Liang Shiyu, Zhang Yue, Lyu Liting, Wang Shuang, Zhao Zongbao K
Laboratory of Biotechnology, Dalian Institute of Chemical Physics, CAS, 457 Zhongshan Road, Dalian, 116023, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
Bioresour Bioprocess. 2023 Mar 2;10(1):16. doi: 10.1186/s40643-023-00639-2.
Lipids produced by oleaginous yeasts are considered as sustainable sources for the production of biofuels and oleochemicals. The red yeast Rhodosporidium toruloides can accumulate lipids to over 70% of its dry cell mass. To facilitate lipid extraction, a recombinant β-1,3-glucomannanase, MAN5C, has been applied to partially breakdown R. toruloides cell wall. In this study, R. toruloides NP11 was engineered for secretory expression of MAN5C to simplify the lipid extraction process. Specifically, a cassette contained a codon-optimized gene MAN5C was integrated into the genome of R. toruloides by Agrobacterium-mediated transformation. The engineered strain NP11-MAN5C was found with proper expression and secretion of active MAN5C, yet no notable compromise in terms of cell growth and lipid production. When NP11-MAN5C cell cultures were extracted with ethyl acetate without any pretreatment, 20% of total lipids were recovered, 4.3-fold higher than that of the parental strain NP11. When the cells were heat-treated followed by extraction with ethyl acetate in the presence of the culture broth supernatants, up to 93% of total lipids were recovered, confirming beneficial effects of MAN5C produced in situ. This study provides a new strategy to engineer oleaginous yeasts for more viable lipid extraction and down-stream processes.
产油酵母产生的脂质被认为是生产生物燃料和油脂化学品的可持续来源。红酵母 Rhodosporidium toruloides 能将脂质积累至其干细胞质量的 70% 以上。为便于脂质提取，一种重组β-1,3-葡甘露聚糖酶 MAN5C 已被用于部分分解 Rhodosporidium toruloides 的细胞壁。在本研究中，对 Rhodosporidium toruloides NP11 进行了工程改造，以实现 MAN5C 的分泌表达，从而简化脂质提取过程。具体而言，通过农杆菌介导的转化，将一个包含密码子优化基因 MAN5C 的盒式结构整合到 Rhodosporidium toruloides 的基因组中。发现工程菌株 NP11-MAN5C 能正确表达和分泌活性 MAN5C，且在细胞生长和脂质生产方面没有明显受损。当 NP11-MAN5C 细胞培养物未经任何预处理用乙酸乙酯提取时，回收了 20% 的总脂质，比亲本菌株 NP11 高 4.3 倍。当细胞进行热处理，然后在培养液上清液存在的情况下用乙酸乙酯提取时，总脂质回收率高达 93%，证实了原位产生的 MAN5C 的有益作用。本研究提供了一种工程改造产油酵母的新策略，以实现更可行的脂质提取和下游工艺。
