Recent advances in microbial and enzymatic engineering for the biodegradation of micro- and nanoplastics.

This review examines the escalating issue of plastic pollution, specifically highlighting the detrimental effects on the environment and human health caused by microplastics and nanoplastics. The extensive use of synthetic polymers such as polyethylene (PE), polyethylene terephthalate (PET), and polystyrene (PS) has raised significant environmental concerns because of their long-lasting and non-degradable characteristics. This review delves into the role of enzymatic and microbial strategies in breaking down these polymers, showcasing recent advancements in the field. The intricacies of enzymatic degradation are thoroughly examined, including the effectiveness of enzymes such as PETase and MHETase, as well as the contribution of microbial pathways in breaking down resilient polymers into more benign substances. The paper also discusses the impact of chemical composition on plastic degradation kinetics and emphasizes the need for an approach to managing the environmental impact of synthetic polymers. The review highlights the significance of comprehending the physical characteristics and long-term impacts of micro- and nanoplastics in different ecosystems. Furthermore, it points out the environmental and health consequences of these contaminants, such as their ability to cause cancer and interfere with the endocrine system. The paper emphasizes the need for advanced analytical methods and effective strategies for enzymatic degradation, as well as continued research and development in this area. This review highlights the crucial role of enzymatic and microbial strategies in addressing plastic pollution and proposes methods to create effective and environmentally friendly solutions.

Water quality seasonal variation assessment of the Gongji and Yaksa streams, Chuncheon, South Korea.

Gongji Stream flows into Lake Uiam, a potable water source for the capital region of Chuncheon, South Korea. Algal blooms often occur downstream of the Gongji stream in combination with drastic flow rate variations. Downstream water quality may also be affected by Yaksa stream. Yaksa stream joins Gongji stream before it reaches Uiam Lake, which is a drinking water source for the city. Limited data exists on the Yaksa stream water quality. Therefore, water quality parameters (pH, electrical conductivity (EC), biological oxygen demand (BOD), total nitrogen (T-N), total phosphorous (T-P), chlorophyll-a (Chl-a), total coliforms, and Escherichia coli (E. coli) concentration) were sampled from Gongji (at sites GJ1 and GJ2) and Yaksa (at sites YS1 and YS2) streams from May to September, 2022. The results revealed the overall water quality of both streams was good (BOD = 0.27-3.66 mg/L; TP = 0.003-0.074 mg/L), except on August 3. On August 3, the concentrations of BOD, TP, total coliforms, and E. coli were elevated, with the highest concentrations in samples from GJ2. The recent heavy rainfall potentially caused sewage inflows near GJ2. The correlation analysis revealed positive linear relationships in the 1-day cumulative precipitation with BOD (r = 0.503), total coliforms (r = 0.547), and TP (r = 0.814). The Yaksa stream may be an Anabaena sp. source, which contaminated samples from YS1, YS2, and GJ2, but not at GJ1 (upstream of the tributary).

Adsorption behavior of triclosan on microplastics and their combined acute toxicity to D. magna.

Microplastics (MP) have been recently identified as emerging water contaminants in worldwide. Owing to its physicochemical properties, MP have been considered as a vector of other micropollutants and may affect their fate and ecological toxicity in the water environment. In this study, triclosan (TCS), which is a widely-used bactericide, and three frequently found types of MP (PS-MP, PE-MP, and PP-MP) were investigated. The adsorption behavior of TCS on MP was investigated by the effect of reaction time, initial concentration of TCS, and other water chemistry factors. Elovich model and Temkin model are the most fitted well with kinetics and adsorption isotherms, respectively. The maximum TCS adsorption capacities were calculated for PS-MP (9.36 mg/g), PP-MP (8.23 mg/g), and PE-MP (6.47 mg/g). PS-MP had higher affinity to TCS owing to hydrophobic and π-π interaction. The TCS adsorption on PS-MP was inhibited by decreasing concentrations of cations, and increasing concentration of anion, pH, and NOM concentration. At pH 10, only 0.22 mg/g of adsorption capacity was obtained because of the isoelectric point (3.75) of PS-MP and pKa (7.9) of TCS. And almost no TCS adsorption occurred at NOM concentration of 11.8 mg/L. Only PS-MP had no acute toxic effect on D. magna, whereas TCS showed acute toxicity (EC50,24h of TCS = 0.36 ± 0.4 mg/L). Although survival rate increased when TCS with PS-MP due to lower the TCS concentration in solution via adsorption, PS-MP was observed in intestine and body surface of D. magna. Our findings can contribute to understanding the combined potential effects of MP fragment and TCS to aquatic biota.

