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Stabilization of cytochrome P450 reductase

A technology of cytochrome and reductase, applied in the direction of oxidoreductase, enzyme, bacteria, etc.

Inactive Publication Date: 2017-02-22
DSM IP ASSETS BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, heterologous expression of membrane-anchored cytochrome P450 enzymes (CYPs) in microbial hosts applied to biocatalytic processes remains challenging

Method used

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  • Stabilization of cytochrome P450 reductase
  • Stabilization of cytochrome P450 reductase
  • Stabilization of cytochrome P450 reductase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0097] 1. A recombinant host cell capable of expressing cytochrome P450 enzymes (CYP) and cytochrome P450 reductase (CPR) and capable of overexpressing the amino acid sequence comprising SEQ ID NO: 2 or having at least 50% thereof Ice2p of sequences of sequence identity.

[0098] 2. The recombinant host cell according to embodiment 1, wherein the CYP is the tofu spirodiene oxygenase CYP71D55 (HPO) from Hyoscymaus muticus, optionally in which mutations V482I and A484I have been carried out, from Mentha piperita (-)-limonene-3-hydroxylase (PM17) or human cytochrome P450 2D6 (CYP2D6).

[0099] 3. The recombinant host cell according to embodiment 1 or 2, wherein the CRP is cytochrome P450 reductase from A. thaliana or human reductase.

[0100] 4. The recombinant host cell according to any one of the preceding embodiments, which is capable of producing a compound of interest.

[0101] 5. The recombinant host cell according to embodiment 4, wherein the compound of interest is a st...

Embodiment 1

[0173] Example 1: Screening for effectors of CYP / CPR function in recombinant S. cerevisiae strains

[0174] (+)-valencene is a by-product of orange juice production and is of low commercial importance. However, many attempts have been made to convert (+)-valencene into the attractive flavor and aroma compound (+)-narone by various biocatalytic pathways (by (Fraatz et al., 2009) review). As highly stereoselective and regioselective catalysts, soluble and membrane-attached cytochrome P450 enzymes (CYPs) have been tested for this reaction (Cankar et al., 2011; Gavira et al., 2013; Girhard et al., 2009; Takahashi et al., 2007). There are solid data emphasizing that CYPs indeed hydroxylate (+)-valencene to produce nootropes ( figure 1 ), but the oxidation of the latter compound to narone is carried out by the endogenous activity of S. cerevisiae (Gavira et al., 2013) or Pichia pastoris (Wriessnegger et al., revised manuscript). An important feature of CYPs is their requiremen...

Embodiment 2

[0175] Example 2: Overexpression of ICE2 in S. cerevisiae W303

[0176] Among the various S. cerevisiae host strains for CYP / CPR expression and CYP-mediated biotransformation, especially the W303 strain proved to be the most suitable host (Loeper et al., 1998; Pompon et al., 1996; Truan et al. et al., 1993; Urban et al., 1994). Many standard laboratory strains of S. cerevisiae, but not the W303 strain, carry deletions in the HAP1 gene. Haplp is a transcription factor responsible for regulating genes that control intracellular heme abundance in response to changes in available oxygen levels (B.S.J. Davies and Jasper Rine, 2006; Ihrig et al., 2010). The Δhap1 genotype strains are said to be less favorable for CYP expression and function because these strains are strongly dependent on optimal heme and oxygen supply. To test host strain effects, HPO / CPR-mediated (+)-valencene conversion of resting cells was performed in parallel in BY4742 and W303 yeast strains with pYES-HPO-C...

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Abstract

The invention relates to stabilization of cytochrome P450 reductase. The present invention relates to a recombinant microorganism which is capable of expressing a cytochrome P450 reductase (CPR) and, optionally, a cytochrome P450 enzyme (CYP) and which is capable of overexpressing a Ice2p comprising the amino acid sequence set out in SEQ ID NO: 2 or a sequence having at least 50% sequence identity thereto. The invention further relates to use of Ice2p to stabilize expression of a cytochrome P450 reductase in a recombinant microorganism.

Description

technical field [0001] The present invention relates to recombinant host cells in which the expression of cytochrome P450 reductase is stabilized. The invention further relates to methods for producing compounds of interest in recombinant host cells, and to methods for producing compounds of interest in biocatalytic reactions. The invention also relates to the use of the protein for stabilizing cytochrome P450 reductase. Background technique [0002] The cytochrome P450 monooxygenase superfamily (formally abbreviated CYP) is a large and diverse group of enzymes that catalyze the oxidation of organic substances. Substrates for CYP enzymes include metabolic intermediates such as lipids and steroid hormones, as well as xenobiotics such as drugs and other toxic chemicals. CYPs are the major enzymes involved in drug metabolism and bioactivation, accounting for about 75% of the total number of different metabolic reactions. [0003] CYP enzymes have been identified in all domai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N1/15C12N1/21C12P19/56C12P33/00C12P7/02C12P7/26
CPCC12N9/0042C12N9/0071C12P7/02C12P7/26C12P19/56C12P33/00C07K14/395C12N9/0073C12Y106/02004C12Y114/13121C12N15/81
Inventor 安尼塔·爱默斯托菲尔塔玛拉·威斯内格尔米丽娅姆·威默-特鲁本巴谢尔哈拉德·皮希勒莫尼卡·米勒依沃娜·卡伦扎马丁·舒尔曼
Owner DSM IP ASSETS BV
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