Production Of Useful Metabolites Research Articles

Involvement of LaeA and Velvet Proteins in Regulating the Production of Mycotoxins and Other Fungal Secondary Metabolites.

Fungi are rich sources of secondary metabolites of agrochemical, pharmaceutical, and food importance, such as mycotoxins, antibiotics, and antitumor agents. Secondary metabolites play vital roles in fungal pathogenesis, growth and development, oxidative status modulation, and adaptation/resistance to various environmental stresses. LaeA contains an S-adenosylmethionine binding site and displays methyltransferase activity. The members of velvet proteins include VeA, VelB, VelC, VelD and VosA for each member with a velvet domain. LaeA and velvet proteins can form multimeric complexes such as VosA-VelB and VelB-VeA-LaeA. They belong to global regulators and are mainly impacted by light. One of their most important functions is to regulate gene expressions that are responsible for secondary metabolite biosynthesis. The aim of this mini-review is to represent the newest cognition of the biosynthetic regulation of mycotoxins and other fungal secondary metabolites by LaeA and velvet proteins. In most cases, LaeA and velvet proteins positively regulate production of fungal secondary metabolites. The regulated fungal species mainly belong to the toxigenic fungi from the genera of Alternaria, Aspergillus, Botrytis, Fusarium, Magnaporthe, Monascus, and Penicillium for the production of mycotoxins. We can control secondary metabolite production to inhibit the production of harmful mycotoxins while promoting the production of useful metabolites by global regulation of LaeA and velvet proteins in fungi. Furthermore, the regulation by LaeA and velvet proteins should be a practical strategy in activating silent biosynthetic gene clusters (BGCs) in fungi to obtain previously undiscovered metabolites.

Melatonin Treatment Enhances the Growth and Productivity of Useful Metabolites in the In Vitro Culture of Spirodela polyrhiza.

Spirodela polyrhiza (Araceae family) is a duckweed species that serves as a potential resource for feed, food, bioremediation, and pharmaceutical applications. In this study, we assessed the effects of different concentrations of melatonin (0, 0.1, 1, and 10 μM) on the growth of S. polyrhiza during in vitro culture and the metabolic profiles and productivities of useful metabolites using gas chromatography-mass spectrometry coupled with multivariable statistical analysis. We found that exogenous melatonin significantly improved the total dry weight and altered the metabolic profiles of S. polyrhiza cultures. Melatonin significantly enhanced the cellular production of useful metabolites, such as γ-aminobutyric acid, dopamine, threonine, valine, and phytosterols. The volumetric productivities (mg/L) of γ-aminobutyric acid, dopamine, campesterol, β-sitosterol, and stigmasterol were the highest in the presence of 10 μM melatonin on day 12. Moreover, the productivities of ascorbic acid and serotonin were the highest in the presence of 1 μM melatonin on day 12. Therefore, melatonin could be used to enhance the production of biomass and useful metabolites during large-scale S. polyrhiza cultivation in cosmetic, food/feed, and pharmaceutical industries.

In vitro regeneration and its histological characteristics of Dioscorea nipponica Makino

