Production Of Aromatic Compounds Research Articles

Mejorando la biosíntesis de acetato de isoamilo por Pichia fermentans

Microbial production of aroma compounds is a promising alternative to extracting plants or chemical synthesis. The yeast Pichia fermentans ITD-00165 has demonstrated a high potential to produce isoamyl acetate. The yeast was cultured in a bioreactor on a chemically defined medium to evaluate the influence of airflow (0.5, 0.75, and 1.0 vvm) and agitation speed (150, 175, and 200 rpm) on the isoamyl acetate production. Isoamyl acetate was retained using an in situ entrapping system, which was obtained, adding a cap of n-decane in a ratio of 80:20 (medium:decane). This work demonstrated the relevance of well-controlled aeration to enhance isoamyl acetate production using isoamyl alcohol as a precursor. Culture operating near to 0.71 vvm and 168 rpm (kLa = 0.16 min-1) achieved production of 2.14 g L-1, which means an almost 2.5-fold increase in respect to the higher value previously reported. On the other hand, the minimum production occurred at the best aeration conditions (kLa = 0.22 min-1), meaning that excessive aeration could negatively affect the aroma production. Nevertheless, it appears that the concomitant ethanol production negatively affects aroma production.

One-pot enzymatic synthesis of 2-deoxy-scyllo-inosose from d-glucose and polyphosphate.

2-Deoxy-scyllo-inosose (2DOI, [2S,3R,4S,5R]-2,3,4,5-tetrahydroxycyclohexan-1-one) is a biosynthetic intermediate of 2-deoxystreptamine-containing aminoglycoside antibiotics, including butirosin, kanamycin, and neomycin. In producer microorganisms, 2DOI is constructed from d-glucose 6-phosphate (G6P) by 2-deoxy-scyllo-inosose synthase (DOIS) with the oxidized form of nicotinamide adenine dinucleotide (NAD+). 2DOI is also known as a sustainable biomaterial for production of aromatic compounds and a chiral cyclohexane synthon. In this study, a one-pot enzymatic synthesis of 2DOI from d-glucose and polyphosphate was investigated. First, 3 polyphosphate glucokinases (PPGKs) were examined to produce G6P from d-glucose and polyphosphate. A PPGK derived from Corynebacterium glutamicum (cgPPGK) was found to be suitable for G6P production under ordinary enzymatic conditions. Next, 7 DOISs were examined for the one-pot enzymatic reaction. As a result, cgPPGK and BtrC, the latter of which is a DOIS derived from the butirosin producer Bacillus circulans, achieved nearly full conversion of d-glucose to 2DOI in the presence of polyphosphate.

Towards aromatics from biomass: Prospective Life Cycle Assessment of bio-based aniline

Bio-based chemicals are seen as a potential stepping-stone towards a more sustainable chemical industry. However, while bio-based routes are already available for various organic alcohols and acids, the direct bio-based production of aromatic compounds has been difficult so far. Recently, the first bio-based process for the production of the important aromatic aniline has been realized. The process produces bio-based aniline via two-stages: first, sugar is fermented by Corynebacterium Glutamicum to aminobenzoic acid, which is then catalytically decarboxylated to aniline. In this study, we present a prospective Life Cycle Assessment for bio-based aniline production to evaluate the environmental potential compared to fossil-based aniline production. Our results suggest that the bio-based production could reduce the global warming impacts of aniline from cradle-to-grave by 35–69% relative to fossil-based production, depending on the type of biomass feedstock. However, bio-based aniline could also substantially increase eutrophication and acidification, a trade-off commonly observed for bio-based processes. Thus, the novel production route is promising and expands the scope of bio-based chemicals towards aromatics. • First Life Cycle Assessment of bio-based aniline production. • Bio-based production chain in three world regions considering four feedstocks. • Significantly lower global warming impacts possible by bio-based aniline. • Reduced global warming impacts robust regarding indirect land-use change. • Feedstock demand for global aniline demand could be satisfied.

