increased precision and recall compared to individual tools. Next, Architect uses these annotations to construct a high-quality metabolic network which is then gap-filled based on likelihood scores from the ensemble approach. The resulting metabolic model is output in SBML format, suitable for constraints-based analyses. Through comparisons of enzyme annotations and curated metabolic models, we demonstrate improved performance of Architect over other state-of-the-art tools, notably with higher precision and recall on the eukaryote C. elegans and when compared to UniProt annotations in two bacterial species. Code for Architect is available at https://github.com/ParkinsonLab/Architect. For ease-of-use, Architect can be readily set up and utilized using its Docker image, maintained on Docker Hub.