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CN-122012485-A - Isopentenyl diphosphate isomerase mutant, method for improving lupeol production of yarrowia lipolytica and application

CN122012485ACN 122012485 ACN122012485 ACN 122012485ACN-122012485-A

Abstract

The invention relates to an isopentenyl diphosphate isomerase mutant, a method for improving lupeol production by yarrowia lipolytica and application thereof, and belongs to the technical field of genetic engineering. The isopentenyl diphosphate isomerase mutant is obtained by rational design and screening based on the amino acid sequence shown in SEQ ID NO. 1. The invention constructs a recombinant strain Po1FK01 for producing lupeol on the basis of a yarrowia lipolytica Po1f strain, converts the constructed isopentenyl diphosphate isomerase mutants into the recombinant strain for expression, and the activity of each mutant is characterized by the yield of lupeol. The yield of lupeol in yarrowia lipolytica engineering strain overexpressing the isopentenyl diphosphate isomerase mutant YlIDI (K78R+S129D) was increased by about 86% compared to the control strain, i.e., the unmutated strain. The invention provides powerful enzyme and strain for biosynthesis of lupeol, and has important significance for production of lupeol.

Inventors

  • MA JINGBO
  • ZHU FUCHENG
  • LI XIAOLI
  • ZHAO KEXUE
  • XU PENG
  • DU ZIXIN
  • Shi Duwen
  • LV ZHIXUAN

Assignees

  • 皖西学院

Dates

Publication Date
20260512
Application Date
20260316

Claims (10)

  1. 1. A mutant of isopentenyl diphosphate isomerase, characterized in that the mutant of isopentenyl diphosphate isomerase is one of the following amino acid mutations based on a wild-type isopentenyl diphosphate isomerase derived from yarrowia lipolytica shown in SEQ ID No.1 ：K78R、S129D、E161M、E192V、H93Y、Q111L、R112Y、Y197F、E194I、C128W、K78R+H93Y、K78R+C128W、K78R+S129D、S129D+E161M、C128W+S129D、C128W+E192V.
  2. 2. A nucleic acid encoding the isopentenyl diphosphate isomerase mutant according to claim 1.
  3. 3. A recombinant vector or recombinant strain comprising the nucleic acid of claim 2.
  4. 4. The recombinant vector of claim 3, wherein the expression plasmid employed is pYLXP'.
  5. 5. The recombinant strain of claim 3, wherein the host cell employed is yarrowia lipolytica.
  6. 6. The recombinant strain of claim 5, wherein the starting strain of the recombinant strain is yarrowia lipolytica Po1f and the isopentenyl diphosphate isomerase mutant of claim 1 is expressed using a pYLXP' plasmid.
  7. 7. A process for producing lupeol, comprising fermenting the recombinant strain of claim 3 or 6 to produce lupeol.
  8. 8. The method of claim 7, wherein the fermentation culture is carried out at a temperature of 25-35 ℃ and under shaking conditions of 90-150 h and a shaking speed of 150-400 rpm.
  9. 9. Use of the recombinant vector or recombinant strain according to claim 3 for the production of lupeol.
  10. 10. Use of the isopentenyl diphosphate isomerase mutant according to claim 1 for enhancing the mevalonate pathway or for producing lupeol.