Dioscorea nipponica Makino is an optimal candidate to develop the diosgenin industry in North China. Due to its increasing demand in the medicine industry, it is urgent to apply new biotechnological tools to foster breeds with desirable traits and enhanced secondary metabolite production. The production of useful metabolites by the in vitro cultured rhizomes can be explored successfully for utilization by various food and drug industries. In this study, we reported callus formation and plantlet regeneration of the medicinal plant D. nipponica. Explants of leaves, stem segments and rhizomes of aseptic seedlings were cultured on Murashige and Skoog (MS) medium containing various combinations of auxin and cytokinin to find the optimal PGRs of each type of explant for callus induction and shoot regeneration of D. nipponica. The paraffin section technique was also used to observe of the morphogenesis of callus and adventitious bud. Explants of seeds and rhizomes formed calli at high frequency in all lines we examined. However, the explant of leaves rarely formed callus. Three kinds of callus were detected during the induction phase. Here, we describe three types of callus (Callus I–III) with different structure characteristics. Greenish in color and a nodule-like protrusion surface (Callus type III) were arranged more closely of cells with less interstitial substance, cell differentiation ability stronger than other callus types. The optimum combination was the maximum shoot differentiation frequency of 90% in callus derived from seeds cultured on MS medium with 2.0 mg L−16-BA + 0.2 mg L−1NAA. The shoot differentiation frequency (88.57%) of rhizome-induced callus was obtained by the combination of MS medium supplemented with 3.0 mg L−16-BA + 2.0 mg L−1NAA. 1/2 MS medium plus 0.5 mg L−1NAA resulted in a higher root regeneration frequency of 86.70%. In vitro propagated plantlets with healthy roots were domesticated and transplanted into small plastic pots containing sterile soil rite under greenhouse conditions with 80% survivability. Bud differentiation is mostly of exogenous origin, mostly occurring on the near callus surface. Therefore, it may be surmised that in vitro morphogenesis of D. nipponica is mainly caused by indirect organogenesis (adventitious bud).

Simultaneous production of cellulase and amylase by Aspergillus fumigatus IB-A1

Utilization of agricultural wastes for production of useful metabolites requires hydrolysis using both cellulase and amylase enzymes. We isolated Aspergillus fumigatus IB-A1 and evaluated its ability to simultaneously produce both cellulase and amylase. Although the isolate could produce both cellulase and amylase from either soluble starch or carboxymethyl cellulose, amylase activity was higher with soluble starch while cellulase activity was higher when carboxymethyl cellulose was used as the sole carbon source. With a mixture of carboxymethyl cellulose and soluble starch, both the amylase and cellulase activities increased with increase in the ratio of soluble starch. The optima ratio of carboxymethyl cellulose to soluble starch for cellulase and amylase activities were 0.7:0.3, and 0.4 to 0.6 respectively. For practical application, the optimum ratio of carboxymethyl cellulose to soluble starch in the production medium depends on the relative composition of cellulose and starch in the substrate to be hydrolyzed. The isolate was also able to efficiently produce both amylase and cellulase from cassava peel. With 10 g/L cassava peel, the cellulase and amylase activities were 6.122± 0.320 U/ml/min and 4.342± 0.210 U/ml/min respectively. When the cells were immobilized on loofa sponge and subjected to alternating air phase- liquid phase culture, cellulase and amylase production from cassava peel increased to 8.106± 0.620 U/ml/min and 5.206± 1.24 U/ml/min respectively. The optimum ratio of the air phase to the liquid phase was 3 hours of air phase to 21 hours of liquid phase.

The Genomic and Transcriptomic Analyses of Floccularia luteovirens, a Rare Edible Fungus in the Qinghai-Tibet Plateau, Provide Insights into the Taxonomy Placement and Fruiting Body Formation.

Floccularia luteovirens is a famous and precious edible mushroom (Huang Mogu) on the Qinghai–Tibet plateau that has a unique flavor and remarkable medical functions. Herein, we report a reference-grade 27 Mb genome of F. luteovirens containing 7068 protein-coding genes. The genome component and gene functions were predicted. Genome ontology enrichment and pathway analyses indicated the potential production capacity for terpenoids, polyketides and polysaccharides. Moreover, 16 putative gene clusters and 145 genes coding for secondary metabolites were obtained, including guadinomine and melleolides. In addition, phylogenetic and comparative genomic analyses shed light on the precise classification of F. luteovirens suggesting that it belongs to the genus Floccularia instead of Armillaria. RNA-sequencing and comparative transcriptomic analysis revealed differentially expressed genes during four developmental stages of F. luteovirens, that of which helps to identify important genes regulating fruiting body formation for strain modification. This study will provide insight into artificial cultivation and increase the production of useful metabolites.

Transfection of Arctic Bryum sp. KMR5045 as a Model for Genetic Engineering of Cold-Tolerant Mosses.

