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Diterpene production in yarrowia

a technology of yarrowia and yarrow, which is applied in the direction of lysine, transferase, enzymology, etc., can solve the problems of large land area, long time, and variable yields

Inactive Publication Date: 2019-07-25
DSM IP ASSETS BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent describes a process for producing diterpene or glycosylated diterpene using a microorganism of the genus Yarrowia. The process involves fermenting the microorganism in a suitable medium at a temperature of about 29°C or higher. The microorganism has been genetically modified to produce the desired diterpene or glycosylated diterpene. The diterpene or glycosylated diterpene can then be physically or chemically recovered from the microorganism or fermentation medium. The invention provides a method for efficiently producing diterpene or glycosylated diterpene using a microorganism of the genus Yarrowia.

Problems solved by technology

However, yields may be variable and affected by agriculture and environmental conditions.
Also, Stevia cultivation requires substantial land area, a long time prior to harvest, intensive labour and additional costs for the extraction and purification of the glycosides.

Method used

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  • Diterpene production in yarrowia
  • Diterpene production in yarrowia
  • Diterpene production in yarrowia

Examples

Experimental program
Comparison scheme
Effect test

example 1

ion of Steviol Producina Strains of Recombinant Yarrowia lipolytica

[0260]Plasmids pMB6754, pMB6761, and pMB6762 (see Table 1 and FIGS. 1, 2 and 3) encoding genes for the synthesis of steviol were constructed as follows. Open reading frames for tCPS (SEQ ID NO: 182), tKS (SEQ ID NO: 183), CPSKS (SEQ ID NO: 184), KOGib (SEQ ID NO: 186), KAH4 (SEQ ID NO: 185), CPR1 (SEQ ID NO: 187) and CPR3 (SEQ ID NO: 188) were codon pair optimized using codon pair optimisation technology as disclosed in PCT / EP2007 / 05594, for expression in Yarrowia lipolytica.

[0261]The optimized sequences, flanked by 60 bp of the desired promoter and terminator, were synthesized by GenScript (SEQ ID NOS: 182-188), and amplified by PCR using appropriate primers. DNA fragments encoding terminator-promoter sequences, TPI promoter, or Yarrowia lipolytica markers were amplified by PCR from existing constructs (SEQID 193-197 and 199). Vector DNA (SEQID 198), consisting of the S. cerevisiae centromere-based URA3 plasmid YC...

example 2

ion of RebA Producina Strains of Recombinant Yarrowia lipolytica

[0263]Plasmid pMB6775 (see Table 1 and FIG. 4) encoding genes for the synthesis of RebA was constructed as follows. Open reading frames for a UGT1, UGT3, UGT4, and UGT2 were codon pair optimized using codon pair optimisation technology as disclosed in PCT / EP2007 / 05594, for expression in Yarrowia lipolytica. The optimized sequences, flanked by 60 bp of the desired promoter and terminator, were synthesized by GenScript (SEQ ID NOs 189-192), and amplified by PCR using appropriate primers. DNA fragments encoding terminator-promoter sequences or Yarrowia lipolytica markers were amplified by PCR from existing constructs (SEQ ID NOS: 194-196,199 and 200). The vector (SEQ ID NO: 198), consisting of the S. cerevisiae centromere-based URA3 plasmid YCp50 (Rose et al., supra) with ENOp from Yarrowia replacing the tet gene using standard techniques, was prepared from E. coli and digested with XbaI and SnaBI. All fragments were puri...

example 3

n of RebA by Yarrowia

[0266]The Y. lipolytica strain ML13113 constructed as described above, was cultivated in shake-flasks (2 l with 200 ml medium) for 1 days at 30° C. and 200 rpm. The medium was based on Verduyn et al. (Verduyn C, Postma E, Scheffers W A, Van Dijken J P. Yeast, 1992 July; 8(7):501-517), with modifications in the carbon and nitrogen sources, as described in Tables 12 and 13.

TABLE 13Preculture medium composition Y. lipolytica strain ML13113ConcentrationRaw materialFormula(g / kg)Glucose.1aqC6H12O6•1H2O22.0Urea(NH2)2CO2.3Potassium dihydrogen phosphateKH2PO43.0Magnesium sulphateMgSO4•7H2O0.5Trace element solution1Vitamin solution1ConcentrationComponentFormula(g / kg)aTrace elements solutionEDTAC10H14N2Na2O8•2H2O15.00Zincsulphate•7H2OZnSO4•7H2O4.50Manganesechloride•2H2OMnCl2•2H2O0.84Cobalt (II) chloride•6H2OCoCl2•6H2O0.30Cupper (II) sulphate•5H2OCuSO4•5H2O0.30Sodium molybdenum•2H2ONa2MoO4•2H2O0.40Calciumchloride•2H2OCaCl2•2H2O4.50Ironsulphate•7H2OFeSO4•7H2O3.00Boric acidH...

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Abstract

The present invention relates to a method for the production of a diterpene or a glycosylated diterpene, which method comprises: a. fermenting a recombinant microorganism of the genus Yarrowia in a suitable fermentation medium at a temperature of about 29° C. or higher, wherein the microorganism comprises one or more nucleotide sequence(s) encoding: a polypeptide having ent-copalyl pyrophosphate synthase activity; a polypeptide having ent-Kaurene synthase activity; a polypeptide having ent-Kaurene oxidase activity; and a polypeptide having kaurenoic acid 13-hydroxylase activity and whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least steviol; and b. recovering the diterpene or glycosylated diterpene.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a Continuation of U.S. application Ser. No. 14 / 906,497, filed 20 Jan. 2016, which is a § 371 National Stage Application of PCT / EP2014 / 065858, filed 23 Jul. 2014, which claims priority to U.S. Ser. No. 13 / 949,079, filed 23 Jul. 2013. The disclosures of the priority applications are incorporated in their entirety herein by reference.REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE (.TXT)[0002]Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (see MPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-compliant text file (entitled “Sequence_Listing_2919208-368001_ST25.txt” created on 8 Mar. 2019, and 1,060,395 bytes in size) is submitted concurrently with the instant application, and the entire contents of the Sequence Listing are incorporated herein by reference.BACKGROUNDField of the Invention[0003]The present invention relates to a process for the production of a diterpene an...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P19/56C12P15/00C12N9/02C12N9/10C07H15/256C07C62/32A23L27/30A23L2/60C12N15/52C12N9/90C12N9/88
CPCC12P19/56C12P15/00C12N9/0042C12N9/0073C12N9/0083C12N9/1051C07H15/256C07C62/32A23L27/36A23L2/60C12N15/52C12N9/90C12N9/88C12Y114/13079C12Y114/13078C12Y402/03019C12Y505/01013C07C2603/86
Inventor BROERS, NICOLETTE JASMIJNBOER, VIKTOR MARIUSLAWRENCE, ADAM G.
Owner DSM IP ASSETS BV
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