Description

Isopentenyl diphosphate isomerase mutant, method for improving lupeol production of yarrowia lipolytica and application Technical Field The invention belongs to the technical field of genetic engineering, and particularly relates to an isopentenyl diphosphate isomerase mutant, a method for improving lupeol production of yarrowia lipolytica and application thereof. Background Lupeol (Lupeol) is a pentacyclic triterpene compound widely found in mango, olive and birch bark. Lupeol has physiological activities of antioxidation, anti-inflammation, anti-tumor, blood sugar reduction and the like, and is widely focused in the field of medicines. The biosynthesis of lupeol occurs mainly in two pathways, the Mevalonate (MVA) pathway and the methylerythrose-4-phosphate (MEP) pathway. Among them, the MVA pathway exists mainly in the cell fluid of eukaryotes and higher plants, and the MEP pathway exists mainly in plant plastids, bacteria, algae, and the like. In yarrowia lipolytica (Yarrowia lipolytica), isopentenyl diphosphate isomerase (Isopentenyl diphosphate isomerase, ylIDI 1) is one of the key regulatory enzymes of the intracellular terpene synthesis pathway, catalyzing one of the key reactions in the Mevalonate (MVA) pathway, namely the isomerization of isopentenyl pyrophosphate (IPP) with dimethylallyl pyrophosphate (DMAPP), which is critical to maintaining the balance of IPP and DMAPP in the cell, which are also precursors for the synthesis of many important natural products, such as terpenes, sterols, etc. However, ylIDI1 is limited by low enzyme activity and low substrate affinity in the natural state, so that improvement of YlIDI1 activity through protein engineering is of great importance. The activity of each YlIDI mutant was characterized by lupeol yield. Site-directed mutagenesis is a technique used in molecular biology to make precise, directed modifications to a particular DNA sequence. The desired changes, including addition, deletion, point mutation, etc., of bases are introduced into the target DNA fragment by the Polymerase Chain Reaction (PCR) method. The site-directed mutagenesis can efficiently and accurately improve the properties and characterization of target proteins expressed by DNA, is a very useful means in the research work of genetic engineering, and is a powerful tool for researching complex relations between protein structures and functions. Disclosure of Invention In order to solve the problems, the invention provides an isopentenyl diphosphate isomerase mutant, a method for improving lupeol production of yarrowia lipolytica and application thereof. Through rational design of wild type isopentenyl diphosphate isomerase from yarrowia lipolytica, efficient mutants are successfully obtained, so that the yield of lupeol in engineering strains is remarkably improved. One of the technical schemes provided by the invention is an isopentenyl diphosphate isomerase mutant, which is one of the following amino acid mutations based on the wild-type isopentenyl diphosphate isomerase of yarrowia lipolytica source shown in SEQ ID No.1 ：K78R、S129D、E161M、E192V、H93Y、Q111L、R112Y、Y197F、E194I、C128W、K78R+H93Y、K78R+C128W、K78R+S129D、S129D+E161M、C128W+S129D、C128W+E192V. The second technical scheme provided by the invention is nucleic acid (namely gene) for encoding the isopentenyl diphosphate isomerase mutant; The third technical scheme provided by the invention is a recombinant vector (i.e. a recombinant plasmid carrying the above gene) containing the above nucleic acid; Further, the recombinant vector adopts an expression vector pYLXP'. The fourth technical scheme provided by the invention is a recombinant strain containing the nucleic acid (namely a recombinant strain expressing the mutant); Further, the host cell employed by the recombinant strain is yarrowia lipolytica. Furthermore, the original strain of the recombinant strain is yarrowia lipolytica Po1f, and the pYLXP' plasmid is adopted to express YlIDI mutant genes. The fifth technical scheme provided by the invention provides a method for producing lupeol, which comprises the steps of fermenting and culturing the recombinant yarrowia lipolytica to obtain a fermentation product lupeol. Preferably, the fermentation culture temperature is 25-35 ℃, the culture is 90-150 h under the shaking condition, and the shaking rotation speed is 150-400 rpm. More preferably, the culture is carried out under shaking conditions at a shaking speed of 250 rpm at a temperature of 30 ℃ and a culture time of 120 h ℃. The sixth technical scheme provided by the invention is the application of the recombinant strain in lupeol production. The seventh technical scheme provided by the invention is the application of the isopentenyl diphosphate isomerase mutant in enhancing mevalonate pathway or producing lupeol. The invention has the beneficial effects that: The invention takes yarrowia lipolytica endogenous isopentenyl diphosphate isomerase as a research object, and uses ratio