Mosses number about 13,000 species and are an important resource for the study of the plant evolution that occurred during terrestrial colonization by plants. Recently, the physiological and metabolic characteristics that distinguish mosses from terrestrial plants have received attention. In the Arctic, in particular, mosses developed their own distinct physiological features to adapt to the harsh environment. However, little is known about the molecular mechanisms by which Arctic mosses survive in extreme environments due to the lack of basic knowledge and tools such as genome sequences and genetic transfection methods. In this study, we report the axenic cultivation and transfection of Arctic Bryum sp. KMR5045, as a model for bioengineering of Arctic mosses. We also found that the inherent low-temperature tolerance of KMR5045 permitted it to maintain slow growth even at 2°C, while the model moss species Physcomitrium patens failed to grow at all, implying that KMR5045 is suitable for studies of cold-tolerance mechanisms. To achieve genetic transfection of KMR5045, some steps of the existing protocol for P. patens were modified. First, protoplasts were isolated using 1% driselase solution. Second, the appropriate antibiotic was identified and its concentration was optimized for the selection of transfectants. Third, the cell regeneration period before transfer to selection medium was extended to 9 days. As a result, KMR5045 transfectants were successfully obtained and confirmed transfection by detection of intracellular Citrine fluorescence derived from expression of a pAct5:Citrine transgene construct. This is the first report regarding the establishment of a genetic transfection method for an Arctic moss species belonging to the Bryaceae. The results of this study will contribute to understanding the function of genes involved in environmental adaptation and to application for production of useful metabolites derived from stress-tolerant mosses.

Comparative Primary Metabolic and Lipidomic Profiling of Freshwater and Marine Synechocystis Strains Using by GC-MS and NanoESI-MS Analyses

Synechocystis strains are considered as valuable resources for production of useful metabolites. Comparative primary metabolic and lipidomic profiling of freshwater and marine Synechocystis strains is needed to understand the physiological characteristics of each strain at molecular level. In this study, we performed comparative primary metabolic and lipidomic profiling of freshwater Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803) and marine Synechocystis sp. PCC 7338 (hereafter Synechocystis 7338) by using gas chromatography-mass spectrometry and nano electrospray ionization-mass spectrometry. Neophytadiene, phytol, monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG) were significantly higher in Synechocystis 6803. Levels of alanine, aspartic acid, valine, linoleic acid, linolenic acid, oleic acid, plamitic acid, stearic acid, glucosylglycerol, sucrose, phosphatidylglycerol (PG), and diacylglyceryltrimethylhomoserine (DGTS) (only detected in Synechocystis 7338) were significantly higher in Synechocystis 7338. Our results provide basic information to choose Synechocystis strains for various industrial usages, and also provide fundamental information on the future development of novel Synechocystis strains to produce high levels of specific metabolites by metabolic pathway modulation.

Immobilization of planktonic algal spores by inkjet printing

The algal cell immobilization is a commonly used technique for treatment of waste water, production of useful metabolites and management of stock culture. However, control over the size of immobilized droplets, the population of microbes, and production rate in current techniques need to be improved. Here, we use drop-on-demand inkjet printing to immobilize spores of the alga Ecklonia cava within alginate microparticles for the first time. Microparticles with immobilized spores were generated by printing alginate-spore suspensions into a calcium chloride solution. We demonstrate that the inkjet technique can control the number of spores in an ejected droplet in the range of 0.23 to 1.87 by varying spore densities in bioink. After the printing-based spore encapsulation, we observe initial sprouting and continuous growth of thallus until 45 days of culture. Our study suggest that inkjet printing has a great potential to immobilize algae, and that the ability to control the number of encapsulated spores and their microenvironments can facilitate research into microscopic interactions of encapsulated spores.

