Biosynthesis of Polyketides in Heterologous Hosts

Pfeifer, Blaine A.; Khosla, Chaitan

doi:10.1128/mmbr.65.1.106-118.2001

Cited by 238 publications

(144 citation statements)

References 88 publications

(77 reference statements)

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“…Along with their semi-synthetic derivatives they now play a vital role as human and veterinary medicine therapeutic agents and agricultural agents including antibiotic, anticancer, antiparasitic, immunosuppressant and insecticide compounds (O'Hagan, 1991;Hopwood, 1997;Pereda et al, 1998;Peiru et al, 2005). Other functions include serving as defensive molecules in microbes as well as having a role in organism-to-organism signaling (Rawlings, 1997;Pfeifer and Khosla, 2001;Staunton and Weissman, 2001). Polyketide biosynthesis mechanistically resembles that of fatty acids.…”

Section: Introductionmentioning

confidence: 99%

Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase

Fritzler

Zhu

2007

International Journal for Parasitology

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The apicomplexan Cryptosporidium parvum possesses a unique 1,500-kDa polyketide synthase (CpPKS1) comprised of 29 enzymes for synthesizing a yet undetermined polyketide. This study focuses on the biochemical characterization of the 845-amino acid loading unit containing acyl-[ACP] ligase (AL) and acyl carrier protein (ACP). The CpPKS1-AL domain has a substrate preference for long chain fatty acids, particularly for the C20:0 arachidic acid. When using [ 3 H] palmitic acid and CoA as co-substrates, the AL domain displayed allosteric kinetics towards palmitic acid (Hill coefficient, h = 1.46, K 50 = 0.751 μM, V max = 2.236 μmol mg −1 min −1 ) and CoA (h = 0.704, K 50 = 5.627 μM, V max = 0.557 μmol mg −1 min −1 ), and biphasic kinetics towards adenosine 5′-triphosphate (K m1 = 3.149 μM, V max1 = 373.3 nmol mg −1 min −1 , K m2 = 121.0 μM, and V max2 = 563.7 nmol mg −1 min −1 ). The AL domain is Mg 2+ -dependent and its activity could be inhibited by triacsin C (IC 50 = 6.64 μM). Furthermore, the ACP domain within the loading unit could be activated by the C. parvum surfactin production element-type phosphopantetheinyl transferase. After attachment of the fatty acid substrate to the AL domain for conversion into the fatty-acyl intermediate, the AL domain is able to transfer palmitic acid to the activated holo-ACP in vitro. These observations ultimately validate the function of the CpPKS1-AL-ACP unit, and make it possible to further dissect the function of this megasynthase using recombinant proteins in a stepwise procedure.

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Section: Introductionmentioning

confidence: 99%

Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase

Fritzler

Zhu

2007

International Journal for Parasitology

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“…Readers are also encouraged to read through other excellent reviews on polyketides in general [178] and heterologous polyketides production. [10f, 177,179] …”

Section: Introductionmentioning

confidence: 99%

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

Park

Yang

et al. 2017

Adv. Biosys.

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Natural products have been attracting much interest around the world for their diverse applications, especially in drug and food industries. Plants have been a major source of many different natural products. However, plants are affected by weather and environmental conditions and their successful extraction is rather limited. Chemical synthesis is inefficient due to the complexity of their chemical structures involving enantioselectivity and regioselectivity. For these reasons, an alternative means of overproducing valuable natural products using microorganisms has emerged. In recent years, various metabolic engineering strategies have been developed for the production of natural products by microorganisms. Here, the strategies taken to produce natural products are reviewed. For convenience, natural products are classified into four main categories: terpenoids, phenylpropanoids, polyketides, and alkaloids. For each product category, the strategies for establishing and rewiring the metabolic network for heterologous natural product biosynthesis, systems approaches undertaken to optimize production hosts, and the strategies for fermentation optimization are reviewed. Taken together, metabolic engineering has enabled microorganisms to serve as a prominent platform for natural compounds production. This article examines both the conventional and novel strategies of metabolic engineering, providing general strategies for complex natural compound production through the development of robust microbial‐cell factories.

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“…Moreover, proper folding of such multidomain enzymes and their proper posttranslational modification in foreign host is not predictable (27,32,33). None of these obstacles have been encountered in this study; the three bacitracin peptide synthetases BacA (598 kDa), BacB (297 kDa), and BacC (723 kDa) were heterologously produced at high levels and were posttranslationally modified to their active holo-forms (see Figs.…”

Section: Discussionmentioning

confidence: 99%

Engineered Biosynthesis of the Peptide Antibiotic Bacitracin in the Surrogate Host Bacillus subtilis

Eppelmann

Doekel

Marahiel

2001

Journal of Biological Chemistry

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Nonribosomal peptides are processed on multifunctional enzymes called nonribosomal peptide synthetases (NRPSs), whose modular multidomain arrangement allowed the rational design of new peptide products. However, the lack of natural competence and efficient transformation methods for most of nonribosomal peptide producer strains prevented the in vivo manipulation of these biosynthetic gene clusters. In this study, we present methods for the construction of a genetically engineered Bacillus subtilis surrogate host for the integration and heterologous expression of foreign NRPS genes. In the B. subtilis surrogate host, we deleted the resident 26-kilobase srfA gene cluster encoding the surfactin synthetases and subsequently used the same chromosomal location for integration of the entire 49-kilobase bacitracin biosynthetic gene cluster from Bacillus licheniformis by a stepwise homologous recombination method. Synthesis of the branched cyclic peptide antibiotic bacitracin in the engineered B. subtilis strain was achieved at high level, indicating a functional production and proper posttranslational modification of the bacitracin synthetases BacABC, as well as the expression of the associated bacitracin self-resistance genes. This engineered and genetically amenable B. subtilis strain will facilitate the rational design of new bacitracin derivatives.

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Biosynthesis of Polyketides in Heterologous Hosts

Cited by 238 publications

References 88 publications

Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase

Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

Engineered Biosynthesis of the Peptide Antibiotic Bacitracin in the Surrogate Host Bacillus subtilis

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