Biosynthesis of Structurally Novel Carotenoids in Escherichia coli

Lee, Pyung Cheon; Momen, Abu Zafar Ruhul; Mijts, Benjamin N.; Schmidt‐Dannert, Claudia

doi:10.1016/s1074-5521(03)00103-0

Cited by 103 publications

(81 citation statements)

References 26 publications

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“…4, C and D). This confirms that AldH has a broad substrate specificity like other carotenoid enzymes (7,10,26).…”

Section: Figure 5 Allelic Replacement Of Aldh In S Aureus For Functsupporting

confidence: 72%

“…The resulting pCL-ALDKO plasmid was electrotransformed into S. aureus RN4220 (24) and incubated at 43°C on B-mediumagar plates containing 10 g/ml erythromycin (BM-agar Erm 10 plates) for 2 days. Colonies were restreaked onto BM-agar Erm 10 plates and incubated at 43°C for 2 days. A single colony was inoculated into 30 ml of B-medium without antibiotics and sequentially diluted (1:1000) into 30 ml of B-medium each day at 30°C for 5 days.…”

Section: Methodsmentioning

confidence: 99%

“…A single colony was inoculated into 30 ml of B-medium without antibiotics and sequentially diluted (1:1000) into 30 ml of B-medium each day at 30°C for 5 days. After 5 days, 1:10 6 diluted cells were spread onto BM-agar Erm 10 plates, and colonies were visually screened. Deletion mutants that developed an orange color were confirmed by PCR and carotenoid profiling.…”

Section: Methodsmentioning

confidence: 99%

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Functional Expression and Extension of Staphylococcal Staphyloxanthin Biosynthetic Pathway in Escherichia coli

Kim

Lee

2012

Journal of Biological Chemistry

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Background:The biosynthetic pathway for staphyloxanthin has previously been proposed to consist of five enzymes. Results: A sixth pathway enzyme, 4,4Ј-diaponeurosporen-aldehyde dehydrogenase, was identified using a synthetic module approach. Conclusion:The complete staphyloxanthin biosynthetic pathway consists of six enzymes in Staphylococcus aureus. Significance: This is the first report demonstrating the complete staphyloxanthin pathway.The biosynthetic pathway for staphyloxanthin, a C 30 carotenoid biosynthesized by Staphylococcus aureus, has previously been proposed to consist of five enzymes (CrtO, CrtP, CrtQ, CrtM, and CrtN). Here, we report a missing sixth enzyme, 4,4-diaponeurosporen-aldehyde dehydrogenase (AldH), in the staphyloxanthin biosynthetic pathway and describe the functional expression of the complete staphyloxanthin biosynthetic pathway in Escherichia coli. When we expressed the five known pathway enzymes through artificial synthetic operons and the wild-type operon (crtOPQMN) in E. coli, carotenoid aldehyde intermediates such as 4,4-diaponeurosporen-4-al accumulated without being converted into staphyloxanthin or other intermediates. We identified an aldH gene located 670 kilobase pairs from the known staphyloxanthin gene cluster in the S. aureus genome and an aldH gene in the non-staphyloxanthin-producing Staphylococcus carnosus genome. These two putative enzymes catalyzed the missing oxidation reaction to convert 4,4-diaponeurosporen-4-al into 4,4-diaponeurosporenoic acid in E. coli. Deletion of the aldH gene in S. aureus abolished staphyloxanthin biosynthesis and caused accumulation of 4,4-diaponeurosporen-4-al, confirming the role of AldH in staphyloxanthin biosynthesis. When the complete staphyloxanthin biosynthetic pathway was expressed using an artificial synthetic operon in E. coli, staphyloxanthin-like compounds, which contained altered fatty acid acyl chains, and novel carotenoid compounds were produced, indicating functional expression and coordination of the six staphyloxanthin pathway enzymes.

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“…4, C and D). This confirms that AldH has a broad substrate specificity like other carotenoid enzymes (7,10,26).…”

Section: Figure 5 Allelic Replacement Of Aldh In S Aureus For Functsupporting

confidence: 72%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Functional Expression and Extension of Staphylococcal Staphyloxanthin Biosynthetic Pathway in Escherichia coli

Kim

Lee

2012

Journal of Biological Chemistry

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“…For example, the introduction of an algal carotenoid biosynthetic gene into tobacco resulted in the accumulation of 3 0 -hydroxyechinenone [50], the cyanobacterial carotenoid found in the OCP. Furthermore, the importance of carotenoids as biological anti-oxidants has led to intensive efforts to express them in heterologous systems such as Escherichia coli [51]. To fully utilize this technology, it must be complemented by an understanding of how to integrate carotenoids into protein carriers which, for many applications, must be water-soluble.…”

Section: Variants Of Ocp and Photoprotectionmentioning

confidence: 99%

Water-soluble carotenoid proteins of cyanobacteria

Kerfeld¹

2004

Archives of Biochemistry and Biophysics

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In photosynthetic organisms, carotenoids function in light harvesting and in photoprotection. In cyanobacteria, there have been numerous reports of proteins that bind exclusively carotenoids. Perhaps the best characterized of these proteins are the 35 kDa water-soluble orange carotenoid proteins (OCPs). Structural, biochemical, and genomic data on the OCP and its paralogs are gradually revealing how these proteins function in photoprotection.

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“…The pathway of a chosen mutant was further modified by introducing a library of shuffled cyclase genes. The engineering of the carotenoid pathway represents a fine example of how directed evolution can be used to redesign a complex pathway (68,147,167,175,176,178,205,206,257,262,263,305,320,324).…”

Section: Directed Evolution Of Metabolic Pathwaysmentioning

confidence: 99%

Laboratory-Directed Protein Evolution

Yuan

Kurek

English

et al. 2005

Microbiol Mol Biol Rev

175

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Systematic approaches to directed evolution of proteins have been documented since the 1970s. The ability to recruit new protein functions arises from the considerable substrate ambiguity of many proteins. The substrate ambiguity of a protein can be interpreted as the evolutionary potential that allows a protein to acquire new specificities through mutation or to regain function via mutations that differ from the original protein sequence. All organisms have evolutionarily exploited this substrate ambiguity. When exploited in a laboratory under controlled mutagenesis and selection, it enables a protein to “evolve” in desired directions. One of the most effective strategies in directed protein evolution is to gradually accumulate mutations, either sequentially or by recombination, while applying selective pressure. This is typically achieved by the generation of libraries of mutants followed by efficient screening of these libraries for targeted functions and subsequent repetition of the process using improved mutants from the previous screening. Here we review some of the successful strategies in creating protein diversity and the more recent progress in directed protein evolution in a wide range of scientific disciplines and its impacts in chemical, pharmaceutical, and agricultural sciences

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Biosynthesis of Structurally Novel Carotenoids in Escherichia coli

Cited by 103 publications

References 26 publications

Functional Expression and Extension of Staphylococcal Staphyloxanthin Biosynthetic Pathway in Escherichia coli

Functional Expression and Extension of Staphylococcal Staphyloxanthin Biosynthetic Pathway in Escherichia coli

Water-soluble carotenoid proteins of cyanobacteria

Laboratory-Directed Protein Evolution

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