Current Advances in the Bacterial Toolbox for the Biotechnological Production of Monoterpene-Based Aroma Compounds

Monoterpenes are plant secondary metabolites, widely used in industrial processes as precursors of important aroma compounds, such as vanillin and (−)-menthol. However, the physicochemical properties of monoterpenes make difficult their conventional conversion into value-added aromas. Biocatalysis, either by using whole cells or enzymes, may overcome such drawbacks in terms of purity of the final product, ecological and economic constraints of the current catalysis processes or extraction from plant material. In particular, the ability of oxidative enzymes (e.g., oxygenases) to modify the monoterpene backbone, with high regio- and stereo-selectivity, is attractive for the production of “natural” aromas for the flavor and fragrances industries. We review the research efforts carried out in the molecular analysis of bacterial monoterpene catabolic pathways and biochemical characterization of the respective key oxidative enzymes, with particular focus on the most relevant precursors, β-pinene, limonene and β-myrcene. The presented overview of the current state of art demonstrates that the specialized enzymatic repertoires of monoterpene-catabolizing bacteria are expanding the toolbox towards the tailored and sustainable biotechnological production of values-added aroma compounds (e.g., isonovalal, α-terpineol, and carvone isomers) whose implementation must be supported by the current advances in systems biology and metabolic engineering approaches.

Secondary Aroma: Influence of Wine Microorganisms in Their Aroma Profile.

Aroma profile is one of the main features for the acceptance of wine. Yeasts and bacteria are the responsible organisms to carry out both, alcoholic and malolactic fermentation. Alcoholic fermentation is in turn, responsible for transforming grape juice into wine and providing secondary aromas. Secondary aroma can be influenced by different factors; however, the influence of the microorganisms is one of the main agents affecting final wine aroma profile. Saccharomyces cerevisiae has historically been the most used yeast for winemaking process for its specific characteristics: high fermentative metabolism and kinetics, low acetic acid production, resistance to high levels of sugar, ethanol, sulfur dioxide and also, the production of pleasant aromatic compounds. Nevertheless, in the last years, the use of non-saccharomyces yeasts has been progressively growing according to their capacity to enhance aroma complexity and interact with S. cerevisiae, especially in mixed cultures. Hence, this review article is aimed at associating the main secondary aroma compounds present in wine with the microorganisms involved in the spontaneous and guided fermentations, as well as an approach to the strain variability of species, the genetic modifications that can occur and their relevance to wine aroma construction.

Effects of antioxidant and elemental sulfur additions at crushing on aroma profiles of Pinot Gris, Chardonnay and Sauvignon Blanc wines

The antioxidants sulfur dioxide (50 ppm) and ascorbic acid (100 ppm) were added to grapes soon after harvest at crushing. The chemical composition and sensory profile of Sauvignon Blanc, Pinot Gris and Chardonnay wines were examined, made from grapes collected at three different sites for each variety. With good antioxidant protection of the juices, reflected in low absorbances at 420 nm, remarkable increases in the polyfunctional mercaptans, 3-mercaptohexanol (3MH) and its acetate ester (3MHA), were seen in the wines. Moreover, high levels of these compounds were produced in the Pinot Gris and Chardonnay wines, equally high as with Sauvignon Blanc. The Pinot Gris wines maintained varietal characteristics in sensory profiles, even with high levels of polyfunctional mercaptans. When elemental sulfur was included with the grapes at crushing, extra increases in polyfunctional mercaptans were observed. However, this led to the production of unwanted reductive aroma compounds in some wines.

Combinatorial analysis of population dynamics, metabolite levels and malolactic fermentation in Saccharomyces cerevisiae/ Lachancea thermotolerans mixed fermentations

The outcome of co- or sequential inoculation of Lachancea thermotolerans in winemaking remains unpredictable due to a lack of integrated data regarding the impact of grape juice composition on L. thermotolerans fermentation behaviour. Here, we investigate the impact of nitrogen composition on fermentation characteristics and aroma compound production in grape juice sequentially inoculated with commercial L. thermotolerans and S. cerevisiae strains. Subsequently, all treatments were subjected to malolactic fermentation (MLF) using two commercial strains of Oenococcus oeni. Addition of amino acids led to faster growth for S. cerevisiae fermentations, compared to the nitrogen-equivalent addition of diammonium phosphate (DAP). L. thermotolerans persistence in the mixed fermentations was significantly higher following DAP addition, with higher glycerol and lactic acid production. Interestingly, the lower total Nitrogen content in DAP-treated musts compared to other treatments did not alter the subsequent growth of S. cerevisiae. MLF was more similar between musts fermented with L. thermotolerans, regardless of nutrient regime, whereas significant differences in MLF completion times were observed for different nitrogen treatments in S. cerevisiae fermentations. Collectively, the data present an integrated view of the impact of nitrogen treatment on multispecies co-inoculation (growth kinetics and aromatic outcomes) and the downstream impact on MLF.

