Additional file 1 of Improved consolidated bioprocessing for itaconic acid production by simultaneous optimization of cellulase and metabolic pathway of Neurospora crassa
Creators: name: Zhao, Chen
name: Zhao, Jiajia
name: Han, Jingjing
name: Mei, Yaojie
name: Fang, Hao
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Molecular Biology
subject: Physiology
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Ecology
subject: Biological Sciences not elsewhere classified
subject: Plant Biology

description: Additional file 1: Figure S1. Main plasmids used in this study. (A) Plasmids pMF-272-Pccg1/Peas/Pcbh1/Pgh6-2/Pgh11-2/Ptef1/Pgpd/Ppda-CAD were used to compare the expression of CAD in N. crassa. (B) Plasmids pMF-272-Pccg1-CBH1/GH6-2/GH5-1/GH3-4/AsBGA/TrCBH2 were used to compare the effects of different cellulases. (C) Plasmids pMF-272-Pccg1-CAD-Pcbh1-CBH1/GH6-2/GH5-1/GH3-4/AsBGA/TrCBH2 were used to compare the effects of different cellulase and CAD co-expression. (D) Plasmid pMF-272-Pccg1-MTK was used to verify the expression of MTK in N. crassa. The plasmids pUC19-MTK-HPH (F) and pMF-272-Pccg1-CAD-Pes-MCL (E) or pMF-272-Pccg1-CAD-Pcbh1-MTTA-Pes-MCL (G) were used to construct N. crassa PMF-CAD-rGS or N. crassa PMF-CAD-MTTA-rGS. (H) Plasmid pMF-272-Pccg1-CAD-Pcbh1-MTTA-Pcbh1-TrCBH2 was used to construct N. crassa PMF-CAD-MTTA-TrCBH2. Figure S2. PCR amplified the promoter sequence. Figure S3. Strain construction process using Pcbh1 as the CAD promoter. (A) Pcbh1 promoter sequence was amplified by PCR. M:Trans2K Plus DNA Marker, 1–6:Pcbh1 (B) PCR identification of vector Blunt-Pcbh1. 1–22: Blunt-Pcbh1 (C) Identification of recombinant plasmid pMF272-CAD. 1–6: pMF272-CAD. (D) Double enzyme digestion of pMF272-CAD recombinant plasmid. (E) Cloning vector Blunt-Pcbh1 double enzyme digestion. (F) Colony PCR identification of recombinant plasmid pMF-CAD-Pcbh1. Figure S4. Construction of cellulase overexpression strain. (A) PCR amplification of Pcbh-1 promoter sequence (1 and 2), gh3-4 sequence (4), and cbh1 sequence (B, 1 and 2). (C) Identification of expression vector containing cbh1 gene. (D) PCR screening of gh3-4 gene expression vectors. (E) Genome PCR for vector transformation screening 1,2,3: cbh1; 4,5: gh3-4. Figure S5. Construction of MTK, MCL expression strain. (A) PCR amplification of MTK (lines 1 ~ 3). (B) Colony PCR for identification of MTK expression cassette (C) PCR amplification of GFP (1) and terminator fragments (2). Identification of expression vector containing cbh1 gene. Genome PCR for MTK expression (D) and MCL expression (E) vector transformation screening. Figure S6. Construction of CAD, MTK and MCL co-expression strain. (A) PCR amplification of 5′ fragment (lines 1 ~ 3). (B) PCR amplification of 3′ fragment (lines 1 ~ 3) and hph fragment (lines 4 and 5). (C) PCR amplification of MTK cassette. (D) Identification of expression vector containing 5′ fragment and hph fragment. Table S1. Plasmids used in this study. Table S2. Strains used in this study. Table S3. Primer list of CAD expression and promoter optimization. Table S4. Primer list of cellulase expression. Table S5. Primer list of CAD and cellulase co-expression. Table S6. Primer list of MTK, MCL and MTTA expression. Table S7. RT-PCR Primers.