Bioproduction of astaxanthin using mutant carotenoid ketolase and carotenoid hydroxylase genes

Inactive Publication Date: 2006-06-29
EI DU PONT DE NEMOURS & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0016] The present invention provides nucleic acid molecules encoding CrtO ketolases and CrtZ hydroxylases useful for producing astaxanthin in recombinant host cells engineered to produce β-carotene. A crtOZ gene combination

Problems solved by technology

Because animals are unable to synthesize carotenoids de novo, they must obtain them by dietary means.
Depending upon the activity and substrate specificity of both the ketolase and hydroxylase used, it is often difficult to predict those combinations that will result in optimal production of astaxanthin.
For example, it has been reported that hydroxylases from cyanobacteria are not able to accept echinenone or canthaxanthin as substrates for hydroxylation.
In such an instance, the majority of the β-carotene would be expected to be converted into hydroxylated carotenoids (such as zeaxanthin) that may not be recognized by the ketolase, resulting in less than optimal production of astaxanthin.
A further problem to be solved is to provide a method to produce matched pairs of carotenoid ketolases and carotenoid hydroxylases having astaxanthin biosynthesis activity.

Method used

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  • Bioproduction of astaxanthin using mutant carotenoid ketolase and carotenoid hydroxylase genes

Examples

Experimental program
Comparison scheme
Effect test

example 1

Selection of the crtOZ Plasmid for Protein Engineering Construction of two plasmids containing different crtOZ genes

[0196] The crtO gene isolated from Rhodococcus erythropolis AN12 has previously been engineered (U.S. Ser. No. 60 / 577,970) to increase the ketolase activity. One of the mutants, crtO-SHU001, produced over 90% canthaxanthin in E. coli when coexpressed with {overscore (β)}-carotene synthesis genes. This crtO was chosen to pair with crtZ for astaxanthin production. The crtO-SHU001 gene (SEQ ID NOs: 1 and 3) was PCR-amplified from pDCQ320-SHU001 plasmid DNA (U.S. Ser. No. 60 / 577,970), using forward primer crtO-SHU001-F 5′-ACTAGTAAGGAGGAATAAACCATGAGCGCA-3′ (SEQ ID NO: 3) and reverse primer crtO-SHU001-R 5′-TGTACAGCTAGCTCACGAGCGGCTCGAACGACGCAT-3′ (SEQ ID NO: 4). Underlined are restriction sites for Spe I, Nhe I and BrsG I. The ˜1.6 kb PCR product was gel purified and cloned into pTrcHis2-Topo vector, resulting in plasmid pDCQ353. The ˜1.6 kb SpeI I / BrsG I fragment of pDCQ35...

example 2

Making Mutant Libraries

Error-Prone PCR:

[0201] The plasmid pDCQ356 was used as a template for error-prone PCR. The insert containing the crtOZ genes (SEQ ID NO: 1 and SEQ ID NO: 5; respectively) can be removed from the construct using BsrG I and Spe I digestion. A random mutant library targeting the crtOZ genes was made using error-prone PCR. The following primers were used to amplify the inserts by error-prone PCR:

(SEQ ID NO: 13)334F1 5′-GCA GCG TGC AGC TCA TGC AGT TC-3′(SEQ ID NO: 14)334r1 5′-CCA GAC CGT TCA GCT GGA TAT TAC-3′

[0202] A Clontech mutagenesis kit (Clontech Laboratories, Inc., Palo Alto, Calif.) was used for performing error-prone PCR. The following condition was used for preparing error-prone PCR reaction mixture:

TABLE 1Conditions for Error-prone PCR using Clontech Mutagenesis KitVolumes (μL)PCR grade water3710x AdvanTaq Plus Buff.5MnSO4 (8 mM)3dGTP (2 mM)150x Diversify dNTP Mix1Primer mix0Template DNA1AdvanTaq Plus Polym.1

[0203] The thermal cycling reaction was...

example 3

Screening the Mutant Libraries and Identifying the Hits

[0207] The color of cells containing pDCQ356 was light yellow. The color of the cells producing astaxanthin is red-orange. The cells that make different percentages of astaxanthin show slightly different levels of pigmentation. Therefore, the mutant colonies that produce different amounts of astaxanthin can be distinguished visually. Approximately 10,000-20,000 mutant colonies from the mutant library were screened visually. Nine putative hits were streaked on Agar plates.

[0208] A follow-up confirmation assay was performed by HPLC analysis. E. coli 10G cells containing pDCQ356 and its mutant derivatives were grown in 25 ml LB with 50 μg / mL kanamycin at 30° C.; shaking for two days. Cells were harvested by centrifugation and extracted with 50% acetone and 50% methanol. HPLC analysis of the carotenoids was performed as described in Example 1. Two of the nine crtOZ mutants produced astaxanthin as shown in Table 2.

TABLE 2HPLC Con...

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Abstract

Protein engineered nucleic acid fragments encoding a CrtO ketolase and a CrtZ hydroxylase are provided with increased astaxanthin synthesis activity. Methods using the present nucleic acid fragments are also provided for increasing or altering astaxanthin production in suitable production hosts.

Description

FIELD OF THE INVENTION [0001] This invention is in the field of microbiology, molecular biology, and the use of carotenoid ketolases and carotenoid hydroxlyases to produce astaxanthin. More specifically, nucleic acid molecules encoding both a CrtO carotenoid ketolase and a CrtZ carotenoid hydroxylase are provided that are characterized by improved astaxanthin production. Methods for recombinant production of astaxanthin using the present nucleic acid molecules are also provided. BACKGROUND OF THE INVENTION [0002] Carotenoids are pigments that are ubiquitous throughout nature and synthesized by all photosynthetic organisms, and in some heterotrophic growing bacteria and fungi. Carotenoids provide color for flowers, vegetables, insects, fish and birds. Colors of carotenoid range from yellow to red with variations of brown and purple. As precursors of vitamin A, carotenoids are fundamental components in our diet and they play additional important role in human health. Industrial uses o...

Claims

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Application Information

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IPC IPC(8): C12P23/00C07H21/04C12P21/06C12N9/02C12N1/18C12N15/74
CPCC12N9/0069C12N9/0071C12P23/00Y10S435/858
Inventor TANG, XIAO-SONGCHENG, QIONGSHYR, JOANNETAO, LUAN
Owner EI DU PONT DE NEMOURS & CO
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