Zhan Yangyang, Zhou Menglin, Wang Huan, Chen Lixia, Li Zhi, Cai Dongbo, Wen Zhiyou, Ma Xin, Chen Shouwen
State Key Laboratory of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, College of Life Sciences, Hubei University, 368 Youyi Avenue, Wuhan, 430062, Hubei, People's Republic of China.
Department of Food Science and Human Nutrition, Iowa State University, Ames, IA, 50011, USA.
Appl Microbiol Biotechnol. 2020 Sep;104(17):7507-7520. doi: 10.1007/s00253-020-10740-7. Epub 2020 Jul 11.
2-Phenylethanol is a valuable flavoring agent with many applications. Although the bioproduction of 2-phenylethanol has been achieved by microbial fermentation, the low titer and high cost hinder its industrial-scale production. The goal of this study is to develop an efficient process for high-level production of 2-phenylethanol from L-phenylalanine. Firstly, candidate hosts for 2-phenylethanol synthesis were screened by evaluating their tolerance to 2-phenylethanol, and Bacillus licheniformis DW2 was proven to be a promising strain for 2-phenylethanol production. Subsequently, phenylpyruvate decarboxylase and alcohol dehydrogenase from different hosts were screened, and the combination of KivD from Lactococcus lactis and YqhD from Escherichia coli owned the best performance on 2-phenylethanol synthesis, and the attained strain DE4 produced 3.04 g/L 2-phenylethanol from 5.00 g/L L-phenylalanine using glucose as carbon source. Furthermore, the fermentation process was optimized using molasses as carbon source, and 2-phenylethanol titer was increased to 4.41 g/L. In fed-batch fermentation, the maximum 2-phenylethanol titer reached 5.16 g/L, with a yield of 0.65 g/g on L-phenylalanine and productivity of 0.12 g/(L.h), which was the highest 2-phenylethnol titer reported to date when molasses was used as carbon source. Collectively, this study develops a robust strain as well as the cost-efficient process for 2-phenylethanol production, which lays a substantial foundation for industrial production of 2-phenylethanol. Key points •Bacillus licheniformis is an excellent 2-PE stress-tolerant strain. •Coexpressed kivD and yqhD is most suitable for 2-PE production in B. licheniformis. •High-level production of 2-PE (5.16 g/L) was obtained by engineered strain DE4.
2-苯乙醇是一种具有多种用途的重要调味剂。尽管通过微生物发酵已实现2-苯乙醇的生物生产,但低产量和高成本阻碍了其工业化规模生产。本研究的目标是开发一种从L-苯丙氨酸高效生产2-苯乙醇的工艺。首先,通过评估它们对2-苯乙醇的耐受性筛选出用于2-苯乙醇合成的候选宿主,地衣芽孢杆菌DW2被证明是一种有前景的2-苯乙醇生产菌株。随后,筛选了来自不同宿主的苯丙酮酸脱羧酶和乙醇脱氢酶,乳酸乳球菌的KivD和大肠杆菌的YqhD组合在2-苯乙醇合成方面表现最佳,所得菌株DE4以葡萄糖为碳源,从5.00 g/L L-苯丙氨酸中产生了3.04 g/L 2-苯乙醇。此外,使用糖蜜作为碳源对发酵过程进行了优化,2-苯乙醇产量提高到4.41 g/L。在分批补料发酵中,2-苯乙醇的最大产量达到5.16 g/L,对L-苯丙氨酸的产率为0.65 g/g,生产力为0.12 g/(L·h),这是迄今为止以糖蜜为碳源报道的最高2-苯乙醇产量。总体而言,本研究开发了一种强大的菌株以及用于2-苯乙醇生产的经济高效工艺,为2-苯乙醇的工业化生产奠定了坚实基础。要点 •地衣芽孢杆菌是一种优秀的耐2-PE菌株。 •共表达的kivD和yqhD最适合在地衣芽孢杆菌中生产2-PE。 •工程菌株DE4实现了2-PE的高产(5.16 g/L)。