Search

CN-122012363-A - Construction method and application of Streptomyces mobaraensis engineering strain for producing phospholipase A1

CN122012363ACN 122012363 ACN122012363 ACN 122012363ACN-122012363-A

Abstract

The invention discloses a construction method and application of a Streptomyces mobaraensis engineering strain for producing phospholipase A1, and belongs to the technical fields of enzyme engineering and food processing. The invention provides a Streptomyces mobaraensis engineering strain for efficiently synthesizing phospholipase A1, which carries out heterologous expression on a coding gene of phospholipase A1 after codon optimization on the basis of smYS-delta G11-delta G8-delta tg of Streptomyces mobaraensis. The invention also discloses an enzyme produced by utilizing the engineering bacterium, and the enzymology property of saPLA after purification and the application in degumming soybean crude oil. The recombinant saPLA1 can reduce the phosphorus content of soybean crude oil from 113.01mg/kg to below 7.36 mg/kg. The food-grade expression system constructed by the invention has the advantages of high safety, high expression level, low production cost and the like, and the produced saPLA degumming efficiency is high, thereby providing a new scheme for industrially producing the food-grade PLA1 and realizing high-efficiency oil degumming.

Inventors

  • LIU SONG
  • GUO JIAMING
  • XUE HONGJIAN
  • GUO XIAODONG
  • FAN WEI

Assignees

  • 江南大学
  • 广东众汇食品科技有限公司

Dates

Publication Date
20260512
Application Date
20260414

Claims (10)

  1. 1. The Streptomyces mobaraensis chassis cell is characterized in that Streptomyces mobaraensis smY2019 is taken as a starting strain, and genome island GIs genes, endogenous CRISPR/Cas system genes and NHEJ repair pathway genes on the genome of the Streptomyces mobaraensis chassis cell are knocked out to obtain smY2019 ΔG5 C3, smY, 2019 ΔG5 Knocking out the GI-8 gene and the GI-11 gene on the genome on the basis of C3; The numbers of the GI-8 genes on NCBI are respectively as follows: K7I03_23670, K7I03_23675, K7I03_23680, K7I03_23685, K7I03_23690, K7I03_23695, K7I03_23700, the numbers of the GI-11 genes on NCBI are respectively as follows: the GI-11 gene is at NCBI the numbers are respectively as follows.
  2. 2. The Streptomyces mobaraensis chassis cell according to claim 1, wherein said chassis cell further comprises the improvement of integration PkasO at the attb site on the genome The bldD gene overexpressed by Sp44 promoter; The nucleotide sequence of the attb site is shown as SEQ ID NO.1, pkasO × The nucleotide sequence of the Sp44 promoter is shown as SEQ ID NO.2, and the nucleotide sequence of the bldD gene is shown as SEQ ID NO. 3.
  3. 3. A preparation method of TGase enzyme is characterized in that the TGase enzyme is prepared by fermenting the Streptomyces mobaraensis chassis cells according to claim 1 or 2.
  4. 4. A method for expressing a target protein, which is characterized in that the method comprises using the Streptomyces mobaraensis chassis cell according to claim 1 or 2 as a host cell to express the target protein.
  5. 5. The recombinant Streptomyces mobaraensis is characterized in that the recombinant Streptomyces mobaraensis takes the Streptomyces mobaraensis chassis cell as described in claim 1 or 2 as a host cell, the TGase enzyme on the genome is knocked out, the GenBank number is UBI36310.1, and the attb site on the genome is integrated with the saPLA1 gene expression frame shown in SEQ ID NO.4, and the nucleotide sequence of the attb site is shown in SEQ ID NO. 1.
  6. 6. A recombinant Streptomyces mobaraensis is characterized in that the recombinant Streptomyces mobaraensis takes the Streptomyces mobaraensis chassis cell as described in claim 1 or 2 as a host cell, the TGase enzyme on the genome is knocked out, the GenBank number is UBI36310.1, and the attb site on the genome is integrated with saPLA1 gene expression frame shown in SEQ ID NO.4 and PkasO # The bldD gene overexpressed by Sp44 promoter; The nucleotide sequence of the attb site is shown as SEQ ID NO.1, pkasO × The nucleotide sequence of the Sp44 promoter is shown as SEQ ID NO.2, and the nucleotide sequence of the bldD gene is shown as SEQ ID NO. 3.
  7. 7. A method for degumming soybean crude oil, which is characterized in that recombinant Streptomyces mobaraensis according to claim 5 or 6 or recombinant enzyme obtained by fermenting recombinant Streptomyces mobaraensis according to claim 5 or 6 is added into soybean crude oil for reaction and degumming.
  8. 8. The method according to claim 7, wherein the reaction conditions are 250 rpm and the reaction time of pH 4.5-7.0 is 2.5-3 h.
  9. 9. The method according to claim 7 or 8, wherein the amount of the recombinase added is 200U/100 g-350U/100g.
  10. 10. Use of the Streptomyces mobaraensis chassis cell according to claim 1 or 2, or the recombinant Streptomyces mobaraensis according to claim 5 or 6, for degumming or for the preparation of a product for degumming a crude soybean oil.

