Tandem Integration of Biological and Electrochemical Catalysis for Efficient Polyester Upcycling under Ambient Conditions.

Excessive production of waste polyethylene terephthalate (PET) poses an ecological challenge, which necessitates developing technologies to extract the values from end-of-life PET. Upcycling has proven effective in addressing the low profitability of current recycling strategies, yet existing upcycling technologies operate under energy-intensive conditions. Here we report a cascade strategy to steer the transformation of PET waste into glycolate in an overall yield of 92.6% under ambient conditions. The cascade approach involves setting up a robust hydrolase with 95.6% PET depolymerization into ethylene glycol (EG) monomer within 12 h, followed by an electrochemical process initiated by a CO-tolerant Pd/Ni(OH)2 catalyst to convert the EG intermediate into glycolate with high Faradaic efficiency of 97.5%. Techno-economic analysis and life cycle assessment indicate that, compared with the widely adopted electrochemical technology that heavily relies on alkaline pretreatment for PET depolymerization, our designed enzymatic-electrochemical approach offers a cost-effective and low-carbon pathway to upgrade PET.

Identification and characterization of a fungal cutinase-like enzyme CpCut1 from Cladosporium sp. P7 for polyurethane degradation.

Plastic degradation by biological systems emerges as a prospective avenue for addressing the pressing global concern of plastic waste accumulation. The intricate chemical compositions and diverse structural facets inherent to polyurethanes (PU) substantially increase the complexity associated with PU waste management. Despite the extensive research endeavors spanning over decades, most known enzymes exhibit a propensity for hydrolyzing waterborne PU dispersion (i.e., the commercial Impranil DLN-SD), with only a limited capacity for the degradation of bulky PU materials. Here, we report a novel cutinase (CpCut1) derived from Cladosporium sp. P7, which demonstrates remarkable efficiency in the degrading of various polyester-PU materials. After 12-h incubation at 55°C, CpCut1 was capable of degrading 40.5% and 20.6% of thermoplastic PU film and post-consumer foam, respectively, while achieving complete depolymerization of Impranil DLN-SD. Further analysis of the degradation intermediates suggested that the activity of CpCut1 primarily targeted the ester bonds within the PU soft segments. The versatile performance of CpCut1 against a spectrum of polyester-PU materials positions it as a promising candidate for the bio-recycling of waste plastics.IMPORTANCEPolyurethane (PU) has a complex chemical composition that frequently incorporates a variety of additives, which poses significant obstacles to biodegradability and recyclability. Recent advances have unveiled microbial degradation and enzymatic depolymerization as promising waste PU disposal strategies. In this study, we identified a gene encoding a cutinase from the PU-degrading fungus Cladosporium sp. P7, which allowed the expression, purification, and characterization of the recombinant enzyme CpCut1. Furthermore, this study identified the products derived from the CpCut1 catalyzed PU degradation and proposed its underlying mechanism. These findings highlight the potential of this newly discovered fungal cutinase as a remarkably efficient tool in the degradation of PU materials.

Isolation and characterization of polyester polyurethane-degrading bacterium Bacillus sp. YXP1.

Microorganisms have the potential to be applied for the degradation or depolymerization of polyurethane (PU) and other plastic waste, which have attracted global attention. The appropriate strain or enzyme that can effectively degrade PU is the key to treat PU plastic wastes by biological methods. Here, a polyester PU-degrading bacterium Bacillus sp. YXP1 was isolated and identified from a plastic landfill. Three PU substrates with increasing structure complexities, including Impranil DLN, poly (1,4-butylene adipate)-based PU (PBA-PU), and polyester PU foam, were used to evaluate the degradation capacity of Bacillus sp. YXP1. Under optimal conditions, strain YXP1 could completely degrade 0.5% Impranil DLN within 7 days. After 30 days, the weight loss of polyester PU foam by strain YXP1 was as high as 42.1%. In addition, PBA-PU was applied for degradation pathway analysis due to its clear composition and chemical structure. Five degradation intermediates of PBA-PU were identified, including 4,4'-methylenedianiline (MDA), 1,4-butanediol, adipic acid, and two MDA derivates, indicating that strain YXP1 could depolymerize PBA-PU by the hydrolysis of ester and urethane bonds. Furthermore, the extracellular enzymes produced by strain YXP1 could hydrolyze PBA-PU to generate MDA. Together, this study provides a potential bacterium for the biological treatment of PU plastic wastes and for the mining of functional enzymes.

