生物转化法生产β-苯乙醇

β-苯乙醇是一种多功能的精细化学品,在食品、日化和轻工等领域有着广泛的应用,全球每年近万吨的β苯乙醇产品基本都是采用化学方法合成,随着人们对天然添加剂的日益需求,迫切需要开发其可以替代的新型生产技术,生物转化法是获得天然β-苯乙醇的最佳途径。概述了生物转化法生产β-苯乙醇的方法,着重介绍β苯乙醇合成的代谢途径、转化的微生物种类和β苯乙醇对酵母细胞毒性问题。     

酵母2-苯乙醇耐受性的研究进展

2-苯乙醇(2-phenylethanol, 2-PE)是一种可食用且有玫瑰香味的高级芳香醇,常用于食品、化妆品和药品行业。由于物理和化学法制备2-PE得率低,不适用于工业生产。而作为单细胞真核微生物的酵母具有高效合成“天然” 2-PE的潜力,因此酵母作为底盘微生物合成2-PE的策略深受研究者青睐。然而,在酵母进行2-PE发酵过程中不免会受到2-PE毒害作用影响。因此,亟须研究酵母耐受2-PE的机制为生产实际提供理论基础,这也有助于选育具有较高2-PE耐受性的酵母菌株。本文综述了酵母2-PE耐受性的研究进展,从酵母2-PE合成途径、2-PE耐受性机理等方面进行阐述,主要说明提升酵母2-PE耐受性的方法。掌握酵母2-PE耐受机制,最终提升酵母2-PE产量及转化效率是今后研究的重中之重。     

酵母菌合成2-苯乙醇的研究进展

2-苯乙醇是一种具有令人愉悦的玫瑰风味的芳香醇,在食品、化妆品和药品等领域具有广泛的应用。本文对酵母菌合成2-苯乙醇的代谢途径及其调控过程、以及提高2-苯乙醇产量的国内外研究进展进行了综述,并对通过微生物转化法合成2-苯乙醇目前存在的不足及进一步研究方向进行了讨论。     

微生物合成2-苯乙醇研究进展*

2-苯乙醇(2-PE)是一种具有广阔应用前景的高级芳香醇。由于化学合成的复杂性和天然提取的高昂成本,近年来,利用微生物发酵合成2-PE受到广泛关注。许多微生物有天然合成2-PE的能力,但产量相对较低,并不适合大规模生产。在最近几年的研究中,利用代谢工程和合成生物学技术,通过上调限速酶基因表达水平,改善前体转运,提高 2-PE耐受性等多方面优化,2-PE的微生物产量有了大幅度的提高。综述微生物合成2-PE的相关研究进展,分析关键代谢调控的机制,并就目前存在的问题提出了改进建议。     

2-苯乙醇对酿酒酵母生理生化特性影响

【目的】研究在不同浓度2-苯乙醇作用下,酵母生理生化特性的变化规律,为优化2-苯乙醇生物合成过程提供重要依据。【方法】透射电镜观察细胞形态;流式细胞术检测细胞膜渗透性、胞内ROS浓度、线粒体膜电位;实时荧光定量PCR检测关键酶基因表达。【结果】随着2-苯乙醇浓度增加(从0到4.0 g/L),酵母细胞分解代谢能力、细胞膜渗透性及aro10基因表达量逐渐降低;线粒体膜电位逐渐增加;胞内ROS浓度先增加后减少。当2-苯乙醇浓度从2.4 g/L增加到3.0 g/L,酵母的分解代谢能力、细胞膜渗透性、aro10基因表达水平等生理生化特性都发生较为显著的变化。【结论】产物原位转移过程中水相2-苯乙醇浓度可考虑控制在2.4 3.0 g/L。     

两相体系中菌体转化法制备2-苯乙醇的研究

目的:利用酿酒酵母(Saccharomyces cerevisiae CICIMY008 6)菌体在油酸-水两 相体系中转化L-苯丙氨酸生成2-苯乙醇,以期解除产物抑制的同时降低萃取相油酸对转化的 不利影响,提高2-苯乙醇产量.方法:对2-苯乙醇的生成与菌体生长的关系进行考察,以确 定菌体转化法的可行性;通过单因素试验和正交设计试验获得转化培养基最佳配方;对菌体 转化条件进行优化.结果:向装液量为25mL/250mL转化培养基中加入0.6g 酵母湿菌体,30℃ 、100r/min条件下转化,9h加入等体积油酸,催化27h,产物浓度达4.55g/L.结 论:2-苯乙醇的制备可以使用菌体转化法,该法可在一定程度上克服两相转化体系中油酸的毒性影响.     

