Department of Chemical and Biomolecular Engineering, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.
U.S. Department of Energy Center for Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL, 61801, USA.
Microb Cell Fact. 2022 Feb 19;21(1):26. doi: 10.1186/s12934-022-01750-3.
The oleaginous, carotenogenic yeast Rhodotorula toruloides has been increasingly explored as a platform organism for the production of terpenoids and fatty acid derivatives. Fatty alcohols, a fatty acid derivative widely used in the production of detergents and surfactants, can be produced microbially with the expression of a heterologous fatty acyl-CoA reductase. Due to its high lipid production, R. toruloides has high potential for fatty alcohol production, and in this study several metabolic engineering approaches were investigated to improve the titer of this product.
Fatty acyl-CoA reductase from Marinobacter aqueolei was co-expressed with SpCas9 in R. toruloides IFO0880 and a panel of gene overexpressions and Cas9-mediated gene deletions were explored to increase the fatty alcohol production. Two overexpression targets (ACL1 and ACC1, improving cytosolic acetyl-CoA and malonyl-CoA production, respectively) and two deletion targets (the acyltransferases DGA1 and LRO1) resulted in significant (1.8 to 4.4-fold) increases to the fatty alcohol titer in culture tubes. Combinatorial exploration of these modifications in bioreactor fermentation culminated in a 3.7 g/L fatty alcohol titer in the LRO1Δ mutant. As LRO1 deletion was not found to be beneficial for fatty alcohol production in other yeasts, a lipidomic comparison of the DGA1 and LRO1 knockout mutants was performed, finding that DGA1 is the primary acyltransferase responsible for triacylglyceride production in R. toruloides, while LRO1 disruption simultaneously improved fatty alcohol production, increased diacylglyceride and triacylglyceride production, and increased glucose consumption.
The fatty alcohol titer of fatty acyl-CoA reductase-expressing R. toruloides was significantly improved through the deletion of LRO1, or the deletion of DGA1 combined with overexpression of ACC1 and ACL1. Disruption of LRO1 surprisingly increased both lipid and fatty alcohol production, creating a possible avenue for future study of the lipid metabolism of this yeast.
产油、类胡萝卜素的酵母罗氏油脂酵母（Rhodotorula toruloides）越来越多地被探索作为萜类化合物和脂肪酸衍生物生产的平台生物。脂肪酸醇是一种广泛用于洗涤剂和表面活性剂生产的脂肪酸衍生物，可以通过表达异源脂肪酸酰基辅酶 A 还原酶在微生物中产生。由于其高脂质产量，罗氏油脂酵母具有生产脂肪酸醇的巨大潜力，在这项研究中，研究了几种代谢工程方法来提高该产物的产量。
来自海生不动杆菌（Marinobacter aqueolei）的脂肪酸酰基辅酶 A 还原酶与 SpCas9 在罗氏油脂酵母 IFO0880 中共表达，探索了一组基因过表达和 Cas9 介导的基因缺失，以提高脂肪酸醇的产量。两个过表达靶点（ACL1 和 ACC1，分别提高细胞质乙酰辅酶 A 和丙二酰辅酶 A 的产生）和两个缺失靶点（酰基转移酶 DGA1 和 LRO1）导致脂肪酸醇产量在培养管中显著增加（1.8 到 4.4 倍）。在生物反应器发酵中对这些修饰的组合探索导致 LRO1Δ 突变体的脂肪酸醇产量达到 3.7g/L。由于在其他酵母中 LRO1 的缺失对脂肪酸醇的生产没有益处，因此对 DGA1 和 LRO1 敲除突变体进行了脂质组学比较，发现 DGA1 是罗氏油脂酵母中三酰基甘油产生的主要酰基转移酶，而 LRO1 的破坏同时提高了脂肪酸醇的产量，增加了二酰基甘油和三酰基甘油的产量，并增加了葡萄糖的消耗。
通过删除 LRO1 或删除 DGA1 并过表达 ACC1 和 ACL1，表达脂肪酸酰基辅酶 A 还原酶的罗氏油脂酵母的脂肪酸醇产量显著提高。令人惊讶的是，LRO1 的缺失同时增加了脂质和脂肪酸醇的产量，为进一步研究该酵母的脂质代谢提供了可能的途径。
