Alcohol Acyltransferase Is Involved in the Biosynthesis of C6 Esters in Apricot (Prunus armeniaca L.) Fruit

Short-chain esters derived from fatty acid contribute to the characteristic flavor of apricot fruit, and the biosynthesis of these compounds in fruit is catalyzed by alcohol acyltransferase (AAT). In this work, we investigated the AAT gene family via genome-wide scanning, and three AAT loci were identified in different linkage groups (LGs), with PaAAT1 (PARG22907m01) in LG7, PaAAT2 (PARG15279m01) in LG4, and PaAAT3 (PARG22697m01) in LG6. Phylogenetic analysis showed that PaAAT1 belongs to clade 3, while PaAAT2 and PaAAT3 belong to clade 1 and clade 2, respectively. In contrast, the three AAT genes present different expression patterns. Only PaAAT1 exhibited distinct patterns of fruit-specific expression, and the expression of PaAAT1 sharply increased during fruit ripening, which is consistent with the abundance of C4–C6 esters such as (E)-2-hexenyl acetate and (Z)-3-hexenyl acetate. The transient overexpression of PaAAT1 in Katy (KT) apricot fruit resulted in a remarkable decrease in hexenol, (E)-2-hexenol, and (Z)-3-hexenol levels while significantly increasing the corresponding acetate production (p < 0.01). A substrate assay revealed that the PaAAT1 protein enzyme can produce hexenyl acetate, (E)-2-hexenyl acetate, and (Z)-3-hexenyl acetate when C6 alcohols are used as substrates for the reaction. Taken together, these results indicate that PaAAT1 plays a crucial role in the production of C6 esters in apricot fruit during ripening.

Developing Clostridia as Cell Factories for Short- and Medium-Chain Ester Production

Short- and medium-chain volatile esters with flavors and fruity fragrances, such as ethyl acetate, butyl acetate, and butyl butyrate, are usually value-added in brewing, food, and pharmacy. The esters can be naturally produced by some microorganisms. As ester-forming reactions are increasingly deeply understood, it is possible to produce esters in non-natural but more potential hosts. Clostridia are a group of important industrial microorganisms since they can produce a variety of volatile organic acids and alcohols with high titers, especially butanol and butyric acid through the CoA-dependent carbon chain elongation pathway. This implies sufficient supplies of acyl-CoA, organic acids, and alcohols in cells, which are precursors for ester production. Besides, some Clostridia could utilize lignocellulosic biomass, industrial off-gas, or crude glycerol to produce other branched or straight-chain alcohols and acids. Therefore, Clostridia offer great potential to be engineered to produce short- and medium-chain volatile esters. In the review, the efforts to produce esters from Clostridia via in vitro lipase-mediated catalysis and in vivo alcohol acyltransferase (AAT)-mediated reaction are comprehensively revisited. Besides, the advantageous characteristics of several Clostridia and clostridial consortia for bio-ester production and the driving force of synthetic biology to clostridial chassis development are also discussed. It is believed that synthetic biotechnology should enable the future development of more effective Clostridia for ester production.

Transcriptomic and Metabolomic Studies Reveal Mechanisms of Effects of CPPU-Mediated Fruit-Setting on Attenuating Volatile Attributes of Melon Fruit