Palmitoleic acid-rich oleaginous yeast Scheffersomyces segobiensis DSM 27193 exerts anti-obesity effects by ameliorating hepatic steatosis and adipose tissue hypertrophy.

BACKGROUND: Yeast biomass, encompassing fatty acids, terpenoids, vitamins, antioxidants, enzymes, and other bioactive compounds have been extensively utilized in food-related fields. The safety and potential bioactivities of Scheffersomyces segobiensis DSM 27193, an oleaginous yeast strain, are unclear. RESULTS: Scheffersomyces segobiensis DSM 27193 accumulated large palmitoleic acid (POA) levels (43.4 g kg-1 biomass) according to the results of whole-cell components. We annotated the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and predicted the categories and host of the pathogen-host interactions (PHI) genes in S. segobiensis DSM 27193. However, S. segobiensis DSM 27193 did not exert toxic effects in mice. Administration of S. segobiensis DSM 27193 led to substantial weight reduction by diminishing food intake in an obesity mouse model. Additionally, it reversed hepatic steatosis and adipose tissue hypertrophy, and improved abnormalities in serum biochemical profiles such as triglyceride, total cholesterol, low-density lipoprotein cholesterol, lipopolysaccharide, tumor necrosis factor-α, interleukin-1ß, and interleukin-6. CONCLUSION: This study is the first to illustrate the safety and effects of S. segobiensis DSM 27193 against obesity and offers a scientific rationale for its application in functional food supplements. © 2023 Society of Chemical Industry.

Enzymatic Upcycling of PET Waste to Calcium Terephthalate for Battery Anodes.

Biotechnological recycling offers a promising solution to address the environmental concerns associated with waste plastics, particularly polyethylene terephthalate (PET), widely utilized in packaging materials and textiles. To advance the development of a bio-based circular plastic economy, innovative upcycling strategies capable of generating higher-value products are needed. In this study, we enhanced the enzymatic depolymerization of waste PET by incorporating highly concentrated calcium ions (up to 1 m) to the hydrolytic reaction catalyzed by the best currently known enzyme LCCICCG . The presence of calcium ions not only improved the thermal stability and activity of the biocatalyst but also significantly reduced the consumption of base required to maintain optimal pH levels. Employing optimized conditions at 80 °C for 12 h, we successfully converted ≈84 % of the waste PET (200 g L-1 ) into solid hydrated calcium terephthalate (CaTP ⋅ 3H2 O) as the primary product instead of soluble terephthalate salt. CaTP ⋅ 3H2 O was easily purified and employed as a raw material for battery electrode production, exhibiting an initial reversible specific capacity of 164.2 mAh g-1 . Through techno-economic analysis, we conclusively demonstrated that the one-pot biocatalysis-based synthesis of CaTP is a superior PET upcycling strategy than the secondary synthesis method employing recycled terephthalic acid.

Sulfur-Rich Norbornadiene-Derived Infrared Transparent Polymers by Inverse Vulcanization.

Infrared (IR) transparent polymer materials prepared by inverse vulcanization, as a promising candidate to replace inorganic materials, are new materials for constructing key devices in IR optics. However, it is difficult to achieve a balance between infrared optical and thermal properties in polymers due to the intrinsic infrared absorption of organic materials. Herein, our strategy is to construct a high boiling point symmetrical molecular norbornadiene derivative cross-linking agent (DMMD) which can be inverse vulcanized with molten sulfur, and obtain Poly(S-r-DMMD) with different sulfur content by controlling the feed ratio of sulfur. With the rigid core and low IR activity in DMMD, the prepared polymers exhibit tunable thermal properties (Tg: 98.3-119.8 °C) and high IR transmittance (medium-wave infrared region (MWIR): 42.9-52.6 %; long-wave infrared region (LWIR): 1.5-5.29 %). In addition, Poly(S-r-DMMD) can be used to prepare large-size free-standing Fresnel lenses for IR imaging by simple hot-pressing, which provides flexibility in the design and production of IR fine lenses. This study provides a novel strategy for balancing the thermal and optical properties of IR transparent polymer materials, while providing relevant references for balancing the IR optical and thermal properties of polymer materials.

