Engineering of molybdenum-cofactor-dependent nitrate assimilation in Yarrowia lipolytica

ABSTRACT Engineering a new metabolic function in a microbial host can be limited by the availability of the relevant cofactor. For instance, in Yarrowia lipolytica, the expression of a functional nitrate reductase is precluded by the absence of molybdenum cofactor (Moco) biosynthesis. In this study, we demonstrated that the Ogataea parapolymorpha Moco biosynthesis pathway combined with the expression of a high affinity molybdate transporter could lead to the synthesis of Moco in Y. lipolytica. The functionality of Moco was demonstrated by expression of an active Moco-dependent nitrate assimilation pathway from the same yeast donor, O. parapolymorpha. In addition to 11 heterologous genes, fast growth on nitrate required adaptive laboratory evolution which, resulted in up to 100-fold increase in nitrate reductase activity and in up to 4-fold increase in growth rate, reaching 0.13h-1. Genome sequencing of evolved isolates revealed the presence of a limited number of non-synonymous mutations or small insertions/deletions in annotated coding sequences. This study that builds up on a previous work establishing Moco synthesis in S. cerevisiae demonstrated that the Moco pathway could be successfully transferred in very distant yeasts and, potentially, to any other genera, which would enable the expression of new enzyme families and expand the nutrient range used by industrial yeasts.

Saccharomyces cerevisiae as host for the recombinant production of polyketides and nonribosomal peptides

As a robust, fast growing and genetically tractable organism, the budding yeast Saccharomyces cerevisiae is one of the most widely used hosts in biotechnology. Its applications range from the manufacturing of vaccines and hormones to bulk chemicals and biofuels. In recent years, major efforts have been undertaken to expand this portfolio to include structurally complex natural products, such as polyketides and nonribosomally synthesized peptides. These compounds often have useful pharmacological properties, which make them valuable drugs for the treatment of infectious diseases, cancer, or autoimmune disorders. In nature, polyketides and nonribosomal peptides are generated by consecutive condensation reactions of short chain acyl-CoAs or amino acids, respectively, with the substrates and reaction intermediates being bound to large, multidomain enzymes. For the reconstitution of these multistep catalytic processes, the enzymatic assembly lines need to be functionally expressed and the required substrates must be supplied in reasonable quantities. Furthermore, the production hosts need to be protected from the toxicity of the biosynthetic products. In this review, we will summarize and evaluate the status quo regarding the heterologous production of polyketides and nonribosomal peptides in S. cerevisiae. Based on a comprehensive literature analysis, prerequisites for a successful pathway reconstitution could be deduced, as well as recurring bottlenecks in this microbial host.

NLRP1B and NLRP3 control the host response following colonization by the commensal protist Tritrichomonas musculis.

Commensal intestinal protozoa, unlike their pathogenic relatives, are neglected members of the mammalian microbiome. These microbes have a significant impact on host intestinal immune homeostasis, typically by elevating anti-microbial host defense. Tritrichomonas musculis (T. mu), a protozoan gut commensal, strengthens the intestinal host defense against enteric Salmonella infections through Asc- and Il1r1-dependent Th1 and Th17 cell activation. However, the underlying inflammasomes mediating this effect remain unknown. Here, we report that colonization with T. mu results in an increase in luminal extracellular ATP, elevated levels of IL-1b, and increased numbers of IL-18 receptor-expressing Th1 and Th17 cells in the colon. Mice deficient in either Nlrp1b or Nlrp3 failed to display these protozoan-driven immune changes and lost resistance to enteric Salmonella infections even in the presence of T. mu. These findings demonstrate that T. mu-mediated host protection requires sensors of extra and intracellular ATP to confer full resistance to enteric Salmonella infections.

Structure-function characterization of Streptococcus intermedius surface antigen Pas