Optimization of Cell Free Protein Synthesis: Development of a Fusion Protein Fitness Strategy

Our undergraduate researchers work in the field of metabolic engineering to harness metabolic pathways for the production of useful metabolites with applications in the biofuel, pharmaceutical, or industrial chemical industries. We published a metabolic engineering approach called programmed evolution that introduces variation in regulatory elements for the expression of enzymes that control orthogonal metabolic pathways. Programmed evolution employs a riboswitch‐based fitness module to transduce the production of a desired metabolite into antibiotic resistance. We validated programmed evolution by optimizing the conversion of caffeine to theophylline by caffeine demethylase, but we recognized several limitations to the widespread use of our approach, including crosstalk between orthogonal and native bacterial metabolism, potential toxicity of metabolites, and poor representation of large variation spaces. To avoid these limitations, we are developing an in vitro approach to programmed evolution. For cell‐free protein synthesis (CFPS), we found supercoiled plasmid was a much better template than linear PCR amplicon. We designed, constructed, and have begun testing a novel fitness module that responds to the production of theophylline produced by the action of caffeine demethylase produced by CFPS. The fitness module encodes a fusion protein composed of three functional units. The amino portion contains a Gal4 DNA binding domain that will target binding sites on a library of DNA regulatory elements for the expression of caffeine demethylase. The carboxyl terminus includes streptavidin which will bind to avidin beads for physical separation of selected DNA regulatory elements. Sandwiched in between these two domains is GFP, which serves as a biosensor. Using this in vitro selection strategy, we plan to pursue the optimization of CFPS for the discovery of efficient metabolic pathways of interest.Support or Funding InformationNSF RUI grant MCB‐1613203 to Davidson College and MCB‐1613281 to Missouri Western State University, Davidson College Martin Genomics ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Optimization of Cell Free Protein Synthesis: Development of a Restriction Endonuclease Fitness Strategy

Our undergraduate researchers are interested in advancing the field of metabolic engineering for the production of useful metabolites that serve as biofuels, pharmaceuticals, or industrial chemical feedstocks. We published an approach to the metabolic engineering of bacteria called programmed evolution, which introduces variation in regulatory elements for the production of enzymes that control orthogonal metabolic pathways. Programmed evolution uses in vivo fitness modules that enable selection by employing riboswitches to transduce the production of desired metabolites into antibiotic resistance. Because of limitations imposed by in vivo programmed evolution such as crosstalk between orthogonal and native bacterial metabolism, the toxicity of some metabolites, and poor representation of large variation spaces, we want to reinvent programmed evolution as an in vitro strategy. This preliminary report describes our first steps of comparing lysates from two different strains of E. coli and using them to carry out cell‐free protein synthesis (CFPS). We also designed, constructed, and have begun testing a fitness module based on the expression of the restriction enzyme SmaI. We are working toward a proof‐of‐concept system in which combinations of DNA regulatory elements that enhance the production of caffeine demethylase are selected for their ability to produce theophylline. Theophylline production will turn on a riboswitch that leads to a burst of SmaI expression. Localized SmaI activity is expected to preferentially cleave successful control elements over unsuccessful ones, allowing purification and identification of previously unknown requirements for CFPS‐controlled metabolism. The successful development of in vitro programmed evolution would enable researchers to exploit the power of selection to discover combinations of control elements for the optimal yield of metabolites that are not easily produced in vivo.Support or Funding InformationNSF RUI grant MCB‐1613203 to Davidson College and MCB‐1613281 to Missouri Western State University, Davidson College Martin Genomics ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Grid-based computational methods for the design of constraint-based parsimonious chemical reaction networks to simulate metabolite production: GridProd

