Heterologous expression and biochemical characterisation of cyanotoxin biosynthesis 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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Heterologous Expression and Manipulation of Three Tetracycline Biosynthetic Pathways

Angewandte Chemie ◽

10.1002/ange.201205426 ◽

2012 ◽

Vol 124 (44) ◽

pp. 11298-11302 ◽

Cited By ~ 3

Author(s):

Peng Wang ◽

Woncheol Kim ◽

Lauren B. Pickens ◽

Xue Gao ◽

Yi Tang

Keyword(s):

Heterologous Expression ◽

Biosynthetic Pathways

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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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Natural product biosyntheses in cyanobacteria: A treasure trove of unique enzymes

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.7.191 ◽

2011 ◽

Vol 7 ◽

pp. 1622-1635 ◽

Cited By ~ 75

Author(s):

Jan-Christoph Kehr ◽

Douglas Gatte Picchi ◽

Elke Dittmann

Keyword(s):

Natural Products ◽

Synthetic Biology ◽

Natural Product ◽

Bioactive Compounds ◽

Polyketide Synthase ◽

Biosynthetic Pathways ◽

Peptide Synthetase ◽

Biochemical Studies ◽

Terrestrial Habitats ◽

The Individual

Cyanobacteria are prolific producers of natural products. Investigations into the biochemistry responsible for the formation of these compounds have revealed fascinating mechanisms that are not, or only rarely, found in other microorganisms. In this article, we survey the biosynthetic pathways of cyanobacteria isolated from freshwater, marine and terrestrial habitats. We especially emphasize modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) pathways and highlight the unique enzyme mechanisms that were elucidated or can be anticipated for the individual products. We further include ribosomal natural products and UV-absorbing pigments from cyanobacteria. Mechanistic insights obtained from the biochemical studies of cyanobacterial pathways can inspire the development of concepts for the design of bioactive compounds by synthetic-biology approaches in the future.

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Heterologous expression and biochemical studies of a thermostable glucose tolerant β-glucosidase from Methylococcus capsulatus ( bath strain )

International Journal of Biological Macromolecules ◽

10.1016/j.ijbiomac.2017.04.078 ◽

2017 ◽

Vol 102 ◽

pp. 805-812 ◽

Cited By ~ 7

Author(s):

Sneha S. Sathe ◽

Surabhi Soni ◽

Vikas P. Ranvir ◽

Vikram G. Choudhari ◽

Annamma A. Odaneth ◽

...

Keyword(s):

Heterologous Expression ◽

Methylococcus Capsulatus ◽

Biochemical Studies ◽

Glucose Tolerant

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Anatomical and biochemical characterisation of a barrier to radial O2 loss in adventitious roots of two contrasting Hordeum marinum accessions

Functional Plant Biology ◽

10.1071/fp16327 ◽

2017 ◽

Vol 44 (9) ◽

pp. 845 ◽

Cited By ~ 14

Author(s):

Lukasz Kotula ◽

Lukas Schreiber ◽

Timothy D. Colmer ◽

Mikio Nakazono

Keyword(s):

Adventitious Roots ◽

Sectional Area ◽

Cross Sectional ◽

Anatomical Studies ◽

Root Zones ◽

Tolerant Plants ◽

Biochemical Characterisation ◽

Biochemical Studies ◽

Almost All ◽

Combined Measurements

A barrier to radial O2 loss (ROL) in roots is an adaptive trait of waterlogging-tolerant plants. Hordeum marinum Huds. is a waterlogging-tolerant species that, in contrast to its waterlogging-sensitive cultivated relatives, forms a tight barrier to ROL in basal root zones. To evaluate the nature of the barrier to ROL in roots, we combined measurements of ROL with histochemical and biochemical studies of two contrasting H. marinum accessions. H21 formed greater aerenchyma (up to 38% of cross-sectional area) and a tight barrier to ROL when grown under deoxygenated stagnant conditions, whereas the barrier was only partially formed in roots of H90 and aerenchyma was up to 26%. A tight barrier to ROL in roots of H21 corresponded with strong suberisation of the exodermis. In agreement with anatomical studies, almost all aliphatic suberin quantities were greater in roots of H21 grown under stagnant conditions compared with roots from aerated controls, and also to those in H90. By contrast to suberin, no differences in root lignification were observed between the two accessions raised in either aerated or stagnant conditions. These findings show that in adventitious roots of H. marinum, suberisation rather than lignification restricts ROL from the basal root zones.

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