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。     

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.     

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。     

Biotechnological production of 2-phenylethanol

2-Phenylethanol (2-PE) is an important flavour and fragrance compound with a rose-like odour. Most of the world's annual production of several thousand tons is synthesised by chemical means but, due to increasing demand for natural flavours, alternative production methods are being sought. Harnessing the Ehrlich pathway of yeasts by bioconversion of L-phenylalanine to 2-PE could be an option, but in situ product removal is necessary due to product inhibition. This review describes the microbial production of 2-PE, and also summarizes the chemical syntheses and the market situation.     

Bioprocesses for 2-phenylethanol and 2-phenylethyl acetate production: current state and perspectives

2-Phenylethanol (2-PE) and 2-phenethyl acetate (2-PEA) are valuable generally recognized as safe flavoring agents widely used in industry. Perfumes, pharmaceuticals, polishes, and personal care products, are some of the final products using these compounds as additives due to their rose-like odor. Also, 2-PE is used in disinfectants, pest control, and cleaning products due to its biocide capability. Although most of these additives production are derived from chemical synthesis, the current trend of consumers to prefer natural products has contributed to the development of biotechnological approaches as an alternative way to obtain natural 2-PE and 2-PEA. The most efficient route to bioproduce these compounds is through the bioconversion of L-phenylalanine via the Ehrlich pathway, and most of the advances have been focused on the development of this process. This review compiles the most recent developments in the biotechnological production of 2-PE and 2-PEA, indicating the most studied strains producing 2-PE and 2-PEA, the current advances in the in situ product recovery in liquid systems, an overview of the strain developments, and the progress in the use of residue-based systems. Future research should address the need for more sustainable and economic systems such as those using wastes as raw materials, as well as the scale-up of the proposed technologies.

     

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.     

Advances in 2-phenylethanol production from engineered microorganisms

2-Phenylethanol (2-PE) is an important flavor ingredient with a rose-like odor. Due to concerns about the toxic byproducts potentially found in 2-PE from chemical synthesis, consumers prefer the natural aroma compound, promoting the biosynthesis of 2-PE. Various microorganisms produce 2-PE naturally with low yield. Recent metabolic engineering strategies in yeasts and Escherichia coli have achieved great success in improving 2-PE bioproduction, including the alleviation of feed-back inhibition, improvement of precursor transport, enhancing activities of crucial enzymes, and reduction of by-products. Here, we review the metabolic engineering strategies applied to microorganisms for increasing bioproduction of 2-PE, address current problems, and propose further improvements.     

生物转化法合成2-苯乙醇的研究进展

2-苯乙醇(2-phenylethanol,2-PE)是具有玫瑰香味的芳香醇,在食品、化妆品以及药品领域有着广泛的应用。但是从植物花卉中提取的天然2-苯乙醇产量低,成本高。目前,用微生物转化生产"天然"2-苯乙醇越来越受到关注。本文对近年来国内外报道的提高微生物转化合成2-苯乙醇产量的研究,尤其是生产菌株选育和发酵工程优化的研究进行了综述,并提出今后研究的新思路,旨在为利用微生物发酵进行2-苯乙醇生产提供参考。     

Screening of Yeasts Isolated from Brazilian Environments for the 2-Phenylethanol (2-PE) Production

Phenylethanol alcohol, or 2-phenylethanol (2-PE) production by yeasts has been considered a promising alternative to its chemical synthesis. In order to evaluate the potential of yeast strains isolated from different Brazilian environments, we evaluated the 2-PE production of 267 strains. Among them, the Kluyveromyces marxianus CCT 7735 yeast stood out as being the best 2-PE producer. The K. marxianus CCT 7735 growth was impaired by 2-PE; nevertheless, this effect is less pronounced than the inhibition reported for certain Saccharomyces cerevisiae strains. The maximum 2-PE titer obtained under optimized conditions was 3.44 g/L, 28% higher than the titer achieved under unoptimized conditions. The optimized conditions were: 30ºC, and glucose and L-phe concentrations of 3.0 and 4.0 g/L, respectively. Moreover, the specific production rate of 2-PE increased twofold compared to the unoptimized conditions.     

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。     

An aqueous–organic two-phase bioprocess for efficient production of the natural aroma chemicals 2-phenylethanol and 2-phenylethylacetate with yeast

The natural aroma chemicals 2-phenylethanol (2-PE) and 2-phenylethylacetate (2-PEAc) are of high industrial relevance and can be produced from l-phenylalanine in a yeast-based process with growth-associated product formation. Due to product inhibition, in situ product removal is mandatory to obtain economically interesting concentrations. A fed-batch approach using polypropylene glycol 1200 as in situ extractant and the precursor in a saturated concentration led to the highest 2-PE productivity reported for a bioprocess so far. With Kluyveromyces marxianus CBS 600, 26.5 g/l 2-PE and 6.1 g/l 2-PEAc in the organic phase were obtained, corresponding to space–time yields of 0.33 and 0.08 g/l h, respectively.     

