Clostridium scindens secretome suppresses virulence gene expression of Clostridioides difficile in a bile acid-independent manner.

Clostridioides difficile infection (CDI) is a major health concern and one of the leading causes of hospital-acquired diarrhea in many countries. C. difficile infection is challenging to treat as C. difficile is resistant to multiple antibiotics. Alternative solutions are needed as conventional treatment with broad-spectrum antibiotics often leads to recurrent CDI. Recent studies have shown that specific microbiota-based therapeutics such as bile acids (BAs) are promising approaches to treat CDI. Clostridium scindens encodes the bile acid-induced (bai) operon that carries out 7-alpha-dehydroxylation of liver-derived primary BAs to secondary BAs. This biotransformation is thought to increase the antibacterial effects of BAs on C. difficile. Here, we used an automated multistage fermentor to study the antibacterial actions of C. scindens and BAs on C. difficile in the presence/absence of a gut microbial community derived from healthy human donor fecal microbiota. We observed that C. scindens inhibited C. difficile growth when the medium was supplemented with primary BAs. Transcriptomic analysis indicated upregulation of C. scindens bai operon and suppressed expression of C. difficile exotoxins that mediate CDI. We also observed BA-independent antibacterial activity of the secretome from C. scindens cultured overnight in a medium without supplementary primary BAs, which suppressed growth and exotoxin expression in C. difficile mono-culture. Further investigation of the molecular basis of our observation could lead to a more specific treatment for CDI than current approaches. IMPORTANCE There is an urgent need for new approaches to replace the available treatment options against Clostridioides difficile infection (CDI). Our novel work reports a bile acid-independent reduction of C. difficile growth and virulence gene expression by the secretome of Clostridium scindens. This potential treatment combined with other antimicrobial strategies could facilitate the development of alternative therapies in anticipation of CDI and in turn reduce the risk of antimicrobial resistance.

Quantitative differences in synthetic gut microbial inoculums do not affect the final stabilized in vitro community compositions.

In vitro studies of synthetic gut microbial communities (SGMCs) can provide valuable insights into the ecological structure and function of gut microbiota. However, the importance of the quantitative composition of an SGMC inoculum and its effect on the eventual stable in vitro microbial community has not been studied. To address this, we constructed two 114-member SGMCs differing only in their quantitative composition-one reflecting the average human fecal microbiome and another mixed in equal proportions based on cell counts. We inoculated each in an automated anaerobic multi-stage in vitro gut fermentor simulating two different colonic conditions, mimicking proximal and distal colons. We replicated this setup with two different nutrient media, periodically sampled the cultures for 27 days, and profiled their microbiome compositions using 16S rRNA gene amplicon sequencing. While nutrient medium explained 36% of the variance in microbiome composition, initial inoculum composition failed to show a statistically significant effect. Under all four conditions, paired fecal and equal SGMC inoculums converged to reach stable community compositions resembling each other. Our results have broad implications for simplifying in vitro SGMC investigations. IMPORTANCE In vitro cultivation of synthetic gut microbial communities (SGMCs) can provide valuable insights into the ecological structure and function of gut microbiota. However, it is currently not known whether the quantitative composition of the initial inoculum can influence the eventual stable in vitro community structure. Hence, using two SGMC inoculums consisting of 114 unique species mixed in either equal proportions (Eq inoculum) or resembling proportions in an average human fecal microbiome (Fec inoculum), we show that initial inoculum compositions did not influence the final stable community structure in a multi-stage in vitro gut fermentor. Under two different nutrient media and two different colon conditions (proximal and distal), both Fec and Eq communities converged to resemble each other's community structure. Our results suggest that the time-consuming preparation of SGMC inoculums may not be needed and has broad implications for in vitro SGMC studies.

Oral supplementation of nicotinamide riboside alters intestinal microbial composition in rats and mice, but not humans.

