Exploiting the Feedstock Flexibility of the Emergent Synthetic Biology Chassis Vibrio natriegens for Engineered Natural Product Production

A recent goal of synthetic biology has been to identify new chassis that provide benefits lacking in model organisms. Vibrio natriegens is a marine Gram-negative bacterium which is an emergent synthetic biology chassis with inherent benefits: An extremely fast growth rate, genetic tractability, and the ability to grow on a variety of carbon sources (“feedstock flexibility”). Given these inherent benefits, we sought to determine its potential to heterologously produce natural products, and chose beta-carotene and violacein as test cases. For beta-carotene production, we expressed the beta-carotene biosynthetic pathway from the sister marine bacterium Vibrio campbellii, as well as the mevalonate biosynthetic pathway from the Gram-positive bacterium Lactobacillus acidophilus to improve precursor abundance. Violacein was produced by expressing a biosynthetic gene cluster derived from Chromobacterium violaceum. Not only was V. natriegens able to heterologously produce these compounds in rich media, illustrating its promise as a new chassis for small molecule drug production, but it also did so in minimal media using a variety of feedstocks. The ability for V. natriegens to produce natural products with multiple industrially-relevant feedstocks argues for continued investigations into the production of more complex natural products in this chassis.

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mSphere of Influence: Synthetic Biology of Natural Product Biosynthesis

mSphere ◽

10.1128/msphere.00954-19 ◽

2020 ◽

Vol 5 (1) ◽

Author(s):

Mark C. Walker

Keyword(s):

Natural Products ◽

Synthetic Biology ◽

Natural Product ◽

Gene Cluster ◽

Biosynthetic Gene Cluster ◽

Biosynthetic Gene ◽

Natural Product Biosynthesis ◽

Antibiotic Compounds ◽

Approaches To Study

ABSTRACT Mark Walker studies the biosynthesis and engineering of bacterial natural products with the long-term goal of identifying new antibiotic compounds. In this mSphere of Influence, he reflects on how “Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A” by K. Yamanaka, K. A. Reynolds, R. D. Kersten, K. S. Ryan, et al. (Proc Natl Acad Sci USA 111:1957–1962, 2014, https://doi.org/10.1073/pnas.1319584111) impacted his thinking on using synthetic biology approaches to study natural product biosynthesis.

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Genome Mining and Metabolic Profiling Uncover Polycyclic Tetramate Macrolactams from Streptomyces koyangensis SCSIO 5802

Marine Drugs ◽

10.3390/md19080440 ◽

2021 ◽

Vol 19 (8) ◽

pp. 440

Author(s):

Wenjuan Ding ◽

Jiajia Tu ◽

Huaran Zhang ◽

Xiaoyi Wei ◽

Jianhua Ju ◽

...

Keyword(s):

Natural Products ◽

Gene Cluster ◽

Metabolic Profiling ◽

Biosynthetic Pathway ◽

Genome Mining ◽

Biosynthetic Gene Cluster ◽

Open Reading Frames ◽

Bioactive Metabolites ◽

Bioinformatics Analyses ◽

Reading Frames

We have previously shown deep-sea-derived Streptomyces koyangensis SCSIO 5802 to produce two types of active secondary metabolites, abyssomicins and candicidins. Here, we report the complete genome sequence of S. koyangensis SCSIO 5802 employing bioinformatics to highlight its potential to produce at least 21 categories of natural products. In order to mine novel natural products, the production of two polycyclic tetramate macrolactams (PTMs), the known 10-epi-HSAF (1) and a new compound, koyanamide A (2), was stimulated via inactivation of the abyssomicin and candicidin biosynthetic machineries. Detailed bioinformatics analyses revealed a PKS/NRPS gene cluster, containing 6 open reading frames (ORFs) and spanning ~16 kb of contiguous genomic DNA, as the putative PTM biosynthetic gene cluster (BGC) (termed herein sko). We furthermore demonstrate, via gene disruption experiments, that the sko cluster encodes the biosynthesis of 10-epi-HSAF and koyanamide A. Finally, we propose a plausible biosynthetic pathway to 10-epi-HSAF and koyanamide A. In total, this study demonstrates an effective approach to cryptic BGC activation enabling the discovery of new bioactive metabolites; genome mining and metabolic profiling methods play key roles in this strategy.

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New from old: discovery of the novel antibiotic actinomycin L in Streptomyces sp. MBT27

10.1101/2021.10.12.464064 ◽

2021 ◽

Author(s):

Nataliia Machushynets ◽

Somayah S. Elsayed ◽

Chao Du ◽

Maxime A. Siegler ◽

Mercedes de la Cruz ◽

...