Dry-aged beef manufactured in Japan: Microbiota identification and their effects on product characteristics

We aimed to determine the mold, yeast, and bacterial distributions in dry-aged beef (DAB) manufactured in Hokkaido, Japan, and to study their effects on meat quality compared to wet-aged beef (WAB). Two rump blocks from Holstein steer were dry- and wet-aged for 35 days at 2.9 °C and 90% RH. The psychrophilic molds Mucor flavus and Helicostylum pulchrum and other fungi (Penicillium sp. and Debaryomyces sp.) appeared on the crust of DAB, while lactic acid bacteria and coliforms were suppressed in the inner part of the meat. The composition of C16:0, C18:0, and C18:1 fatty acids did not differ between DAB and WAB, while more C17:0 fatty acids were detected in DAB. Dry aging suppressed acids and increased the production of various aroma compounds with mushroom-like, nutty, and other pleasant flavors. The meat quality and free amino acid (FAA) contents of DAB and WAB did not differ significantly. In this study, we identified major molds on DAB, which might contribute to an increase in aroma. Keywords: dry-aged beef; Mucor flavus; Helicostylum pulchrum; psychrophilic mold; meat quality; volatile aroma compounds.

The non-Saccharomyces yeast Pichia kluyveri for the production of aromatic volatile compounds in alcoholic fermentation.

Alcoholic fermentation is influenced by yeast strain, culture media, substrate concentration and fermentation conditions, which contribute to taste and aroma. Some non-Saccharomyces yeasts are recognized as volatile compound producers that enrich aromatic profile of alcoholic beverages. In this work, 21 strains of Pichia kluyveri isolated from different fermentative processes and regions were evaluated. A principal component analysis (PCA) showed statistical differences between strains mainly associated with the variety and concentration of the compounds produced. From the PCA, two strains (PK1 and PK8) with the best volatile compound production were selected to evaluate the impact of culture media (M12 medium and Agave tequilana juice), stirring speeds (100 and 250rpm) and temperatures (20°C, 25°C and 30°C). Increased ester production was observed at 250rpm. Greatest effect in alcohols and ester production was found with A. tequilana, identifying PK1 as higher alcohol producer, and PK8 as better ester producer. Regarding temperature, PK1 increased ester production with decreased fermentation temperature. PK8 presented maximum levels of ethyl acetate and ethyl dodecanoate at 20°C, and finally isoamyl acetate increased its production at 30°C. Therefore,P. kluyveri strains are of great interest to produce different aromatic profiles that are affected by factors including medium, agitation and temperature.

Recent Research Progress in the Microbial Production of Aromatic Compounds Derived from L-Tryptophan

Recent Research Progress in the Microbial Production of Aromatic Compounds Derived from L-Tryptophan

The Selective Conversion of Methyl and Ethyl Acetate to High Content Alkyl Aromatic Hydrocarbons over H-ZSM5

This research is devoted to a catalytic process using the H-ZSM5 catalyst for the conversion of methyl and ethyl acetate to hydrocarbon aromatics. These reactions are carried out in a fixed bed reactor under atmospheric pressure at 370°C. The distribution of products was measured by GC-Mass spectrometer. The variation of weight hourly space velocity (WHSV) on the conversion of these esters to aromatic hydrocarbons showed a significant effect on carbon distribution. The deactivation catalyst by time was monitored using product selectivity and conversion. The production of alkyl and poly alkyl aromatic compounds was formed under controlled conditions. The advantages of these methods are the formation of a higher concentration of octane number booster poly alkyl aromatic compounds (mono-aromatics) from esters as starting materials. Moreover, the catalyst lifetime on stream was investigated and exhibited longer catalyst lifetime for ethyl acetate conversion than methyl acetate.

