Recombinant
The disclosure discloses recombinant Pseudomonas plecoglossicida for producing L-xylose and application thereof, and belongs to the technical field of bioengineering. According to the disclosure, a synthesized 2-ketogluconate reductase gene and a 2,5-diketogluconate reductase gene derived from Corynebaterium ATCC 31090 and a pyruvate decarboxylase gene derived from Saccharomyces cerevisiae are successfully expressed in a host P. plecoglossicida by a double plasmid system, and an obtained genetically engineered strain is fermented for 56 h in a shake flask, where the yield of L-xylose reaches 16.2 g/L, and the transformation rate reaches 20.3%; the obtained genetically engineered strain is fermented for 48 h and 44 h in 3 L and 15 L fermentors, respectively, where the yields of L-xylose reach 37.6 g/L and 45.8 g/L, respectively, and the glucose transformation rates are 47.0% and 57.3%, respectively. The method has the advantages of low raw material cost, no pollution to the environment, simple operation, and important economic and social benefits.
1. A recombinant strain for producing L-xylose, comprising nucleic acid sequences for expressing a 2-ketogluconate dehydrogenase gene, a 2,5-diketogluconate reductase gene, and a pyruvate decarboxylase gene, wherein the recombinant strain uses Pseudomonas plecoglossicida as a host strain, wherein the 2-ketogluconate dehydrogenase gene comprises nucleic acid sequences for three subunits, which are set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, wherein the 2,5-diketogluconate reductase gene is from Corynebaterium ATCC 31090, and wherein the pyruvate decarboxylase gene is from Saccharomyces cerevisiae.
2. The recombinant strain according to claim 1 , comprising a double plasmid expression system to express the 2-ketogluconate dehydrogenase gene, the 2,5-diketogluconate reductase gene and the pyruvate decarboxylase gene, and the double plasmid expression system comprises a plasmid pME6032 and a plasmid pBBR1MCS-2.
3. The recombinant strain according to claim 2 , wherein the plasmid pME6032 is used for expressing the 2-ketogluconate dehydrogenase gene, and the plasmid pBBR1MCS-2 is used for expressing the 2,5-diketogluconate reductase gene and the pyruvate decarboxylase gene.
4. The recombinant strain according to claim 1 , wherein the host strain of Pseudomonas plecoglossicida is any one selected from a group consisting of Pseudomonas plecoglossicida CGMCC 7150, Pseudomonas plecoglossicida CGMCC 1.16111, Pseudomonas plecoglossicida CGMCC 1.12685, and Pseudomonas plecoglossicida CGMCC 1.761.
5. A method for constructing the recombinant strain according to claim 1 , comprising the following steps:
(1) simultaneously ligating nucleic acid sequences of gene subunits set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 to a vector plasmid to form a first recombinant plasmid;
(2) transforming the first recombinant plasmid obtained in step (1) into Pseudomonas plecoglossicida to obtain single plasmid-containing recombinant Pseudomonas plecoglossicida;
(3) separately amplifying a 2,5-diketogluconate reductase gene and a pyruvate decarboxylase gene, and simultaneously ligating amplified nucleic acid sequences of the 2,5-diketogluconate reductase gene and the pyruvate decarboxylase gene to an expression vector to form a second recombinant plasmid; and
(4) transforming the second recombinant plasmid obtained in step (3) into the Pseudomonas plecoglossicida containing the first recombinant plasmid obtained in step (2) to obtain double plasmid-containing recombinant Pseudomonas plecoglossicida.
6. A method of synthesizing L-xylose, comprising: culturing the recombinant strain of claim 1 in a fermentation medium until OD 650 is 0.6 to 0.8; inducing expression of the 2-ketogluconate dehydrogenase gene, the 2,5-diketogluconate reductase gene and the pyruvate decarboxylase gene; and adding glucose as a substrate; and fermenting for sufficient time for production of L-xylose.
7. The method according to claim 6 , wherein the fermentation medium comprises 75.0 to 85.0 g/L of glucose, 3.0 to 4.0 g/L of corn steep liquor, 1.5 to 2.5 g/L of urea, 1.5 to 2.5 g/L of KH 2 PO 4 , 0.4 to 0.6 g/L of MgSO 4 .7H 2 O, and 9.0 to 11.0 g/L of CaCO 3 , and has a pH value of 6.8.
8. The method according to claim 6 , comprising culturing the recombinant strain on a seed medium before fermentation, and the seed medium comprises 14.0 to 15.0 g/L of glucose, 3.5 to 4.0 g/L of corn steep liquor, 1.5 to 2.5 g/L of urea, 1.5 to 2.5 g/L of KH 2 PO 4 , 0.4 to 0.6 g/L of MgSO 4 .7H 2 O, and 0.8 to 1.2 g/L of CaCO 3 , and has a pH value of 7.0.