BackgroundConstraint-based metabolic flux analysis of knockout strategies is an efficient method to simulate the production of useful metabolites in microbes. Owing to the recent development of technologies for artificial DNA synthesis, it may become important in the near future to mathematically design minimum metabolic networks to simulate metabolite production.ResultsWe have developed a computational method where parsimonious metabolic flux distribution is computed for designated constraints on growth and production rates which are represented by grids. When the growth rate of this obtained parsimonious metabolic network is maximized, higher production rates compared to those noted using existing methods are observed for many target metabolites. The set of reactions used in this parsimonious flux distribution consists of reactions included in the original genome scale model iAF1260. The computational experiments show that the grid size affects the obtained production rates. Under the conditions that the growth rate is maximized and the minimum cases of flux variability analysis are considered, the developed method produced more than 90% of metabolites, while the existing methods produced less than 50%. Mathematical explanations using examples are provided to demonstrate potential reasons for the ability of the proposed algorithm to identify design strategies that the existing methods could not identify.ConclusionWe developed an efficient method for computing the design of minimum metabolic networks by using constraint-based flux balance analysis to simulate the production of useful metabolites. The source code is freely available, and is implemented in MATLAB and COBRA toolbox.

Effects of coronatine elicitation on growth and metabolic profiles of Lemna paucicostata culture.

In this study, the effects of coronatine treatment on the growth, comprehensive metabolic profiles, and productivity of bioactive compounds, including phenolics and phytosterols, in whole plant cultures of Lemna paucicostata were investigated using gas chromatography-mass spectrometry (GC-MS) coupled with multivariate statistical analysis. To determine the optimal timing of coronatine elicitation, coronatine was added on days 0, 23, and 28 after inoculation. The total growth of L. paucicostata was not significantly different between the coronatine treated groups and the control. The coronatine treatment in L. paucicostata induced increases in the content of hydroxycinnamic acids, such as caffeic acid, isoferulic acid, ρ-coumaric acid, sinapic acid, and phytosterols, such as campesterol and β-sitosterol. The productivity of these useful metabolites was highest when coronatine was added on day 0 and harvested on day 32. These results suggest that coronatine treatment on day 0 activates the phenolic and phytosterol biosynthetic pathways in L. paucicostata to a greater extent than in the control. To the best of our knowledge, this is the first report to investigate the effects of coronatine on the alteration of metabolism in L. paucicostata based on GC-MS profiling. The results of this research provide a foundation for designing strategies for enhanced production of useful metabolites for pharmaceutical and nutraceutical industries by cultivation of L. paucicostata.

Lactobacillus plantarum induces apoptosis in oral cancer KB cells through upregulation of PTEN and downregulation of MAPK signalling pathways.

Introduction: The oral tumor is the sixth most prevalent type of cancer worldwide and the second leading cause of cancer-related mortality. Although chemotherapy and immunotherapy are the main strategies for the treatment of oral cancer, an emergence of inevitable resistance to these treatment modalities is the major drawback that causes recurrence of the disease. Nowadays, probiotics have been suggested as adjunctive and complementary treatment modalities for improving the impacts of chemotherapy and immunotherapy agents. Probiotics, the friendly microflora in our bodies, contribute to the production of useful metabolites with positive effects on the immune system against various diseases such as cancer. Methods: Lactobacillus plantarum is one of the most important bacteria, which commensally live in the human oral system. In the current study, the impacts of L. plantarum on maintaining oral system health were investigated, and the molecular mechanisms of inhibition of oral cancer KB cells mediated by L. plantarum were evaluated using real-time polymerase chain reaction (PCR) and FACS flow cytometry analyses. Results: Our findings showed that L. plantarum is effective in the signal transduction of the oral cancer cells through upregulation and downregulation of PTEN and MAPK pathways, respectively. Conclusion: Based on the biological effects of oral candidate probiotics candidate bacterium L. plantarum on functional expression of PTEN and MAPK pathways, this microorganism seems to play a key role in controlling undesired cancer development in the oral system. Taken all, L. plantarum is proposed as a potential candidate for probiotics cancer therapy.

Analysis of the genomic sequences and metabolites of Serratia surfactantfaciens sp. nov. YD25T that simultaneously produces prodigiosin and serrawettin W2.