Combination of microbial and chemical synthesis for the sustainable production of ß-elemene, a promising plant-extracted anticancer compound.

Beta-elemene, a class of sesquiterpene derived from the Chinese medicinal herb Curcuma wenyujin, is widely used in clinical medicine due to its broad-spectrum antitumor activity. However, the unsustainable plant extraction prompted the search for environmentally friendly strategies for ß-elemene production. In this study, we designed a Yarrowia lipolytica cell factory that can continuously produce germacrene A, which is further converted into ß-elemene with 100% yield through a Cope rearrangement reaction by shifting the temperature to 250°C. First, the productivity of four plant-derived germacrene A synthases was evaluated. After that, the metabolic flux of the precursor to germacrene A was maximized by optimizing the endogenous mevalonate pathway, inhibiting the competing squalene pathway, and expressing germacrene A synthase gene in multiple copies. Finally, the most promising strain achieved the highest ß-elemene titer reported to date with 5.08 g/L. This sustainable and green method has the potential for industrial ß-elemene production.

Characterization of a novel esterase and construction of a Rhodococcus-Burkholderia consortium capable of catabolism bis (2-hydroxyethyl) terephthalate.

Bis (2-hydroxyethyl) terephthalate (BHET) is one of the main compounds produced by enzymatic hydrolysis or chemical depolymerization of polyethylene terephthalate (PET). However, the lack of understanding on BHET microbial metabolism is a main factor limiting the bio-upcycling of PET. In this study, BHET-degrading strains of Rhodococcus biphenylivorans GA1 and Burkholderia sp. EG1 were isolated and identified, which can grow with BHET as the sole carbon source. Furthermore, a novel esterase gene betH was cloned from strain GA1, which encodes a BHET hydrolyzing esterase with the highest activity at 30 °C and pH 7.0. In addition, the co-culture containing strain GA1 and strain EG1 could completely degrade high concentration of BHET, eliminating the inhibition on strain GA1 caused by the accumulation of intermediate metabolite ethylene glycol (EG). This work will provide potential strains and a feasible strategy for PET bio-upcycling.

Biodegradation of Poly(ethylene terephthalate) by Bacillus safensis YX8.

Due to the extensive utilization of poly (ethylene terephthalate) (PET), a significant amount of PET waste has been discharged into the environment, endangering both human health and the ecology. As an eco-friendly approach to PET waste treatment, biodegradation is dependent on efficient strains and enzymes. In this study, a screening method was first established using polycaprolactone (PCL) and PET nanoparticles as substrates. A PET-degrading strain YX8 was isolated from the surface of PET waste. Based on the phylogenetic analysis of 16S rRNA and gyrA genes, this strain was identified as Bacillus safensis. Strain YX8 demonstrated the capability to degrade PET nanoparticles, resulting in the production of terephthalic acid (TPA), mono (2-hydroxyethyl) terephthalic acid (MHET), and bis (2-hydroxyethyl) terephthalic acid (BHET). Erosion spots on the PET film were observed after incubation with strain YX8. Furthermore, the extracellular enzymes produced by strain YX8 exhibited the ability to form a clear zone on the PCL plate and to hydrolyze PET nanoparticles to generate TPA, MHET, and BHET. This work developed a method for the isolation of PET-degrading microorganisms and provides new strain resources for PET degradation and for the mining of functional enzymes.

Preparation and Characterization of a Novel Morphosis of Dextran and Its Derivatization with Polyethyleneimine.

Dextran, a variant of α-glucan with a significant proportion of α-(1,6) bonds, exhibits remarkable solubility in water. Nonetheless, the precipitation of dextran has been observed in injection vials during storage. The present study aimed to establish a technique for generating insoluble dextran and analyze its structural properties. Additionally, the potential for positively ionizing IS-dextran with polyethyleneimine was explored, with the ultimate objective of utilizing IS-dextran-PEI as a promising support for enzyme immobilization. As a result, IS-dextran was obtained by the process of slow evaporation with an average molecular weight of 6555 Da and a yield exceeding 60%. The calculated crystallinity of IS-dextran, which reaches 93.62%, is indicative of its irregular and dense structure, thereby accounting for its water insolubility. Furthermore, positive charge modification of IS-dextran, coupled with the incorporation of epichlorohydrin, resulted in all zeta potentials of IS-dextran-PEIs exceeding 30 mV, making it a promising supporting factor for enzyme immobilization.