N1-(2-chloro-4-pyridyl)-N3-phenylurea (CPPU), a synthetic cytokinin-active compound, is widely applied to induce parthenocarpic fruit set and enhance melon fruit enlargement (Cucumis melo L.). CPPU may also influence fruit quality; however, the mechanisms through which this occurs remain unknown. We investigated the differences in volatile emissions between parthenocarpic fruit set by CPPU (C) and seeded fruit set by artificial pollination (P). Gas chromatography–mass spectrometry (GC–MS) analysis revealed that six volatile organic compounds (VOCs) emitted by the P-group fruits were not detected in C-group fruits. The relative abundances of another 14 VOCs emitted by the CPPU-treated fruits were less than those in the P-group fruits. RNA sequencing analysis indicated that a total of 1027, 994, and 743 differentially expressed genes (DEGs) were detected in the C20 (treatment with 20 mg·L–1 CPPU) vs. P, P-C20 (pollination followed by 20 mg·L−1 CPPU treatment) vs. P, and P-C20 vs. C20 treatments, respectively. Compared with the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, the DEGs related to fatty acid degradation and metabolism, which contribute to volatile production, were enriched. In particular, DEGs such as carotenoid cleavage dioxygenase (CCD)-, lipoxygenase (LOX)-, alcohol dehydrogenase (ADH)-, and alcohol acyltransferase (ATT)-related genes were closely related to the formation of volatiles. In summary, our study provides a metabolic and transcriptomic atlas, reveals the impact of CPPU on VOCs, and enhances our understanding of the mechanisms of CPPU that contribute towards generally reducing the quality of melon fruit.

Site-directed mutagenesis identified the key active site residues of alcohol acyltransferase PpAAT1 responsible for aroma biosynthesis in peach fruits

The aroma of peach fruit is predominantly determined by the accumulation of γ-decalactone and ester compounds. A previous study showed that the biosynthesis of these aroma compounds in peach fruit is catalyzed by PpAAT1, an alcohol acyltransferase. In this work, we investigated the key active site residues responsible for γ-decalactone and ester biosynthesis. A total of 14 candidate amino acid residues possibly involved in internal esterification and 9 candidate amino acid residues possibly involved in esterification of PpAAT1 were assessed via site-directed mutagenesis. Analyses of the in vitro enzyme activities of PpAAT1 and its site-directed mutant proteins (PpAAT1-SMs) with different amino acid residue mutations as well as the contents of γ-decalactone in transgenic tobacco leaves and peach fruits transiently expressing PpAAT1 and PpAAT1-SMs revealed that site-directed mutation of H165 in the conserved HxxxD motif led to lost enzymatic activity of PpAAT1 in both internal esterification and its reactions, whereas mutation of the key amino acid residue D376 led to the total loss of γ-decalactone biosynthesis activity of PpAAT1. Mutations of 9 and 7 other amino acid residues also dramatically affected the enzymatic activity of PpAAT1 in the internal esterification and esterification reactions, respectively. Our findings provide a biochemical foundation for the mechanical biosynthesis of γ-decalactone and ester compounds catalyzed by PpAAT1 in peach fruits, which could be used to guide the molecular breeding of new peach species with more favorable aromas for consumers.

Effects of Melatonin Treatment of Postharvest Pear Fruit on Aromatic Volatile Biosynthesis

Aroma affects the sensory quality of fruit and, consequently, consumer satisfaction. Melatonin (MT) is a plant growth regulator used to delay senescence in postharvest fruit during storage; however, its effect on aroma of pear fruit remains unclear. In this study, we assessed the effects of 0.1 mmol L−1 MT on volatiles and associated gene expression in the fruit of pear cultivars ‘Korla’ (Pyrus brestschneideri Rehd) and ‘Abbé Fetel’ (Pyrus communis L.). MT mainly affected the production of C6 aromatic substances in the two varieties. In ‘Korla’, MT inhibited expression of PbHPL, and reduced hydroperoxide lyase (HPL) activity and content of hexanal and (E)-hex-2-enal. In contrast, MT inhibited activity of lipoxygenase (LOX), reduced expression of PbLOX1 and PbLOX2, promoted PbAAT gene expression, increased alcohol acyltransferase (AAT) activity, and increased propyl acetate, and hexyl acetate content in ‘Abbé Fetel’ that similarly led to the reduction in content of hexanal and (E)-hex-2-enal. Content of esters in ‘Abbé Fetel’ pear increased with increasing postharvest storage period. Although mechanisms differed between the two varieties, effects on aroma volatiles mediated by MT were driven by expression of genes encoding LOX, HPL, and AAT enzymes.