Streptococcus intermedius , an oral commensal bacterium, is found at various sites including subgingival dental plaque, purulent infections, and in cystic fibrosis lungs. Oral streptococci utilize proteins on their surface to adhere to tissues and/or surfaces localizing the bacteria, which subsequently leads to the development of biofilms, colonization and infection. Among the 19 genomically annotated cell-wall attached surface proteins on S. intermedius , Pas is an adhesin that belongs to the Antigen I/II (AgI/II) family. Here we have structurally and functionally characterized Pas, particularly focusing on its microbial-host as well as microbial-microbial interactions. The crystal structures of V Pas and C 123 Pas show high similarity with AgI/II of S. mutans . V Pas hosts a conserved metal binding site, and likewise the C 123 Pas structure retains its conserved metal binding sites and isopeptide bonds within its three DEv-IgG domains. Pas interacts with nanomolar affinity to lung alveolar glycoprotein 340 (Gp340), its scavenger receptor cysteine rich domains (SRCRs) and with fibrinogen. Both Candida albicans and Pseudomonas aeruginosa , the opportunistic pathogens that cohabitate with S. intermedius in the lungs of CFTR patients were studied in dual-species biofilm studies. The Pas deficient mutant (Δ pas ) displayed significant reduction in dual biofilm formation with C. albicans . In similar studies with P. aeruginosa , Pas did not mediate the biofilm formation with either the acute isolate (PAO1), or the chronic isolate (FRD1). However, the Sortase A deficient mutant (Δ srtA ) displayed reduced biofilm formation with both C. albicans and P. aeruginosa FRD1. Taken together, our findings highlight the role of Pas in both microbial-host and interkingdom interactions and expose its potential role in disease outcomes. Importance Streptococcus intermedius , an oral commensal bacterium, has been clinically observed in subgingival dental plaque, purulent infections, and in cystic fibrosis lungs. In this study, we have (a) determined the crystal structure of the V- and C-regions of Pas; (b) shown that its surface protein Pas adheres to fibrinogen, which could potentially ferry the microbe through the blood stream from the oral cavity; (c) characterized Pas’s high affinity adherence to lung alveolar protein Gp340 that could fixate the microbe on lung epithelial cells; and (d) most importantly shown that these surface proteins on the oral commensal S. intermedius enhances biofilms of known pathogens Candida albicans and Pseudomonas aeruginosa .

Escherichia coli as a platform microbial host for systems metabolic engineering

Bio-based production of industrially important chemicals and materials from non-edible and renewable biomass has become increasingly important to resolve the urgent worldwide issues including climate change. Also, bio-based production, instead of chemical synthesis, of food ingredients and natural products has gained ever increasing interest for health benefits. Systems metabolic engineering allows more efficient development of microbial cell factories capable of sustainable, green, and human-friendly production of diverse chemicals and materials. Escherichia coli is unarguably the most widely employed host strain for the bio-based production of chemicals and materials. In the present paper, we review the tools and strategies employed for systems metabolic engineering of E. coli. Next, representative examples and strategies for the production of chemicals including biofuels, bulk and specialty chemicals, and natural products are discussed, followed by discussion on materials including polyhydroxyalkanoates (PHAs), proteins, and nanomaterials. Lastly, future perspectives and challenges remaining for systems metabolic engineering of E. coli are discussed.

Classical macrophage polarisation is limited by human β-defensin 3 via an autocrine IL-4 dependent process.

Human β-defensin 3 (HBD3), is an anti-microbial host-defence peptide, that can rapidly enter macrophages to modulate TLR4 responses to lipopolysaccharide. However, the molecular mechanisms by which HBD3 exerts this anti-inflammatory influence remain unclear. Here, we show mice deleted for the orthologue of HBD3 have an increased acute lipopolysaccharide response in vivo. Furthermore, we found that HBD3 limited the response of macrophages to classical activation, and contemporaneously drove expression of IL-4. An increase in markers of alternative activation, and a change in metabolic flux was also observed. Consistent with these results, HBD3 enhanced the IL-4 activation of macrophages. Finally, we demonstrate that the ability of HBD3 to limit macrophage classical activation requires IL-4Rα. These data reveal a previously unrecognised role for HBD3 in influencing the polarisation state of macrophages to enable a state conducive for repair and resolution.

Surface Topography, Bacterial Carrying Capacity, and the Prospect of Microbiome Manipulation in the Sea Anemone Coral Model Aiptasia

