Optimization of flavanonols heterologous biosynthesis in Streptomyces albidoflavus, and generation of auronols.

Aromadendrin and taxifolin are two flavanonols (derived from the precursor naringenin) displaying diverse beneficial properties for humans. The carbon skeleton of these flavonoids may be transformed by the human gastrointestinal microbiota into other compounds, like auronols, which exert different and interesting biological activities. While research in flavonoids has become a certainly extensive field, studies about auronols are still scarce. In this work, different versions of the key plant enzyme for flavanonols biosynthesis, The flavanone 3-hydroxylase (F3H), has been screened for selecting the best one for the de novo production of these compounds in the bacterial factory Streptomyces albidoflavus UO-FLAV-004-NAR, a naringenin overproducer strain. This screening has rendered 2.6 µg/L of aromadendrin and 2.1 mg/L of taxifolin final production titers. Finally, the expression of the chalcone isomerase (CHI) from the gut bacterium Eubacterium ramulus has rendered a direct conversion (after feeding experiments) of 38.1% of (+)-aromadendrin into maesopsin and 74.6% of (+)-taxifolin into alphitonin. Moreover, de novo heterologous biosynthesis of 1.9 mg/L of alphitonin was accomplished by means of a co-culture strategy of a taxifolin producer S. albidoflavus and a CHI-expressing Escherichia coli, after the observation of the high instability of alphitonin in the culture medium. This study addresses the significance of culture time optimization and selection of appropriate enzymes depending on the desired final product. To our knowledge, this is the first time that alphitonin de novo production has been accomplished.

Antitumor Effect and Gut Microbiota Modulation by Quercetin, Luteolin, and Xanthohumol in a Rat Model for Colorectal Cancer Prevention.

Colorectal cancer stands as the third most prevalent form of cancer worldwide, with a notable increase in incidence in Western countries, mainly attributable to unhealthy dietary habits and other factors, such as smoking or reduced physical activity. Greater consumption of vegetables and fruits has been associated with a lower incidence of colorectal cancer, which is attributed to their high content of fiber and bioactive compounds, such as flavonoids. In this study, we have tested the flavonoids quercetin, luteolin, and xanthohumol as potential antitumor agents in an animal model of colorectal cancer induced by azoxymethane and dodecyl sodium sulphate. Forty rats were divided into four cohorts: Cohort 1 (control cohort), Cohort 2 (quercetin cohort), Cohort 3 (luteolin cohort), and Cohort 4 (xanthohumol cohort). These flavonoids were administered intraperitoneally to evaluate their antitumor potential as pharmaceutical agents. At the end of the experiment, after euthanasia, different physical parameters and the intestinal microbiota populations were analyzed. Luteolin was effective in significantly reducing the number of tumors compared to the control cohort. Furthermore, the main significant differences at the microbiota level were observed between the control cohort and the cohort treated with luteolin, which experienced a significant reduction in the abundance of genera associated with disease or inflammatory conditions, such as Clostridia UCG-014 or Turicibacter. On the other hand, genera associated with a healthy state, such as Muribaculum, showed a significant increase in the luteolin cohort. These results underline the anti-colorectal cancer potential of luteolin, manifested through a modulation of the intestinal microbiota and a reduction in the number of tumors.

Gut Microbiota and Inflammation Modulation in a Rat Model for Ulcerative Colitis after the Intraperitoneal Administration of Apigenin, Luteolin, and Xanthohumol.

Ulcerative colitis (UC) is a chronic inflammatory disorder affecting the colon, with symptomatology influenced by factors including environmental, genomic, microbial, and immunological interactions. Gut microbiota dysbiosis, characterized by bacterial population alterations, contributes to intestinal homeostasis disruption and aberrant immune system activation, thereby exacerbating the inflammatory state. This study assesses the therapeutic efficacy of intraperitoneal (IP) injected flavonoids (apigenin, luteolin, and xanthohumol) in the reduction of inflammatory parameters and the modulation of the gut microbiota in a murine model of ulcerative colitis. Flavonoids interact with gut microbiota by modulating their composition and serving as substrates for the fermentation into other anti-inflammatory bioactive compounds. Our results demonstrate the effectiveness of luteolin and xanthohumol treatment in enhancing the relative abundance of anti-inflammatory microorganisms, thereby attenuating pro-inflammatory species. Moreover, all three flavonoids exhibit efficacy in the reduction of pro-inflammatory cytokine levels, with luteolin strongly demonstrating utility in alleviating associated physical UC symptoms. This suggests that this molecule is a potential alternative or co-therapy to conventional pharmacological interventions, potentially mitigating their adverse effects. A limited impact on microbiota is observed with apigenin, and this is attributed to its solubility constraints via the chosen administration route, resulting in its accumulation in the mesentery.