Keyword(s):

Natural Products ◽

Metabolic Pathways ◽

Chemical Space ◽

Carbon Sources ◽

Growth Conditions ◽

Biosynthetic Gene Cluster ◽

The Novel ◽

Product Families ◽

Antibiotic Discovery ◽

The Impact

ABSTRACTStreptomycetes are major producers of bioactive natural products, including the majority of the antibiotics. While much if the low-hanging fruit has been discovered, it is predited that less than 5% of the chemical space has been mined. Here, we describe the novel actinomycins L1 and L2, which are produced by Streptomyces sp. MBT27. The molecules were discovered via metabolic analysis combined with molecular networking of cultures grown with different combinations of carbon sources. Actinomycins L1 and L2 are diastereoisomers, and the structure of actinomycin L2 was resolved using NMR and single crystal X-ray crystallography. Actinomycin L is formed via a unique spirolinkage of anthranilamide to the 4-oxoproline moiety of actinomycin X2, prior to the condensation of the actinomycin halves. Feeding anthranilamide to cultures of Streptomyces antibioticus, which has the same biosynthetic gene cluster as Streptomyces sp. MBT27 but only produces actinomycin X2, resulted in the production of actinomycin L. This shows that actinomycin L results from joining two distinct metabolic pathways, namely those for actinomycin X2 and for anthranilamide. Actinomycins L1 and L2 showed significant antimicrobial activity against Gram- positive bacteria. Our work shows how new molecules can still be identified even in the oldest of natural product families.IMPORTANCEActinomycin was the first antibiotic discovered in an actinobacterium by Selman Waksman and colleagues, as early as 1940. This period essentially marks the start of the ‘golden era’ of antibiotic discovery. Over time, emerging antimicrobial resistance (AMR) and the declining success rate of antibiotic discovery resulted in the current antibiotic crisis. We surprisingly discovered that under some growth conditions, Streptomyces sp. MBT27 can produce actinomycins that are significantly different from those that have been published so far. The impact of this work is not only that we have discovered a novel molecule with very interesting chemical modifications in one of the oldest antibiotics ever described, but also that this requires the combined action of primary and secondary metabolic pathways, namely the biosynthesis of anthranilamide and of actinomycin X2, respectively. The implication of the discovery is that even the most well-studied families of natural products may still have surprises in store for us.

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Biosynthesis of dihydroquercetin in Escherichia coli from glycerol

10.1101/2020.11.27.401000 ◽

2020 ◽

Author(s):

Seon Young Park ◽

Dongsoo Yang ◽

Shin Hee Ha ◽

Sang Yup Lee

Keyword(s):

Escherichia Coli ◽

Natural Products ◽

Biosynthetic Pathway ◽

Carbon Sources ◽

Aromatic Amino Acids ◽

Engineering Model ◽

Producer Strain ◽

E Coli ◽

One Step ◽

First Time

AbstractPhenylpropanoids are a group of diverse natural products derived from aromatic amino acids. Although their demands are high both as drugs and nutraceuticals, their production mainly depends on inefficient extraction from plants. To achieve sustainable production of phenylpropanoids, engineering model microorganisms such as Escherichia coli has been sought, but most strains require supplementation of expensive precursors. Here, we report one-step production of a representative phenylpropanoid, dihydroquercetin (DHQ), from simple carbon sources in E. coli for the first time. The best DHQ producer strain capable of producing 239.4 mg/L of DHQ from glycerol was obtained by optimizing the biosynthetic pathway and engineering the signal peptide of cytochrome P450 (TT7) from Arabidopsis thaliana. The engineered plant P450 could produce a significantly higher titer of DHQ than a bacterial monooxygenase, showing the potential of employing plant P450s for the production of diverse natural products that has been previously difficult in bacterial hosts. This study will serve as a guideline for industrial production of pharmaceutically important yet complex natural products.

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Enzymatic dimerization in the biosynthetic pathway of microbial natural products

Natural Product Reports ◽

10.1039/d0np00063a ◽

2021 ◽

Author(s):

Jiawang Liu ◽

Anan Liu ◽

Youcai Hu

Keyword(s):

Natural Products ◽

Biosynthetic Pathway ◽

Structural Diversity ◽

Cytochrome P450s ◽

Microbial Natural Products

Cytochrome P450s, laccases, and intermolecular [4 + 2] cyclases, along with other enzymes were utilized to catalyze varied dimerization of matured natural products so as to create the structural diversity and complexity in microorganisms.

