title: Effects of nitrogen availability on polymalic acid biosynthesis in the yeast-like fungus Aureobasidium pullulans.
abstract: 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.
authors.affiliation: College of Pharmaceutical Sciences, Chongqing Engineering Research Center for Pharmaceutical Process and Quality Control, Southwest University, 2 Tian Sheng Road, Beibei, Chongqing, 400715, People's Republic of China.
authors.forename: Yongkang
authors.initials: Y
authors.lastname: Wang
authors.affiliation: College of Pharmaceutical Sciences, Chongqing Engineering Research Center for Pharmaceutical Process and Quality Control, Southwest University, 2 Tian Sheng Road, Beibei, Chongqing, 400715, People's Republic of China.
authors.forename: Xiaodan
authors.initials: X
authors.lastname: Song
authors.affiliation: Biotechnology Research Center, Southwest University, Chongqing, 400715, People's Republic of China.
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Ministry of Education, Key Laboratory of Biorheological Science and Technology (Chongqing University), Chongqing, 400044, People's Republic of China.
authors.forename: Bochu
authors.initials: B
authors.lastname: Wang
authors.affiliation: College of Pharmaceutical Sciences, Chongqing Engineering Research Center for Pharmaceutical Process and Quality Control, Southwest University, 2 Tian Sheng Road, Beibei, Chongqing, 400715, People's Republic of China. zhx1030@swu.edu.cn.|Ministry of Education, Key Laboratory of Biorheological Science and Technology (Chongqing University), Chongqing, 400044, People's Republic of China. zhx1030@swu.edu.cn.
authors.forename: Xiang
authors.initials: X
authors.lastname: Zou
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology; D001203:Ascomycota / Q000187:drug effects / Q000254:growth & development* / Q000378:metabolism*; D019149:Bioreactors; D053898:Biosynthetic Pathways / Q000235:genetics*; D020869:Gene Expression Profiling; D005800:Genes, Fungal; D006868:Hydrolysis; D008293:Malates / Q000032:analysis / Q000378:metabolism*; D009584:Nitrogen / Q000032:analysis* / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism*; D040901:Proteomics; D012270:Ribosomes / Q000235:genetics / Q000502:physiology; D015398:Signal Transduction / Q000235:genetics; D020123:Sirolimus / Q000494:pharmacology
chemical_list: D000935:Antifungal Agents; D008293:Malates; D011108:Polymers; C121060:poly(malic acid); C030298:malic acid; D009584:Nitrogen; D020123:Sirolimus
keywords: Aureobasidium pullulans; Nitrogen availability; Polymalic acid; Proteomics; TOR signaling pathway; Transcriptomics