BackgroundGram-negative bacteria of the genus Serratia are potential producers of many useful secondary metabolites, such as prodigiosin and serrawettins, which have potential applications in environmental bioremediation or in the pharmaceutical industry. Several Serratia strains produce prodigiosin and serrawettin W1 as the main bioactive compounds, and the biosynthetic pathways are co-regulated by quorum sensing (QS). In contrast, the Serratia strain, which can simultaneously produce prodigiosin and serrawettin W2, has not been reported. This study focused on analyzing the genomic sequence of Serratia sp. strain YD25T isolated from rhizosphere soil under continuously planted burley tobacco collected from Yongding, Fujian province, China, which is unique in producing both prodigiosin and serrawettin W2.ResultsA hybrid polyketide synthases (PKS)-non-ribosomal peptide synthetases (NRPS) gene cluster putatively involved in biosynthesis of antimicrobial serrawettin W2 was identified in the genome of YD25T, and its biosynthesis pathway was proposed. We found potent antimicrobial activity of serrawettin W2 purified from YD25T against various pathogenic bacteria and fungi as well as antitumor activity against Hela cells. Subsequently, comparative genomic analyses were performed among a total of 133 Serratia species. The prodigiosin biosynthesis gene cluster in YD25T belongs to the type I pig cluster, which is the main form of pig-encoding genes existing in most of the pigmented Serratia species. In addition, a complete autoinducer-2 (AI-2) system (including luxS, lsrBACDEF, lsrGK, and lsrR) as a conserved bacterial operator is found in the genome of Serratia sp. strain YD25T. Phylogenetic analysis based on concatenated Lsr and LuxS proteins revealed that YD25T formed an independent branch and was clearly distant from the strains that solely produce either prodigiosin or serrawettin W2. The Fe (III) ion reduction assay confirmed that strain YD25T could produce an AI-2 signal molecule. Phylogenetic analysis using the genomic sequence of YD25T combined with phylogenetic and phenotypic analyses support this strain as a member of a novel and previously uncharacterized Serratia species.ConclusionGenomic sequence and metabolite analysis of Serratia surfactantfaciens YD25T indicate that this strain can be further explored for the production of useful metabolites. Unveiling the genomic sequence of S. surfactantfaciens YD25T benefits the usage of this unique strain as a model system for studying the biosynthesis regulation of both prodigiosin and serrawettin W2 by the QS system.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3171-7) contains supplementary material, which is available to authorized users.

Metabolite Profiling of In Vitro Cultured and Field Grown Rhizomes of Acorus calamus from Mongolia Using GC–MS

Acorus calamus (sweet flag) is used in the traditional Chinese and Indian medicines for various ailments. Due to its extensive use in herbal medicine, natural resources from the world’s forests are being depleted at an alarming rate. In the present study, an in vitro cell culture technique is being explored as an alternative to field grown A. calamus with respect to the metabolite profile, antioxidant properties, total phenol, and total flavonoid content. Gas chromatography mass spectrometry (GC–MS) was utilized to compare the metabolite profiling between methanolic extracts of in vitro and field grown rhizome tissues of A. calamus. A statistical analysis indicated an upregulation of α-selinene, which is representative of sesquiterpene ketones, and a cyclic polyol, d-pinitol, which has an insulin mimicking effect in the in vitro cultivated rhizome tissue when compared to field grown rhizomes. Significantly higher free-radical scavenging activity (IC50 69.32 μg mL−1), total phenolic content (71.60 mg GAE g−1), and total flavonoid content (42.34 mg CE g−1) were observed in in vitro rhizome tissues compared with those from field grown rhizomes. These observations suggest that the in vitro cultivation of Acorus rhizomes could be exploited as an alternative to field grown A. calamus, as it is an endangered medicinal plant. The production of useful metabolites by the in vitro cultured rhizomes can be explored successfully for utilization by various food and drug industries.

Editorial - Synthetic Biology: Engineering Complexity and Refactoring Cell Capabilities.