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Biosynthesis and synthetic biology of psychoactive natural products

Chemical Society Reviews ◽

10.1039/d1cs00065a ◽

2021 ◽

Author(s):

Cooper S. Jamieson ◽

Joshua Misa ◽

Yi Tang ◽

John M. Billingsley

Keyword(s):

Natural Products ◽

Synthetic Biology ◽

Biological Activities ◽

Engineering Applications ◽

Pathway Discovery

The biosynthetic logic employed by Nature in the construction of psychoactive natural products is reviewed, in addition to biological activities, methodologies enabling pathway discovery, and engineering applications.

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Development of synthetic biology tools to engineer Pichia pastoris as a chassis for the production of natural products

Synthetic and Systems Biotechnology ◽

10.1016/j.synbio.2021.04.005 ◽

2021 ◽

Vol 6 (2) ◽

pp. 110-119

Author(s):

Jucan Gao ◽

Lihong Jiang ◽

Jiazhang Lian

Keyword(s):

Natural Products ◽

Synthetic Biology ◽

Pichia Pastoris

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Identification, heterologous production and bioactivity of lentinulin A and dendrothelin A, two natural variants of backbone N-methylated peptide macrocycle omphalotin A

Scientific Reports ◽

10.1038/s41598-021-83106-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Emmanuel Matabaro ◽

Hannelore Kaspar ◽

Paul Dahlin ◽

Daniel L. V. Bader ◽

Claudia E. Murar ◽

...

Keyword(s):

Natural Products ◽

Lentinula Edodes ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Precursor Protein ◽

Fungal Genome ◽

Homologous Gene ◽

C Terminus ◽

Natural Variants ◽

Recombinant Peptides

AbstractBackbone N-methylation and macrocyclization improve the pharmacological properties of peptides by enhancing their proteolytic stability, membrane permeability and target selectivity. Borosins are backbone N-methylated peptide macrocycles derived from a precursor protein which contains a peptide α-N-methyltransferase domain autocatalytically modifying the core peptide located at its C-terminus. Founding members of borosins are the omphalotins from the mushroom Omphalotus olearius (omphalotins A-I) with nine out of 12 L-amino acids being backbone N-methylated. The omphalotin biosynthetic gene cluster codes for the precursor protein OphMA, the protease prolyloligopeptidase OphP and other proteins that are likely to be involved in other post-translational modifications of the peptide. Mining of available fungal genome sequences revealed the existence of highly homologous gene clusters in the basidiomycetes Lentinula edodes and Dendrothele bispora. The respective borosins, referred to as lentinulins and dendrothelins are naturally produced by L. edodes and D. bispora as shown by analysis of respective mycelial extracts. We produced all three homologous peptide natural products by coexpression of OphMA hybrid proteins and OphP in the yeast Pichia pastoris. The recombinant peptides differ in their nematotoxic activity against the plant pathogen Meloidogyne incognita. Our findings pave the way for the production of borosin peptide natural products and their potential application as novel biopharmaceuticals and biopesticides.

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Biosynthetic pathways and enzymes involved in the production of phosphonic acid natural products

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbaa052 ◽

2021 ◽

Vol 85 (1) ◽

pp. 42-52

Author(s):

Taro Shiraishi ◽

Tomohisa Kuzuyama

Keyword(s):

Natural Products ◽

Biosynthetic Pathway ◽

Organophosphorus Compounds ◽

Genome Mining ◽

Biological Molecules ◽

Antibacterial Drug ◽

Gene Encoding ◽

Quarter Century ◽

Lead Compounds ◽

Biosynthetic Machinery

Abstract Phosphonates are organophosphorus compounds possessing a characteristic C−P bond in which phosphorus is directly bonded to carbon. As phosphonates mimic the phosphates and carboxylates of biological molecules to potentially inhibit metabolic enzymes, they could be lead compounds for the development of a variety of drugs. Fosfomycin (FM) is a representative phosphonate natural product that is widely used as an antibacterial drug. Here, we review the biosynthesis of FM, which includes a recent breakthrough to find a missing link in the biosynthetic pathway that had been a mystery for a quarter-century. In addition, we describe the genome mining of phosphonate natural products using the biosynthetic gene encoding an enzyme that catalyzes C–P bond formation. We also introduce the chemoenzymatic synthesis of phosphonate derivatives. These studies expand the repertoires of phosphonates and the related biosynthetic machinery. This review mainly covers the years 2012-2020.

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