The gut microbiota impacts systemic levels of multiple metabolites including NAD+ precursors through diverse pathways. Nicotinamide riboside (NR) is an NAD+ precursor capable of regulating mammalian cellular metabolism. Some bacterial families express the NR-specific transporter, PnuC. We hypothesized that dietary NR supplementation would modify the gut microbiota across intestinal sections. We determined the effects of 12 weeks of NR supplementation on the microbiota composition of intestinal segments of high-fat diet-fed (HFD) rats. We also explored the effects of 12 weeks of NR supplementation on the gut microbiota in humans and mice. In rats, NR reduced fat mass and tended to decrease body weight. Interestingly, NR increased fat and energy absorption but only in HFD-fed rats. Moreover, 16S rRNA gene sequencing analysis of intestinal and fecal samples revealed an increased abundance of species within Erysipelotrichaceae and Ruminococcaceae families in response to NR. PnuC-positive bacterial strains within these families showed an increased growth rate when supplemented with NR. The abundance of species within the Lachnospiraceae family decreased in response to HFD irrespective of NR. Alpha and beta diversity and bacterial composition of the human fecal microbiota were unaltered by NR, but in mice, the fecal abundance of species within Lachnospiraceae increased while abundances of Parasutterella and Bacteroides dorei species decreased in response to NR. In conclusion, oral NR altered the gut microbiota in rats and mice, but not in humans. In addition, NR attenuated body fat mass gain in rats, and increased fat and energy absorption in the HFD context.

Ecological Adaptation and Succession of Human Fecal Microbial Communities in an Automated In Vitro Fermentation System.

Longitudinal studies of gut microbiota following specific interventions are vital for understanding how they influence host health. However, robust longitudinal sampling of gut microbiota is a major challenge, which can be addressed using in vitro fermentors hosting complex microbial communities. Here, by employing 16S rRNA gene amplicon sequencing, we investigated the adaptation and succession of human fecal microbial communities in an automated multistage fermentor. We performed two independent experiments using different human donor fecal samples, one configured with two units of three colon compartments each studied for 22 days and another with one unit of two colon compartments studied for 31 days. The fermentor maintained a trend of increasing microbial alpha diversity along colon compartments. Within each experiment, microbial compositions followed compartment-specific trajectories and reached independent stable configurations. While compositions were highly similar between replicate units, they were clearly separated between different experiments, showing that they maintained the individuality of fecal inoculum rather than converging on a fermentor-specific composition. While some fecal amplicon sequence variants (ASVs) were undetected in the fermentor, many ASVs undetected in the fecal samples flourished in vitro. These bloomer ASVs accounted for significant proportions of the population and included prominent health-associated microbes such as Bacteroides fragilis and Akkermansia muciniphila. Turnover in community compositions is likely explained by feed composition and pH, suggesting that these communities can be easily modulated. Our results suggest that in vitro fermentors are promising tools to study complex microbial communities harboring important members of human gut microbiota. IMPORTANCE In vitro fermentors that can host complex gut microbial communities are promising tools to investigate the dynamics of human gut microbiota. In this work, using an automated in vitro gut fermentor consisting of different colon compartments, we investigated the adaptation dynamics of two different human fecal microbial communities over 22 and 31 days. By observing the temporal trends of different community members, we found that many dominant members of the fecal microbiota failed to maintain their dominance in vitro, and some of the low-abundance microbes undetected in the fecal microbiota successfully grew in the in vitro communities. Microbiome compositional changes and blooming could largely be explained by feed composition and pH, suggesting that these communities can be modulated to desired compositions via optimizing culture conditions. Thus, our results open up the possibility of modulating in vitro microbial communities to predefined compositions by optimizing feed composition and culture conditions.

Responses of Synechocystis sp. PCC 6803 to heterologous biosynthetic pathways.