Aiptasia is an emerging model organism to study cnidarian symbioses due to its taxonomic relatedness to other anthozoans such as stony corals and similarities of its microalgal and bacterial partners, complementing the existing Hydra (Hydrozoa) and Nematostella (Anthozoa) model systems. Despite the availability of studies characterizing the microbiomes of several natural Aiptasia populations and laboratory strains, knowledge on basic information, such as surface topography, bacterial carrying capacity, or the prospect of microbiome manipulation is lacking. Here we address these knowledge gaps. Our results show that the surface topographies of the model hydrozoan Hydra and anthozoans differ substantially, whereas the ultrastructural surface architecture of Aiptasia and stony corals is highly similar. Further, we determined a bacterial carrying capacity of ∼104 and ∼105 bacteria (i.e., colony forming units, CFUs) per polyp for aposymbiotic and symbiotic Aiptasia anemones, respectively, suggesting that the symbiotic status changes bacterial association/density. Microbiome transplants from Acropora humilis and Porites sp. to gnotobiotic Aiptasia showed that only a few foreign bacterial taxa were effective colonizers. Our results shed light on the putative difficulties of transplanting microbiomes between cnidarians in a manner that consistently changes microbial host association at large. At the same time, our study provides an avenue to identify bacterial taxa that exhibit broad ability to colonize different hosts as a starting point for cross-species microbiome manipulation. Our work is relevant in the context of microbial therapy (probiotics) and microbiome manipulation in corals and answers to the need of having cnidarian model systems to test the function of bacteria and their effect on holobiont biology. Taken together, we provide important foundation data to extend Aiptasia as a coral model for bacterial functional studies.

Microbially guided discovery and biosynthesis of biologically active natural products

The design of small molecules that inhibit disease-relevant proteins represents a longstanding challenge of medicinal chemistry. Here, we describe an approach for encoding this challenge—the inhibition of a human drug target—into a microbial host and using it to guide the discovery and biosynthesis of targeted, biologically active natural products. This approach identified two previously unknown terpenoid inhibitors of protein tyrosine phosphatase 1B (PTP1B), an elusive therapeutic target for the treatment of diabetes and cancer. Both inhibitors appear to target an allosteric site, which confers selectivity, and can inhibit PTP1B in living cells. A screen of 24 uncharacterized terpene synthases from a pool of 4,464 genes uncovered additional hits, demonstrating a scalable discovery approach, and the incorporation of different PTPs into the microbial host yielded alternative PTP-specific detection systems. Findings illustrate the potential for using microbes to discover and build natural products that exhibit precisely defined biochemical activities yet possess unanticipated structures and/or binding sites.

Efficient Production of Pyruvate Using Metabolically Engineered Lactococcus lactis

Microbial production of commodity chemicals has gained increasing attention and most of the focus has been on reducing the production cost. Selecting a suitable microorganism, which can grow rapidly on cheap feedstocks, is of key importance when developing an economically feasible bioprocess. We chose Lactococcus lactis, a well-characterized lactic acid bacterium, as our microbial host to produce pyruvate, which is a commodity chemical with various important applications. Here we report the engineering of Lactococcus lactis into becoming an efficient microbial platform for producing pyruvate. The strain obtained, FS1076 (MG1363 Δ3ldh Δpta ΔadhE Δals), was able to produce pyruvate as the sole product. Since all the competitive pathways had been knocked out, we achieved growth-coupled production of pyruvate with high yield. More than 80 percent of the carbon flux was directed toward pyruvate, and a final titer of 54.6 g/L was obtained using a fed-batch fermentation setup. By introducing lactose catabolism into FS1076, we obtained the strain FS1080, which was able to generate pyruvate from lactose. We then demonstrated the potential of FS1080 for valorizing lactose contained in dairy side-streams, by achieving a high titer (40.1 g/L) and high yield (78.6%) of pyruvate using residual whey permeate (RWP) as substrate. The results obtained, show that the L. lactis platform is well-suited for transforming lactose in dairy waste into food-grade pyruvate, and the yields obtained are the highest reported in the literature. These results demonstrate that it is possible to achieve sustainable bioconversion of waste products from the dairy industry (RWP) to valuable products.

Microbially guided discovery and biosynthesis of biologically active natural products

The design of small molecules that inhibit disease-relevant proteins represents a longstanding challenge of medicinal chemistry. Here, we describe an approach for encoding this challenge—the inhibition of a human drug target—into a microbial host and using it to guide the discovery and biosynthesis of targeted, biologically active natural products. This approach identified two previously unknown terpenoid inhibitors of protein tyrosine phosphatase 1B (PTP1B), an elusive therapeutic target for the treatment of diabetes and cancer. Both inhibitors target an allosteric site, which confers unusual selectivity, and can inhibit PTP1B in living cells. A screen of 24 uncharacterized terpene synthases from a pool of 4,464 genes uncovered additional hits, demonstrating a scalable discovery approach, and the incorporation of different PTPs into the microbial host yielded alternative PTP-specific detection systems. Findings illustrate the potential for using microbes to discover and build natural products that exhibit precisely defined biochemical activities yet possess unanticipated structures and/or binding sites.