Overexpression of Mutant Galactose Permease (ScGal2_N376F) Effective for Utilization of Glucose/Xylose or Glucose/ Galactose Mixture by Engineered Kluyveromyces marxianus.

Mutant sugar transporter ScGAL2-N376F was overexpressed in Kluyveromyces marxianus for efficient utilization of xylose, which is one of the main components of cellulosic biomass. K. marxianus ScGal2_N376F, the ScGAL2-N376F-overexpressing strain, exhibited 47.04 g/l of xylose consumption and 26.55 g/l of xylitol production, as compared to the parental strain (24.68 g/l and 7.03 g/l, respectively) when xylose was used as the sole carbon source. When a mixture of glucose and xylose was used as the carbon source, xylose consumption and xylitol production rates were improved by 195% and 360%, respectively, by K. marxianus ScGal2_N376F. Moreover, the glucose consumption rate was improved by 27% as compared to that in the parental strain. Overexpression of both wild-type ScGAL2 and mutant ScGAL2-N376F showed 48% and 52% enhanced sugar consumption and ethanol production rates, respectively, when a mixture of glucose and galactose was used as the carbon source, which is the main component of marine biomass. As shown in this study, ScGAL2-N376F overexpression can be applied for the efficient production of biofuels or biochemicals from cellulosic or marine biomass.

Alleviation of catabolite repression in Kluyveromyces marxianus: the thermotolerant SBK1 mutant simultaneously coferments glucose and xylose.

BACKGROUND: Simultaneous cofermentation of glucose and xylose mixtures would be a cost-effective solution for the conversion of cellulosic biomass to high-value products. However, most yeasts ferment glucose and xylose sequentially due to glucose catabolite repression. A well known thermotolerant yeast, Kluyveromyces marxianus, was selected for this work because it possesses cost-effective advantages over Saccharomyces cerevisiae for biofuel production from cellulosic biomass. RESULTS: In the present study, we employed a directed evolutionary approach using 2-deoxyglucose to develop a thermotolerant mutant capable of simultaneous cofermentation of glucose and xylose by alleviating catabolite repression. The selected mutant, K. marxianus SBK1, simultaneously cofermented 40 g/L glucose and 28 g/L xylose to produce 23.82 g/L ethanol at 40 °C. This outcome corresponded to a yield of 0.35 g/g and productivity of 0.33 g/L h, representing an 84% and 129% improvement, respectively, over the parental strain. Interestingly, following mutagenesis the overall transcriptome of the glycolysis pathway was highly downregulated in K. marxianus SBK1, except for glucokinase-1 (GLK1) which was 21-fold upregulated. Amino acid sequence of GLK1 from K. marxianus SBK1 revealed three amino acid mutations which led to more than 22-fold lower enzymatic activity compared to the parental strain. CONCLUSIONS: We herein successfully demonstrated that the cofermentation of a sugar mixture is a promising strategy for the efficient utilization of cellulosic biomass by K. marxianus SBK1. Through introduction of additional biosynthetic pathways, K. marxianus SBK1 could become a chassis-type strain for the production of fuels and chemicals from cellulosic biomass.

Overexpression of Endogenous Xylose Reductase Enhanced Xylitol Productivity at 40 °C by Thermotolerant Yeast Kluyveromyces marxianus.

