Plant cell packs: a scalable platform for recombinant protein production and metabolic engineering

Industrial plant biotechnology applications include the production of sustainable fuels, complex metabolites and recombinant proteins, but process development can be impaired by a lack of reliable and scalable screening methods. Here, we describe a rapid and versatile expression system which involves the infusion of Agrobacterium tumefaciens into three‐dimensional, porous plant cell aggregates deprived of cultivation medium, which we have termed plant cell packs (PCPs). This approach is compatible with different plant species such as Nicotiana tabacum BY2, Nicotiana benthamiana or Daucus carota and 10‐times more effective than transient expression in liquid plant cell culture. We found that the expression of several proteins was similar in PCPs and intact plants, for example, 47 and 55 mg/kg for antibody 2G12 expressed in BY2 PCPs and N. tabacum plants respectively. Additionally, the expression of specific enzymes can either increase the content of natural plant metabolites or be used to synthesize novel small molecules in the PCPs. The PCP method is currently scalable from a microtiter plate format suitable for high‐throughput screening to 150‐mL columns suitable for initial product preparation. It therefore combined the speed of transient expression in plants with the throughput of microbial screening systems. Plant cell packs therefore provide a convenient new platform for synthetic biology approaches, metabolic engineering and conventional recombinant protein expression techniques that require the multiplex analysis of several dozen up to hundreds of constructs for efficient product and process development.     

Fathoming Aspergillus oryzae metabolomes in formulated growth matrices

The stochasticity of Aspergillus oryzae (Trivially: the koji mold) pan-metabolomes commensurate with its ubiquitously distributed landscapes, i.e. growth matrices have been seemed uncharted since its food fermentative systems are mostly being investigated. In this review, we explicitly have discussed the likely tendencies of A. oryzae metabolomes pertaining to its growth milieu formulated with substrate matrices of varying nature, composition, texture, and associated physicochemical parameters. We envisaged typical food matrices, namely, meju, koji, and moromi as the semi-natural cultivation models toward delineating the metabolomic patterns of the koji mold, which synergistically influences the organoleptic and functional properties of the end products. Further, we highlighted how tailored conditions in sub-natural growth matrices, i.e. synthetic cultivation media blends, inducers, and growth surfaces, may influence A. oryzae metabolomes and targeted phenotypes. In general, the sequential or synchronous growth of A. oryzae on formulated matrices results in a number of metabolic tradeoffs with its immediate microenvironment influencing its adaptive and regulatory metabolomes. In broader context, evaluating the metabolic plasticity of A. oryzae relative to the tractable variables in formulated growth matrices might help approximate its growth and metabolism in the more complex natural matrices and environs. These approaches may considerably help in the design and manipulation of hybrid cultivation systems towards the efficient harnessing of commercial molds.     

Synthetic biology and metabolic engineering of actinomycetes for natural product discovery

Actinomycetes are one of the most valuable sources of natural products with industrial and medicinal importance. After more than half a century of exploitation, it has become increasingly challenging to find novel natural products with useful properties as the same known compounds are often repeatedly re-discovered when using traditional approaches. Modern genome mining approaches have led to the discovery of new biosynthetic gene clusters, thus indicating that actinomycetes still harbor a huge unexploited potential to produce novel natural products. In recent years, innovative synthetic biology and metabolic engineering tools have greatly accelerated the discovery of new natural products and the engineering of actinomycetes. In the first part of this review, we outline the successful application of metabolic engineering to optimize natural product production, focusing on the use of multi-omics data, genome-scale metabolic models, rational approaches to balance precursor pools, and the engineering of regulatory genes and regulatory elements. In the second part, we summarize the recent advances of synthetic biology for actinomycetal metabolic engineering including cluster assembly, cloning and expression, CRISPR/Cas9 technologies, and chassis strain development for natural product overproduction and discovery. Finally, we describe new advances in reprogramming biosynthetic pathways through polyketide synthase and non-ribosomal peptide synthetase engineering. These new developments are expected to revitalize discovery and development of new natural products with medicinal and other industrial applications.     

Coumarins and other components of Daphne oleoides Schreb. subsp. oleoides from Majella National Park

The present study reports about the phytochemical analysis of a sample of Daphne oleoides Schreb. subsp. oleoides (family Thymelaeaceae) collected from the Majella National Park. Twenty components, belonging to different classes of natural products, have been identified by means of spectroscopic and spectrometric techniques: [monomeric (4), bis- (5, 7–10, 20) and trimeric coumarins (11), including aglycones and glycosides, coumarinolignoid (6); flavonoids (16–19); glycosidic furolignans (14, 15), glucosidic phenylpropanoids (12, 13), cyclic tetrapyrrole derivatives (2, 3), unsaturated triglyceride (1)]. Besides the chemosystematic markers of the genus (4–10, 12, 14–18, 20) several other components were identified for the first time in the species (17, 20) and/or in the Daphne genus (1–3, 13, 19). The observed composition was discussed from the chemotaxonomic standpoint and compared with those recognized from a Sardinian accession. It was observed a pronounced difference in the two metabolites patterns, most probably attributable to geographic isolation of the studied populations and, in some extent, also by the different environmental conditions, evidencing a possible tendency of D. oleoides to the infraspecific chemovariability. Considering the wide traditional uses of Daphne spp. in ethnomedicine of several countries, also the pharmacologic potential of the identified secondary metabolites is discussed.     

