Metabolic engineering of an acid-tolerant yeast strain Pichia kudriavzevii for itaconic acid production
Metabolic engineering of an acid-tolerant yeast strain Pichia kudriavzevii for itaconic acid production
复制标题
DOI：
10.1016/j.mec.2020.e00124
复制
发表时间：
2020-02
影响因子：
5.2
通讯作者：
Wan Sun;Ana Vila-Santa;Na Liu;T. Prozorov;D. Xie;N. T. Faria;F. Ferreira;N. Mira;Zengyi Shao
中科院分区：
文献类型：
--
作者：
Wan Sun;Ana Vila-Santa;Na Liu;T. Prozorov;D. Xie;N. T. Faria;F. Ferreira;N. Mira;Zengyi Shao
衣康酸(IA)或2-亚甲基琥珀酸在生物聚合物工业中有广泛的应用，因为其结构中存在一个乙烯基键和两个酸基。它的聚合机理与丙烯酸相似，但额外的β酸基可以加入额外的官能团。目前，IA的工业化生产主要依靠丝状真菌土曲霉的发酵。然而，丝状真菌进行发酵的困难以及OFA的致病潜力给工业化生产带来了严重的挑战。近年来，人们对开发发酵过程的替代生产宿主越来越感兴趣，这些生产宿主在生产有机酸时更加均匀。毕赤酵母是一种非传统酵母，在低pH值下对有机酸具有高度的耐酸性，这是一种非常可取的特性，因为它简化了下游加工。我们将At_cad基因(命名为At_cad)导入该酵母菌，建立了IA的初始滴度为135±105mg/L。随后过量表达天然线粒体三羧酸转运蛋白(这里命名为Pk_mtA)可能将顺式乌头酸高效地输送到胞浆中，使IA产量加倍。通过将新发明的CRISPR-Cas9系统引入到库德里亚夫采维氏酵母中，我们成功地敲除了编码异柠檬酸脱氢酶(ICD)基因的两个拷贝，目的是提高顺式乌头酸酯的利用率。结果表明，不含ICD、高表达Pk_mtt A和At_Cad的枯草杆菌在摇瓶发酵中的IA产量为505±17.7mg/L，在补料分批发酵中的IA产量为1232±和mg/L。由于在发酵过程中使用耐酸菌种不需要调节pH值，这项工作展示了将P.kudriavzevii设计为生产有机酸的工业底盘的巨大潜力。
Itaconic acid (IA), or 2-methylenesuccinic acid, has a broad spectrum of applications in the biopolymer industry owing to the presence of one vinyl bond and two acid groups in the structure. Its polymerization can follow a similar mechanism as acrylic acid but additional functionality can be incorporated into the extra beta acid group. Currently, the bio-based production of IA in industry relies on the fermentation of the filamentous fungusAspergillus terreus. However, the difficulties associated with the fermentation undertaken by filamentous fungi together with the pathogenic potential ofA. terreuspose a serious challenge for industrial-scale production. In recent years, there has been increasing interest in developing alternative production hosts for fermentation processes that are more homogenous in the production of organic acids.Pichia kudriavzeviiis a non-conventional yeast with high acid tolerance to organic acids at low pH, which is a highly desirable trait by easing downstream processing. We introducedcis-aconitic acid decarboxylase gene (cad) fromA. terreus(designatedAt_cad) into this yeast and established the initial titer of IA at 135 ± 5 mg/L. Subsequent overexpression of a native mitochondrial tricarboxylate transporter (herein designatedPk_mttA) presumably deliveredcis-aconitate efficiently to the cytosol and doubled the IA production. By introducing the newly invented CRISPR-Cas9 system intoP.kudriavzevii, we successfully knocked out both copies of the gene encoding isocitrate dehydrogenase (ICD), aiming to increase the availability ofcis-aconitate. The resultingP. kudriavzeviistrain, devoid ofICDand overexpressingPk_mttAandAt_cadon its genome produced IA at 505 ± 17.7 mg/L in shake flasks, and 1232 ± 64 mg/L in fed-batch fermentation. Because the usage of an acid-tolerant species does not require pH adjustment during fermentation, this work demonstrates the great potential of engineeringP.kudriavzeviias an industrial chassis for the production of organic acid.