Yarrowia lipolytica: the novel and promising 2-phenylethanol producer

This is the first report on the ability of Yarrowia lipolytica strains to produce 2-phenylethanol (2-PE), which has not been identified for this species to date. 2-PE is a valuable aroma compound of rose-like odor. Its isolation from the other than microbial source—rose petals, is limited by the substrate availability. Thus, this chemical compound constitutes an attractive product for biotechnological conversions. To date, the ability to produce 2-PE has been described for such genera as Saccharomyces sp., Kluyveromyces sp., Geotrichum sp., and Pichia sp. This report provides evidence that Y. lipolytica is a novel 2-PE producer. Moreover, the titers of 2-PE obtained in Y. lipolytica NCYC3825 non-optimized cultures, nearly 2 g/l, are competitive to titers obtained by the other species.     

Tailoring of microbes for the production of high value plant-derived compounds: From pathway engineering to fermentative production

Plant natural products have been an attracting platform for the isolation of various active drugs and other bioactives. However large-scale extraction of these compounds is affected by the difficulty in mass cultivation of these plants and absence of strategies for successful extraction. Even though, synthesis by chemical method is an alternative method; it is less efficient as their chemical structure is highly complex which involve enantio-selectivity. Thus an alternate bio-system for heterologous production of plant natural products using microbes has emerged. Advent of various omics, synthetic and metabolic engineering strategies revolutionised the field of heterologous plant metabolite production. In this context, various engineering methods taken to synthesise plant natural products are described with an additional focus to fermentation strategies.     

脱落酸生物合成研究进展

脱落酸作为一种抑制生长的植物激素,是平衡植物内源激素和调节生长代谢的关键因子。脱落酸具有提高作物抗旱耐盐、减少果实褐变的作用,同时可降低疟疾发病率、刺激胰岛素分泌,因此在农业和医药领域有着广阔的应用前景。相较于传统的植物提取法和化学合成法,利用微生物合成脱落酸是一种经济、可持续的来源方式。目前利用天然微生物如灰葡萄孢霉菌、蔷薇色尾孢菌等合成脱落酸的研究已经取得了诸多进展,而脱落酸的异源微生物合成研究相对较少。酿酒酵母、解脂耶氏酵母、大肠杆菌等工程菌株作为天然产物异源合成的常用宿主,具有遗传背景清晰、易于操作、便于工业化生产等优势,因此利用微生物异源合成脱落酸是一种更具潜力的生产方式。本文着重从底盘细胞的选择、关键酶的筛选与表达强化、辅因子的调节、增强前体供应及促进脱落酸外排5个方面对微生物异源合成脱落酸的研究进行综述。最后,对该领域的未来发展方向进行了展望。     

Efficient phase separation and product recovery in organic‐aqueous bioprocessing using supercritical carbon dioxide

Biphasic hydrocarbon functionalizations catalyzed by recombinant microorganisms have been shown to be one of the most promising approaches for replacing common chemical synthesis routes on an industrial scale. However, the formation of stable emulsions complicates downstream processing, especially phase separation. This fact has turned out to be a major hurdle for industrial implementation. To overcome this limitation, we used supercritical carbon dioxide (scCO₂) for both phase separation and product purification. The stable emulsion, originating from a stereospecific epoxidation of styrene to (S)‐styrene oxide, a reaction catalyzed by recombinant Escherichia coli, could be destabilized efficiently and irreversibly, enabling complete phase separation within minutes. By further use of scCO₂ as extraction agent, the product (S)‐styrene oxide could be obtained with a purity of 81% (w/w) in one single extraction step. By combining phase separation and product purification using scCO₂, the number of necessary workup steps can be reduced to one. This efficient and easy to use technique is generally applicable for the workup of biphasic biocatalytic hydrocarbon functionalizations and enables a cost effective downstream processing even on a large scale. Biotechnol. Bioeng. 2010;107:642–651. © 2010 Wiley Periodicals, Inc.     

酿酒酵母合成木栓酮及其衍生物的研究现状及展望

木栓酮及其衍生物在植物中普遍存在且种类繁多,具有丰富的生理药理学活性。木栓酮衍生物是以木栓酮为骨架经细胞色素氧化酶P450(cytochromeP450,CYP450)及UDP葡萄糖醛酸转移酶(UDP-glucuronosyltransferase, UGT)修饰而来。植物中天然木栓酮及其衍生物的含量极低,传统的萃取分离和化学合成效率低、能耗高且污染环境,因此,利用酿酒酵母作为宿主菌生产木栓酮及其衍生物是一种高效且环保的策略。本文从增加前体含量、提高酶活性和产物合成的亚细胞定位等方面介绍并展望了木栓酮在酿酒酵母中高效生产的策略,并介绍了目前几种常见的木栓酮衍生物研究现状,从根据碳骨架相似性挖掘CYP450、蛋白质工程改造CYP450和合成代谢基因簇的挖掘等方面展望了木栓酮衍生物的合成途径解析的新思路。     