Metabolic engineering in Streptomyces albidoflavus for the biosynthesis of the methylated flavonoids sakuranetin, acacetin, and genkwanin.

Flavonoids are important plant secondary metabolites showing antioxidant, antitumor, anti-inflammatory, and antiviral activities, among others. Methylated flavonoids are particularly interesting compared to non-methylated ones due to their greater stability and intestinal absorption, which improves their oral bioavailability. In this work we have stablished a metabolic engineered strain of Streptomyces albidoflavus with enhanced capabilities for flavonoid production, achieving a 1.6-fold increase in the biosynthesis of naringenin with respect to the parental strain. This improved strain, S. albidoflavus UO-FLAV-004, has been used for the heterologous biosynthesis of the methylated flavonoids sakuranetin, acacetin and genkwanin. The achieved titers of sakuranetin and acacetin were 8.2 mg/L and 5.8 mg/L, respectively. The genkwanin titers were 0.8 mg/L, with a bottleneck identified in this producing strain. After applying a co-culture strategy, genkwanin production titers reached 3.5 mg/L, which represents a 4.4-fold increase. To our knowledge, this study presents the first biosynthesis of methylated flavonoids in not only any Streptomyces species, but also in any Gram-positive bacteria.

Metabolic engineering strategies for naringenin production enhancement in Streptomyces albidoflavus J1074.

BACKGROUND: Naringenin is an industrially relevant compound due to its multiple pharmaceutical properties as well as its central role in flavonoid biosynthesis. RESULTS: On our way to develop Streptomyces albidoflavus J1074 as a microbial cell factory for naringenin production, we have significantly increased the yields of this flavanone by combining various metabolic engineering strategies, fermentation strategies and genome editing approaches in a stepwise manner. Specifically, we have screened different cultivation media to identify the optimal production conditions and have investigated how the additive feeding of naringenin precursors influences the production. Furthermore, we have employed genome editing strategies to remove biosynthetic gene clusters (BGCs) associated with pathways that might compete with naringenin biosynthesis for malonyl-CoA precursors. Moreover, we have expressed MatBC, coding for a malonate transporter and an enzyme responsible for the conversion of malonate into malonyl-CoA, respectively, and have duplicated the naringenin BGC, further contributing to the production improvement. By combining all of these strategies, we were able to achieve a remarkable 375-fold increase (from 0.06 mg/L to 22.47 mg/L) in naringenin titers. CONCLUSION: This work demonstrates the influence that fermentation conditions have over the final yield of a bioactive compound of interest and highlights various bottlenecks that affect production. Once such bottlenecks are identified, different strategies can be applied to overcome them, although the efficiencies of such strategies may vary and are difficult to predict.

Optimized De Novo Eriodictyol Biosynthesis in Streptomyces albidoflavus Using an Expansion of the Golden Standard Toolkit for Its Use in Actinomycetes.

Eriodictyol is a hydroxylated flavonoid displaying multiple pharmaceutical activities, such as antitumoral, antiviral or neuroprotective. However, its industrial production is limited to extraction from plants due to its inherent limitations. Here, we present the generation of a Streptomyces albidoflavus bacterial factory edited at the genome level for an optimized de novo heterologous production of eriodictyol. For this purpose, an expansion of the Golden Standard toolkit (a Type IIS assembly method based on the Standard European Vector Architecture (SEVA)) has been created, encompassing a collection of synthetic biology modular vectors (adapted for their use in actinomycetes). These vectors have been designed for the assembly of transcriptional units and gene circuits in a plug-and-play manner, as well as for genome editing using CRISPR-Cas9-mediated genetic engineering. These vectors have been used for the optimization of the eriodictyol heterologous production levels in S. albidoflavus by enhancing the flavonoid-3'-hydroxylase (F3'H) activity (by means of a chimera design) and by replacing three native biosynthetic gene clusters in the bacterial chromosome with the plant genes matBC (involved in extracellular malonate uptake and its intracellular activation into malonyl-CoA), therefore allowing more malonyl-CoA to be devoted to the heterologous production of plant flavonoids in this bacterial factory. These experiments have allowed an increase in production of 1.8 times in the edited strain (where the three native biosynthetic gene clusters have been deleted) in comparison with the wild-type strain and a 13 times increase in eriodictyol overproduction in comparison with the non-chimaera version of the F3'H enzyme.

Terpenoids and Polyphenols as Natural Antioxidant Agents in Food Preservation.

