Bacterium and constructing method and application thereof

A construction method, a strain technology, applied in the field of bioengineering

Active Publication Date: 2016-12-07
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

coproduction means simultaneous production

Method used

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  • Bacterium and constructing method and application thereof
  • Bacterium and constructing method and application thereof
  • Bacterium and constructing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1: Screening and identification of strains with 1,3-PD and LAC as main products

[0113] Weigh 2 g of the soil sample and add it to 50 mL of glycerol liquid medium, and place it in a shaker at 37° C. for 24 hours, and the shaker rotates at 200 rpm. Then the culture solution was diluted with sterile physiological saline, diluted 10-fold, 100-fold, 1000-fold and 10000-fold respectively, and then applied to a petri dish containing glycerol solid medium, and cultured at 37°C for 24 hours. After a single colony grows, select the colony with the largest colony area and the area of ​​the acid-producing transparent zone, inoculate it into a glycerol liquid medium, and place it in a shaker for 24 hours at 37°C with a shaker speed of 200rpm. Centrifuge the culture medium, measure the production of 1,3-PD and LAC in the culture medium, and pick a strain with high production of 1,3-PD and LAC and easy to centrifuge.

[0114] The above strains were separated and purified by...

Embodiment 2

[0118] Example 2: Deficiency of the α-acetolactate decarboxylase gene (budA) and the α-acetolactate synthase gene (budB) in Klebsiella oxytoca PDL-0

[0119] (1) Construction of a vector for partially deleting the budA gene in Klebsiella oxytoca PDL-0

[0120] Primers were designed according to the budA gene sequence (as shown in SEQ ID NO: 1), and the upstream and downstream homologous fragments of the budA gene were amplified by PCR. Using the genomic DNA of Klebsiella oxytoca PDL-0 as a template, primer budA-1: 5'-ACATGATTACGAATTCATGAACCATTCTGCTGAATG-3' (shown in SEQ ID NO: 10) and primer budA-2: 5'-AACGGGCTGGCATCACCGCGAAGGGCGTGC were used -3' (shown in SEQ ID NO:11) was amplified by PCR to obtain an upstream homologous fragment; using primer budA-3: 5'-CGCGGTGATGCCAGCCCGTTTTCCGCTTCA-3' (shown in SEQ ID NO:12) and primer budA -4: 5'-TACCGAGCTCGAATTCTTAGTTTTCGACTGAGCGAA-3' (shown in SEQ ID NO: 13) was amplified by PCR to obtain downstream homologous fragments. The PCR ampl...

Embodiment 3

[0140] Example 3: Deficiency of the aldehyde dehydrogenase gene (adhE) in Klebsiella oxytoca PDL-1

[0141] (1) Construction of a vector for partially deleting the adhE gene in Klebsiella oxytoca PDL-1

[0142] Primers were designed according to the adhE gene sequence (as shown in SEQ ID NO: 3), and the upstream and downstream homologous fragments of the adhE gene were amplified by PCR. Using the genomic DNA of Klebsiella oxytoca PDL-1 as a template, primer adhE-1: 5'-ACATGATTACGAATTCATGGCTGTTACTAATGTCGC-3' (as shown in SEQ ID NO: 18) and primer adhE-2: 5'-TGCTGTCTGTTGGCGTTACGGGTCTTCAGG were used -3' (shown in SEQ ID NO:19) was amplified by PCR to obtain an upstream homologous fragment; using primer adhE-3: 5'-CGTAACGCCAACAGACAGCATTCAGCCAGT-3' (shown in SEQ ID NO:20) and primer adhE -4: 5'-TACCGAGCTCGAATTCTTAAGCGGATTTTTTCGCTT-3' (shown in SEQ ID NO: 21) was amplified by PCR to obtain downstream homologous fragments. The PCR amplification conditions were: 95°C for 5 minutes; ...

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Abstract

The invention discloses a bacterium and a constructing method and application thereof. The bacterium features co-operation of 1,3-propylene glycol and L-lactic acid. Further, the bacterium is acid-producing Klebsiella oxytoca and includes acid-producing Klebsiella oxytoca PLL CCTCC M2016186. The constructing method of the bacterium refers to genetic engineering modification for wild fungi. The bacterium and the constructing method and application thereof have the advantages that it is possible to co-produce 1,3-propylene glycol and L-lactic acid by fermenting the bacterium, both the two products are high in molar conversion rate and concentration with fewer types of byproducts and low byproduct concentration, product extraction process can be simplified, efficient biological production of 1,3-propylene glycol and L-lactic acid can be achieved, and the bacterium has promising industrial application prospect.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a bacterium and its construction method and application. Background technique [0002] Petroleum-based polymers have brought great convenience to people's production and life. However, with the decrease of petrochemical resources and the environmental problems caused by the use of petrochemical resources, the search for new environmentally friendly non-petroleum-based polymers has received more and more attention. Bio-based materials have the advantages of easy biodegradation, wide source of raw materials and easy chemical improvement, which make them have important applications in many fields. Currently, polytrimethylene terephthalate (PTT) and polylactic acid (PLA) are two bio-based materials that have received a lot of attention. PTT is a new type of polyester fiber with excellent performance. It is formed by polycondensation of terephthalic acid and 1,3-propylene glycol (1,3-PD)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/18C12P7/56C12R1/22
CPCC12N9/0006C12N9/0008C12N9/001C12N9/1022C12N9/1029C12N9/1217C12N9/88C12P7/18C12P7/56C12Y101/01027C12Y101/01028C12Y102/0101C12Y102/03003C12Y103/01006C12Y202/01006C12Y203/01008C12Y207/02001C12Y401/01005
Inventor 陶飞王钰辛波唐鸿志马翠卿许平
Owner SHANGHAI JIAO TONG UNIV
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