combinatorial biosynthesis

BACKGROUND: Isoquercitrin (quercetin-3-O-β-D-glucopyranoside) has exhibited promising therapeutic potentials as cardioprotective, anti-diabetic, anti-cancer, and anti-viral agents. However, its structural complexity and limited natural abundance make both bulk chemical synthesis and extraction from medical plants difficult. Microbial biotransformation through heterologous expression of glycosyltransferases offers a safe and sustainable route for its production. Despite several attempts reported in microbial hosts, the current production levels of isoquercitrin still lag behind industrial standards...

Natural products are small molecules synthesized by fungi, bacteria and plants, which historically have had a profound effect on human health and quality of life. These natural products have evolved over millions of years resulting in specific biological functions that may be of interest for pharmaceutical, agricultural, or nutraceutical use. Often natural products need to be structurally modified to make them suitable for specific applications. Combinatorial biosynthesis is a method to alter the composition of enzymes needed to synthesize a specific natural product resulting in structurally diversified molecules...

Vitamin B12 is a complex compound synthesized by microorganisms. The industrial production of vitamin B12 relies on specific microbial fermentation processes. E. coli has been utilized as a host for the de novo biosynthesis of vitamin B12 , incorporating approximately 30 heterologous genes. However, a metabolic imbalance in the intricate pathway significantly limits vitamin B12 production. In this study, we employed multivariate modular metabolic engineering to enhance vitamin B12 production in E. coli by manipulating two modules comprising a total of 10 genes within the vitamin B12 biosynthetic pathway...

Terpenoids are a vast class of plant specialized metabolites (PSMs) manufactured by plants and are involved in their interactions with environment. In addition, they add health benefits to human nutrition and are widely used as pharmaceutically active compounds. However, native plants produce a limited amount of terpenes restricting metabolite yield of terpene-related metabolites. Exponential growth in the plant metabolome data and the requirement of alternative approaches for producing the desired amount of terpenoids, has redirected plant biotechnology research to plant metabolic engineering, which requires in-depth knowledge and precise expertise about dynamic plant metabolic pathways and cellular physiology...

Arabidopsis thaliana Mitogen-activated protein Kinase Phosphatase 1 (MKP1) negatively balances production of reactive oxygen species (ROS) triggered by Microbe-Associated Molecular Patterns (MAMPs) through uncharacterized mechanisms. Accordingly, ROS production is enhanced in mkp1 mutant after MAMP treatment. Moreover, mkp1 plants show a constitutive activation of immune responses and enhanced disease resistance to pathogens with distinct colonization styles, like the bacterium Pseudomonas syringae pv. tomato DC3000, the oomycete Hyaloperonospora arabidopsidis Noco2 and the necrotrophic fungus Plectosphaerella cucumerina BMM...

BACKGROUND: The emergence of the pathogenic yeast Candida auris is of global concern due to its ability to cause hospital outbreaks and develop resistance against all antifungal drug classes. Based on published data for baker's yeast Saccharomyces cerevisiae, sphingolipid biosynthesis, which is essential for maintaining membrane fluidity and formation of lipid rafts, could offer a target for additive treatment. METHODS: We analysed the susceptibility of C. auris to myriocin, which is an inhibitor of the de novo synthesis of sphingolipids in eukaryotic cells in comparison to other Candida species...

Glycosides make up a biomedically important class of secondary metabolites. Most naturally occurring glycosides were isolated from plants and bacteria; however, the chemical diversity of glycosylated natural products in fungi remains largely unexplored. Herein, we present a paradigm to specifically discover diverse and bioactive glycosylated natural products from fungi by combining tailoring enzyme-guided genome mining with mass spectrometry (MS)-based metabolome analysis. Through in vivo genes deletion and heterologous expression, the first fungal C -glycosyltransferase AuCGT involved in the biosynthesis of stromemycin was identified from Aspergillus ustus ...

Arylalkylamine N-acetyltransferase (AANAT) serves as a key enzyme in the biosynthesis of melatonin by transforming 5-hydroxytryptamine (5-HT) to N-acetyl-5-hydroxytryptamine (NAS), while its low activity may hinder melatonin yield. In this study, a novel AANAT derived from Sus scrofa (SsAANAT) was identified through data mining using 5-HT as a model substrate, and a rational design of SsAANAT was conducted in the quest to improving its activity. After four rounds of mutagenesis procedures, a triple combinatorial dominant mutant M3 was successfully obtained...

This review focuses on discussing natural products (NPs) that contain higher homologs of amino acids (homoAAs) in the structure as well as the proposed and characterized biosynthesis of these non-proteinogenic amino acids. Homologation of amino acids includes the insertion of a methylene group into its side chain. It is not a very common modification found in NP biosynthesis as approximately 450 homoAA-containing NPs have been isolated from four bacterial phyla (Cyanobacteria, Actinomycetota, Myxococcota, and Pseudomonadota), two fungal phyla (Ascomycota and Basidiomycota), and one animal phylum (Porifera), except for a few examples...

Introduction: Daunorubicin and doxorubicin, two anthracycline polyketides produced by S. peucetius , are potent anticancer agents that are widely used in chemotherapy, despite severe side effects. Recent advances have highlighted the potential of producing improved derivatives with reduced side effects by incorporating l-rhodosamine, the N,N -dimethyl analogue of the native amino sugar moiety. Method: In this study, we aimed to produce N,N -dimethylated anthracyclines by engineering the doxorubicin biosynthetic pathway in the industrial Streptomyces peucetius strain G001...