Xylitol is a valuable substance utilized by food and biochemical industries. NAD(P)H-dependent xylose reductase (XR)-encoded by the yeast KmXYL1 gene-is the key enzyme which facilitates reduction of xylose to xylitol. Multi-copy integration of a mutant KmXYL1 (mKmXYL1) gene was carried out using thermotolerant yeast Kluyveromyces marxianus KCTC17555ΔURA3, in order to enhance xylitol production. After multi-copy integration, the highest xylitol producing strain was isolated and named K. marxianus 17555-JBP2. This strain exhibited 440% higher xylitol production than the parental strain at 30 °C. Due to a multi-copy integration of the mKmXYL1 gene, various additional differences between K. marxianus 17555-JBP2 and the parental strain were observed, including a 66% increase in NAD(P)H-dependent XR activity at high temperature (45 °C). Quantitative real-time PCR and transcriptome analysis demonstrated that, relative to the parent strain, K. marxianus 17555-JBP2 exhibited two more copies of mKmXY1 genes and a 9.63-fold elevation in transcription of NAD(P)H-dependent XR. After optimization of bioreactor fermentation conditions (agitation speed), high-temperature (40 °C) xylitol productivity of K. marxianus 17555-JBP2 exhibited an 81% improvement relative to the parental strain. In this study, we demonstrated that the overexpression of endogenous XR could enhance xylitol productivity at 40 °C by thermotolerant K. marxianus.

Ethanol production from xylose is highly increased by the Kluyveromyces marxianus mutant 17694-DH1.

Directed evolutionary approach and random mutagenesis were performed on thermotolerant yeast Kluyveromyces marxianus KCTC17694 for isolating a yeast strain producing ethanol from xylose efficiently. The isolated mutant strain, K. marxianus 17694-DH1, showed 290% and 131% improvement in ethanol concentration and ethanol production yield from xylose, respectively, as compared with the parental strain. Sequencing of the KmXYL1 gene of K. marxianus 17694-DH1 revealed substitutions of arginine and tryptophan with lysine and leucine at positions 25 and 202, respectively, as compared to the parental strain. In addition, sequencing of the KmXYL2 gene uncovered a substitution of glutamate with leucine at position 232. When enzymatic assays of xylose reductase (XR) and xylitol dehydrogenase (XDH) from the parental strain and K. marxianus 17694-DH1 were performed, XR activities were not significantly different whereas XDH activities were significantly improved in the mutant strain up to 50 °C of reaction temperatures. RNA-Seq based transcriptome analysis showed that alcohol dehydrogenases and glucose transporters were up-regulated while TCA cycle involved enzymes were down-regulated in K. marxianus 17694-DH1.

Characterization of divergent pseudo-sucrose isomerase from Azotobacter vinelandii: Deciphering the absence of sucrose isomerase activity.

Among members of the glycoside hydrolase (GH) family, sucrose isomerase (SIase) and oligo-1,6-glucosidase (O16G) are evolutionarily closely related even though their activities show different specificities. A gene (Avin_08330) encoding a putative SIase (AZOG: Azotobacterglucocosidase) from the nitrogen-fixing bacterium Azotobacter vinelandii is a type of pseudo-SIase harboring the "RLDRD" motif, a SIase-specific region in 329-333. However, neither sucrose isomerization nor hydrolysis activities were observed in recombinant AZOG (rAZOG). The rAZOG showed similar substrate specificity to Bacillus O16G as it catalyzes the hydrolysis of isomaltulose and isomaltose, which contain α-1,6-glycosidic linkages. Interestingly, rAZOG could generate isomaltose from the small substrate methyl-α-glucoside (MαG) via intermolecular transglycosylation. In addition, sucrose isomers isomaltulose and trehalulose were produced when 250 mM fructose was added to the MαG reaction mixture. The conserved regions I and II of AZOG are shared with many O16Gs, while regions III and IV are very similar to those of SIases. Strikingly, a shuffled AZOG, in which the N-terminal region of SIase containing conserved regions I and II was exchanged with the original enzyme, exhibited a production of sucrose isomers. This study demonstrates an evolutionary relationship between SIase and O16G and suggests some of the main regions that determine the specificity of SIase and O16G.