Synthetic Biology is now in its second decade and many goals have been achieved toward the rational design of biological systems. This Research Topic features and reviews some of the latest progress in Synthetic Biology with a focus on research at the intersection between rational design and natural complexity with a potential outcome to concrete biotechnological applications. Kelwick et al. (2014) summarize the great expansion in the genetic toolkit and DNA assembly techniques that are currently available for synthetic biologists. These tools will advance the implementation of new functions and the production of useful metabolites in living cells in a controlled fashion. Using engineering formality, Synthetic Biology aims to identify biological design principles that can be used for practical applications. As one of the results, metabolic engineering is now becoming feasible to introduce novel functions and properties into an increasing number of microbial hosts. Examples come from Yu et al. (2014) and Heider et al. (2014) that describe the production of fatty-acid-derived chemicals and astaxanthin in microbes, respectively. Furthermore, bacteria can be engineered for the conversion of waste into renewable products, as Nieves et al. (2015) demonstrate with the bioconversion of lignocellulose. Along with its great successes, Synthetic Biology is also encountering new challenges, represented by emerging behaviors in modified host cells (chassis) that are difficult to predict. Limitations in the robust prediction of gene networks arise from the lack of a proper understanding of the living systems used in synthetic biology. For instance, Akhtar and Jones (2014) appropriately present the evidence that the failure of a number of pathway engineering strategies are often due the lack of co-factors needed for the proper activity of the key enzymes. Co-factor production needs to be integrated in the system’s design to achieve proper enzymatic activity. As synthetic network designs become more complex, emerging evidence shows that elements within these networks can exhibit crosstalk and lead to non-specific behavior. As presented in Davis et al. (2015), bacterial quorum sensing pathways, which are widely used in Synthetic Biology, exhibit crosstalk that can limit the number of nodes in a network, and therefore stifle efforts to build sophisticated systems. New efforts are needed to better understand the behavior of composable parts and to develop new orthogonal elements. Lastly, Beal (2015) addresses unresolved questions in the area of cell-based information processors and noise. He proposes a quantitative signal-to-noise ratio-based standard to assess circuit performance. An important aspect in the engineering of living cells that only recently has been investigated in detail by the synthetic biology community is the interaction between the system and the chassis. The exploitation of the cell’s resources for the operation of heterologous systems has proven to be deleterious, leading to non-robust gene expression and inefficient cellular performance with decreased population growth. In that direction, Moya (2014) reflects on the need of controllable systems in synthetic entities preventing obsolescence, similarly as how living cells exhibit self-maintenance. In Feher et al. (2015) the observation of the dynamic response of a malonyl-CoA biosensor in Escherichia coli was used to understand the toxicity of the overproduction of a synthetic compound, which interfered with the system’s behavior. Martinez-Garcia et al. (2014) present the development of new broad host range Tn5 vectors in order to relieve the burden of PHB production on the health of gram negative bacteria (E. coli). These studies address the need to investigate and develop controlled production of the molecule of interest to avoid burden-related negative feedback from the chassis. As illustrated by the works in this Research Topic, now is a critical moment for Synthetic Biology, where the initial enthusiasm for the major achievements attained gives way to a deeper and better understanding of the complexity of biological systems. Advancing in this direction will significantly improve the applicability of design principles for living organisms.

Cell Factories of Higher Fungi for Useful Metabolite Production.

Higher fungi or called as macro-fungi, consisting of the divisions ascomycetes, basidiomycetes, and imperfect fungi, are receiving great interest around the world, because studies of higher fungi help us not only to find new edible and officinal resources but also to understand their complicated biology. In recent decades, a large number of useful substances from higher fungi have been isolated, identified, and characterized, which have important biological functions, such as reducing blood pressure, enhancing immunity, and possessing anti-cancer and anti-HIV and other pharmacological activities. This chapter will review the genetic manipulation tools for higher fungi, omics analysis of higher-fungus cell factories, and production of useful metabolites by higher fungi, including those of terpenoids, heterocyclics, polysaccharides, and polyketides. Trends in future development of cell factories of higher fungi for useful metabolite production will also be analyzed. Graphical Abstract Strategies for improving cell factories of higher fungi for useful metabolite production.

