Czajka Jeffrey J, Dai Ziyu, Radivojević Tijana, Kim Joonhoon, Deng Shuang, Lemmon Teresa, Swita Marie, Burnet Meagan C, Munoz Nathalie, Gao Yuqian, Kim Young-Mo, Hofstad Beth, Magnuson Jon K, Garcia Martin Hector, Burnum-Johnson Kristin E, Pomraning Kyle R
Energy and Environment Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA.
DOE Agile BioFoundry, Emeryville, CA, 94608, USA.
Microb Cell Fact. 2025 May 21;24(1):117. doi: 10.1186/s12934-025-02705-0.
Lipomyces starkeyi is an oleaginous yeast with a native metabolism well-suited for production of lipids and biofuels from complex lignocellulosic and waste feedstocks. Recent advances in genetic engineering tools have facilitated the development of L. starkeyi into a microbial chassis for biofuel and chemical production. However, the feasibility of redirecting L. starkeyi lipid flux away from lipids and towards other products remains relatively unexplored. Here, we engineer the native metabolism to produce malic acid by introducing the reductive TCA pathway and a C4-dicarboxylic acid transporter to the yeast.
Heterogeneous expression of two genes, the Aspergillus oryzae malate transporter and malate dehydrogenase, enabled L. starkeyi malic acid production. Overexpression of a third gene, the native pyruvate carboxylase, allowed titers to reach approximately 10 g/L during shaking flasks cultivations, with production of malic acid inhibited at pH values less than 4. Corn-stover hydrolysates were found to be well-tolerated, and controlled bioreactor fermentations on the real hydrolysate produced 26.5 g/L of malic acid. Proteomic, transcriptomic and metabolomic data from real and mock hydrolysate fermentations indicated increased levels of a S. cerevisiae hsp9/hsp12 homolog (proteinID: 101453), glutathione dependent formaldehyde dehydrogenases (proteinIDs: 2047, 278215), oxidoreductases, and expression of efflux pumps and permeases during growth on the real hydrolysate. Simultaneously, machine learning based medium optimization improved production dynamics by 18% on mock hydrolysate and revealed lower tolerance to boron (a trace element included in the standard cultivation medium) than other yeasts.
Together, this work demonstrated the ability to produce organic acids in L. starkeyi with minimal byproducts. The fermentation characterization and omic analyses provide a rich dataset for understanding L. starkeyi physiology and metabolic response to growth in hydrolysates. Identified upregulated genes and proteins provide potential targets for overexpression for improving growth and tolerance to concentrated hydrolysates, as well as valuable information for future L. starkeyi engineering work.
斯达氏油脂酵母是一种产油酵母，其天然代谢非常适合从复杂的木质纤维素和废料原料生产脂质和生物燃料。基因工程工具的最新进展推动了将斯达氏油脂酵母开发成用于生物燃料和化学品生产的微生物底盘。然而，将斯达氏油脂酵母的脂质通量从脂质转向其他产品的可行性仍相对未被探索。在此，我们通过向酵母中引入还原性三羧酸循环途径和一种C4-二羧酸转运蛋白来改造其天然代谢以生产苹果酸。
米曲霉苹果酸转运蛋白和苹果酸脱氢酶这两个基因的异源表达使斯达氏油脂酵母能够生产苹果酸。第三个基因——天然丙酮酸羧化酶的过表达使得在摇瓶培养期间产量达到约10 g/L，在pH值小于4时苹果酸的产生受到抑制。发现玉米秸秆水解产物具有良好的耐受性，在实际水解产物上进行的受控生物反应器发酵产生了26.5 g/L的苹果酸。来自实际和模拟水解产物发酵的蛋白质组学、转录组学和代谢组学数据表明，在实际水解产物上生长期间，酿酒酵母hsp9/hsp12同源物（蛋白质ID：101453）、谷胱甘肽依赖性甲醛脱氢酶（蛋白质ID：2047、278215）、氧化还原酶以及外排泵和通透酶的表达水平增加。同时，基于机器学习的培养基优化使模拟水解产物的生产动力学提高了18%，并揭示出其对硼（标准培养基中包含的一种微量元素）的耐受性低于其他酵母。
总之，这项工作证明了在斯达氏油脂酵母中以最少副产物生产有机酸的能力。发酵特性和组学分析提供了丰富的数据集，用于了解斯达氏油脂酵母的生理学以及对水解产物中生长的代谢反应。鉴定出的上调基因和蛋白质为过表达以改善生长和对浓缩水解产物的耐受性提供了潜在靶点，也为未来斯达氏油脂酵母的工程工作提供了有价值的信息。
