Effects of nitrogen availability on polymalic acid biosynthesis in the yeast-like fungus Aureobasidium pullulans.
Effects of nitrogen availability on polymalic acid biosynthesis in the yeast-like fungus Aureobasidium pullulans.
复制标题
氮利用率对类酵母真菌出芽短梗霉聚苹果酸生物合成的影响
DOI：
10.1186/s12934-016-0547-y
复制
发表时间：
2016-08-22
影响因子：
6.4
通讯作者：
Zou X
中科院分区：
文献类型：
--
作者：
Wang Y;Song X;Zhang Y;Wang B;Zou X
背景：聚苹果酸（PMA）是一种新型的聚酯聚合物，在医疗和食品工业中有着广泛的应用。它的单体l -苹果酸也是一种潜在的C4平台化学物质。然而，对酵母样真菌普鲁兰毛霉（auobasidium pululans）中PMA生物合成的机制知之甚少。本研究通过比较转录组学和蛋白质组学分析研究了不同氮浓度对细胞生长和PMA生物合成的影响，并对相关信号通路进行了评价。结果：在低氮浓度（2 g/L NH4NO3）条件下，5 L发酵罐中PMA的最终效价为44.00±3.65 g/L（水解后苹果酸为49.9±4.14 g/L），比高氮浓度（10 g/L NH4NO3）条件下的产率提高了18.3%。对比转录组学分析显示，在氮充足条件下，与核糖体、核糖体生物发生、蛋白酶体和氮代谢相关的一组基因显著上调或下调，这些基因可能受到TOR信号通路的调控。通过蛋白质组学分析鉴定出14个蛋白点，发现这些蛋白点与细胞分裂和生长、能量代谢和糖酵解途径相关。qRT-PCR进一步证实了PMA生物合成途径的关键基因GLK、CS、FUM、DAT和MCL以及TOR信号通路的关键基因GS、TOR1、Tap42和Gat1的表达水平因氮限制而上调。在雷帕霉素胁迫下，PMA的生物合成明显受到剂量依赖性抑制，TOR1、MCL和DAT的转录水平也下调。结论：氮水平可调节细胞生长和PMA的生物合成。低浓度氮有利于PMA的生物合成，可以上调PMA生物合成途径中关键基因的表达。细胞生长和PMA生物合成可能通过TOR信号通路响应氮。该研究将有助于我们深入了解PMA生物合成的分子机制，并为PMA和苹果酸类化学品的生产开发有效的工艺。
Background:Polymalic acid (PMA) is a novel polyester polymer that has been broadly used in the medical and food industries. Its monomer, L-malic acid, is also a potential C4 platform chemical. However, little is known about the mechanism of PMA biosynthesis in the yeast-like fungus, Aureobasidium pullulans. In this study, the effects of different nitrogen concentration on cell growth and PMA biosynthesis were investigated via comparative transcriptomics and proteomics analyses, and a related signaling pathway was also evaluated.Results:A high final PMA titer of 44.00 ± 3.65 g/L (49.9 ± 4.14 g/L of malic acid after hydrolysis) was achieved in a 5-L fermentor under low nitrogen concentration (2 g/L of NH4NO3), which was 18.3 % higher yield than that obtained under high nitrogen concentration (10 g/L of NH4NO3). Comparative transcriptomics profiling revealed that a set of genes, related to the ribosome, ribosome biogenesis, proteasome, and nitrogen metabolism, were significantly up- or down-regulated under nitrogen sufficient conditions, which could be regulated by the TOR signaling pathway. Fourteen protein spots were identified via proteomics analysis, and were found to be associated with cell division and growth, energy metabolism, and the glycolytic pathway. qRT-PCR further confirmed that the expression levels of key genes involved in the PMA biosynthetic pathway (GLK, CS, FUM, DAT, and MCL) and the TOR signaling pathway (GS, TOR1, Tap42, and Gat1) were upregulated due to nitrogen limitation. Under rapamycin stress, PMA biosynthesis was obviously inhibited in a dose-dependent manner, and the transcription levels of TOR1, MCL, and DAT were also downregulated.Conclusions:The level of nitrogen could regulate cell growth and PMA biosynthesis. Low concentration of nitrogen was beneficial for PMA biosynthesis, which could upregulate the expression of key genes involved in the PMA biosynthesis pathway. Cell growth and PMA biosynthesis might be mediated by the TOR signaling pathway in response to nitrogen. This study will help us to deeply understand the molecular mechanisms of PMA biosynthesis, and to develop an effective process for the production of PMA and malic acid chemicals.