Synthetic antioxidant food additives, such as BHA, BHT and TBHQ, are going through a difficult time, since these products generate a negative perception in consumers. This has generated an increased pressure on food manufacturers to search for safer natural alternatives like phytochemicals (such as polyphenols, including flavonoids, and essential oils rich in terpenoids, including carotenoids). These plant bioactive compounds have antioxidant activities widely proven in in vitro tests and in diverse food matrices (meat, fish, oil and vegetables). As tons of food are wasted every year due to aesthetic reasons (lipid oxidation) and premature damage caused by inappropriate packaging, there is an urgent need for natural antioxidants capable of replacing the synthetic ones to meet consumer demands. This review summarizes industrially interesting antioxidant bioactivities associated with terpenoids and polyphenols with respect to the prevention of lipid oxidation in high fat containing foods, such as meat (rich in saturated fat), fish (rich in polyunsaturated fat), oil and vegetable products, while avoiding the generation of rancid flavors and negative visual deterioration (such as color changes due to oxidized lipids). Terpenoids (like monoterpenes and carotenoids) and polyphenols (like quercetin and other flavonoids) are important phytochemicals with a broad range of antioxidant effects. These phytochemicals are widely distributed in fruits and vegetables, including agricultural waste, and are remarkably useful in food preservation, as they show bioactivity as plant antioxidants, able to scavenge reactive oxygen and nitrogen species, such as superoxide, hydroxyl or peroxyl radicals in meat and other products, contributing to the prevention of lipid oxidation processes in food matrices.

Reconstruction of a Genome-Scale Metabolic Model of Streptomyces albus J1074: Improved Engineering Strategies in Natural Product Synthesis.

Streptomyces albus J1074 is recognized as an effective host for heterologous production of natural products. Its fast growth and efficient genetic toolbox due to a naturally minimized genome have contributed towards its advantage in expressing biosynthetic pathways for a diverse repertoire of products such as antibiotics and flavonoids. In order to develop precise model-driven engineering strategies for de novo production of natural products, a genome-scale metabolic model (GEM) was reconstructed for the microorganism based on protein homology to model species Streptomyces coelicolor while drawing annotated data from databases and literature for further curation. To demonstrate its capabilities, the Salb-GEM was used to predict overexpression targets for desirable compounds using flux scanning with enforced objective function (FSEOF). Salb-GEM was also utilized to investigate the effect of a minimized genome on metabolic gene essentialities in comparison to another Streptomyces species, S. coelicolor.

Repositioning microbial biotechnology against COVID-19: the case of microbial production of flavonoids.

Coronavirus-related disease 2019 (COVID-19) became a pandemic in February 2020, and worldwide researchers try to tackle the disease with approved drugs of all kinds, or to develop novel compounds inhibiting viral spreading. Flavonoids, already investigated as antivirals in general, also might bear activities specific for the viral agent causing COVID-19, SARS-CoV-2. Microbial biotechnology and especially synthetic biology may help to produce flavonoids, which are exclusive plant secondary metabolites, at a larger scale or indeed to find novel pharmaceutically active flavonoids. Here, we review the state of the art in (i) antiviral activity of flavonoids specific for coronaviruses and (ii) results derived from computational studies, mostly docking studies mainly inhibiting specific coronaviral proteins such as the 3CL (main) protease, the spike protein or the RNA-dependent RNA polymerase. In the end, we strive towards a synthetic biology pipeline making the fast and tailored production of valuable antiviral flavonoids possible by applying the last concepts of division of labour through co-cultivation/microbial community approaches to the DBTL (Design, Build, Test, Learn) principle.

Dissection of Nidogen function in Drosophila reveals tissue-specific mechanisms of basement membrane assembly.

Basement membranes (BMs) are thin sheet-like specialized extracellular matrices found at the basal surface of epithelia and endothelial tissues. They have been conserved across evolution and are required for proper tissue growth, organization, differentiation and maintenance. The major constituents of BMs are two independent networks of Laminin and Type IV Collagen in addition to the proteoglycan Perlecan and the glycoprotein Nidogen/entactin (Ndg). The ability of Ndg to bind in vitro Collagen IV and Laminin, both with key functions during embryogenesis, anticipated an essential role for Ndg in morphogenesis linking the Laminin and Collagen IV networks. This was supported by results from cultured embryonic tissue experiments. However, the fact that elimination of Ndg in C. elegans and mice did not affect survival strongly questioned this proposed linking role. Here, we have isolated mutations in the only Ndg gene present in Drosophila. We find that while, similar to C.elegans and mice, Ndg is not essential for overall organogenesis or viability, it is required for appropriate fertility. We also find, alike in mice, tissue-specific requirements of Ndg for proper assembly and maintenance of certain BMs, namely those of the adipose tissue and flight muscles. In addition, we have performed a thorough functional analysis of the different Ndg domains in vivo. Our results support an essential requirement of the G3 domain for Ndg function and unravel a new key role for the Rod domain in regulating Ndg incorporation into BMs. Furthermore, uncoupling of the Laminin and Collagen IV networks is clearly observed in the larval adipose tissue in the absence of Ndg, indeed supporting a linking role. In light of our findings, we propose that BM assembly and/or maintenance is tissue-specific, which could explain the diverse requirements of a ubiquitous conserved BM component like Nidogen.