BACKGROUND: There are an increasing number of studies regarding genetic manipulation of cyanobacteria to produce commercially interesting compounds. The majority of these works study the expression and optimization of a selected heterologous pathway, largely ignoring the wholeness and complexity of cellular metabolism. Regulation and response mechanisms are largely unknown, and even the metabolic pathways themselves are not fully elucidated. This poses a clear limitation in exploiting the rich biosynthetic potential of cyanobacteria. RESULTS: In this work, we focused on the production of two different compounds, the cyanogenic glucoside dhurrin and the diterpenoid 13R-manoyl oxide in Synechocystis PCC 6803. We used genome-scale metabolic modelling to study fluxes in individual reactions and pathways, and we determined the concentrations of key metabolites, such as amino acids, carotenoids, and chlorophylls. This allowed us to identify metabolic crosstalk between the native and the introduced metabolic pathways. Most results and simulations highlight the metabolic robustness of cyanobacteria, suggesting that the host organism tends to keep metabolic fluxes and metabolite concentrations steady, counteracting the effects of the heterologous pathway. However, the amino acid concentrations of the dhurrin-producing strain show an unexpected profile, where the perturbation levels were high in seemingly unrelated metabolites. CONCLUSIONS: There is a wealth of information that can be derived by combining targeted metabolite identification and computer modelling as a frame of understanding. Here we present an example of how strain engineering approaches can be coupled to 'traditional' metabolic engineering with systems biology, resulting in novel and more efficient manipulation strategies.

Extending the biosynthetic repertoires of cyanobacteria and chloroplasts.

Chloroplasts in plants and algae and photosynthetic microorganisms such as cyanobacteria are emerging hosts for sustainable production of valuable biochemicals, using only inorganic nutrients, water, CO2 and light as inputs. In the past decade, many bioengineering efforts have focused on metabolic engineering and synthetic biology in the chloroplast or in cyanobacteria for the production of fuels, chemicals and complex, high-value bioactive molecules. Biosynthesis of all these compounds can be performed in photosynthetic organelles/organisms by heterologous expression of the appropriate pathways, but this requires optimization of carbon flux and reducing power, and a thorough understanding of regulatory pathways. Secretion or storage of the compounds produced can be exploited for the isolation or confinement of the desired compounds. In this review, we explore the use of chloroplasts and cyanobacteria as biosynthetic compartments and hosts, and we estimate the levels of production to be expected from photosynthetic hosts in light of the fraction of electrons and carbon that can potentially be diverted from photosynthesis. The supply of reducing power, in the form of electrons derived from the photosynthetic light reactions, appears to be non-limiting, but redirection of the fixed carbon via precursor molecules presents a challenge. We also discuss the available synthetic biology tools and the need to expand the molecular toolbox to facilitate cellular reprogramming for increased production yields in both cyanobacteria and chloroplasts.

Transfer of the cytochrome P450-dependent dhurrin pathway from Sorghum bicolor into Nicotiana tabacum chloroplasts for light-driven synthesis.

Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1-0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons.

Metabolic engineering of light-driven cytochrome P450 dependent pathways into Synechocystis sp. PCC 6803.

Solar energy provides the energy input for the biosynthesis of primary and secondary metabolites in plants and other photosynthetic organisms. Some secondary metabolites are high value compounds, and typically their biosynthesis requires the involvement of cytochromes P450s. In this proof of concept work, we demonstrate that the cyanobacterium Synechocystis sp. PCC 6803 is an eminent heterologous host for expression of metabolically engineered cytochrome P450-dependent pathways exemplified by the dhurrin pathway from Sorghum bicolor comprising two membrane bound cytochromes P450s (CYP79A1 and CYP71E1) and a soluble glycosyltransferase (UGT85B1). We show that it is possible to express multiple genes incorporated into a bacterial-like operon by using a self-replicating expression vector in cyanobacteria. We demonstrate that eukaryotic P450s that typically reside in the endoplasmic reticulum membranes can be inserted in the prokaryotic membranes without affecting thylakoid membrane integrity. Photosystem I and ferredoxin replaces the native P450 oxidoreductase enzyme as an efficient electron donor for the P450s both in vitro and in vivo. The engineered strains produced up to 66mg/L of p-hydroxyphenylacetaldoxime and 5mg/L of dhurrin in lab-scale cultures after 3 days of cultivation and 3mg/L of dhurrin in V-shaped photobioreactors under greenhouse conditions after 9 days cultivation. All the metabolites were found to be excreted to the growth media facilitating product isolation.