Production of podophyllotoxin from Podophyllum hexandrum: a potential natural product for clinically useful anticancer drugs

Podophyllum hexandrum Royle of family Berberidaceae is an endangered medicinal plant. Rhizome ofP.hexandrum contains several lignans which posses antitumor activity. Podphyllotoxin is the most active cytotoxic natural product. It is used as starting compound for the synthesis of anticancer drug etoposide and teniposide. Podophyllotoxin acts as an inhibitor of microtubule assembly. These drugs are used for lung cancer, testicular cancer, neuroblastoma, hepatoma and other tumors. Besides this, it also shows antiviral activities by interfering with some critical viral processes. Availabilityof podophyllotoxin from plants has its limitations because of its intense collection from nature and lack of organized cultivation. The chemical synthesis of podophyllotoxin is considered to be very complicated as yet. The use of biotechnological approaches for the production of podophyllotoxin using cell cultures, organ cultures, and biotransformation route or by manipulating biosynthetic pathway proves to be an attractive alternative for production of podophyllotoxin. The present paper discusses the current status of research, limitations and future prospects for theproduction of podophyllotoxinin vitro.     

New insights and novel developments in clostridial acetone/butanol/isopropanol fermentation

Clostridial acetone/butanol fermentation used to rank second only to ethanol fermentation by yeast in its scale of production and thus is one of the largest biotechnological processes known. Its decline since about 1950 has been caused by increasing substrate costs and the availability of much cheaper feedstocks for chemical solvent synthesis by the petrochemical industry. The so-called oil crisis in 1973 led to renewed interest in novel fermentation and product recovery technologies as well as in the metabolism and genetics of the bacterial species involved. As a consequence, almost all of the enzymes leading to solvent formation are known, their genes have been sequenced (in fact, Clostridium acetobutylicum has been recently included in the microbial genome sequencing project), the regulatory mechanisms controlling solventogenesis have begun to emerge and recombinant DNA techniques have been developed for these clostridia to construct specific production strains. In parallel, cheap agricultural-waste-based feedstocks have been exploited for their potential as novel substrates, continuous culture methods have been successfully established and new on-line product recovery technologies are now available, such as gas stripping, liquid/liquid extraction, and membrane-based methods. In combination with these achievements, a reintroduction of acetone/butanol fermentation on an industrial scale seems to be economically feasible, a view that is supported by a new pilot plant in Austria recently coming into operation. Received: 18 December 1997 / Received revision: 27 January 1998 / Accepted: 27 January 1998     

Production of 2-phenylethanol and 2-phenylethylacetate from L-phenylalanine by coupling whole-cell biocatalysis with organophilic pervaporation

An integrated bioprocess for the production of the natural rose-like aroma compounds, 2-phenylethanol (2-PE) and 2-phenylethylacetate (2-PEAc), from L-phenylalanine (L-phe) with yeasts was investigated. The hydrophobicity of the products leads to product inhibition, which can be compensated by in situ product removal (ISPR). An organophilic pervaporation unit, equipped with a polyoctylmethylsiloxane (POMS) membrane, was coupled via a bypass to a bioreactor and proved to be a suitable technique for the in situ removal of high-boiling products from culture broth. With batch cultures of the thermotolerant yeast Kluyveromyces marxianus CBS 600 in a standard medium at 35 degrees C, the use of pervaporation resulted in a double 2-PE concentration (2.2 g/L) and 1.3 g/L 2-PEAc, which only accumulated transiently in low concentrations during cultivation without ISPR. Using a previously optimized medium, the variation of the temperature from 30 degrees C to 40 degrees C caused an increase in the total conversion yield from 63% to 79%, corresponding to total product concentrations of 5.23 and 5.85 g/L, respectively. In the 40 degrees C batch experiment, the volumetric productivity (2-PE + 2-PEAc) during the exponential phase was 5.2 mmol/L h. While for 2-PE, there is still potential for further optimization, the more hydrophobic 2-PEAc was nearly completely removed from the aqueous culture broth (enrichment factor >400), resulting in highly aroma-enriched permeates. Due to the temperature-correlated performance of the pervaporation, the bioconversion was still efficient even at 45 degrees C (conversion yield: 69%). Surprisingly, at 45 degrees C, the molar ratio of the two products inverted and 2-PEAc turned out to be the main product (4.0 g/L), which opens easy control of the reaction's selectivity by external means. Retrofitting the process with interim heating and cooling equipment to use different temperature levels for cultivation and pervaporation resulted in a decreased yield and product concentration caused by multiple stress factors. The medium composition affected the pervaporation efficiency with molasses acting detrimental.