酿酒酵母B5不对称还原制备手性药物中间体R-2′-氯-1-苯乙醇的研究

从 11株微生物中筛选出 4株具有不对称还原 2′ 氯 苯乙酮能力的酵母 ,其中酿酒酵母B5的还原产率与对映体选择性最佳。确定了酿酒酵母B5对 2′ 氯 苯乙酮还原的最佳反应时间为 2 4h ;最佳pH 8 0 ;最佳反应温度为2 5℃ ;最佳共底物为 5 % (体积比 )乙醇。同时研究了底物浓度、微生物的量、微生物的培养条件等对反应产率和立体选择性的影响。细胞浓度为 10 75mg mL(细胞干重 反应体积 )的酿酒酵母B5可将 6 47mmol L的 2′ 氯 苯乙酮10 0 %地转化为R 2′ 氯 1 苯乙醇 ,其对映体选择性为 10 0 %。酿酒酵母B5可重复利用的特点可提高产物的产量。     

酿酒酵母B5不对称还原制备手性药物中间体R-2′-氯-1-苯乙醇的研究

从11株微生物中筛选出4株具有不对称还原2′-氯-苯乙酮能力的酵母,其中酿酒酵母B5的还原产率与对映体选择性最佳。确定了酿酒酵母B5对2′-氯-苯乙酮还原的最佳反应时间为24h;最佳pH 8.0;最佳反应温度为25℃;最佳共底物为5%（体积比）乙醇。同时研究了底物浓度、微生物的量、微生物的培养条件等对反应产率和立体选择性的影响。细胞浓度为10.75mg/mL（细胞干重/反应体积）的酿酒酵母B5可将647mmol/L的2′-氯-苯乙酮100%地转化为R-2′-氯-1-苯乙醇,其对映体选择性为100%。酿酒酵母B5可重复利用的特点可提高产物的产量。     

生物基乳酸生物转化研究进展

乳酸的发酵生产技术已取得了长足的进步,作为一种重要生物基化学品,乳酸除了可用于食品工业及生产聚乳酸外,亦可作为一种重要的平台化合物,用于生产丙烯酸、丙酮酸、1,2-丙二醇、乳酸酯等。文中重点综述了以生物基乳酸为原料经脱水、脱氢、还原及酯化反应生产乳酸衍生物的生物转化工艺,对该领域的发展趋势进行了展望。     

Cofactor self-sufficient whole-cell biocatalysts for the production of 2-phenylethanol

The efficiency of biocatalysis is often affected by an insufficient supply and regeneration of cofactors and redox equivalents. To alleviate this shortcoming, a cofactor self-sufficient system was developed for enhanced production of 2-phenylethanol (2-PE) in E. coli. A “bridge” between the amino acid and its corresponding alcohol was designed in the system using glutamate dehydrogenase. By coupling glutamate dehydrogenase with transaminase and alcohol dehydrogenase, the cosubstrate (2-oxoglutarate) and redox equivalents (NAD(P)H) were regenerated simultaneously, so that no external cofactor or redox source was required. Thus, a cofactor self-sufficient system was developed, which improved the biocatalyst efficiency 3.8-fold. The ammonium generated in this process was removed using zeolite, which further improved the biosynthetic efficiency and resulted in a cleaner system. To the best of our knowledge, this system yielded the highest titer of 2-PE ever obtained in E. coli. Additionally, the wider applicability of this self-sufficient strategy was demonstrated in the production of D-phenyllactic acid. This study thus offers a new method to resolve the cofactor/redox imbalance problem and demonstrates the feasibility of the cofactor self-sufficient strategy for enhanced production of diverse chemicals.     