N-acyl-homoserine lactones in extracellular polymeric substances from sludge for enhanced chloramphenicol-degrading anode biofilm formation in microbial fuel cells.

Exploring an efficient acclimation strategy to obtain robust bioanodes is of practical significance for antibiotic wastewater treatment by bioelectrochemical systems (BESs). This study investigated the effects of two acclimation conditions on chloramphenicol (CAP)-degrading anode biofilm formation in microbial fuel cells (MFCs). The one was continuously added the extracellular polymeric substances (EPS) extracted from anaerobic sludge and increasing concentrations of CAP after the first start-up phase, while the other was added the EPS-1 (N-acyl-homoserine lactones, namely AHLs were extracted from the EPS) at the same conditions. The results demonstrated that AHLs in the sludge EPS played a crucial role for enhanced CAP-degrading anode biofilm formation in MFCs. The AHL-regulation could not only maintain stable voltage outputs but also significantly accelerate CAP removal in the EPS MFC. The maximum voltage of 653.83 mV and CAP removal rate of 1.21 ± 0.05 mg/L·h were attained from the EPS MFC at 30 mg/L of CAP, which were 0.84 and 1.57 times higher than those from the EPS-1 MFC, respectively. These improvements were largely caused by the thick and 3D structured biofilm, strong and homogeneous cell viability throughout the biofilm, and high protein/polysaccharide ratio along with more conductive contents in the biofilm EPS. Additionally, AHLs facilitated the formation of a biofilm with rich biodiversity and balanced bacterial proportions, leading to more beneficial mutualism among different functional bacteria. More bi-functional bacteria (for electricity generation and antibiotic resistance/degradation) were specifically enriched by AHLs as well. These findings provide quorum sensing theoretical knowledge and practical instruction for rapid antibiotic-degrading electrode biofilm acclimation in BESs.

Photonic Plasticines with Uniform Structural Colors, High Processability, and Self-Healing Properties.

Despite the vast variety of colloidal superstructures available in soft matter photonics, it remains challenging to balance the trade-off between their optical microstructures and material processability. By synergizing colloidal photonics and dynamic chemistry, a type of photonic "plasticine" with characteristics of uniform structural colors, high processability, and self-healing is demonstrated. Specifically, a boronate ester bond-based macromonomer is first prepared through complexation between the diols of polyvinyl alcohol and the boronic acid group of 3-(acrylamido) phenylboronic acid in the presence of concentrated silica colloids. Upon photopolymerization, the dynamic photonic plasticine is formed in situ as the result of the crosslinking of the boronate ester bonded networks. The randomly packed colloids inside the plasticine compose the amorphous photonic crystals, giving rise to angle-independent structural colors that would not compromise during subsequent processing steps; the reversible nature of the boronate ester bonds endows the plasticine with self-adaptable and self-healing properties. Further, the plasticine is also compatible with common shaping methods, that is, cutting, molding, and carving, and thus can be facilely processed into 3D structural colored objects, holding great potentials in fields such as bio-encoding, optical filters, anti-counterfeiting, etc.

Metabolic engineering of Pichia pastoris for malic acid production from methanol.

The application of rational design in reallocating metabolic flux to accumulate desired chemicals is always restricted by the native regulatory network. In this study, recombinant Pichia pastoris was constructed for malic acid production from sole methanol through rational redistribution of metabolic flux. Different malic acid accumulation modules were systematically evaluated and optimized in P. pastoris. The recombinant PP-CM301 could produce 8.55 g/L malic acid from glucose, which showed a 3.45-fold increase compared to the parent strain. To improve the efficiency of site-directed gene knockout, NHEJ-related protein Ku70 was destroyed, whereas leading to the silencing of heterogenous genes. Hence, genes related to by-product generation were deleted via a specially designed FRT/FLP system, which successfully reduced succinic acid and ethanol production. Furthermore, a key node in the methanol assimilation pathway, glucose-6-phosphate isomerase was knocked out to liberate metabolic fluxes trapped in the XuMP cycle, which finally enabled 2.79 g/L malic acid accumulation from sole methanol feeding with nitrogen source optimization. These results will provide guidance and reference for the metabolic engineering of P. pastoris to produce value-added chemicals from methanol.

Droplet microfluidics on analysis of pathogenic microbes for wastewater-based epidemiology.

