The Studies in Constructing Yeast Cell Factories for the Production of Fatty Acid Alkyl Esters

Fatty acid alkyl esters have broad applications in biofuels, lubricant formulas, paints, coatings, and cosmetics. Traditionally, these esters are mostly produced through unsustainable and energy-intensive processes. In contrast, microbial production of esters from renewable and sustainable feedstocks may provide a promising alternative and has attracted widespread attention in recent years. At present, yeasts are used as ideal hosts for producing such esters, due to their availability for high-density fermentation, resistance to phage infection, and tolerance against toxic inhibitors. Here, we summarize recent development on the biosynthesis of alkyl esters, including fatty acid ethyl esters (FAEEs), fatty acid short-branched chain alkyl esters (FASBEs), and wax esters (WEs) by various yeast cell factories. We focus mainly on the enzyme engineering strategies of critical wax ester synthases, and the pathway engineering strategies employed for the biosynthesis of various ester products. The bottlenecks that limit productivity and their potential solutions are also discussed in this review.

Exposure to binge ethanol and fatty acid ethyl esters exacerbates chronic ethanol-induced pancreatic injury in hepatic alcohol dehydrogenase deficient deer mice

Alcoholic chronic pancreatitis (ACP) is a fibroinflammatory disease of the pancreas. However, metabolic basis of ACP is not clearly understood. In this study, we evaluated differential pancreatic injury in hepatic alcohol dehydrogenase deficient (ADH-) deer mice fed chronic ethanol (EtOH), chronic plus binge EtOH, and chronic plus binge EtOH and fatty acid ethyl esters (FAEEs, nonoxidative metabolites of EtOH) to understand the metabolic basis of ACP. Hepatic ADH- and ADH normal (ADH+) deer mice were fed Lieber-DeCarli liquid diet containing 3% (w/v) EtOH for three months. One week before the euthanization, chronic EtOH fed mice were further administered with an oral gavage of binge EtOH with/without FAEEs. Blood alcohol concentration (BAC), pancreatic injury and inflammatory markers were measured. Pancreatic morphology, ultrastructural changes, endoplasmic reticulum (ER)/oxidative stress were examined using H & E staining, electron microscopy, immunostaining, and/or Western blot, respectively. Overall, BAC was substantially increased in chronic EtOH fed groups of ADH- vs. ADH+ deer mice. A significant change in pancreatic acinar cell morphology, with mild to moderate fibrosis and ultrastructural changes evident by dilatations and disruption of ER cisternae, ER/oxidative stress along with increased levels of inflammatory markers were observed in the pancreas of chronic EtOH fed groups of ADH- vs. ADH+ deer mice. Furthermore, chronic plus binge EtOH and FAEEs exposure elevated BAC, enhanced ER/oxidative stress and exacerbated chronic EtOH-induced pancreatic injury in ADH- deer mice suggesting a role of increased body burden of EtOH and its metabolism under reduced hepatic ADH in initiation and progression of ACP.

Ethanolysis of Waste Cooking oils using KOH Catalyst

The transesterification of waste cooking oils (WCO) with ethanol was investigated by means of potassium hydroxide (KOH) as catalyst. This work aimed to study the influences of catalyst concentration, temperature, ethanol to WCO molar ratio, reaction time, and stirring rate on the biodiesel conversion. Gas chromatography (GC) was used during the process of transesterification to determine the evolution of ethyl esters concentration with time. Biodiesel with maximum yield was obtained (92.5%) when 2 wt% KOH, temperature of 75°C, and ethanol/oil molar ratio of 11:1 were utilized.

Characterization and Evolution of Volatile Compounds of Cabernet Sauvignon Wines from Two Different Clones during Oak Barrel Aging

Xinjiang is a major wine-making region in China, but its hot climate in summer and intense sun exposure negatively affect the aroma quality of Cabernet Sauvignon wine. The aim of this study was to characterize and differentiate the volatile composition of Cabernet Sauvignon wines from two clones (169 and 191) in Xinjiang, and to study their aromatic profile evolution during 12-month oak barrel aging period. Results showed that before aging, clone 169 wine contained higher concentrations of several alcohols and ethyl esters, while acetate esters and furanic compounds were higher in clone 191 wine. After aging, levels of many terpenes, norisoprenoids, volatile phenols and phenolic aldehydes were significantly higher in clone 169 wine than 191 wine. Aroma series analysis revealed that clone 169 wine exhibited higher floral and roasty aromas after aging, while clone 191 wine had stronger chemical aroma. Principal component analysis indicated that aging process played a primary role in the alteration of volatile profile in these wines. Clone played a secondary role and oak barrel had a tertiary contribution to the variation. The present work indicates that clone 169 is a better choice for producing high-quality aged Cabernet Sauvignon wine with intense and elegant aroma in Xinjiang.