Description

Construction method and application of Streptomyces mobaraensis engineering strain for producing phospholipase A1 Technical Field The invention relates to a construction method and application of a Streptomyces mobaraensis engineering strain for producing phospholipase A1, and belongs to the technical fields of enzyme engineering and food processing. Background Phospholipase A1 (EC 3.1.1.32, abbreviated as PLA 1) is a class of esterases capable of specifically catalyzing the hydrolysis of the ester acyl bond at the Sn-1 position of a phospholipid molecule to form free fatty acids and lysophospholipids. The enzyme has important application value in enzymatic oil degumming, and is widely applied to the fields of phospholipid modification, preparation of natural emulsifying agent (lysophospholipid), biosensing detection and the like. Currently, phospholipase A1 (PLA 1) has a distribution in various organisms in nature, and its main industrial production sources are focused on Serratia and Aspergillus. However, the fermentation level of wild strains is generally low, the yield is more than 5U/mL, and the fungal expression system is at risk of glycosylation heterogeneity and unstable enzyme activity. To increase yield, heterologous expression systems based on ESCHERICHIA COLI, pichia pastoris and Bacillus subtilis have been developed. However, the above systems have the remarkable defects that E.coli is easy to produce endotoxin and form inclusion bodies, P.pastoris is induced by methanol with potential safety hazard, and the enzyme activity is low when expressed in a food-grade host B.subilis. Although research has been conducted to achieve higher enzyme activities in Streptomyces lividans, the fermentation of this strain requires the addition of antibiotics, resulting in high costs and adverse food applications. Although Streptomyces mobaraensis (Streptomyces mobaraensis) is used as a food-grade host authenticated by GRAS, has a mature fermentation process and high-efficiency secretion capacity, and has been successfully applied to the production of Transglutaminase (TGase), the efficient expression of PLA1 by directly using the same is still challenging. The mutant strain obtained by mutagenesis and the chassis strain smYS-Deltatg constructed by gene editing are studied in the early stage, which lays a foundation for the expression of PLA1, but the expression efficiency, the process stability and the final fermentation level of the strain still need to be further optimized and improved. Therefore, how to realize efficient, stable and low-cost industrial production of PLA1 in the safe host is a core problem to be solved in order to break the technical bottleneck and meet the requirements of the food industry. Disclosure of Invention The invention provides a Streptomyces mobaraensis chassis cell, which takes Streptomyces mobaraensis smY2019 as an initial strain, and knocks out genome island GIs genes, endogenous CRISPR/Cas system genes and NHEJ repair pathway genes on the genome of the initial strain to obtain smY2019ΔG5C3 (described in the text of Chinese patent application publication No. CN 120230690A); in smY2019ΔG5Knocking out the GI-8 gene and the GI-11 gene on the genome on the basis of C3; The numbers of the GI-8 genes on NCBI are respectively as follows: K7I03_23670, K7I03_23675, K7I03_23680, K7I03_23685, K7I03_23690, K7I03_23695, K7I03_23700, the numbers of the GI-11 genes on NCBI are respectively as follows: the GI-11 gene is at NCBI the numbers are respectively as follows. The NCBI numbers of the genome island GIs genes are K7I03_28245、K7I03_28250、K7I03_28255、K7I03_28260、K7I03_28265、K7I03_28270、K7I03_28275、K7I03_28280、K7I03_28285、K7I03_28290、K7I03_28295、K7I03_28300、K7I03_28305、K7I03_28310、K7I03_28315、K7I03_28320、K7I03_28325、K7I03_28330、K7I03_28335、K7I03_28340、K7I03_28345、K7I03_28350、K7I03_28355、K7I03_28360、K7I03_28365、K7I03_28370、K7I03_28375、K7I03_28380、K7I03_28385、K7I03_28390、K7I03_28395、K7I03_28400、K7I03_28405、K7I03_28410、K7I03_28415、K7I03_28420、K7I03_28425、K7I03_28430、K7I03_28435、K7I03_28835、K7I03_28840、K7I03_28845、K7I03_28850、K7I03_28855、K7I03_28860、K7I03_28865、K7I03_28870、K7I03_28875、K7I03_28880、K7I03_28885、K7I03_28890、K7I03_28895、K7I03_28900、K7I03_28905、K7I03_28910、K7I03_28915、K7I03_28920、K7I03_28925、K7I03_28930、K7I03_28935、K7I03_28940、K7I03_28945、K7I03_28950、K7I03_28955、K7I03_28960、K7I03_28965、K7I03_28970、K7I03_28975、K7I03_28980、K7I03_28985、K7I03_28990、K7I03_28995、K7I03_29000、K7I03_29005、K7I03_18425、K7I03_18430、K7I03_18435、K7I03_18440、K7I03_18445、K7I03_18450、K7I03_18455、K7I03_18460、K7I03_18465、K7I03_18470、K7I03_18475、K7I03_18480、K7I03_18485、K7I03_18490、K7I03_18495、K7I03_18500、K7I03_18505、K7I03_18510、K7I03_18515、K7I03_18520、K7I03_18525、K7I03_18530、K7I03_18535、K7I03_18540、K7I03_18545、K7I03_18550、K7I03_18555、K7I03_18560、K7I03_18565、K7I03_18570、K7I03_18575、K7I03_18580、K7I03_18585、K7I03_18590、K7I03_18595、K7I03_18600、K7I03_18605、K7I03_18610、K7I03_03350、K7I03_03355、K7I03_03360、K7I03_03365、K7I03_0337