Infectious diseases caused by pathogenic microbes have posed a major health issue for the public, such as the ongoing COVID-19 global pandemic. In recent years, wastewater-based epidemiology (WBE) is emerging as an effective and unbiased method for monitoring public health. Despite its increasing importance, the advancement of WBE requires more competent and streamlined analytical platforms. Herein we discuss the interactions between WBE and droplet microfluidics, focusing on the analysis of pathogens in droplets, which is hard to be tackled by traditional analytical tools. We highlight research works from three aspects, namely, quantitation of pathogen biomarkers in droplets, single-cell analysis in droplets, and living cell biosensors in droplets, as well as providing future perspectives on the synergy between WBE and droplet microfluidics.

Quantitative proteomic analysis to reveal expression differences for butanol production from glycerol and glucose by Clostridium sp. strain CT7.

Clostridium sp. strain CT7 is a new emerging microbial cell factory with high butanol production ratio owing to its non-traditional butanol fermentation mode with uncoupled acetone and 1,3-propanediol formation. Significant changes of metabolic products profile were shown in glycerol- and glucose-fed strain CT7, especially higher butanol and lower volatile fatty acids (VFAs) production occurred from glycerol-fed one. However, the mechanism of this interesting phenomenon was still unclear. To better elaborate the bacterial response towards glycerol and glucose, the quantitative proteomic analysis through iTRAQ strategy was performed to reveal the regulated proteomic expression levels under different substrates. Proteomics data showed that proteomic expression levels related with carbon metabolism and solvent generation under glycerol media were highly increased. In addition, the up-regulation of hydrogenases, ferredoxins and electron-transferring proteins may attribute to the internal redox balance, while the earlier triggered sporulation response in glycerol-fed media may be associated with the higher butanol production. This study will pave the way for metabolic engineering of other industrial microorganisms to obtain efficient butanol production from glycerol.

Luteimonas wenzhouensis Sp. Nov., A Chitinolytic Bacterium Isolated from a Landfill Soil.

A Gram-staining-negative, aerobic, non-motile, rod-shaped bacterium with degradation ability of chitin, designated strain YD-1 T, was isolated from landfill soil sample collected in Wenzhou, Zhejiang province, China. The growth of strain YD-1 T occurred optimally in the tryptone soy broth (TSB) with 1.0% NaCl at pH 7.0-8.0, 30 °C. Ubiquinone-8 (Q-8) was the predominant quinone. The polar lipids of strain YD-1 T consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, five glycolipids and four lipids. The major fatty acids were iso-C15:0 (30.7%), iso-C17:1ω9c (23.2%), iso-C11:0 (18.9%), iso-C11:0 3-OH (6.8%) and iso-C17:0 (5.9%). Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain YD-1 T was affiliated to the genus Luteimonas with the highest similarity to Luteimonas marina KCTC 12327 T (97.3%), followed by Luteimonas aquatica DSM 22088 T (96.5%) and Luteimonas composti CCUG 53595 T (96.4%). The genomic DNA G + C content of strain YD-1 T was 71.8 mol%. Average nucleotide identity (ANI) and the digital DNA-DNA hybridizations (dDDH) for draft genomes between strain YD-1 T and Luteimonas marina KCTC 12327 T were 82.7% and 26.1%, respectively. On the basis of genotypic, phenotypic and chemotaxonomic data, strain YD-1 T is considered to represent a novel species to degrade chitin in the genus Luteimonas, for which the name Luteimonas wenzhouensis sp. nov. is proposed, with YD-1 T (= KCTC 72425 T = CCTCC AB 2019153 T) as the type strain.

Pollutant Degrading Enzyme: Catalytic Mechanisms and Their Expanded Applications.

The treatment of environmental pollution by microorganisms and their enzymes is an innovative and socially acceptable alternative to traditional remediation approaches. Microbial biodegradation is often characterized with high efficiency as this process is catalyzed via degrading enzymes. Various naturally isolated microorganisms were demonstrated to have considerable ability to mitigate many environmental pollutants without external intervention. However, only a small fraction of these strains are studied in detail to reveal the mechanisms at the enzyme level, which strictly limited the enhancement of the degradation efficiency. Accordingly, this review will comprehensively summarize the function of various degrading enzymes with an emphasis on catalytic mechanisms. We also inspect the expanded applications of these pollutant-degrading enzymes in industrial processes. An in-depth understanding of the catalytic mechanism of enzymes will be beneficial for exploring and exploiting more degrading enzyme resources and thus ameliorate concerns associated with the ineffective biodegradation of recalcitrant and xenobiotic contaminants with the help of gene-editing technology and synthetic biology.

