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Phosphite dehydrogenase marker for Aspergillus host strains

IP.com Disclosure Number: IPCOM000248070D
Publication Date: 2016-Oct-24
Document File: 1 page(s) / 162K

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Title: Phosphite dehydrogenase marker for Aspergillus host strains

Hiromi Akeboshi (HAhi) & Kiyomi Kagami (KyiK)

29-AUG-2016

Purpose: A possibility of using Pseudomonas stutzeri WM88 phosphite dehydrogenase as a new marker for transformation of Aspergillus strain was investigated.

Results and discussion

   Transformation of Aspergillus was done with the amdS marker (phahi022 or 049), the ptxd marker (phahi054 or 052) or both amdS and ptxd markers (phahi022 & 054; or phahi049 & 052). As expected, growth was observed only with the strains which had both plasmids integrated. The pictures of transformation plates after Enzyme A integration (by phahi022 & 054) are shown in Figure 1. Only co- transformed strains are able to grow on the COVE-PO3 plate.

Figure 1 Transformants on COVE-PO3 (TF)

Integrated marker genes

# of days after TF

  AmdS (phahi022)

  ptxd (phahi054)

  AmdS+ptxd (phahi022+054)

No DNA

3

7-10

   Twenty clones were randomly selected from co-transformation plates and were analyzed by spore PCR, gDNA PCR, and southern blot. 12 clones showed proper integrations of both phahi054 & 022 DNA fragments, 2 clones had dropped either the Enzyme A or marker gene in a locus, and 6 clones had some failure in integrations (Table 3-I). Similar results were obtained with Enzyme B-amdS & -ptxd co- transformants (phahi049 & 052, Table 3-II). To confirm that the integration is not affected by marker difference, Emzyme B-amdS and Enzyme C-pyrG co-transformants (phahi042 & 033) were also analyzed (Table 3-III, ELN-15-HAHI-0025). In all cases, 60-70% of the transformants had properly integrated DNA fragments, while 30-40% of them had some failure with integrations. The integration tendency was not largely affected by using ptxd (Table 3-I and -II) instead of other marker such as pyrG (Table 3-III).

Table 3 Integration accuracy by co-transformation

I. Enzyme A-amdS (022) + Enzyme A-ptxd (054)

II. Enzyme B-amdS (049) + Enzyme B-ptxd (052)

III. Enzyme B-amdS (042) + Enzyme C-pyrG (033)

Clone #

%

Clone #

%

Clone #

%

Proper integration

12


60.0

7


46.7

8


53.3

Partially dropped

2


10.0

2


13.3

1


6.7

No proper integration

6


30.0

6


40.0

6


40.0

Total

20

100

15

100

15

100

   It was also possible to obtain transformants when ptxd was used as a dominant marker. However, the number of clones obtained was less than those obtained by co-transformation (data not shown, ELN-16- HAHI-0007). Also, due to the plasmid design, ptxd was placed in between 2 indentical terminators and most of the strains had looped out ptxd. But all the strains analyzed detected at least 1 copy of ptxd left
in its genome (ELN-16-HAHI-0008). With refinem...