Electronic Nose for Monitoring Odor Changes of Lactobacillus Species during Milk Fermentation and Rapid Selection of Probiotic Candidates.

Probiotic bacteria have been associated with a unique production of aroma compounds in fermented foods but rapid methods for discriminating between foods containing probiotic, moderately probiotic, or non-probiotic bacteria remain aloof. An electronic nose (e-nose) is a high-sensitivity instrument capable of non-invasive volatile measurements of foods. In our study, we applied the e-nose to differentiate probiotic, moderately probiotic, and non-probiotic Lactobacillus bacteria strains at different fermentation time points (0th, 4th, and 11th) of milk fermentation. The pH of the changing milk medium was monitored with their corresponding increase in microbial cell counts. An e-nose with two gas chromatographic columns was used to develop classification models for the different bacteria groups and time points and to monitor the formation of the aromatic compounds during the fermentation process. Results of the e-nose showed good classification accuracy of the different bacteria groups at the 0th (74.44% for column 1 and 82.78% for column 2), the 4th (89.44% for column 1 and 92.22% for column 2), and the 11th (81.67% for column 1 and 81.67% for column 2) hour of fermentation. The loading vectors of the classification models showed the importance of some specific aroma compounds formed during the fermentation. Results show that aroma monitoring of the fermentation process with the e-nose is a promising and reliable analytical method for the rapid classification of bacteria strains according to their probiotic activity and for the monitoring of aroma changes during the fermentation process.

A Multi-Enzyme Cascade for the Production of High-Value Aromatic Compounds

Cascade reactions are the basis of life in nature and are adapted to research and industry in an increasing manner. The focus of this study is the production of the high-value aromatic ester cinnamyl cinnamate, which can be applied in flavors and fragrances. A three-enzyme cascade was established to realize the synthesis, starting from the corresponding aldehyde with in situ cofactor regeneration in a two-phase system. After characterization of the enzymes, a screening with different organic solvents was carried out, whereby xylene was found to be the most suitable solvent for the second phase. The reaction stability of the formate dehydrogenase (FDH) from Candida boidinii is the limiting step during cofactor regeneration. However, the applied enzyme cascade showed an overall yield of 54%. After successful application on lab scale, the limitation by the FDH was overcome by immobilization of the enzymes and an optimized downstream process, transferring the cascade into a miniplant. The upscaling resulted in an increased yield for the esterification, as well as overall yields of 37%.

Evolving the core microbial community in pit mud based on bioturbation of fortified Daqu.

Directional stress is an effective measure to change the community structure and improve the bioactivity of pit mud (PM). In this study, the addition of fortified Daqu to artificial PM (APM) was intended to disturb the microbial community and further affect metabolites. To evaluate the effect of fortified Daqu on culturing APM, the microbial communities of APM with or without the addition of fortified Daqu were investigated by fluorescence in situ hybridization and Illumina MiSeq. The results indicated that microbes (Clostridium sp., Clostridium kluyveri, hydrogenotrophic methanogens, and acetotrophic methanogens) related to the production of key aroma compounds increased notably when fortified Daqu was added. In particular, the hydrogenotrophic and acetotrophic methanogens increased by 6.19- and 4.63-fold after 30days of culture. Subsequently, metabolites (organic acids, volatile compounds) were also analyzed by HPLC (high-performance liquid chromatography) and HS-SPME-GC-MS (headspace solid phase microextraction - gas chromatography - mass spectrometry). The results showed that the content of butyric acid and hexanoic acid was significantly higher when fortified Daqu was added to APM. In addition, the proportion of esters and phenols was also higher than in APM without fortified Daqu. A survey of the microbial compositions of APMs with or without added fortified Daqu indicated that the microbial community evolves into a functional community favoring liquor brewing. We have developed a novel process by disturbing the community diversity.

Metabolic Responses to Waterlogging Differ between Roots and Shoots and Reflect Phloem Transport Alteration in Medicago truncatula.