As the field of plant synthetic biology continues to grow, Agrobacterium-mediated transient expression has become an essential method to rapidly test pathway candidate genes in a combinatorial fashion. This is especially important when elucidating and engineering more complex pathways to produce commercially relevant chemicals like many terpenoids, a widely diverse class of natural products of often industrial relevance. Agrobacterium-mediated transient expression has facilitated multiplex expression of recombinant and modified enzymes, including synthetic biology approaches to compartmentalize the biosynthesis of terpenoids subcellularly...

Microbial production of genistein, an isoflavonoid primarily found in soybeans, is gaining prominence in the food industry due to its significant nutritional and health benefits. However, challenges arise in redesigning strains due to intricate regulatory nodes between cell growth and genistein production and in systematically exploring core enzymes involving genistein biosynthesis. To address this, this study devised a strategy that simultaneously and precisely rewires flux at both acetyl-CoA and malonyl-CoA nodes toward genistein synthesis...

Surfactin biosynthesis in Bacillus subtilis is intricately regulated by environmental conditions. In the present study, addition of nitrate, a nitrogen source, increased the production of surfactin in B. subtilis ATCC 21332, whereas its absence resulted in minimal or no surfactin production. Proteomics revealed the mechanism underlying nitrate-induced surfactin overproduction, identifying three key differential proteins (preprotein translocase subunit SecA, signal recognition particle receptor FtsY, and cell division adenosine triphosphate-binding protein FtsE) relevant to surfactin transport and regulation...

Piperazic acid is a cyclic nonproteinogenic amino acid that contains a hydrazine N-N bond formed by a piperazate synthase (KtzT-like). This amino acid, found in bioactive natural products synthesized by non-ribosomal peptide synthetases (NRPSs), confers conformational constraint to peptides, an important feature for their biological activities. Genome mining of Streptomyces strains has been revealed as a strategy to identify biosynthetic gene clusters (BGCs) for potentially active compounds. Moreover, the isolation of new strains from underexplored habitats or associated with other organisms has allowed to uncover new BGCs for unknown compounds...

Diterpenoids form a diverse group of natural products, many of which are or could become pharmaceuticals or industrial chemicals. The modular character of diterpene biosynthesis and the promiscuity of the enzymes involved make combinatorial biosynthesis a promising approach to generate libraries of diverse diterpenoids. Here, we report on the combinatorial assembly in yeast of ten diterpene synthases producing (+)-copalyl diphosphate-derived backbones and four cytochrome P450 oxygenases (CYPs) in diverse combinations...

Balancing relative expression of pathway genes to minimize flux bottlenecks and metabolic burden is one of the key challenges in metabolic engineering. This is especially relevant for iterative pathways, such as reverse β-oxidation (rBOX) pathway, which require control of flux partition at multiple nodes to achieve efficient synthesis of target products. Here, we develop a plasmid-based inducible system for orthogonal control of gene expression (referred to as the TriO system) and demonstrate its utility in the rBOX pathway...

Polyketides with the isochroman-3-one pharmacophore are rare among fungal natural products as their biosynthesis requires an unorthodox S-type aromatic ring cyclization. Genome mining uncovered a conserved gene cluster in select leotiomycetous fungi that encodes the biosynthesis of cytosporones, including isochroman-3-one congeners. Combinatorial biosynthesis in total biosynthetic and biocatalytic formats in Saccharomyces cerevisiae and in vitro reconstitution of key reactions with purified enzymes revealed how cytosporone structural and bioactivity diversity is generated...

Currently, most maytansine-containing antibody-drug conjugates (ADCs) in clinical trials are prepared with DM1 or DM4, which in turn is synthesized mainly from ansamitocin P-3 (AP-3), a bacterial maytansinoid, isolated from Actinosynnema pretiosum . However, due to the high self-toxicity of AP-3 to A. pretiosum , the yield of AP-3 has been difficult to improve. Herein, a new maytansinoid with much lower self-toxicity to A. pretiosum , 3- O -carbamoylmaytansinol (CAM, 3 ), was designed and generated by introducing the 3- O -carbamoyltransferase gene asc21b together with the N -methyltransferase genes from exogenous maytansinoid gene clusters into the 3- O -acyltransferase gene ( asm19 ) deleted mutant HGF052...

BACKGROUND: Acidic lipases with high catalytic activities under acidic conditions have important application values in the food, feed and pharmaceutical industries. However, the availability of acidic lipases is still the main obstacle to their industrial applications. Although a novel acidic lipase Rasamsonia emersonii (LIPR) was heterologously expressed in Escherichia coli, the expression level was unsatisfactory. RESULTS: To achieve the high-efficiency expression and secretion of LIPR in Pichia pastoris GS115, the combinatorial optimization strategy was adopted including gene codon preference, signal peptide, molecular chaperone co-expression and disruption of vacuolar sorting receptor VPS10...

Microbes are increasingly employed as cell factories to produce biomolecules. This often involves the expression of complex heterologous biosynthesis pathways in host strains. Achieving maximal product yields and avoiding build-up of (toxic) intermediates requires balanced expression of every pathway gene. However, despite progress in metabolic modeling, the optimization of gene expression still heavily relies on trial-and-error. Here, we report an approach for in vivo, multiplexed Gene Expression Modification by LoxPsym-Cre Recombination (GEMbLeR)...