Production Of 2-phenylethanol Research Articles

Biosynthesis of 2-phenylethanol from styrene using engineered Escherichia coli whole cells.

Biosynthesis of 2-phenylethanol from styrene using engineered Escherichia coli whole cells.

Effect of the initial glucose concentration on the performance of ice wine fermentation of Vidal grape juice

Ice wine is produced from concentrated grape juice obtained by the natural freezing and pressing of grapes. The high sugar content of this juice has an impact on fermentation. To investigate the impact of the initial sugar concentration on the fermentation of ice wine, the initial sugar concentration of Vidal ice grape juice was adjusted to 370, 450, 500 and 550 g/L by the addition of glucose. The fermentation process was conducted at a temperature of 18 °C with Zymaflore ST yeast. To ensure optimal fermentation, the yeast assimilable nitrogen (YAN) in the grape juice was adjusted to 350 mg/L using a commercial nitrogen source. The initial sugar concentrations (370, 450, 500, and 550 g/L) were significantly negatively correlated with the number of viable yeast during active fermentation, the total consumption of sugar and YAN, and ethanol production (130.26, 117.83, 106.14, and 90.85 g/L) but positively correlated with the production of acetic acid (0.82–2.86 g/L) and acetaldehyde. Fermentation with initial sugar concentrations ≥ 500 g/L (500 and 550 g/L) produced ethanol (106.14 and 90.85 g/L) and excessive acetic acid (2.14 and 2.86 g/L), inhibited the esterification of n-butanol and n-hexanol, and promoted the production of isopentanol, isoamyl acetate, diethyl succinate, ethyl hexanoate and ethyl octanoate while increasing the production of 2-phenylethanol and isobutanol. The initial sugar concentration affected the production of glycerol, isobutanol, ethyl acetate, and diethyl succinate, but it did not affect the final contents of the ice wine. This study examined fermentation dynamics, including substrate consumption, yeast cell production, and the production of important metabolites such as ethanol, acetic acid, higher alcohols, and esters. Appropriate initial concentrations of YAN and sugar (below 500 g/L) and a relatively high ratio of glucose to fructose (between 1:1 and 1.7:1) is preferable for effective ice wine fermentation.

Biotechnological 2-Phenylethanol Production: Recent Developments.

2-Phenylethanol (2-PE) is a key flavor compound with a rose-like scent, used in the cosmetics, perfume, home care and food industries. This aroma compound can be obtained naturally from various flowers, however chemical synthesis is the most used route to meet market demand. The increasing interest in natural products has led to the development of more environmentally friendly alternatives for 2-PE production through biotechnological approaches. The most efficient approach involves the biotransformation of L-phenylalanine into 2-PE via the Ehrlich pathway, a process observed in different microorganisms such as yeasts and bacteria. 2-PE produced by this way can be considered as natural. However, due to the toxicity of the aroma to the producing microorganism, low production yields are typically obtained, motivating efforts to develop production processes that can overcome this bottleneck, enhance 2-PE yields and reduce the production costs. This review presents and discusses the latest advances in the bioproduction of 2-PE through microbial fermentation, in terms of producing strains, the optimization of cultivation processes, strategies to mitigate product toxicity, and the use of low value feedstocks. Novel applications for 2-PE are also highlighted.

Inhibition Control by Continuous Extractive Fermentation Enhances De Novo 2-Phenylethanol Production by Yeast.

