Additional file 1 of Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species
Creators: name: Dai, Ziyu
name: Pomraning, Kyle R.
name: Deng, Shuang
name: Kim, Joonhoon
name: Campbell, Kristen B.
name: Robles, Ana L.
name: Hofstad, Beth A.
name: Munoz, Nathalie
name: Gao, Yuqian
name: Lemmon, Teresa
name: Swita, Marie S.
name: Zucker, Jeremy D.
name: Kim, Young-Mo
name: Burnum-Johnson, Kristin E.
name: Magnuson, Jon K.
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Space Science
subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Physiology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Ecology
subject: Immunology
subject: FOS: Clinical medicine
subject: Inorganic Chemistry
subject: FOS: Chemical sciences
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Detailed Description of transgene vector construction for gene overexpression or disruption in A. pseudoterreus or A. niger. The vector prepared are: 3HP4025, 3HP4028, 3HP4069, 3HP4070, 3HP4071, 3HP4074, 3HP4076, 3HP4077, 3HP4102, 3HP4103, 3HP4104, 3HP4108, 3HP4109, 3HP4114, 3HP4126, 3HP4134, 3HP4136, 3HP4140, 3HP4144, and 3HP4145. Table S1. Oligos used for transgene vector constructions of the gene overexpressions or disruptions. Figure S1. The diagram of the β-alanine 3HP pathway transgene expression cassette with A. pseudoterreus cad1 gene locus targeting and Southern blotting analyses of transgenic A. pseudoterreus. (A) the diagram of the β-alanine 3HP pathway (3HP4028, Ap3HP); (B) the diagram of the β-alanine 3HP pathway with the pattern of restriction endonuclease BamHI, EcoRV, and HindIII; (C) the restriction fragment length polymorphism of BamHI or EcoRV in selected transgenic strains; (D) the restriction fragment length polymorphism of HindIII in selected transgenic strains. Figure S2. 3-hydroxypropionic acid and itaconic acid production in the selected individual transgenic strains of A. pseudoterreus with overexpression of the β-alanine 3HP pathway transgene expression cassette in the modified RDM medium at 30 °C and 200 rpm for 7 days. Figure S3. The diagram of β-alanine 3HP pathway transgene expression cassette with two identical copies of β-alanine 3HP pathway (3HP4046, 2 × 3HP). Figure S4. The diagram of β-alanine 3HP pathway transgene expression cassette with E. coli hygromycin B phosphotransferase (hph) marker gene (3HP4070). Figure S5. The diagram of β-alanine 3HP pathway along with an additional aat1 transgene overexpression under the control of A. pseudoterreus tef1 gene promoter (3HP4071). Figure S6. Southern blot analysis confirmed the β-alanine 3HP pathway random integrations into the chromosomes of A. niger in the single spore isolates of transgenic strain An3HP5, An3HP9, An3HP10, and An2 × 3HP1 with multiple copies of chromosomal insertions. (A). restriction map of the plasmid DNA fragments containing the β-alanine 3HP pathway used for random integration with restriction endonucleases of BamHI (B), or HindIII (C). Figure S7. The diagram of transgene overexpression cassettes of A. niger aspartate aminotransferase (aat1), pyruvate carboxylase (pyc) or their combination; (A). the ble, the bacterial bleomycin resistance gene; Tef1P, A. niger tef1 gene promoter; A. niger aat1, aspartate aminotransferase; Tpgk, A. niger pgk transcriptional terminator (3HP4074); (B). &amp; (C) mbf1P, A. niger mbf1 gene promoter; pyc, A. niger pyc gene without aat1 (B, 3HP4076) &amp; with aat1 (C, 3HP4077); (D). the nat1 (Streptomyces noursei nourseothricin N-acetyl transferase optimized for the codon usage of Saccharomyces cerevisiae) selection marker under the control of A. nidulans trpC promoter and A. niger trpC transcriptional terminator (3HP4114); (E) the nat, nourseothricin N-acetyl transferase marker gene; the mct1, A. niger monocarboxylate transporter; the Tmct1, A. niger mct1 transcriptional terminator (3HP4126). Figure S8. 3-hydroxypropionic acid production in the selected individual transgenic strains of A. niger with overexpression of cytosolic aspartate aminotransferase (aat, A), pyruvate carboxylase (pyc, B), or aspartate aminotransferase + pyruvate carboxylase (aat-pyc, C) in An3HP9 strain grown in modified RDM medium at 30 °C and 200 rpm for 7 days. Figure S9. The diagram of gene disruption constructs of A. niger ald6a, ald6b, ald3, uga2, or oahA gene. (A). 5’-oahA and 3’-oahA are upstream and downstream fragments of oahA gene (3HP4102); (B). 5’-ald6a and 3’-ald6a are fragments of upstream and downstream of ald6a gene (3HP4103); (C). 5’-ald6b and 3’-ald6b are fragments of upstream and downstream of ald6b gene (3HP4104); (D). 5’-ald3 and 3’-ald3 are fragments of upstream and downstream of ald3 gene (3HP4108); and (E). 5’-uga2 and 3’-uga2 are upstream and downstream fragments of uga2 gene (3HP4109). Figure S10. The diagram of new β-alanine 3HP pathway transgene expression cassette (3HP4145) with loxP-nptII marker gene recycle for A. niger. The Tet-On/Cre-loxP system (3HP4140) for marker gene recycle conditionally activated by doxycycline, nat1, S. noursei nourseothricin acetyltransferase (resistance) marker gene; ubi1S27p, A. niger ubi1S27 promoter; rtTA2A, the reverse tetracycline transactivator; TetO7, tetracycline resistance operon; Pmn, A. nidulans gpdA minimal promoter; Cre, Cre recombinase; ActrpCt, A. carbonarius trpC transcriptional terminator; (B). the bacterial neomycin-resistance (nptII) marker gene under the control of A. niger malate dehydrogenase (mdhp) promoter and A. nidulans trpC transcriptional terminator (trpCt) (3HP4134); (C). backbone marker gene cassette with 31 bp loxP fragments fused at 5’- and 3’-end of nptII marker gene (3HP3136); (D). the intermediate transgene expression cassette (3HP4144) contains the ubi4p, A. niger ubi4 gene promoter; PAND, T. castaneum aspartate decarboxylase; elf3t, A. niger elongation factor 3 transcriptional terminator; BAPAT, B. cereus β-alanine-pyruvate aminotransferase; and ubiSp, A. niger ubi1S promoter; (E). the final new β-alanine 3HP pathway (3HP4145) was assembled with the HPDH, E. coli 3-hydroxypropionate dehydrogenase under the control of A. niger mbfA promoter (mbfAp) and A. nidulans trpC transcriptional terminator(trpCt). Figure S11. The effects of culture medium and pH on 3-HP production and spore germination in A. niger strain An3HP9 grown at 30 °C and 200 rpm for 7 days. (A) 3-HP and DCW titer and yield and (B) the percentage of spore germination.