Heterologous Expression and Manipulation of Three Tetracycline Biosynthetic Pathways

<p>Antibiotic discovery rates dramatically declined following the “golden age” of the 1940’s to the 1960’s. The platforms that underpinned that age of discovery rested upon laboratory cultivation of a small clade of bacteria, the actinomycetes, primarily isolated from soil environments. Fermentation extracts of these isolated bacteria have provided the majority of antibiotics and anticancer small molecules still used today. By applying modern genetic analysis techniques to these same environmental sources that have previously yielded such success, we can uncover new biosynthetic pathways, and bioactive compounds. The work described in this thesis investigated New Zealand soil metagenomes for this purpose. Four large metagenome libraries were constructed from the microbiomes of diverse soil environments. These were then interrogated by a functional screening approach in a knockout Escherichia coli strain, to recover a large collection of the biosynthetic gene clusters responsible for bacterial secondary metabolite production. Using different modes of bioinformatic analysis, these gene clusters were demonstrated to have both phylogenetic divergence, and functional difference from bacterial biosynthesis pathways previously discovered from culture based studies. Two additional biosynthetic pathways were recovered from one of these metagenome libraries, and in each case found to have novel genetic features. These gene clusters were further studied by heterologous expression within Streptomyces albus production hosts. One of these gene clusters produced small aromatic polyketide compounds, the structure of one of which was solved by chemical analytic techniques, and found to be a new chemical entity. The second gene cluster was demonstrated to have similarity to known aureolic acid biosynthesis gene clusters – a class of potent anticancer natural products. Heterologous expression resulted in the production of many metabolites, two of which were characterised and found to be new members of this chemical class. The research in this thesis both validates the use of metagenomic analysis for future natural product discovery efforts, and adds to a growing body of evidence that understudied clades of bacteria have an untapped biosynthetic potential that can be accessed by metagenomic methods.</p>

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Functional Genome Mining for Metabolites Encoded by Large Gene Clusters through Heterologous Expression of a Whole-Genome Bacterial Artificial Chromosome Library in Streptomyces spp.

Applied and Environmental Microbiology ◽

10.1128/aem.01383-16 ◽

2016 ◽

Vol 82 (19) ◽

pp. 5795-5805 ◽

Cited By ~ 31

Author(s):

Min Xu ◽

Yemin Wang ◽

Zhilong Zhao ◽

Guixi Gao ◽

Sheng-Xiong Huang ◽

...

Keyword(s):

Heterologous Expression ◽

Genome Mining ◽

Gene Clusters ◽

Artificial Chromosome ◽

Functional Screening ◽

Biosynthetic Pathways ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Content Type ◽

Bac Libraries

ABSTRACTGenome sequencing projects in the last decade revealed numerous cryptic biosynthetic pathways for unknown secondary metabolites in microbes, revitalizing drug discovery from microbial metabolites by approaches called genome mining. In this work, we developed a heterologous expression and functional screening approach for genome mining from genomic bacterial artificial chromosome (BAC) libraries inStreptomycesspp. We demonstrate mining from a strain ofStreptomyces rochei, which is known to produce streptothricins and borrelidin, by expressing its BAC library in the surrogate hostStreptomyces lividansSBT5, and screening for antimicrobial activity. In addition to the successful capture of the streptothricin and borrelidin biosynthetic gene clusters, we discovered two novel linear lipopeptides and their corresponding biosynthetic gene cluster, as well as a novel cryptic gene cluster for an unknown antibiotic fromS. rochei. This high-throughput functional genome mining approach can be easily applied to other streptomycetes, and it is very suitable for the large-scale screening of genomic BAC libraries for bioactive natural products and the corresponding biosynthetic pathways.IMPORTANCEMicrobial genomes encode numerous cryptic biosynthetic gene clusters for unknown small metabolites with potential biological activities. Several genome mining approaches have been developed to activate and bring these cryptic metabolites to biological tests for future drug discovery. Previous sequence-guided procedures relied on bioinformatic analysis to predict potentially interesting biosynthetic gene clusters. In this study, we describe an efficient approach based on heterologous expression and functional screening of a whole-genome library for the mining of bioactive metabolites fromStreptomyces. The usefulness of this function-driven approach was demonstrated by the capture of four large biosynthetic gene clusters for metabolites of various chemical types, including streptothricins, borrelidin, two novel lipopeptides, and one unknown antibiotic fromStreptomyces rocheiSal35. The transfer, expression, and screening of the library were all performed in a high-throughput way, so that this approach is scalable and adaptable to industrial automation for next-generation antibiotic discovery.