Consolidated bioprocessing performance of a two-species microbial consortium for butanol production from lignocellulosic biomass.

Consolidated bioprocessing (CBP) by using microbial consortium was considered as a promising approach to achieve direct biofuel production from lignocellulose. In this study, the interaction mechanism of microbial consortium consisting of Thermoanaerobacterium thermosaccharolyticum M5 and Clostridium acetobutylicum NJ4 was analyzed, which could achieve efficient butanol production from xylan through CBP. Strain M5 possesses efficient xylan degradation capability, as 19.73 g/L of xylose was accumulated within 50 hr. The efficient xylose utilization capability of partner strain NJ4 could relieve the substrate inhibition to hydrolytic enzymes of xylanase and xylosidase secreted by strain M5. In addition, the earlier solventogenesis of strain NJ4 was observed due to the existence of butyrate generated by strain M5. The mutual interaction of these two strains finally gave 13.28 g/L of butanol from 70 g/L of xylan after process optimization, representing a relatively high butanol production from hemicellulose. Moreover, 7.61 g/L of butanol was generated from untreated corncob via CBP. This successfully constructed microbial consortium exhibits efficient cooperation performance on butanol production from lignocellulose, which could provide a platform for the emerging butanol production from lignocellulose.

Metabolic engineering of Escherichia coli for L-malate production anaerobically.

BACKGROUND: L-malate is one of the most important platform chemicals widely used in food, metal cleaning, textile finishing, pharmaceuticals, and synthesis of various fine chemicals. Recently, the development of biotechnological routes to produce L-malate from renewable resources has attracted significant attention. RESULTS: A potential L-malate producing strain E. coli BA040 was obtained by inactivating the genes of fumB, frdABCD, ldhA and pflB. After co-overexpression of mdh and pck, BA063 achieved 18 g/L glucose consumption, leading to an increase in L-malate titer and yield of 13.14 g/L and 0.73 g/g, respectively. Meantime, NADH/NAD+ ratio decreased to 0.72 with the total NAD(H) of 38.85 µmol/g DCW, and ATP concentration reached 715.79 nmol/g DCW. During fermentation in 5L fermentor with BA063, 41.50 g/L glucose was consumed within 67 h with the final L-malate concentration and yield of 28.50 g/L, 0.69 g/g when heterologous CO2 source was supplied. CONCLUSIONS: The availability of NAD(H) was correlated positively with the glucose utilization rate and cellular metabolism capacities, and lower NADH/NAD+ ratio was beneficial for the accumulation of L-malate under anaerobic conditions. Enhanced ATP level could significantly enlarge the intracellular NAD(H) pool under anaerobic condition. Moreover, there might be an inflection point, that is, the increase of NAD(H) pool before the inflection point is followed by the improvement of metabolic performance, while the increase of NAD(H) pool after the inflection point has no significant impacts and NADH/NAD+ ratio would dominate the metabolic flux. This study is a typical case of anaerobic organic acid fermentation, and demonstrated that ATP level, NAD(H) pool and NADH/NAD+ ratio are three important regulatory parameters during the anaerobic production of L-malate.

Perspectives for the microbial production of ethyl acetate.

Ethyl acetate is one of the short-chain esters and widely used in the food, beverage, and solvent areas. The ethyl acetate production currently proceeds through unsustainable and energy intensive processes, which are based on natural gas and crude oil. Microbial conversion of biomass-derived sugars into ethyl acetate may provide a sustainable alternative. In this review, the perspectives of bio-catalyzing ethanol and acetic acid to ethyl acetate using lipases in vitro was introduced. Besides, the crucial elements for high yield of ethyl acetate in fermentation was expounded. Also, metabolic engineering in yeasts to product ethyl acetate in vivo using alcohol acyl transferases (AAT) was discussed. KEY POINTS: •The accumulation of acetyl-CoA is crucial for synthesizing ethyl acetate in vivo; AAT-mediated metabolic engineering could efficiently improve ethyl acetate production.