Current microbial cell factory methods for producing chemicals from renewable resources primarily rely on (fed-)batch production systems, leading to the accumulation of the desired product. Industrially relevant chemicals like 2-phenylethanol (2PE), a flavor and fragrance compound, can exhibit toxicity at low concentrations, inhibit the host activity, and negatively impact titer, rate, and yield. Batch liquid-liquid (L-L) In Situ Product Removal (ISPR) was employed to mitigate inhibition effects, but was not found sufficient for industrial-scale application. Here, we demonstrated that continuous selective L-L ISPR provides the solution for maintaining the productivity of de novo produced 2PE at an industrial pilot scale. A unique bioreactor concept called "Fermentation Accelerated by Separation Technology" (FAST) utilizes hydrostatic pressure differences to separate aqueous- and extractant streams within one unit operation, where both production and product extraction take place - allowing for the control of the concentration of the inhibiting compound. Controlled aqueous 2PE levels (0.43 ± 0.02 g kg-1) and extended production times (>100 h) were obtained and co-inhibiting by-product formation was reduced, resulting in a twofold increase of the final product output of batch L-L ISPR approaches. This study establishes that continuous selective L-L ISPR, enabled by FAST, can be applied for more economically viable production of inhibiting products.

Ethanol and 2-phenylethanol production by bee-isolated Meyerozyma caribbica strains

Investigating the biotechnological potential of wild microorganisms is paramount for optimizing bioprocesses. Given this premise, we looked for yeasts in Brazilian native stingless bees, considering the recognized potential of pollinating insect-associated microorganisms for the production of volatile organic compounds (VOCs). Two yeast strains of the species Meyerozyma caribbica were isolated from bees Scaptotrigona postica and evaluated for their fermentative capacity. Both yeasts were capable of fermenting sucrose (the main sugar used in the Brazilian ethanol industry) with over 90% efficiency and yields of up to 0.504 g/g. Through an experimental design analysis (CCD), it was verified that the ethanol productivity of these yeasts can also benefit from high concentrations of sucrose and low pH values, desirable traits for microorganisms in this biofuel production. At the same time, CCD analyses also showed the great capacity of these M. caribbica strains to produce another alcohol of broad biotechnological interest, 2-phenylethanol. Interestingly, the statistical analyses demonstrated that greater production of this compound can occur at high sugar concentrations and low availability of nitrogen sources, which can be easily achieved using residual low-cost feedstocks. Thus, our results suggest that these M. caribbica strains may be efficiently used in both ethanol and 2-phenylethanol production.

Assessment of in situ product recovery techniques to enhance 2-phenylethanol production by Acinetobacter soli ANG344B

Assessment of in situ product recovery techniques to enhance 2-phenylethanol production by Acinetobacter soli ANG344B

Engineering Living Material for Controlled Fragrance Release Utilizing Kluyveromyces marxianus CBS6556 and Adaptive Hydrogel.

The demand for controllable fragrance materials is substantial owing to their potential to impart enduring scents in a variety of applications. However, the practical application of such materials has been limited by challenges in tunable morphogenesis, structural variability, and adaptability to diverse conditions. In our study, we introduce a hybrid living material that integrates a genetically engineered strain of Kluyveromyces marxianus CBS6556 with an adaptive hydrogel. The engineered K. marxianus achieved temperature stability in 2-phenylethanol (2-PE) and 2-phenylethyl acetate (2-PEAc) production by expressing relevant genes in the 2-PE metabolic pathway using the high-temperature preferential promoter SSE1. The enhanced water retention capacity supports the metabolic activities of the encapsulated yeast cells, ensuring their survival and functionality over an extended period. Fragrance-releasing living material (FLM) is designed to controllably emit fragrance 2-PE by adjusting the microbial concentration within the hydrogel matrix. The FLM exhibits versatile adhesion capabilities, effectively binding to a spectrum of surfaces such as wood, textiles, and glass as well as to natural substrates like leaves. This adaptability enhances the material's applicability across various settings. Furthermore, FLM can be crafted into various forms, including microbeads, fibers, and films. This research opens up new horizons for controlled fragrance release of living materials.

