Genetically engineered Zymomonas mobilis ZM4 for producing PHB and uses thereof

An application of genetically engineered strain in simultaneous production of ethanol and poly-p-hydroxybutyric PHB, wherein, the genetically engineered strain is selected from the group consisting of the strains named ZMPtN1-EUP, ZMPtN2-EUP, or ZMPt-FloN2-EUP;

• the strain ZMPtN1-EUP is obtained by transferring plasmid pE39p-PeEUP into strain ZMPtN1, and the ZMPtN1 is obtained by transferring plasmid pEZ-PgN1 into strain ZMPt;
• the ZMPtN2-EUP strain is obtained by transferring the pE39p-PeEUP plasmid into strain ZMPtN2, and the ZMPtN2 is obtained by transferring plasmid pEZ-PgN2 into strain ZMPt;
• the ZMPt-FloN2-EUP strain is obtained by transferring the pE39p-PeEUP plasmid into a strain named ZMPt-FloN2, and the ZMPt-FloN2 is obtained by transferring the pEZ-PgN2 plasmid into a strain named ZMPt-Flo;
• the ZMPt is obtained by replacing the ZMO0038 gene of the Zymomomonas mobilis (Z. mobilis) ZM4 strain genome with an Ptet-phaCAB operon;
• the ZMPt-Flo is obtained by replacing the genome ZMO0038 of Z. mobilis ZM4 strain genome with the Ptet-phaCAB operon and knocking out the 181th nucleotide of ZMO1082 gene of Z. mobilis ZM4 strain genome with thymidine;
• a plasmid named pEZ-Pt is a recombinant plasmid prepared by linking the Ptet-phaCAB operon to the pEZ15A vector;
• the pEZ-Pg is a recombinant plasmid obtained by replacing the inducible promoter Ptet of plasmid pEZ-Pt with a strong promoter Pgap;
• the pEZ-PgN1 is a recombinant plasmid obtained by linking the gene ZMO1329 with a plasmid pEZ-Pg;
• the pEZ-PgN2 is a recombinant plasmid obtained by linking the gene ZM00367 with the plasmid pEZ-Pg;
• the pE39p-PeEUP is a recombinant plasmid obtained by constructing the ada and adh2 genes connected by ribosome binding site (RBS) sequence into a shuttle vector pEZ39p, and the pEZ39p is a recombinant plasmid obtained by replacing the replicon of a plasmid pEZ15A with the replicon from a endogenous plasmid 39-032 in Z. mobilisZM4;
• the Ptet-phaCAB has a nucleotide sequence formed by sequential connection of Ptet, phaC, RBS, phaA, and phaB elements, and the nucleotide sequences of phaC, phaA, and phaB are sequentially shown as SEQ ID NO:1 to 3;
• the nucleotide sequence of the RBS is shown as SEQ ID NO:4;
• the nucleotide sequence of ada and adh2 are shown as SEQ ID NO:
• 39 and SEQ ID NO:
• 40, respectively;
• the nucleotide sequence of the replicon of 39-032 from Z. mobilis ZM4 shown as SEQ ID NO:41;
• the pEZ39p is constructed as follows 1)~2):
• 1) a PCR is presented by amplifying a fragment from the replicon of 39-032 from Z. mobilis ZM4, with Z. mobilis ZM4 strain as the DNA template, 39-032-p-spe-F and 39-032-p-spe-R as primers;
• the nucleotide sequence of 39-032-p-spe-F is shown as SEQ ID NO:42, and the nucleotide sequence of 39-032-p-spe-R is shown as SEQ ID NO:43;
• 2) a PCR is presented by amplifying a reverse fragment from pEZ15A, with the pEZ15A used as the DNA amplification template, and pEZ-dp-anti-F and pEZ-anti-R used as primers;
• the nucleotide sequence of pEZ-dp-anti-F is shown as SEQ ID NO:
• 44, the nucleotide sequence of pEZ-anti-R is shown as SEQ ID NO:
• 45:
• the described strains ZMPtN1-EUP and ZMPtN2-EUP are respectively inoculated into a 100 ml triangular flask containing 80 mL RMG5 medium supplemented with tetracycline inducer at a concentration of 1.2 μg/mL to an initial OD600nm of 0.1;
• and
• then fermented at 30° C. and 100 rpm;
• the described strains ZMPt-FloN2-EUP is inoculated into a 100-ml triangular flask containing 80 mL RMG5 medium supplemented with tetracycline inducer at a concentration of 1.2 μg/ml to an initial OD600nm of 0.1;
• and then fermented at 30° C. and 100 rpm.