艾纳香内生真菌Diaporthe sp.次生代谢产物

【目的】从艾纳香内生菌J1中获得活性次生代谢产物。【方法】对菌株J1进行ITS序列分子鉴定,综合运用多种色谱技术对其发酵产物进行分离纯化,结合波谱学技术对其进行结构表征。【结果】经构建系统进化树,鉴定菌株J1为Diaporthe sp.,从该菌株大米培养基中分离得到7个单体化合物,经鉴定分别为Dicerandrol A (1)、Dicerandrol B (2)、4,6-dihydroxy-1H-isoindole1,3(2H)-dione (3)、Cytochalasin H (4)、Cytochalasin J (5)、4,6-dihydroxy-2,3-dihydro-1H-isoindol-1-one (6)、Cerebroside C (7)。所有化合物均为首次从该菌中分得,化合物1对枯草芽孢杆菌Bacillus subtilis KCTC 1021具有非常强的抑制活性,MIC值为0.125μg/mL。【结论】Diaporthe sp.富含抑菌活性化合物,具有开发成微生物源农药潜力。     

Odorella benthonica gen. & sp. nov. (Pleurocapsales,Cyanobacteria): an odor and prolific toxin producer isolated from a California aqueduct

Pleurocapsales are one of the least understood groups of cyanobacteria in terms of molecular systematics and biochemistry. Considering the high number of cryptic taxa within the Synechococcales and Oscillatoriales, it is likely that such taxa also occur in the Pleurocapsales. The new genus described in our research is the first known pleurocapsalean cryptic taxon. It produces off‐flavor and a large number of bioactive metabolites (n = 38) some of which can be toxic including four known microcystins. Using a polyphasic approach, we propose the establishment of the genus Odorella with the new species O. benthonica from material originally isolated from the California Aqueduct near Los Angeles.     

The effects of 5‐azacytidine and cadmium on global 5‐methylcytosine content and secondary metabolites in the freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus quadricauda

Epigenetic changes are important mechanisms in the regulation of chromatin structure and gene expression. Cytosine methylation is one of the major epigenetic modifications, mediated by DNA methyltransferases, which transfer methyl groups from S‐adenosyl‐L‐methionine (SAM) to the fifth carbon of cytosine. Various external environmental conditions can change the global hypo/hypermethylation pattern of DNA. These alterations may affect the organism's response to stress conditions. In this study, for the first time, we investigated the effects of 5‐azacytidine, a DNA methyltransferase inhibitor, and cadmium, a toxic metal and environmental pollutant, on the growth, biosynthesis of secondary metabolites (phenols, flavonoids, carotenoids), SAM, S‐adenosylhomocysteine, 5′‐methylthioadenosine and global 5‐methylcytosine (5‐mC) in the green microalgae Chlamydomonas reinhardtii and Scenedesmus quadricauda. The studied species showed major differences in 5‐mC content, secondary metabolite content, and antioxidant activity. Cadmium increased GSH (glutathione) content in C. reinhardtii by 60% whereas 5‐azacytidine did not affect GSH. The biosynthesis of GSH in S. quadricauda in response to the stressors was the opposite. Global 5‐mC content of C. reinhardtii was 1%–1.5%, and the content in S. quadricauda was 3.5%. Amount of some investigated methionine cycle metabolites (SAM, S‐adenosyl homocysteine [SAH], methionine) in S. quadricauda distinctly exceeded C. reinhardtii as well. However, chlorophylls a and b, carotenoids, total phenolic content, total flavonoid content and, antioxidant activity were significantly higher in C. reinhardtii than S. quadricauda. Therefore, in further studies it would be advisable to verify whether methylation of cytosine affects the expression of genes encoding certain secondary metabolites.     

The fungus Penicillium variabile Sopp 1912 isolated from permafrost deposits as a producer of rugulovasines

It has been established that relict fungi Penicillium variabile Sopp can synthesize clavine alkaloids, rugulovasines A and B, which are revealed in this species for the first time. Submerged cultivation of the strain-producer revealed several microcycles of conidia formation. The synthesis of alkaloids was also of a cyclic character. The synchronism of cyclic rugulovasine biosynthesis and conidia formation was revealed. Zinc ions stimulated fungal growth but had a negative effect on the biosynthesis of rugulovasines.     

Impaired daily glucocorticoid rhythm in Per1 Brd mice

Biological clocks have evolved in all kinds of organisms in order to anticipate and adjust to the daily light–dark cycle. Within the last decade, the molecular machinery underlying the circadian system was unraveled. In the present study, the impact of the loss of the Per1 or Per2 genes, key components of the core clock oscillator, on body mass, food and water intake, glucose metabolism, and hypothalamic-pituitary-adrenal axis, was investigated in the Per1 ^Brd and Per2 ^Brd mouse models. The results reveal that the lack of Per1 but not Per2 has severe consequences for the regulation of these parameters. Specifically, in Per1 ^Brd animals, we found an impaired daily glucocorticoid rhythm, with markedly elevated levels during the day compared to control animals. In addition, Per1 ^Brd mice showed significant differences in body mass as well as food and water intake. Although the Per1 ^Brd are lighter than wildtype mice, food and water intake per gram body mass is elevated. In addition, the Per1 ^Brd mice exhibit an increased glucose metabolism after i.p. injection with glucose. In conclusion, our study presents first evidence for a link between an altered metabolism in Per1 and Per2 deficient mice, which in the case of the Per1 ^Brd animals might be due to an impaired corticosterone rhythm.