Production and purification of 2-phenylethanol by Saccharomyces cerevisiae using tobacco waste extract as a substrate

2-phenylethanol (2-PE), which is extracted naturally from plant or biotechnology processing, is widely used in the food and cosmetics industries. Due to the high cost of 2-PE production, the valorization of waste carbon to produce 2-PE has gained increasing attention. Here, 2-PE was produced by Saccharomyces cerevisiae using tobacco waste extract (TWE) as the substrate. Considering the toxicity of nicotine and its inhibition of 2-PE, the tolerance of S. cerevisiae was first evaluated. The results suggested that the production of 2-PE by S. cerevisiae in TWEs could be carried out at 2·0 mg ml⁻¹ nicotine concentrations and may be inhibited by 1·0 mg ml⁻¹ 2-PE. Thus, the compounds in the TWEs prepared at different temperatures were detected, and the results revealed that the TWEs prepared at 140°C contained 2·18 mg ml⁻¹ of nicotine, had total sugar concentrations of 26·8 mg ml⁻¹ and were suitable for 2-PE production. Due to feedback regulation, the 2-PE production was only 1·11 mg ml⁻¹, and the remaining glucose concentration remained at 13·78 mg ml⁻¹, which indicated insufficient glucose utilization. Then, in situ product recovery was further implemented to remove this inhibition; the glucose utilization (the remaining concentration decreased to 3·64 mg ml⁻¹) increased, and the 2-PE production increased to 1·65 mg ml⁻¹. The 2-PE produced in the fermentation broth was first isolated by elution from the resin with 75% ethanol and then by removing the impurities with 2·5% activated charcoal, and pure 2-PE was identified by gas chromatography mass spectrometry. The results of this study suggest that TWE could be an alternative carbon source for 2-PE production. This could provide an outlet tobacco waste as well as reducing the price of natural 2-PE, although more strategies need to be explored to improve the production yield of 2-PE by using TWE.     

Recent advances in biotechnological production of 2-phenylethanol

2-Phenylethanol (2-PE) is an important aromatic alcohol with a rose-like fragrance. It has been widely applied in the cosmetic, perfume, and food industries and is mainly produced by chemical synthesis. An alternative method for the production of natural flavors and fragrances is the microbial transformation process, which is attracting increasing attention because it is an environmentally friendly process and the products are considered “natural”. The production of 2-PE from L-phenylalanine by biotransformation is possible through the Ehrlich pathway and considerable progress has been made in the development of this process. The present report reviews recent advances in biotechnological production of 2-PE, with emphasis on the strategies used to increase production and the applications of in situ product removal techniques. Future research should focus on product scale-up and product recovery processes for the industrialization of microbial processes.     

Biosynthesis of floral scent 2-phenylethanol in rose flowers

Plants emit chemically diverse volatile compounds for attracting pollinators or putting up a chemical defense against herbivores. 2-Phenylethanol (2PE) is one of the abundantly emitted scent compounds in rose flowers. Feeding experiments with l-[²H₈]phenylalanine into rose flowers and subsequent analysis using gas chromatography–mass spectrometry analysis revealed the hypothetical biosynthetic intermediates to [²H₈]-2PE, and the biochemical and genetic analyses elucidated the principal pathway to [²H₈]-2PE. We recently found season-specific 2PE pathway producing [²H₇]-2PE from l-[²H₈]phenylalanine. This is a unique example where the dominant pathway to a specific compound changes with the seasons. This review focuses on the biosynthesis of floral volatiles and their regulation to adapt to the changes in the environment.     

Biosynthesis of 2-phenylethanol using tobacco waste as feedstock

Abstract

2-Phenylethanol (2-PE) is an aromatic alcohol with a rose-like odour, which is widely used in the food, drink and cosmetic industry. It also present in cigarette aromas. As a unique renewable biomass, tobacco contains abundant aromatic compounds, but is rarely used as a feedstock for synthesizing bio-based products. In this study, it was found that 2-PE can be produced from tobacco waste by Saccharomyces cerevisiae. Plackett–Burman design, steepest ascent design and Box–Behnken designs were applied to optimize the fermentation bioprocess, and the maximum titre of 2-PE reached 1.55 g/l. The study explored a new method of 2-PE production and also provided a valuable way to utilize tobacco waste.     

Screening of yeasts for the production of the aroma compound 2-phenylethanol in a molasses-based medium

Fourteen yeast strains were screened for production of 2-phenylethanol from l-phenylalanine with molasses as carbon source. Up to 1 g 2-phenylethanol l^–1 was obtained. Using oleyl alcohol as a second phase for in situ product removal to enhance the production of 2-phenylethanol increased the yield to about 3 g 2-phenylethanol l^–1 at 35 °C. The most productive strains were Kluyveromyces marxianus CBS 600 and CBS 397.