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Facile assembly and fluorescence-based screening method for heterologous expression of biosynthetic pathways in fungi

Metabolic Engineering ◽

10.1016/j.ymben.2018.05.014 ◽

2018 ◽

Vol 48 ◽

pp. 44-51 ◽

Cited By ~ 22

Author(s):

Sandra Hoefgen ◽

Jun Lin ◽

Janis Fricke ◽

Maria C. Stroe ◽

Derek J. Mattern ◽

...

Keyword(s):

Heterologous Expression ◽

Screening Method ◽

Biosynthetic Pathways

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Loseolamycins: A Group of New Bioactive Alkylresorcinols Produced after Heterologous Expression of a Type III PKS from Micromonospora endolithica

Molecules ◽

10.3390/molecules25204594 ◽

2020 ◽

Vol 25 (20) ◽

pp. 4594

Author(s):

Constanze Lasch ◽

Nils Gummerlich ◽

Maksym Myronovskyi ◽

Anja Palusczak ◽

Josef Zapp ◽

...

Keyword(s):

Natural Products ◽

Heterologous Expression ◽

Transcriptional Activation ◽

Polyketide Synthase ◽

Biologically Active ◽

Aliphatic Chain ◽

Biosynthetic Pathways ◽

Type Iii Polyketide Synthase ◽

Type Iii ◽

Potential Applications

Natural products are a valuable source of biologically active compounds with potential applications in medicine and agriculture. Unprecedented scaffold diversity of natural products and biocatalysts from their biosynthetic pathways are of fundamental importance. Heterologous expression and refactoring of natural product biosynthetic pathways are generally regarded as a promising approach to discover new secondary metabolites of microbial origin. Here, we present the identification of a new group of alkylresorcinols after transcriptional activation and heterologous expression of the type III polyketide synthase of Micromonospora endolithica. The most abundant compounds loseolamycins A1 and A2 have been purified and their structures were elucidated by NMR. Loseolamycins contain an unusual branched hydroxylated aliphatic chain which is provided by the host metabolism and is incorporated as a starter fatty acid unit. The isolated loseolamycins show activity against gram-positive bacteria and inhibit the growth of the monocot weed Agrostis stolonifera in a germination assay. The biosynthetic pathway leading to the production of loseolamycins is proposed in this paper.

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Null mutation analysis of an afsA-family gene, barX, that is involved in biosynthesis of the γ-butyrolactone autoregulator in Streptomyces virginiae

Microbiology ◽

10.1099/mic.0.032003-0 ◽

2010 ◽

Vol 156 (1) ◽

pp. 206-210 ◽

Cited By ~ 18

Author(s):

Yong Jik Lee ◽

Shigeru Kitani ◽

Takuya Nihira

Keyword(s):

Heterologous Expression ◽

Mutation Analysis ◽

Biosynthetic Pathway ◽

Streptomyces Griseus ◽

Null Mutation ◽

Biosynthetic Pathways ◽

Regulatory Pathway ◽

Streptomyces Virginiae ◽

Family Gene

Virginiae butanolide (VB) is a γ-butyrolactone autoregulator that triggers production of the streptogramin antibiotic virginiamycin in Streptomyces virginiae. Our previous studies suggested that the barX gene, an afsA-family gene, is likely to participate in the regulatory pathway for the production of VB, rather than in the biosynthetic pathway of VB itself, in contrast to the function of other afsA-family genes. Mutation analysis now shows that BarX at least plays an enzymic role in the VB biosynthetic pathway. Heterologous expression of the afsA gene from Streptomyces griseus into the barX mutant partially restored the deficiency of virginiamycin production, suggesting that afsA-family genes have a common ability to synthesize the γ-butyrolactone autoregulators. Taken together with previous works relating to the function of an afsA-family gene, these results support the idea that streptomycetes have two biosynthetic pathways for the γ-butyrolactone autoregulators.

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Reconstructing fungal natural product biosynthetic pathways

Natural Product Reports ◽

10.1039/c4np00084f ◽

2014 ◽

Vol 31 (10) ◽

pp. 1339-1347 ◽

Cited By ~ 36

Author(s):

C. M. Lazarus ◽

K. Williams ◽

A. M. Bailey

Keyword(s):

Molecular Biology ◽

Heterologous Expression ◽

Natural Product ◽

Genome Sequencing ◽

Vital Role ◽

Whole Genome ◽

Biosynthetic Pathways ◽

Expression Systems ◽

Natural Product Research ◽

Heterologous Expression Systems

Molecular biology plays a vital role in contemporary natural product research. Responding to developments in whole genome sequencing, heterologous expression systems are being refined to accommodate whole fungal biosynthetic pathways.