Metabolic and tolerance engineering of Komagataella phaffii for 2-phenylethanol production through genome-wide scanning

Background2-Phenylethanol (2-PE) is one of the most widely used spices. Recently, 2-PE has also been considered a potential aviation fuel booster. However, the lack of scientific understanding of the 2-PE biosynthetic pathway and the cellular response to 2-PE cytotoxicity are the most important obstacles to the efficient biosynthesis of 2-PE.ResultsHere, metabolic engineering and tolerance engineering strategies were used to improve the production of 2-PE in Komagataella phaffii. First, the endogenous genes encoding the amino acid permease GAP1, aminotransferase AAT2, phenylpyruvate decarboxylase KDC2, and aldehyde dehydrogenase ALD4 involved in the Ehrlich pathway and the 2-PE stress response gene NIT1 in K. phaffii were screened and characterized via comparative transcriptome analysis. Subsequently, metabolic engineering was employed to gradually reconstruct the 2-PE biosynthetic pathway, and the engineered strain S43 was obtained, which produced 2.98 g/L 2-PE in shake flask. Furthermore, transcriptional profiling analyses were utilized to screen for novel potential tolerance elements. Our results demonstrated that cells with knockout of the PDR12 and C4R2I5 genes exhibited a significant increase in 2-PE tolerance. To confirm the practical applications of these results, deletion of the PDR12 and C4R2I5 genes in the hyper 2-PE producing strain S43 dramatically increased the production of 2-PE by 18.12%, and the production was 3.54 g/L.ConclusionThis is the highest production of 2-PE produced by K. phaffii via l-phenylalanine conversion. These identified K. phaffii endogenous elements are highly conserved in other yeast species, suggesting that manipulation of these homologues might be a useful strategy for improving aromatic alcohol production. These results also enrich the understanding of aromatic compound biosynthetic pathways and 2-PE tolerance, and provide new elements and strategies for the synthesis of aromatic compounds by microbial cell factories.

Comprehensive investigations of 2-phenylethanol production by the filamentous fungus Annulohypoxylon stygium

2-Phenylethanol (2-PE) is an aromatic compound with a rose-like fragrance that is widely used in food and other industries. Yeasts have been implicated in the biosynthesis of 2-PE; however, few studies have reported the involvement of filamentous fungi. In this study, 2-PE was detected in Annulohypoxylon stygium mycelia grown in both potato dextrose broth (PDB) and sawdust medium. Among the 27 A. stygium strains investigated in this study, the strain “Jinjiling” (strain S20) showed the highest production of 2-PE. Under optimal culture conditions, the concentration of 2-PE was 2.33 g/L. Each of the key genes in Saccharomyces cerevisiae shikimate and Ehrlich pathways was found to have homologous genes in A. stygium. Upon the addition of L-phenylalanine to the medium, there was an upregulation of all key genes in the Ehrlich pathway of A. stygium, which was consistent with that of S. cerevisiae. A. stygium as an associated fungus provides nutrition for the growth of Tremella fuciformis and most spent composts of T. fuciformis contain pure A. stygium mycelium. Our study on the high-efficiency biosynthesis of 2-PE in A. stygium offers a sustainable solution by utilizing the spent compost of T. fuciformis and provides an alternative option for the production of natural 2-PE.Key points• Annulohypoxylon stygium can produce high concentration of 2-phenylethanol.• The pathways of 2-PE biosynthesis in Annulohypoxylon stygium were analyzed.• Spent compost of Tremella fuciformis is a potential source for 2-phenylethanol.

Metabolic and enzymatic engineering approach for the production of 2-phenylethanol in engineered Escherichia coli

Metabolic and enzymatic engineering approach for the production of 2-phenylethanol in engineered Escherichia coli

Highly efficient production of 2-phenylethanol by wild-type Saccharomyces bayanus strain

Highly efficient production of 2-phenylethanol by wild-type Saccharomyces bayanus strain

Integrated biorefinery approach for utilization of wood waste into levulinic acid and 2-Phenylethanol production under mild treatment conditions

Integrated biorefinery approach for utilization of wood waste into levulinic acid and 2-Phenylethanol production under mild treatment conditions

Exploring the biotechnological potential of Acinetobacter soli ANG344B: A novel bacterium for 2-phenylethanol production

Exploring the biotechnological potential of Acinetobacter soli ANG344B: A novel bacterium for 2-phenylethanol production

Multi-mode enhancement and Co-fermentation mechanism of 2-phenylethanol production by Kluyveromyces marxianus

Multi-mode enhancement and Co-fermentation mechanism of 2-phenylethanol production by Kluyveromyces marxianus

Coupling dairy wastewaters for nutritional balancing and water recycling: sustainable heterologous 2-phenylethanol production by engineered cyanobacteria.

