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Establishment of a visual gene knockout system based on CRISPR/Cas9 for the rare actinomycete Nonomuraea gerenzanensis

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Li Tian
Li Tian
Li Tian
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Yan Bingyu at Jiangnan University
Yan Bingyu
Dandan Huo
Dandan Huo
Dandan Huo
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Wenhui Sun
Wenhui Sun
Wenhui Sun
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Abstract and Figures

Objectives To develop a modified CRISPR/Cas9 system with the β-glucuronidase (GusA) reporter and a dual sgRNA cassette for Nonomuraea gerenzanensis (N. gerenzanensis).ResultsWith the aid of a visual GusA reporter, the complicated and tedious process of cloning and gene identification could be abandoned entirely in the genetic editing of N. gerenzanensis. Moreover, introducing a dual sgRNA cassette into the CRISPR/Cas9 system significantly improved gene deletion efficiency compared to the single sgRNA element. Furthermore, the length of the homologous flanking sequences set to the lowest value of 500 bp in this system could still reach the relatively higher conjugation transfer frequency.Conclusions The enhanced CRISPR/Cas9 system could efficiently perform genetic manipulation on the rare actinomycete N. gerenzanensis.
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Biotechnol Lett (2023) 45:401–410
https://doi.org/10.1007/s10529-023-03347-1
ORIGINAL RESEARCH PAPER
Establishment ofavisual gene knockout system based
onCRISPR/Cas9 fortherare actinomycete Nonomuraea
gerenzanensis
LiTian· BingyuYan· DandanHuo· WenhuiSun· SufangCui·
XiaojingLi· XiangmeiZhang· HuijunDong
Received: 7 July 2022 / Revised: 18 November 2022 / Accepted: 5 January 2023 / Published online: 18 January 2023
© The Author(s), under exclusive licence to Springer Nature B.V. 2023
Abstract
Objectives To develop a modified CRISPR/Cas9
system with the β-glucuronidase (GusA) reporter and
a dual sgRNA cassette for Nonomuraea gerenzanen-
sis (N. gerenzanensis).
Results With the aid of a visual GusA reporter,
the complicated and tedious process of cloning and
gene identification could be abandoned entirely in
the genetic editing of N. gerenzanensis. Moreover,
introducing a dual sgRNA cassette into the CRISPR/
Cas9 system significantly improved gene deletion
efficiency compared to the single sgRNA element.
Furthermore, the length of the homologous flanking
sequences set to the lowest value of 500 bp in this
system could still reach the relatively higher conjuga-
tion transfer frequency.
Conclusions The enhanced CRISPR/Cas9 system
could efficiently perform genetic manipulation on the
rare actinomycete N. gerenzanensis.
Keywords A40926· β-Glucuronidase· CRISPR/
Cas9· N. gerenzanensis
Introduction
Nonomuraea gerenzanensis (N. gerenzanensis) is
a rare actinomycete with the biosynthetic ability of
A40926, which is the precursor of the second-gener-
ation glycopeptide antibiotic Dalbavancin (Alduina
etal. 2018; Marschall et al. 2019). The biosynthetic
gene cluster (BGC) for A40926 in N. gerenzanen-
sis has been identified as the dbv gene cluster with
37 open reading frames (ORFs) (Sosio et al. 2003).
A40926 is a mixture of A40926 homologs with dif-
ferent long-chain N-acyl groups, mainly due to the
substrate extensivity of the post-modification acyl-
transferase Dbv8 (Sosio and Donadio 2006). A40926
blend is the main obstacle to improve the quality of
Dalbavancin and to reduce its industrial produc-
tion cost. Previous research had reported that a dou-
ble mutant lacking dbv8 and dbv23 was constructed
to produce A40926 intermediates in order to obtain
the single A40926 component by chemical synthe-
sis method in vitro (Alt et al. 2019). The difference
in our research is that a method of directed evolu-
tion for Dbv8 is considered to eliminate homologous
impurities in vivo. Therefore, in this study, we per-
formed the deletion of the dbv8 gene. In addition,
we constructed a Δ23 mutant of N. gerenzanensis
in our previous study to delete the acetyl moiety of
Li Tian and Bingyu Yan are Co-first authors.
Supplementary Information The online version
contains supplementary material available at https:// doi.
org/ 10. 1007/ s10529- 023- 03347-1.
L.Tian· B.Yan· D.Huo· W.Sun· S.Cui· X.Li·
X.Zhang· H.Dong(*)
School ofPharmaceutical Sciences, Liaocheng
University, No.1 Hunan Road, Dongchangfu District,
Liaocheng252000, China
e-mail: donghuijun_747@163.com
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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