School of Biological Science and Engineering, South China University of Technology, Guangzhou, 510006, People's Republic of China.
College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, People's Republic of China.
Appl Microbiol Biotechnol. 2016 Jul;100(13):5791-803. doi: 10.1007/s00253-016-7324-z. Epub 2016 Feb 2.
Citric acid is produced by an industrial-scale process of fermentation using Aspergillus niger as a microbial cell factory. However, citric acid production was hindered by the non-fermentable isomaltose and insufficient saccharification ability in A. niger when liquefied corn starch was used as a raw material. In this study, A. niger TNA 101ΔagdA was constructed by deletion of the α-glucosidase-encoding agdA gene in A. niger CGMCC 10142 genome using Agrobacterium tumefaciens-mediated transformation. The transformants A. niger OG 1, OG 17, and OG 31 then underwent overexpression of glucoamylase in A. niger TNA 101ΔagdA. The results showed that the α-glucosidase activity of TNA 101ΔagdA was decreased by 62.5 % compared with CGMCC 10142, and isomaltose was almost undetectable in the fermentation broth. The glucoamylase activity of the transformants OG 1 and OG 17 increased by 34.5 and 16.89 % compared with that of TNA 101ΔagdA, respectively. In addition, for the recombinants TNA 101ΔagdA, OG 1 and OG 17, there were no apparent defects in the growth development. Consequently, in comparison with CGMCC 10142, TNA 101ΔagdA and OG 1 decreased the residual reducing sugar by 52.95 and 88.24 %, respectively, and correspondingly increased citric acid production at the end of fermentation by 8.68 and 16.87 %. Citric acid production was further improved by decreasing the non-fermentable residual sugar and increasing utilization rate of corn starch material in A. niger. Besides, the successive saccharification and citric acid fermentation processes were successfully integrated into one step.
柠檬酸是通过使用黑曲霉作为微生物细胞工厂的发酵工业规模过程生产的。然而,当液化玉米淀粉用作原料时,柠檬酸的生产受到黑曲霉中非发酵性异麦芽糖和不足的糖化能力的阻碍。在这项研究中,通过根癌农杆菌介导的转化,在黑曲霉 CGMCC 10142 基因组中缺失编码α-葡萄糖苷酶的 agdA 基因,构建了黑曲霉 TNA 101ΔagdA。然后,转化子黑曲霉 OG 1、OG 17 和 OG 31 在黑曲霉 TNA 101ΔagdA 中过表达了糖化酶。结果表明,与 CGMCC 10142 相比,TNA 101ΔagdA 的α-葡萄糖苷酶活性降低了 62.5%,发酵液中几乎检测不到异麦芽糖。转化子 OG 1 和 OG 17 的糖化酶活性分别比 TNA 101ΔagdA 提高了 34.5%和 16.89%。此外,对于重组子 TNA 101ΔagdA、OG 1 和 OG 17,其生长发育没有明显缺陷。因此,与 CGMCC 10142 相比,TNA 101ΔagdA 和 OG 1 分别将残留还原糖降低了 52.95%和 88.24%,相应地在发酵结束时柠檬酸产量分别提高了 8.68%和 16.87%。通过降低不可发酵的残留糖并提高黑曲霉中玉米淀粉原料的利用率,进一步提高了柠檬酸的产量。此外,成功地将连续糖化和柠檬酸发酵过程集成到一步中。