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Discovery and Biosynthesis of Natural Products from New Zealand Soil Metagenome Libraries

10.26686/wgtn.17147699 ◽

2021 ◽

Author(s):

◽

Luke Stevenson

Keyword(s):

Natural Products ◽

New Zealand ◽

Heterologous Expression ◽

Gene Clusters ◽

Bioinformatic Analysis ◽

Coli Strain ◽

Functional Screening ◽

Functional Difference ◽

Biosynthetic Pathways ◽

New Members

<p>Antibiotic discovery rates dramatically declined following the “golden age” of the 1940’s to the 1960’s. The platforms that underpinned that age of discovery rested upon laboratory cultivation of a small clade of bacteria, the actinomycetes, primarily isolated from soil environments. Fermentation extracts of these isolated bacteria have provided the majority of antibiotics and anticancer small molecules still used today. By applying modern genetic analysis techniques to these same environmental sources that have previously yielded such success, we can uncover new biosynthetic pathways, and bioactive compounds. The work described in this thesis investigated New Zealand soil metagenomes for this purpose. Four large metagenome libraries were constructed from the microbiomes of diverse soil environments. These were then interrogated by a functional screening approach in a knockout Escherichia coli strain, to recover a large collection of the biosynthetic gene clusters responsible for bacterial secondary metabolite production. Using different modes of bioinformatic analysis, these gene clusters were demonstrated to have both phylogenetic divergence, and functional difference from bacterial biosynthesis pathways previously discovered from culture based studies. Two additional biosynthetic pathways were recovered from one of these metagenome libraries, and in each case found to have novel genetic features. These gene clusters were further studied by heterologous expression within Streptomyces albus production hosts. One of these gene clusters produced small aromatic polyketide compounds, the structure of one of which was solved by chemical analytic techniques, and found to be a new chemical entity. The second gene cluster was demonstrated to have similarity to known aureolic acid biosynthesis gene clusters – a class of potent anticancer natural products. Heterologous expression resulted in the production of many metabolites, two of which were characterised and found to be new members of this chemical class. The research in this thesis both validates the use of metagenomic analysis for future natural product discovery efforts, and adds to a growing body of evidence that understudied clades of bacteria have an untapped biosynthetic potential that can be accessed by metagenomic methods.</p>

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Recent advances in the heterologous expression of microbial natural product biosynthetic pathways

Natural Product Reports ◽

10.1039/c3np70034h ◽

2013 ◽

Vol 30 (8) ◽

pp. 1121 ◽

Cited By ~ 133

Author(s):

Sarah E. Ongley ◽

Xiaoying Bian ◽

Brett A. Neilan ◽

Rolf Müller

Keyword(s):

Heterologous Expression ◽

Natural Product ◽

Biosynthetic Pathways ◽

Recent Advances

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Heterologous expression and biochemical characterisation of cyanotoxin biosynthesis pathways

Natural Product Reports ◽

10.1039/c8np00063h ◽

2019 ◽

Vol 36 (8) ◽

pp. 1117-1136 ◽

Cited By ~ 6

Author(s):

Alescia Cullen ◽

Leanne A. Pearson ◽

Rabia Mazmouz ◽

Tianzhe Liu ◽

Angela H. Soeriyadi ◽

...

Keyword(s):

Heterologous Expression ◽

Biosynthetic Pathways ◽

Biochemical Characterisation ◽

Biochemical Studies

This review discusses cyanotoxin biosynthetic pathways and highlights the heterologous expression and biochemical studies used to characterise them.

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Heterologous Expression of the Oxytetracycline Biosynthetic Pathway in Myxococcus xanthus

Applied and Environmental Microbiology ◽

10.1128/aem.02841-09 ◽

2010 ◽

Vol 76 (8) ◽

pp. 2681-2683 ◽

Cited By ~ 25

Author(s):

D. Cole Stevens ◽

Michael R. Henry ◽

Kimberly A. Murphy ◽

Christopher N. Boddy

Keyword(s):

Drug Discovery ◽

Heterologous Expression ◽

Biosynthetic Pathway ◽

Myxococcus Xanthus ◽

Gene Clusters ◽

Biosynthetic Pathways ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Metagenomic Dna ◽

Dna Libraries

ABSTRACT New natural products for drug discovery may be accessed by heterologous expression of bacterial biosynthetic pathways in metagenomic DNA libraries. However, a “universal” host is needed for this experiment. Herein, we show that Myxococcus xanthus is a potential “universal” host for heterologous expression of polyketide biosynthetic gene clusters.

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