Root oxygen deficiency that is induced by flooding (waterlogging) is a common situation in many agricultural areas, causing considerable loss in yield and productivity. Physiological and metabolic acclimation to hypoxia has mostly been studied on roots or whole seedlings under full submergence. The metabolic difference between shoots and roots during waterlogging, and how roots and shoots communicate in such a situation is much less known. In particular, the metabolic acclimation in shoots and how this, in turn, impacts on roots metabolism is not well documented. Here, we monitored changes in the metabolome of roots and shoots of barrel clover (Medicago truncatula), growth, and gas-exchange, and analyzed phloem sap exudate composition. Roots exhibited a typical response to hypoxia, such as γ-aminobutyrate and alanine accumulation, as well as a strong decline in raffinose, sucrose, hexoses, and pentoses. Leaves exhibited a strong increase in starch, sugars, sugar derivatives, and phenolics (tyrosine, tryptophan, phenylalanine, benzoate, ferulate), suggesting an inhibition of sugar export and their alternative utilization by aromatic compounds production via pentose phosphates and phosphoenolpyruvate. Accordingly, there was an enrichment in sugars and a decline in organic acids in phloem sap exudates under waterlogging. Mass-balance calculations further suggest an increased imbalance between loading by shoots and unloading by roots under waterlogging. Taken as a whole, our results are consistent with the inhibition of sugar import by waterlogged roots, leading to an increase in phloem sugar pool, which, in turn, exert negative feedback on sugar metabolism and utilization in shoots.

Multi-modular engineering of Saccharomyces cerevisiae for high-titre production of tyrosol and salidroside.

Tyrosol and its glycosylated product salidroside are important ingredients in pharmaceuticals, nutraceuticals and cosmetics. Despite the ability of Saccharomyces cerevisiae to naturally synthesize tyrosol, high yield from de novo synthesis remains a challenge. Here, we used metabolic engineering strategies to construct S. cerevisiae strains for high-level production of tyrosol and salidroside from glucose. First, tyrosol production was unlocked from feedback inhibition. Then, transketolase and ribose-5-phosphate ketol-isomerase were overexpressed to balance the supply of precursors. Next, chorismate synthase and chorismate mutase were overexpressed to maximize the aromatic amino acid flux towards tyrosol synthesis. Finally, the competing pathway was knocked out to further direct the carbon flux into tyrosol synthesis. Through a combination of these interventions, tyrosol titres reached 702.30 ± 0.41 mg l-1 in shake flasks, which were approximately 26-fold greater than that of the WT strain. RrU8GT33 from Rhodiola rosea was also applied to cells and maximized salidroside production from tyrosol in S. cerevisiae. Salidroside titres of 1575.45 ± 19.35 mg l-1 were accomplished in shake flasks. Furthermore, titres of 9.90 ± 0.06 g l-1 of tyrosol and 26.55 ± 0.43 g l-1 of salidroside were achieved in 5 l bioreactors, both are the highest titres reported to date. The synergistic engineering strategies presented in this study could be further applied to increase the production of high value-added aromatic compounds derived from the aromatic amino acid biosynthesis pathway in S. cerevisiae.

Nitrogen metabolism in three non-conventional wine yeast species: A tool to modulate wine aroma profiles

The positive impact of certain non-Saccharomyces yeasts on the aromatic profile of wines has been well documented in literature and their industrial use in association with S. cerevisiae is now recommended. Competition between non-Saccharomyces species and Saccharomyces cerevisiae for various nutrients, especially nitrogen sources, greatly impacts the production of aroma compounds. In this study, we further explored the impact of different nitrogen nutrition strategies on the production of carbon and sulphur volatile compounds of three non-Saccharomyces strains, namely Pichia burtonii, Kluyveromyces marxianus, Zygoascus meyerae sequentially inoculated with S. cerevisiae in Sauvignon blanc and Shiraz grape musts. Nitrogen additions were implemented according the specific requirement of each species. At the end of fermentation, we observed specific metabolic signatures for each strain in response to the nature of the nitrogen source suggesting strain-specific metabolic fluxes present. Overall, these results confirmed and further explored the interconnection between nitrogen sources and aroma metabolism (including that of higher alcohols, fatty acids, esters and volatile sulphur compounds), and their variations according to species and the nature of the nitrogen source. The knowledge generated provides new insights to modulate the aroma profile of wines produced with non-Saccharomyces species.

