Effects of msn2 Knock-out on the Growth and Kojic Acid Production of Aspergillus oryzae

doi:10.13560/j.cnki.biotech.bull.1985.2021-1492

Abstract

Abstract:

Msn2,as a C2H2-type zinc finger structure transcriptional activator,is involved in regulating biological processes such as fungal growth rate,conidial development and various stress responses. In this thesis,Aspergillus oryzae 3.042 was used as the starting strain to construct the pyrG nutrition-deficient strain. The upstream and downstream sequences of msn2 gene were as homologous recombination arms and the complementary pyrG gene was used as the screening marker. Finally,a mutant strain g-5 with msn2 knockout and the complementary pyrG gene was obtained. Compared with the original strain A. oryzae 3.042,the growth of strain g-5 was inhibited and the spore production significantly reduced by 60%. Strain g-5 could not sporulate in the environment containing 10% or more glucose,but tolerated 30 mmol/LH₂O₂. g-5 achieved 21.55 g/L kojic acid in shake flasks,which was 81% higher than that of A. oryzae 3.042（11.86 g/L）. By RT-qPCR analysis,the msn2 gene was not expressed in strain g-5 compared with A. oryzae 3.042. After 6 d of fermentation,the expressions of kojR and LaeA genes related to kojic acid synthesis increased by 4.13 and 21 times,respectively. The expressions of mycelial growth and sporulation related genes brlA and tps1 in g-5 strain were down-regulated by 63 and 128 folds compared with A. oryzae 3.042,while the expressions of ageB gene were up-regulated by 59 folds.

Key words: Aspergillus oryzae, msn2, gene knock-out, kojic acid

SHI Ya-nan, WANG De-pei, WANG Yi-chuan, ZHOU Hao, XUE Xian-li. Effects of msn2 Knock-out on the Growth and Kojic Acid Production of Aspergillus oryzae[J]. Biotechnology Bulletin, 2022, 38(8): 188-197.

Figures/Tables 8

Table 1 RT-qPCR primers

目的基因Target gene	引物序列Sequence of primer（5'-3'）
brlA	TTATCACAAGGCGGAGAAGG
brlA	CGAACAGTCAGTATGGGTTGC
tps1	GAATACGGGACTGGCCCT
tps1	GGGCTCGAAACATATCTGTGA
ageB	CACCATCTCCAACCACTCG
ageB	CAGCGTAGGTGGGAGACC
LaeA	TCCATTCATAGCAAGAGTTCTGAA
LaeA	TTGCTCCTGCTCATCGC
kojR	CCGCAGACGTTAGCGTAAAT
kojR	GCGATCGGCCATATTCATAC
his2A	GCCTGGTGGAAAGGGAAAG
his2A	CTTTGCGCTGTGGGACTT
msn2	AAGCGAGGCTGTGACTTTG
msn2	AGGCGCTGTCTCTTTTCG

Fig.1 Construction of plasmid pk1 and screening of △msn2 strain A：pk1 plasmid diagram. B：msn2 knock-out diagram. C：Screening of transformants on plate. D：PCR verification of △msn2 strains. M：DL5000;1：negative control with genome template in 3.042（2 965 bp）;2：positive control with pk1 template（3 706 bp）;3-5：transformants,3 refers to that pk1 was randomly inserted in it,4 refers to that pk1 was not inserted in it,5 is positive transformant,and 6 refers to msn2L up stream-pyrG middle（2 602 bp）was validation by PCR with transformant 5 genome as template

Table 2 Growth morphology of Aspergillus oryzae 3.042 and g-5 strain

菌株Strain	24 h	36 h	48 h
3.042
g-5

Table 3 H2O2 tolerance of A. oryzae 3.042 and g-5 strain

时间 Time/h	菌株 Strain	0 mmol·L^-1	15 mmol·L^-1	25 mmol·L^-1	30 mmol·L^-1
36	3.042
36	g-5
48	3.042
48	g-5

Table 4 High sugar tolerance of A. oryzae 3.042 and g-5 strains

时间 Time/h	菌株Strain	2%	10%	15%
36	3.042
36	g-5
48	3.042
48	g-5

Fig. 2 Fermentation results of A. oryzae 3.042 and strain g-5 and observation of mycelial pellet morphology A：Kojic acid yield of A. oryzae 3.042 and g-5 strain. B：Mycelial pellet morphology of A. oryzae 3.042 in at 7 d in fermentation. C：Mycelial pellet morphology of g-5 strain at 7 d of fermentation

Fig. 3 Relative expressions of A. oryzae 3.042 and g-5 strain brlA,tps1 and ageB

Fig. 4 Relative expressions of A. oryzae 3.042 and g-5 strain kojR,msn2 and LaeA

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