Applied Microbiology and Biotechnology Inducer-independent production of pectinases in Aspergillus niger by overexpression of the D-galacturonic acid responsive transcription factor gaaR



Fig ESM_3. 2 Fig ESM_3. 5

 25 Fig ESM_3.2 Radial growth assay on solid MM containing 50 mM monomeric or 1% polymeric carbon sources after 7 days at 30 °C 




  ESM_1 Strains used in this study 


	ESM_2 Primers used in this study. hygP8f AAAGTTCGACAGCGTCTCC	amplification of split marker fragment 3' to create MA342.2	
	hygP9r		GGCGTCGGTTTCCACTATC	amplification of split marker fragment 5' to create MA342.2	
	Primer name	Sequence (5' to 3')	Used for	Remarks
	gaaR comp P1 PscI	CATGACATGTCCCGCCCCCAGGT	amplification of gaaR for the construction of pEA4	PscI site underlined
	gaaR comp P2 BglII	GAAGATCTCAAGGATTCTCCACCTCCA	amplification of gaaR for the construction of pEA4	BglII site underlined
	gaaR SBfor		CCTCGACGCCATTCCAGTT	amplification of Southern blot probe	
	gaaR SBrev		GGTCATGGACACCGCATTG	amplification of Southern blot probe	
	gaaR comp P1		ATGTCCCGCCCCCAGGT	amplification of gaaR for the construction of pEA2 and pEA3	Fusion PCR overlapping region in bold
	gaaR comp P2 BglII	GAAGATCTCAAGGATTCTCCACCTCCA	amplification of gaaR for the construction of pEA2 and pEA3	BglII site underlined
	eGFP P1 NcoI		GATGCCATGGTGAGCAAGGGCGAG	amplification of eGFP for the construction of pEA2 and pEA3	NcoI site underlined
	Strain eGFP P2 gaaR phu Accession CTGACCTGGGGGCGGGACATCTTGTACAGCTCGTCCATG Genotype Number N402 ATCC cspA1 PgaaR P1 NotI AAGGAAAAAAGCGGCCGCTGGGATTGAAGATGTCGATGC MA234.1 CBS cspA1, kusA::DR-amdS-DR PgaaR P2 NcoI CATGCCATGGCATTGCCTGTGCATAGG	Description amplification of eGFP for the construction of pEA2 and pEA3 Derivative of NRRL3 (N400) Fusion PCR overlapping region in bold Reference amplification of PgaaR for the construction of pEA2 NotI site underlined Bos et al. 1988 Restored pyrG in MA169.4 Alazi et al. 2016 amplification of PgaaR for the construction of pEA2 NcoI site underlined
	XY1.1 creA sm p1f	CBS	cspA1, creA::pyrG AAGCAGCCGATCTGGTTCAA	ΔcreA in AB4.1 amplification of creA 5' flank to create TK2.1		Yuan et al. 2006
	MA342.2 creA sm p2r	CBS	cspA1, kusA::DR-amdS-DR, creA::hygB CAATTCCAGCAGCGGCTTGTGAAGCTTGTCCCAAGAC	ΔcreA in MA234.1 amplification of creA 5' flank to create TK2.1	This study Fusion PCR overlapping region in bold
	JN35.1 creA sm p3f	CBS	cspA1, kusA::DR-amdS-DR, gaaR::hygB ACACGGCACAATTATCCATCGTTCGAACATTCTTCAGCCACAC amplification of creA 3' flank to create TK2.1 ΔgaaR in MA234.1	Alazi et al. 2016 Fusion PCR overlapping region in bold
	JN36.1 creA sm p4r	CBS	cspA1, gaaR::hygB GGGAATGGTCTGGTCTCCGT	amdS loopout in JN35.1 amplification of creA 3' flank to create TK2.1		Alazi et al. 2016