Heterologous expression of the isopimaric acid pathway in Nicotiana benthamiana and the effect of N-terminal modifications of the involved cytochrome P450 enzyme.

BACKGROUND: Plant terpenoids are known for their diversity, stereochemical complexity, and their commercial interest as pharmaceuticals, food additives, and cosmetics. Developing biotechnology approaches for the production of these compounds in heterologous hosts can increase their market availability, reduce their cost, and provide sustainable production platforms. In this context, we aimed at producing the antimicrobial diterpenoid isopimaric acid from Sitka spruce. Isopimaric acid is synthesized using geranylgeranyl diphosphate as a precursor molecule that is cyclized by a diterpene synthase in the chloroplast and subsequently oxidized by a cytochrome P450, CYP720B4. RESULTS: We transiently expressed the isopimaric acid pathway in Nicotiana benthamiana leaves and enhanced its productivity by the expression of two rate-limiting steps in the pathway (providing the general precursor of diterpenes). This co-expression resulted in 3-fold increase in the accumulation of both isopimaradiene and isopimaric acid detected using GC-MS and LC-MS methodology. We also showed that modifying or deleting the transmembrane helix of CYP720B4 does not alter the enzyme activity and led to successful accumulation of isopimaric acid in the infiltrated leaves. Furthermore, we demonstrated that a modified membrane anchor is a prerequisite for a functional CYP720B4 enzyme when the chloroplast targeting peptide is added. We report the accumulation of 45-55 µg/g plant dry weight of isopimaric acid four days after the infiltration with the modified enzymes. CONCLUSIONS: It is possible to localize a diterpenoid pathway from spruce fully within the chloroplast of N. benthamiana and a few modifications of the N-terminal sequences of the CYP720B4 can facilitate the expression of plant P450s in the plastids. The coupling of terpene biosynthesis closer to photosynthesis paves the way for light-driven biosynthesis of valuable terpenoids.

Redirecting photosynthetic electron flow into light-driven synthesis of alternative products including high-value bioactive natural compounds.

Photosynthesis in plants, green algae, and cyanobacteria converts solar energy into chemical energy in the form of ATP and NADPH, both of which are used in primary metabolism. However, often more reducing power is generated by the photosystems than what is needed for primary metabolism. In this review, we discuss the development in the research field, focusing on how the photosystems can be used as synthetic biology building blocks to channel excess reducing power into light-driven production of alternative products. Plants synthesize a large number of high-value bioactive natural compounds. Some of the key enzymes catalyzing their biosynthesis are the cytochrome P450s situated in the endoplasmic reticulum. However, bioactive compounds are often synthesized in low quantities in the plants and are difficult to produce by chemical synthesis due to their often complex structures. Through a synthetic biology approach, enzymes with a requirement for reducing equivalents as cofactors, such as the cytochrome P450s, can be coupled directly to the photosynthetic energy output to obtain environmentally friendly production of complex chemical compounds. By relocating cytochrome P450s to the chloroplasts, reducing power can be diverted toward the reactions catalyzed by the cytochrome P450s. This provides a sustainable production method for high-value compounds that potentially can solve the problem of NADPH regeneration, which currently limits the biotechnological uses of cytochrome P450s. We describe the approaches that have been taken to couple enzymes to photosynthesis in vivo and to photosystem I in vitro and the challenges associated with this approach to develop new green production platforms.