Isolation and Identification of Wild Yeast from Malaysian Grapevine and Evaluation of Their Potential Antimicrobial Activity against Grapevine Fungal Pathogens

Pathogenic fungi belonging to the genera Botrytis, Phaeomoniella, Fusarium, Alternaria and Aspergillus are responsible for vines diseases that affect the growth, grapevine yield and organoleptic quality. Among innovative strategies for in-field plant disease control, one of the most promising is represented by biocontrol agents, including wild epiphytic yeast strains of grapevine berries. Twenty wild yeast, isolated and molecularly identified from three different Malaysian regions (Perlis, Perak and Pahang), were evaluated in a preliminary screening test on agar to select isolates with inhibition against Botrytis cinerea. On the basis of the results, nine yeasts belonging to genera Hanseniaspora, Starmerella, Metschnikowia, Candida were selected and then tested against five grape berry pathogens: Aspergillus carbonarius, Aspergillus ochraceus, Fusarium oxysporum, Alternaria alternata and Phaeomoniella chlamydospora. Starmerella bacillaris FE08.05 and Metschnikowia pulcherrima GP8 and Hanseniaspora uvarum GM19 showed the highest effect on inhibiting mycelial growth, which ranged between 15.1 and 4.3 mm for the inhibition ring. The quantitative analysis of the volatile organic compound profiles highlighted the presence of isoamyl and phenylethyl alcohols and an overall higher presence of low-chain fatty acids and volatile ethyl esters. The results of this study suggest that antagonist yeasts, potentially effective for the biological control of pathogenic moulds, can be found among the epiphytic microbiota associated with grape berries.

Synthesis of Highly Functionalised Furo[3,4-b]pyrans: Towards the Fungal Metabolite (−)-TAN-2483B

<p>Carbohydrate-derived cyclopropanes combine both the stereochemical wealth of carbohydrates and the reactivity of cyclopropanes. A diverse variety of reaction modes for these cyclopropyl carbohydrates can be harnessed for the synthesis of natural products and other targets. The natural products (−)-TAN-2483A and (−)-TAN-2483B are fungal secondary metabolites displaying a variety of bioactivities such as inhibition of c-src kinase action and parathyroid hormone-induced bone resorption. This thesis described several synthetic approaches to the natural product (−)-TAN-2483B and analogues of (−)-TAN-2483B employing cyclopropane ring expansion. The synthetic route to (−)-TAN-2483B began with the readily available substrate D-mannose. The pyran ring unsaturation of the natural product was established by a cyclopropanation-ring expansion sequence. A synthetic strategy via dichlorocyclopropane-based intermediates is described in chapter 2. This being unsuccessful, an alternative approach via 2-fomyl-glycal was developed in chapter 3. The chapter 2 and 3 provided a solid background for the achievement of the analogues synthesis illustrated in chapter 4 via dibromocyclopropane. Lewis acid-mediated alkynylation followed by Pdcatalysed carbonylative lactonisation was successfully utilised in the revelation of the furo[3,4-b]pyran ring skeleton. This route afforded analogues of TAN-2483B; the Z-and E-unsaturated ethyl esters 140 and 141 and hydroxy(−)-TAN-2483B 145. The total synthesis of (−)-TAN-2483B was not achieved due to unforeseen obstacles encountered in the deoxygenation of the side arm of 335 (Chapter 4) into the E-propenyl side arm of (−)-TAN-2483B.</p>

Synthesis of Highly Functionalised Furo[3,4-b]pyrans: Towards the Fungal Metabolite (−)-TAN-2483B

