Methods for Improved C4-Dicarboxylic Acid Production in Filamentous Fungi
a technology of dicarboxylic acid and filamentous fungi, which is applied in the field of improving the production of dicarboxylic acid, can solve the problems of increasing the cost of petroleum derived building blocks, increasing the cost of producing large volume industrial chemicals by traditional petrochemical routes, etc., and achieves the effect of increasing the level of dicarboxylic acid
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example 1
Cloning of the Aspergillus clavatus NRRL1 C4-Dicarboxylic Acid Transporter Gene and Construction of Expression Vector pShTh120AcC4T
[0218]The 1179 by C4-dicarboxylic acid transporter gene acc4t (ACLA—058030) was synthetically constructed into pAcC4T (FIG. 1; DNA2.0, Menlo Park, Calif., USA). The acc4t gene was amplified from pAcC4T using primers 069735 and 069736 shown below.
Primer 069735:(SEQ ID NO: 5)5′-GTGTGATAGAACATCGTCCATAATGTTCGAAAATCG-3′Primer 069736:(SEQ ID NO: 6)5′-GTCAGTCACCTCTAGTTAATTAACTAGTCTGCAGCATCCTCATC-3′
[0219]The PCR reaction mixture was composed of 50 ng pAcC4T template, 200 μM dNTP mixture, 50 pM primer 069735, 50 pM primer 069736, 1× Poll reaction buffer (New England Biolabs, MA, USA), and 1 unit Vent Polymerase (New England Biolabs) and deionized water to 50 μl. The PCR reaction was incubated in an EPPENDORF MASTERCYCLER® (Eppendorf Scientific Inc., Westbury, N.Y., USA) programmed for 1 cycle at 94° C. for 3 minutes; 35 cycles at 94° C. for 15 seconds, 59° C. for...
example 2
Cloning of the Aspergillus fumigates Af293 C4-Dicarboxylic Acid Transporter Gene and Construction of Expression Vector pShTh121AfC4T
[0222]The 1182 bp C4-dicarboxylic acid transporter gene sequence afc4t (AFUA—8G04630) was synthetically constructed into pAfC4T (FIG. 5; DNA2.0). The afc4t gene was amplified from pAfC4T using primers 069737 and 069738 shown below.
Primer 069737:(SEQ ID NO: 9)5-GTGTGATAGAACATCGTCCATAATGTTCAACGATCATGATCA-3′Primer 069738:(SEQ ID NO: 10)5′-GTCAGTCACCTCTAGTTAATTAATTAATCTAGCACATCCTCGTC-3′
[0223]The PCR reaction mixture was composed of 50 ng pAtC4T template, 200 μM dNTP mixture, 50 pM primer 069737, 50 pM primer 069738, 1× Poll reaction buffer, 1 unit Vent Polymerase and deionized water to 50 μl. The PCR reaction was incubated in an EPPENDORF MASTERCYCLER® programmed for 1 cycle at 94° C. for 3 minutes; 35 cycles at 94° C. for 15 seconds, 59° C. for 30 seconds, and 72° C. for 1 minute; and 1 cycle at 72° C. for 5 minutes. The PCR product was purified by 1% agar...
example 3
Transformation of Expression Vector Fragments of pShTh120AcC4T and pShTh121AfC4T into Aspergillus oryzae NRRL3488 (ShTh1200 and ShTh1210)
[0226]Protoplast preparation and transformation of Aspergillus oryzae NRRL3488 were performed by inoculating approximately 2×107 spores into 100 ml YEG medium and incubating the flask at 27° C. for 16-18 hours at 140 rpm. Mycelia were collected by pouring the culture through a sterile funnel lined with MIRACLOTH® (Calbiochem, San Diego, Calif., USA) and rinsing with 50 ml of 0.7 M KCl. The washed mycelia were resuspended in a 125 ml flask with 20 ml of protoplasting solution composed of 5 mg of GLUCANEX™ (Novozymes NS, Bagsvrd, Denmark) and 0.5 mg of chitinase (Sigma Chemical Co., St. Louis, Mo., USA) per ml of 0.7 M KCl (filter sterilized) and incubated at 34° C., for 30 minutes with mixing at 80 rpm. The protoplasting solution was poured through a sterile funnel lined with MIRACLOTH® and rinsed with 50 ml of STC buffer (1 M sorbitol-10 mM Tris-HC...
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