代谢工程改造毕赤酵母艾氏途径促进糖蜜合成2-苯乙醇

Metabolic engineering of Ehrlich pathway in Komagataella phaffii improves the synthesis of 2-phenylethanol from sugarcane molasses

摘要:

【背景】2-苯乙醇(2-phenylethanol, 2-PE)是大宗香料之一，已广泛应用于食品、化妆品、抗菌剂及医药产业中。【目的】通过代谢工程改造艾氏途径，利用甘蔗糖蜜为碳源合成2-PE，为构建2-PE工业化生产的毕赤酵母(Komagataella phaffii)菌株奠定基础。【方法】代谢工程改造毕赤酵母菌株PD-TA艾氏途径，构建2-PE高产菌株；优化发酵培养基，提高产量。【结果】以糖蜜为碳源时，重组菌PD-TA的生长是菌株GS115的1.4倍。以菌株PD-TA为出发菌，敲除醛脱氢酶基因ALD3后2-PE产量显著提高了30.7%。评估了13个醇脱氢酶和苯乙醛还原酶催化2-PE合成的效率，过表达酿酒酵母(Saccharomyces cerevisiae)醇脱氢酶基因ScADH5工程菌的2-PE合成量最高，较对照菌提高了29.9%。过表达苯丙酮酸脱羧酶基因ARO10，2-PE产量提高了19.7%。敲除ALD3并共表达ARO10和ScADH5的工程菌P2的2-PE产量较对照菌提高了109.4%，产量为397 mg/L。优化发酵培养基后，菌株P2经摇瓶发酵36 h的2-PE产量提高了218.1%，达到1 263 mg/L。【结论】毕赤酵母具备将廉价糖蜜转化为高附加值产物2-PE的能力。本研究为构建包括2-PE在内的多种杂醇类细胞工厂提供了理论依据和技术基础。

Abstract:

[Background] The organic compound 2-phenylethanol (2-PE) known for its attractive floral fragrance is widely utilized in the cosmetic, pharmaceutical, and food industries. [Objective] To develop an engineered Komagataella phaffii strain for de novo synthesis of 2-PE from sugarcane molasses. [Methods] The Ehrlich pathway was engineered to enhance 2-PE production in the recombinant K. phaffii strain PD-TA, and the fermentation medium was optimized. [Results] With sugarcane molasses as the carbon source, the cell density of PD-TA was 1.4 folds that of the original strain (GS115). The deletion of ALD3 from PD-TA resulted in a 30.7% increase in the 2-PE titer by reducing by-product formation. Furthermore, we investigated 13 candidate genes encoding alcohol dehydrogenase (ADH) or phenylacetaldehyde reductase (PAR) to identify the optimal enzyme. The highest 2-PE titer was observed in the strain overexpressing ScADH5, with an increase of 29.9% compared with that of PD-TA. Additionally, the overexpression of ARO10 led to a 19.7% increase in the 2-PE titer. The simultaneous overexpression of ScADH5 and ARO10, combined with the deletion of ALD3, enabled the engineered strain P2 to achieve a 2-PE titer of 397 mg/L in shake flasks, representing an increase of 109.4% compared with that of PD-TA. To further improve the production of 2-PE, we optimized the fermentation medium, which achieved an increase of 218.1% in the 2-PE titer (1 263 mg/L) after fermentation in shake flasks for 36 h. [Conclusion] This study demonstrates the potential of K. phaffii as a chassis for the de novo synthesis of 2-PE from molasses. Furthermore, it offers both theoretical and technical frameworks for the establishment of microbial cell factories for the production of aromatic chemicals including 2-PE.