A polymer-grade lactic acid monomer producing bacteria, its construction method and lactic acid manufacturing technology
A lactic acid monomer and polymerization-grade technology, which is applied in the field of microbial applications, can solve the problems of complex culture medium, unsatisfactory optical purity of lactic acid, and inability to achieve large-scale production of extremely optically pure D-lactic acid and L-lactic acid, etc., to achieve efficient preparation The effect of the process
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0118] Example 1 - Mutation Cassette ldhA::kan-cI ts 857-p R -p L build
[0119] The ldhA gene fragment ldhA' on the B0013 chromosome was amplified by PCR with primers ldhA1 and ldhA2, and cloned into the pUC18 vector to obtain the recombinant plasmid pUC-ldhA'. Plasmid pPL451 was amplified by inverse PCR with primers PPL1 and PPL2, and combined with the kanamycin resistance gene fragment (plasmid pSK sym Km was digested with SmaI, and the 966bp fragment was recovered by gel) for ligation to obtain the recombinant plasmid pPL-Kan. Plasmid pPL-Kan was amplified by PCR with primers PPL3 and PPL4, and the product was double-digested with EcoRI and EcoRV, and then reverse-PCR amplified with primers Ec-RlA1 and Ec-RlA2 using pUC-ldhA' as a template and used EcoRI digested products were ligated to obtain the recombinant plasmid pUC-ldhAp::kan-cI ts 857-p R -p L . The physical map of the resulting recombinant plasmid is attached figure 1 shown. The recombinant plasmid pUC-l...
Embodiment 2
[0120] Example 2 - Construction of Mutation Cassette thiE::difGm
[0121] Using E. coli B0013 chromosomal DNA as template, ThiE1p and ThiE2p as primers, the partial sequence of thiE gene with a length of 0.6kb was amplified. This was cloned into pUC18 to obtain the recombinant plasmid pUC-thiE. The 1.2kb difGm fragment was cloned into the StuI site in the middle of thiE to obtain the recombinant plasmid pUC-thiE::difGm. The physical map of the resulting recombinant plasmid is attached figure 2 shown. The recombinant plasmid pUC-thiE::difGm was digested with ApaLI, and the linearized plasmid was recovered by gel, and PCR amplification was performed with primers ThiE1p and ThiE2p to obtain the thiE::difGm gene fragment.
Embodiment 3
[0122] Example 3 - Construction of Mutation Cassette dld::difGm
[0123] Using the chromosomal DNA of strain B0013 as a template, the dld' gene fragment (0.9 kb) was amplified by PCR with primers Dld1 and Dld2, and the PCR product was cloned into the SmaI site of the vector pUC18 to obtain the recombinant plasmid pUC-dld'. Digest the recombinant plasmid with EcoRV, remove the 0.4 kb gene fragment in the middle of the dld' gene, and clone into the dif-Gm-dif fragment to obtain the recombinant plasmid pUC-dld'::difGm. The physical map of the resulting recombinant plasmid is attached image 3 shown. The recombinant plasmid pUC-dld'::difGm was digested with EcoRI, and the linearized plasmid was recovered by gel, and PCR amplification was performed with primers Dld1 and Dld2 to obtain the dld'::difGm gene fragment.
PUM
| Property | Measurement | Unit |
|---|---|---|
| optical purity | aaaaa | aaaaa |
| purity | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More