Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Ave, Tianjin Airport Economic Park, Tianjin, 300308, China.
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
Microb Biotechnol. 2021 May;14(3):1130-1147. doi: 10.1111/1751-7915.13781. Epub 2021 Feb 25.
The unconventional yeast Pichia kudriavzevii is renowned for its ability to survive at low pH and has been exploited for the industrial production of various organic acids, especially succinic acid (SA). However, P. kudriavzevii can also utilize the di- and tricarboxylate intermediates of the Krebs cycle as the sole carbon sources for cell growth, which may adversely affect the extracellular accumulation of SA. Because the carboxylic acid transport machinery of P. kudriavzevii remains poorly understood, here, we focused on studying its SA transportation process from the perspective of mining and characterization of dicarboxylate transporters in a newly isolated acid-tolerant P. kudriavzevii strain CY902. Through genome sequencing and transcriptome analysis, two JEN family carboxylate transporters (PkJEN2-1 and PkJEN2-2) were found to be involved in SA transport. Substrate specificity analysis revealed that both PkJEN proteins are active dicarboxylate transporters, that can effectively import succinate, fumarate and L-malate into the cell. In addition, PkJEN2-1 can transport α-ketoglutarate, while PkJEN2-2 cannot. Since PkJEN2-1 shows higher transcript abundance than PkJEN2-2, its role in dicarboxylate transport is more important than PkJEN2-2. In addition, PKJEN2-2 is also responsible for the uptake of citrate. To our best knowledge, this is the first study to show that a JEN2 subfamily transporter is involved in tricarboxylate transport in yeast. A combination of model-based structure analysis and rational mutagenesis further proved that amino acid residues 392-403 of the tenth transmembrane span (TMS-X) of PkJEN2-2 play an important role in determining the specificity of the tricarboxylate substrate. Moreover, these two PkJEN transporters only exhibited inward transport activity for SA, and simultaneous inactivation of both PkJEN transporters reduced the SA influx, resulting in enhanced extracellular accumulation of SA in the late stage of fermentation. This work provides useful information on the mechanism of di-/tricarboxylic acid utilization in P. kudriavzevii, which will help improve the organic acid production performance of this microbial chassis.
非常规酵母毕赤酵母以能够在低 pH 值下生存而闻名，并且已被用于各种有机酸的工业生产，尤其是琥珀酸（SA）。然而，毕赤酵母也可以利用三羧酸循环的二羧酸和三羧酸中间产物作为细胞生长的唯一碳源，这可能会对 SA 的细胞外积累产生不利影响。由于毕赤酵母的羧酸转运机制仍不清楚，因此在这里，我们专注于从新分离的耐酸毕赤酵母菌株 CY902 中挖掘和表征二羧酸转运蛋白的角度来研究其 SA 转运过程。通过基因组测序和转录组分析，发现两种 JEN 家族羧酸转运蛋白（PkJEN2-1 和 PkJEN2-2）参与了 SA 转运。底物特异性分析表明，两种 PkJEN 蛋白都是活性二羧酸转运蛋白，能够有效地将琥珀酸、富马酸和 L-苹果酸导入细胞内。此外，PkJEN2-1 可以转运α-酮戊二酸，而 PkJEN2-2 则不能。由于 PkJEN2-1 的转录丰度高于 PkJEN2-2，因此它在二羧酸转运中的作用比 PkJEN2-2 更重要。此外，PKJEN2-2 还负责摄取柠檬酸。据我们所知，这是首次表明 JEN2 亚家族转运蛋白参与酵母中的三羧酸转运。基于模型的结构分析和合理的突变进一步证明，PkJEN2-2 的第十个跨膜区（TMS-X）的 392-403 个氨基酸残基在决定三羧酸底物的特异性方面起着重要作用。此外，这两种 PkJEN 转运蛋白仅对 SA 表现出内向转运活性，同时失活两种 PkJEN 转运蛋白会降低 SA 的流入量，从而导致发酵后期 SA 的细胞外积累增加。这项工作提供了有关毕赤酵母中二羧酸/三羧酸利用机制的有用信息，这将有助于提高该微生物底盘的有机酸生产性能。
