Sabra Wael, Bommareddy Rajesh Reddy, Maheshwari Garima, Papanikolaou Seraphim, Zeng An-Ping
Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickestrasse 15, 21071, Hamburg, Germany.
Synthetic Biology Research Centre, University of Nottingham, Nottingham, NG7 2RD, UK.
Microb Cell Fact. 2017 May 8;16(1):78. doi: 10.1186/s12934-017-0690-0.
Unlike the well-studied backer yeast where catabolite repression represents a burden for mixed substrate fermentation, Yarrowia lipolytica, an oleaginous yeast, is recognized for its potential to produce single cell oils and citric acid from different feedstocks. These versatilities of Y. lipolytica with regards to substrate utilization make it an attractive host for biorefinery application. However, to develop a commercial process for the production of citric acid by Y. lipolytica, it is necessary to better understand the primary metabolism and its regulation, especially for growth on mixed substrate.
Controlling the dissolved oxygen concentration (pO) in Y. lipolytica cultures enhanced citric acid production significantly in cultures grown on glucose in mono- or dual substrate fermentations, whereas with glycerol as mono-substrate no significant effect of pO was found on citrate production. Growth on mixed substrate with glucose and glycerol revealed a relative preference of glycerol utilization by Y. lipolytica. Under optimized conditions with pO control, the citric acid titer on glucose in mono- or in dual substrate cultures was 55 and 50 g/L (with productivity of 0.6 g/Lh in both cultures), respectively, compared to a maximum of 18 g/L (0.2 g/Lh) with glycerol in monosubstrate culture. Additionally, in dual substrate fermentation, glycerol limitation was found to trigger citrate consumption despite the presence of enough glucose in pO-limited culture. The metabolic behavior of this yeast on different substrates was investigated at transcriptomic and C-based fluxomics levels.
Upregulation of most of the genes of the pentose phosphate pathway was found in cultures with highest citrate production with glucose in mono- or in dual substrate fermentation with pO control. The activation of the glyoxylate cycle in the oxygen limited cultures and the imbalance caused by glycerol limitation might be the reason for the re-consumption of citrate in dual substrate fermentations. This study provides interesting targets for metabolic engineering of this industrial yeast.
与研究充分的酿酒酵母不同，在酿酒酵母中分解代谢物阻遏是混合底物发酵的一个负担，而解脂耶氏酵母作为一种产油酵母，因其能够利用不同原料生产单细胞油和柠檬酸的潜力而受到认可。解脂耶氏酵母在底物利用方面的这些多功能性使其成为生物炼制应用中具有吸引力的宿主。然而，要开发一种利用解脂耶氏酵母生产柠檬酸的商业工艺，有必要更好地了解其初级代谢及其调控，特别是在混合底物上的生长情况。
在单底物或双底物发酵中，控制解脂耶氏酵母培养物中的溶解氧浓度（pO）可显著提高以葡萄糖为底物培养时的柠檬酸产量，而以甘油为单底物时，未发现pO对柠檬酸盐产量有显著影响。在葡萄糖和甘油的混合底物上生长表明解脂耶氏酵母相对更倾向于利用甘油。在优化的pO控制条件下，单底物或双底物培养中以葡萄糖为底物的柠檬酸滴度分别为55和50 g/L（两种培养物的生产率均为0.6 g/Lh），相比之下，单底物培养中以甘油为底物时的最高滴度为18 g/L（0.2 g/Lh）。此外，在双底物发酵中，尽管在pO受限的培养物中有足够的葡萄糖，但发现甘油限制会引发柠檬酸盐的消耗。在转录组学和基于C的通量组学水平上研究了这种酵母在不同底物上的代谢行为。
在单底物或双底物发酵中，通过pO控制使柠檬酸产量最高的培养物中，发现磷酸戊糖途径的大多数基因上调。氧受限培养物中乙醛酸循环的激活以及甘油限制导致的失衡可能是双底物发酵中柠檬酸盐重新消耗的原因。本研究为这种工业酵母的代谢工程提供了有趣的靶点。