Microalgae biotechnology is hampered by the high production costs and the massive usage of water during large-volume cultivations. These drawbacks can be softened by the production of high-value compounds and by adopting metabolic engineering strategies to improve their performances and productivity. Today, the most sustainable approach is the exploitation of industrial wastewaters for microalgae cultivation, which couples valuable biomass production with water resource recovery. Among the food processing sectors, the dairy industry generates the largest volume of wastewaters through the manufacturing process. These effluents are typically rich in dissolved organic matter and nutrients, which make it a challenging and expensive waste stream for companies to manage. Nevertheless, these rich wastewaters represent an appealing resource for microalgal biotechnology. In this study, we propose a sustainable approach for high-value compound production from dairy wastewaters through cyanobacteria. This strategy is based on a metabolically engineered strain of the model cyanobacterium Synechococcus elongatus PCC 7942 (already published elsewhere) for 2-phenylethanol (2-PE). 2-PE is a high-value aromatic compound that is widely employed as a fragrance in the food and cosmetics industry thanks to its pleasant floral scent. First, we qualitatively assessed the impact of four dairy effluents on cyanobacterial growth to identify the most promising substrates. Both tank-washing water and the liquid effluent of exhausted sludge resulted as suitable nutrient sources. Thus, we created an ideal buffer system by combining the two wastewaters while simultaneously providing balanced nutrition and completely avoiding the need for fresh water. The combination of 75% liquid effluent of exhausted sludge and 25% tank-washing water with a fine-tuning ammonium supplementation yielded 180mgL-1 of 2-PE and a biomass concentration of 0.6gDW L-1 within 10days. The mixture of 90% exhausted sludge and 10% washing water produced the highest yield of 2-PE (205mgL-1) and biomass accumulation (0.7gDW L-1), although in 16days. Through these treatments, the phosphates were completely consumed, and nitrogen was removed in a range of 74%-77%. Overall, our approach significantly valorized water recycling and the exploitation of valuable wastewaters to circularly produce marketable compounds via microalgae biotechnology, laying a promising groundwork for subsequent implementation and scale-up.

Gene Editing of Candida glycerinogenes by Designed Toxin-Antitoxin Cassette.

Candida glycerinogenes is an industrial yeast with excellent multistress resistance. However, due to the diploid genome and the lack of meiosis and screening markers, its molecular genetic operation is limited. Here, a gene editing system using the toxin-antitoxin pair relBE from the type II toxin-antitoxin system in Escherichia coli as a screening marker was constructed. The RelBE complex can specifically and effectively regulate cell growth and arrest through a conditionally controlled toxin RelE switch, thereby achieving the selection of positive recombinants. The constructed editing system achieved precise gene deletion, replacement, insertion, and gene episomal expression in C.glycerinogenes. Compared with the traditional amino acid deficiency complementation editing system, this editing system produced higher biomass and the gene deletion efficiency was increased by 3.5 times. Using this system, the production of 2-phenylethanol by C.glycerinogenes was increased by 11.5-13.5% through metabolic engineering and tolerance engineering strategies. These results suggest that the stable gene editing system based on toxin-antitoxin pairs can be used for gene editing of C.glycerinogenes to modify metabolic pathways and promote industrial applications. Therefore, the constructed gene editing system is expected to provide a promising strategy for polyploid industrial microorganisms lacking gene manipulation methods.