	EA21.3 Anid pyrFP1for	CBS	cspA1, gaaR::hygB, amdS AAGCCGCTGCTGGAATTGACTGATTGCGC ACATTGAC + , ectopically integrated PgpdA-gaaR amplification of A. nidulans pyrF fragment 1 to create TK2.1 gaaR overexpression in JN36.1 Fusion PCR overlapping region in bold This study
	EA21.5 EA21.6 Anid pyrFP4rev CBS CBS EA21.8 CBS Anid pyrFP3for TK1.1 CBS Anid pyrFP2rev EA23.6 CBS An02g03830P1f_KIT	cspA1, gaaR::hygB, amdS cspA1, gaaR::hygB, amdS CGCCCTCCTTCTCGATGAT cspA1, gaaR::hygB, amdS GCCGTGAATCGCCCTACTT cspA1, gaaR::hygB, amdS CGATGGATAATTGTGCCGTGATCAGGCACGGTCAGTCCTC + , ectopically integrated PgpdA-gaaR amplification of A. nidulans pyrF fragment 1 to create TK2.1 gaaR overexpression in JN36.1 + , ectopically integrated PgpdA-gaaR gaaR overexpression in JN36.1 + , ectopically integrated PgpdA-gaaR gaaR overexpression in JN36.1 amplification of A. nidulans pyrF fragment 2 to create TK2.1 + , ectopically integrated PgpdA-gaaR gaaR overexpression in JN36.1 amplification of A. nidulans pyrF fragment 2 to create TK2.1 Fusion PCR overlapping region in bold This study This study This study This study cspA1, gaaR::hygB, amdS + , ectopically integrated PgpdA-gaaR, pyrE -Loss of pyrE in EA21.6 This study AGTATTCCACTCAGCTCTGGACTAACCTCTTTAATTCTACTCCG TAATCCCTCTCGAC amplification of creA 5' flank to create MA342.2
	TK2.1 EA19.2 An02g03830P2r_KIT CBS CBS EA20.10 CBS An02g03830P3f_KIT	cspA1, gaaR::hygB, amdS cspA1, gaaR::hygB, amdS TGATGTGTGTGACGTGATGTGTATATCCTATCCCAAGACCGAC + , ectopically integrated PgpdA-gaaR, pyrE + , ectopically integrated PgaaR-eGFP-gaaR -, ΔcreA::pyrF GAGGGTAAAA amplification of creA 5' flank to create MA342.2 ΔcreA in EA23.6 eGFP-gaaR in JN36.1 cspA1, gaaR::hygB, amdS + , ectopically integrated PgpdA-eGFP-gaaR eGFP-gaaR overexpression in JN36.1 TGATGATGGATATATGGAAGCTGGAGGATGTTCGAACATTCT TCAGCCACACGTTG amplification of creA 3' flank to create MA342.2	This study This study This study
	JN126.2 An02g03830P4r_KIT CBS	cspA1, Δku70::amdS, gaaX::nicB, PgaaX-gaaX-eGFP integrated to the pyrG locus TAGCAGGTGAGAACTCACTTGTACTCGACTCCTGCATACTGGT ATATACAGTAAAACCCCATACTATC amplification of creA 3' flank to create MA342.2 gaaX-eGFP in JN125.1		Niu et al. 2017
	An02g03830P5f_KIT	ACACCCTCTCTTCCCGGTCA	amplification of split marker fragment 5' to create MA342.2	
	An02g03830P6r_KIT	AGTCAATCAAGTAAGTCCCC	amplification of split marker fragment 3' to create MA342.2	
	hygP3f		TAGGATATACACATCACGTC	amplification of hygR selection marker	
	hygP4r		CATCCTCCAGCTTCCATATATC	amplification of hygR selection marker	

ESM_3 Supplementary figures
Ebru Alazi, Tim Knetsch, Marcos Di Falco, Ian D Reid, Mark Arentshorst, Jaap Visser, Adrian Tsang, Arthur F J Ram
Applied Microbiology and Biotechnology Inducer-independent production of pectinases in Aspergillus niger by overexpression of the D-galacturonic acid responsive transcription factor gaaR