Enhanced production of Monacolin K by addition of precursors and surfactants in submerged fermentation of Monascus purpureus 9901

The main problem in Monacolin K (MK) production by submerged fermentation of Monascus purpureus is low productivity. In this study, on one hand, addition of precursors was used to activate the biosynthesis of MK. When 4.0 g/L of sodium citrate was supplemented at the 48th H of the fermentation, the final MK production reached to 1,658.9 ± 28.5 mg/L after 20 day of fermentation, which was improved by 52.6% compared with that of the control. On the other hand, addition of surfactants could increase the permeability of cell membrane, thus driving more intracellular metabolites secreted into the fermentation broth and alleviating the product inhibition. When 40.0 g/L of Triton X-100 was added at the beginning of the fermentation, the final MK production reached to 2,026.0 ± 30.4 mg/L after 20 day of fermentation, which was improved by 84.9% compared with that of the control. These results are helpful to provide some new insights into the biosynthetic regulation on MK production; the approach can be applied to other fungal fermentation processes for enhancing production of useful metabolites.

Dynamic Modeling of Metabolic and Gene Regulatory Systems toward Developing Virtual Microbes

In systems biology, computer simulation of the biochemical networks of a microbial cell is a powerful method to predict their function and phenotype under different culture conditions and genetic modifications. Construction of a virtual microbe, a generic mathematical model of cell growth and metabolism, can be an ambitious but realistic project. It would enable the rational design of a biochemical network to enhance the production of useful metabolites and proteins. However, there exist critical problems, such as parameter uncertainty, mechanism complexity and high dimensionality of the model. We would like to review the dynamic models of the biochemical networks with their problems, providing insight into developing a virtual microbe.

Problems, progress and future prospects of improvement of Commiphora wightii (Arn.) Bhandari, an endangered herbal magic, through modern biotechnological tools: a review

Commiphora wightii (Arn.) Bhandari syn. C. mukul Engl. (Burseraceae) is an economically and pharmacologically important slow growing, dioecious, balsamiferous woody, multipurpose shrub heading towards extinction. Commonly known as “Guggul” due to the presence of steroidal compound guggulsterone in the oleo-gum resin, it has been used in treating various ailments and disorders since ancient times (2000 B.C.). Evaluation and confirmation of hypolipidemic effects of guggul based on Ayurvedic text in 1960s provided a new insight into its pharmacological applications. Two bioactive isomers of guggulsterone, E and Z, are responsible for lipid- and cholesterol-lowering activities. Recently, it has been shown to have anti-cancerous activity also. It is found in the dry regions of Indian subcontinent, namely India, Pakistan and Bangladesh. Ruthless and unscientific harvesting of oleo-gum resin from the wild, by local populations, for economic benefits with negligible conservation efforts has made this species endangered and has led to its inclusion in Red Data Book of IUCN. Although this plant has many excellent traits, adequate attention has not been focused on its conservation and improvement. Conventional propagation methods i.e., seeds, cuttings and air layering are in place but have many limitations. Therefore, application of modern biotechnological tools needs to be standardized for harnessing maximum benefits from this pharmaceutically important plant. An efficient regeneration system needs to be in place for improvement of this genus through genetic transformation and production of useful metabolites in cell cultures. Studies are in progress for micropropagation through shoot multiplication and somatic embryogenesis, as well as for secondary metabolite (guggulsterone) production in callus cultures and bioreactors. No selected germplasm is available for C. wightii since it is a wild plant. Breeding programs have not yet been started due to lack of systematic cultivation and conservation programs. Moreover, little information has been gathered regarding the genetic variability in this species using RAPD and ISSR markers. No details are available about genetic makeup and QTL linkage maps. Investigations are in progress to search sex linked markers in this dioecious species. Research is also in progress to decipher the molecular mechanisms underlying various pharmacological actions of guggul. Since the approval of use of guggul as a food supplement by United States Food and Drug Administration in 1994, an exponential increase in research publications on various aspects of research on guggul have been published. Present communication summarizes the problems, progress made and suggests some future directions of research for this important endangered medicinal plant.