TY  - JOUR
A1  - Haeger, Gerrit
A1  - Bongaerts, Johannes
A1  - Siegert, Petra
T1  - A convenient ninhydrin assay in 96-well format for amino acid-releasing enzymes using an air-stable reagent
JF  - Analytical Biochemistry
N2  - An improved and convenient ninhydrin assay for aminoacylase activity measurements was developed using the commercial EZ Nin™ reagent. Alternative reagents from literature were also evaluated and compared. The addition of DMSO to the reagent enhanced the solubility of Ruhemann's purple (RP). Furthermore, we found that the use of a basic, aqueous buffer enhances stability of RP. An acidic protocol for the quantification of lysine was developed by addition of glacial acetic acid. The assay allows for parallel processing in a 96-well format with measurements microtiter plates.
Y1  - 2022
U6  - http://dx.doi.org/10.1016/j.ab.2022.114819
SN  - 1096-0309
IS  - 624
PB  - Elsevier
CY  - Amsterdam
ER  - 
TY  - JOUR
A1  - Haeger, Gerrit
A1  - Grankin, Alina
A1  - Wagner, Michaela
T1  - Construction of an Aspergillus oryzae triple amylase deletion mutant as a chassis to evaluate industrially relevant amylases using multiplex CRISPR/Cas9 editing technology
JF  - Applied Research
N2  - Aspergillus oryzae is an industrially relevant organism for the secretory production of heterologous enzymes, especially amylases. The activities of potential heterologous amylases, however, cannot be quantified directly from the supernatant due to the high background activity of native α-amylase. This activity is caused by the gene products of amyA, amyB, and amyC. In this study, an in vitro CRISPR/Cas9 system was established in A. oryzae to delete these genes simultaneously. First, pyrG of A. oryzae NSAR1 was mutated by exploiting NHEJ to generate a counter-selection marker. Next, all amylase genes were deleted simultaneously by co-transforming a repair template carrying pyrG of Aspergillus nidulans and flanking sequences of amylase gene loci. The rate of obtained triple knock-outs was 47%. We showed that triple knockouts do not retain any amylase activity in the supernatant. The established in vitro CRISPR/Cas9 system was used to achieve sequence-specific knock-in of target genes. The system was intended to incorporate a single copy of the gene of interest into the desired host for the development of screening methods. Therefore, an integration cassette for the heterologous Fpi amylase was designed to specifically target the amyB locus. The site-specific integration rate of the plasmid was 78%, with exceptional additional integrations. Integration frequency was assessed via qPCR and directly correlated with heterologous amylase activity. Hence, we could compare the efficiency between two different signal peptides. In summary, we present a strategy to exploit CRISPR/Cas9 for gene mutation, multiplex knock-out, and the targeted knock-in of an expression cassette in A. oryzae. Our system provides straightforward strain engineering and paves the way for development of fungal screening systems.
KW  - aspergillus
KW  - CRISPR/Cas9
KW  - filamentous fungi
KW  - genome engineering
Y1  - 2023
U6  - http://dx.doi.org/10.1002/appl.202200106
SN  - 2702-4288
IS  - Early View
SP  - 1
EP  - 15
PB  - Wiley-VCH
ER  - 
TY  - RPRT
A1  - Haeger, Gerrit
A1  - Bongaerts, Johannes
A1  - Siegert, Petra
T1  - Abschlussbericht Teil II: Eingehende Darstellung Neue biobasierte Lipopeptide aus nachhaltiger Produktion (LipoPep)
Y1  - 2023
N1  - Förderkennzeichen: 13FH256PA6

Titel: FHprofUnt 2016: Neue biobasierte Lipopeptide aus nachhaltiger Produktion

Laufzeit: 01.02.2019 – 31.10.2022
ER  -