<p>Carbohydrate-derived cyclopropanes combine both the stereochemical wealth of carbohydrates and the reactivity of cyclopropanes. A diverse variety of reaction modes for these cyclopropyl carbohydrates can be harnessed for the synthesis of natural products and other targets. The natural products (−)-TAN-2483A and (−)-TAN-2483B are fungal secondary metabolites displaying a variety of bioactivities such as inhibition of c-src kinase action and parathyroid hormone-induced bone resorption. This thesis described several synthetic approaches to the natural product (−)-TAN-2483B and analogues of (−)-TAN-2483B employing cyclopropane ring expansion. The synthetic route to (−)-TAN-2483B began with the readily available substrate D-mannose. The pyran ring unsaturation of the natural product was established by a cyclopropanation-ring expansion sequence. A synthetic strategy via dichlorocyclopropane-based intermediates is described in chapter 2. This being unsuccessful, an alternative approach via 2-fomyl-glycal was developed in chapter 3. The chapter 2 and 3 provided a solid background for the achievement of the analogues synthesis illustrated in chapter 4 via dibromocyclopropane. Lewis acid-mediated alkynylation followed by Pdcatalysed carbonylative lactonisation was successfully utilised in the revelation of the furo[3,4-b]pyran ring skeleton. This route afforded analogues of TAN-2483B; the Z-and E-unsaturated ethyl esters 140 and 141 and hydroxy(−)-TAN-2483B 145. The total synthesis of (−)-TAN-2483B was not achieved due to unforeseen obstacles encountered in the deoxygenation of the side arm of 335 (Chapter 4) into the E-propenyl side arm of (−)-TAN-2483B.</p>

Performance of Liquid Eversa on Fatty Acid Ethyl Esters Production by Simultaneous Esterification/Transesterification of Low-to-High Acidity Feedstocks

Liquid Eversa was evaluated in hydrolysis of acylglycerols from soybean oil deodorizer distillate (SODD), as well as simultaneous esterification/transesterification of SODD with low-to-high free fatty acids (FFAs) content using ethanol as acyl acceptor. Hydrolysis of SODD at mild temperature (37 °C) and without pH control (water:SODD mass ratio of 4:1) increased its FFAs content from 17.2 wt.% to 72.5 wt.% after 48 h reaction. A cold saponification of SODD allowed a saponification phase (SODD-SP) to be recovered with 93 wt.% saponification index and 2.25 wt.% FFAs content, which was used to find the experimental conditions for simultaneous esterification/transesterification reactions by experimental design. Temperature of 35 °C, enzyme concentration of 8.36 wt.%, and molar ratio of 3.64:1 (ethanol:SODD-SP) were found as the best conditions for fatty acid ethyl esters (FAEEs) production from SODD-SP (86.56 wt.% ester yield after 23 h reaction). Under the same reaction conditions, crude SODD (17.2 wt.% FFAs) and hydrolyzed SODD (72.5 wt.% FFAs) yielded products containing around 80 wt.% FAEEs. Caustic treatment could increase the ester content to around 90 wt.% and reduce the FFAs content to less than 1 wt.%. Our results show the good performance of liquid Eversa in aqueous (hydrolysis reactions) and organic (esterification/transesterification reactions) media.

“Portugizac Mlado vino”; is the aroma of younger red wine more attractive?

“Portugizac Mlado vino” is a local red wine with Protected designation of origin and Traditional term, and it is usually consumed very soon after alcoholic fermentation as a young wine. The maturation and aging of the wine affect the aroma composition, which is generally not pronounced and specific in the case of most red wines, but, as a sensory property, it is important for the perception of quality and consumer choice. The objective of this work was to analyze the most important aroma compounds of esters and higher alcohols, with the unpleasant volatile phenols too, in “Portugizac” wines, related to aging. The 9 young wines “Portugizac”, PDO “Plešivica” were analyzed after 3 and after 15 months of bottle storage at 16 ºC. Ethyl esters of butanoic and hexanoic acid along with isoamyl acetate are considered to be the most important esters in the fruity aroma of wine, and their concentrations in all analyzed “Portugizac” young wine samples were higher than their perception thresholds. The concentration of ethyl acetate was in the range 30-123 mg/L. The concentrations of ethyl esters of octanoic and decanoic acid, as well as 2-phenyl ethyl acetate and diethyl succinate in all analyzed wines, were lower than their perception thresholds. The concentrations of higher alcohols were much higher than their perception threshold. Bottle storage significantly affected the concentrations of analyzed compounds; after 15 months, the concentrations of acetate and ethyl esters (except ethyl acetate and diethyl succinate), as well as terpene (linalool), decreased, while higher alcohols and ethyl phenols slightly increased if compared to three months. In general, the compounds responsible for the desired fresh, fruity aroma tones were altered in an undesirable manner, while, undesirable compounds of 4-ethyl-phenol and 4-ethyl-quaiacol, which were not present in young wines, were detected in aged wines. It can be concluded that the bottle aging affects the aroma profile, undesirable changes were more pronounced than positive ones and therefore the consumption of young wine “Portugizac” might be more sensory attractive.