Endogenous production of 2-phenylethanol by Cunninghamella echinulata inhibits biofilm growth of the fungus

The filamentous fungus Cunninghamella echinulata is a model of mammalian xenobiotic metabolism. Under certain conditions it grows as a biofilm, which is a natural form of immobilisation and enables the fungus to catalyse repeated biotransformations. Putative signalling molecules produced by other Cunninghamella spp., such as 3-hydroxytyrosol and tyrosol, do not affect the biofilm growth of C. echinulata, suggesting that it employs a different molecule to regulate biofilm growth. In this paper we report that 2-phenylethanol is produced in higher concentrations in planktonic cultures of C. echinulata than when the fungus is grown as a biofilm. We demonstrate that exogenously added 2-phenylethanol inhibits biofilm growth of C. echinulata but has no effect on planktonic growth. Furthermore, we show that addition of 2-phenylethanol to established C. echinulata biofilm causes detachment. Therefore, we conclude that this molecule is produced by the fungus to regulate biofilm growth.

A novel toolbox for precise regulation of gene expression and metabolic engineering in Bacillus licheniformis

A novel toolbox for precise regulation of gene expression and metabolic engineering in Bacillus licheniformis

Co-culture of Kluyveromyces marxianus and Meyerozyma guilliermondii with In Situ Product Recovery of 2-Phenylethanol.

Production of 2-phenylethanol (2-PE) via Kluyveromyces marxianus is well-established. However, co-culture with other microbes in combination with in situ product recovery (ISPR) yields improved selectivity and volumetric productivity. Fermentation ofK. marxianus (MUCL 53775) with direct inclusion of absorptive polymer Hytrel3548 achieved ISPR, but accumulation of the byproduct phenylethyl acetate (PEA) was strongly favored. Co-culture of K. marxianus (MUCL 53775) with Meyerozyma guilliermondii (MUCL 28072) with ISPR limited PEA production, thereby improving the 2-PE selectivity from 13 to 90%, compared to a pure culture of K. marxianus (MUCL 53775) under similar conditions. This improved the volumetric productivity by 85% compared to 2-PE ISPR with a pure culture of K. marxianus. This is the first report of co-culture in a two-phase fermentation for 2-PE bioproduction and demonstrates that interactions between co-culture and ISPR techniques can modulate bioproduction between 2-PE and byproduct PEA, and this technique will be explored for other strain combinations and for other high-value molecules of interest.

Dynamic genome-scale modeling of Saccharomyces cerevisiae unravels mechanisms for ester formation during alcoholic fermentation.

Fermentation employing Saccharomyces cerevisiae has produced alcoholic beverages and bread for millennia. More recently, S. cerevisiae has been used to manufacture specific metabolites for the food, pharmaceutical, and cosmetic industries. Among the most important of these metabolites are compounds associated with desirable aromas and flavors, including higher alcohols and esters. Although the physiology of yeast has been well-studied, its metabolic modulation leading to aroma production in relevant industrial scenarios such as winemaking is still unclear. Here we ask what are the underlying metabolic mechanisms that explain the conserved and varying behavior of different yeasts regarding aroma formation under enological conditions? We employed dynamic flux balance analysis (dFBA) to answer this key question using the latest genome-scale metabolic model (GEM) of S. cerevisiae. The model revealed several conserved mechanisms among wine yeasts, for example, acetate ester formation is dependent on intracellular metabolic acetyl-CoA/CoA levels, and the formation of ethyl esters facilitates the removal of toxic fatty acids from cells using CoA. Species-specific mechanisms were also found, such as a preference for the shikimate pathway leading to more 2-phenylethanol production in the Opale strain as well as strain behavior varying notably during the carbohydrate accumulation phase and carbohydrate accumulation inducing redox restrictions during a later cell growth phase for strain Uvaferm. In conclusion, our new metabolic model of yeast under enological conditions revealed key metabolic mechanisms in wine yeasts, which will aid future research strategies to optimize their behavior in industrial settings.