Production of Aromatic Compounds by Catalytic Depolymerization of Technical and Downstream Biorefinery Lignins.

Lignocellulosic materials are promising alternatives to non-renewable fossil sources when producing aromatic compounds. Lignins from Populus salicaceae. Pinus radiata and Pinus pinaster from industrial wastes and biorefinery effluents were isolated and characterized. Lignin was depolymerized using homogenous (NaOH) and heterogeneous (Ni-, Cu- or Ni-Cu-hydrotalcites) base catalysis and catalytic hydrogenolysis using Ru/C. When homogeneous base catalyzed depolymerization (BCD) and Ru/C hydrogenolysis were combined on poplar lignin, the aromatics amount was ca. 11 wt.%. Monomer distributions changed depending on the feedstock and the reaction conditions. Aqueous NaOH produced cleavage of the alkyl side chain that was preserved when using modified hydrotalcite catalysts or Ru/C-catalyzed hydrogenolysis in ethanol. Depolymerization using hydrotalcite catalysts in ethanol produced monomers bearing carbonyl groups on the alkyl side chain. The analysis of the reaction mixtures was done by size exclusion chromatography (SEC) and diffusion ordered nuclear magnetic resonance spectroscopy (DOSY NMR). 31P NMR and heteronuclear single quantum coherence spectroscopy (HSQC) were also used in this study. The content in poly-(hydroxy)-aromatic ethers in the reaction mixtures decreased upon thermal treatments in ethanol. It was concluded that thermo-solvolysis is key in lignin depolymerization, and that the synergistic effect of Ni and Cu provided monomers with oxidized alkyl side chains.

Phenotypic and transcriptional analysis of Saccharomyces cerevisiae during wine fermentation in response to nitrogen nutrition and co-inoculation with Torulaspora delbrueckii

Nitrogen content of grape musts strongly impacts on fermentation performance and wine metabolite production. As nitrogen is a limiting nutrient in most grape musts, nitrogen supplementation is a common practice that ensures yeast growth during fermentation. However, preferred nitrogen sources -as ammonium- repress the genes related to alternative nitrogen sources consumption, usually involved in aromatic compounds production. Here, we describe the effect of high ammonium doses in Saccharomyces cerevisiae fermentation performance and wine properties, and how it is affected by yeast co-inoculation in mixed (S. cerevisiae + Torulaspora delbrueckii) fermentations. In addition, an RNA-seq analysis allowed us to study the S. cerevisiae transcriptional response to ammonium nutrition and yeast interaction, demonstrating that T. delbrueckii presence affects the global S. cerevisiae transcriptional response, reducing ammonium effects at both phenotypic -fermentation kinetics and metabolite production- and transcriptional levels, under experimental conditions.

Metabolic Engineering of Shikimic Acid-Producing Corynebacterium glutamicum From Glucose and Cellobiose Retaining Its Phosphotransferase System Function and Pyruvate Kinase Activities

The production of aromatic compounds by microbial production is a promising and sustainable approach for producing biomolecules for various applications. We describe the metabolic engineering of Corynebacterium glutamicum to increase its production of shikimic acid. Shikimic acid and its precursor-consuming pathways were blocked by the deletion of the shikimate kinase, 3-dehydroshikimate dehydratase, shikimate dehydratase, and dihydroxyacetone phosphate phosphatase genes. Plasmid-based expression of shikimate pathway genes revealed that 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, encoded by aroG, and DHQ synthase, encoded by aroB, are key enzymes for shikimic acid production in C. glutamicum. We constructed a C. glutamicum strain with aroG, aroB and aroE3 integrated. This strain produced 13.1 g/L of shikimic acid from 50 g/L of glucose, a yield of 0.26 g-shikimic acid/g-glucose, and retained both its phosphotransferase system and its pyruvate kinase activity. We also endowed β-glucosidase secreting ability to this strain. When cellobiose was used as a carbon source, the strain produced shikimic acid at 13.8 g/L with the yield of 0.25 g-shikimic acid/g-glucose (1 g of cellobiose corresponds to 1.1 g of glucose). These results demonstrate the feasibility of producing shikimic acid and its derivatives using an engineered C. glutamicum strain from cellobiose as well as glucose.