Changes in antioxidant activities and volatile compounds of mixed berry juice through fermentation by lactic acid bacteria.

The objectives of this study were to analyze antioxidant activities and identify volatile compounds in mixed berry juice after fermentation by lactic acid bacteria (LAB). Antioxidant activity of the mixed berry juice increased significantly from 209.57±2.93 to 268.30±1.75 µmol TE/g after 24 h of fermentation. After LAB fermentation, 34 volatile compounds were identified. Among them, three compounds-benzoic acid, benzaldehyde, and vitispirane-showed significant changes in their concentrations. Peak areas of benzoic acid and benzaldehyde, which are known to possess antioxidant activities, increased by 64 and 188%, respectively, after fermentation. However, the peak area of vitispirane, which is the most abundant terpene compound in berry juices, decreased by 92% after fermentation.

Effects of polysaccharides isolated from Inonotus obliquus against hydrogen peroxide-induced oxidative damage in RINm5F pancreatic ß-cells.

The purpose of the present study was to elucidate the cytoprotective effects of polysaccharides isolated from Inonotus obliquus. The polysaccharides were extracted from the fruiting body of I. obliquus (PFIO) and the liquid culture broth of I. obliquus (PLIO). The effects of PFIO and PLIO on hydrogen peroxide (H2O2)­induced oxidative damage of RINm5F pancreatic ß­cells were comparatively investigated using an MTT assay, immunofluorescent staining, flow cytometry, and western blot analyses in vitro. The results of the present study demonstrated that treatment with PFIO and PLIO decreased DNA fragmentation and the rate of apoptosis. In addition, pretreatment of cells with PFIO and PLIO prior to H2O2 exposure resulted in increased insulin secretion and scavenging activity for intracellular reactive oxygen species, as compared with treatment with H2O2 alone. The results of the present study suggested that PFIO and PLIO may exert protective effects against H2O2­induced oxidative stress via the regulation of mitogen­activated protein kinases, nuclear factor­κB and apoptotic proteins. Therefore, PFIO and PLIO may have potential merit as a medicinal food for the prevention of diabetes.

Polysaccharide isolated from the liquid culture broth of Inonotus obliquus suppresses invasion of B16-F10 melanoma cells via AKT/NF-κB signaling pathway.

A number of polysaccharides exhibit pharmacological activities. Polysaccharides derived from Inonotus obliquus (PLIO) appear to have various potential pharmacological properties, including anti­tumor activity. However, the molecular mechanisms underlying these properties remain to be elucidated. The present study investigated the anti­metastatic potential of PLIO and the underlying signaling pathways in B16­F10 murine melanoma cells using the MTT colorimetric assay, in vitro migration and invasion assays, and flow cytometric and western blot analyses. PLIO inhibited the invasion of B16­F10 cells and suppressed the expression of matrix metalloproteinases. PLIO treatment inhibited nuclear factor­κB (NF­κB) nuclear translocation in B16­F10 cells. In addition, PLIO treatment inhibited the phosphorylation of c-Jun N­terminal kinases and AKT. These results suggest that PLIO may suppress the invasion of highly metastatic melanoma cells via inhibition of the AKT/NF-κB signaling pathways.

Enhanced Xylitol Production by Mutant Kluyveromyces marxianus 36907-FMEL1 Due to Improved Xylose Reductase Activity.

A directed evolution and random mutagenesis were carried out with thermotolerant yeast Kluyveromyces marxianus ATCC 36907 for efficient xylitol production. The final selected strain, K. marxianus 36907-FMEL1, exhibited 120 and 39 % improvements of xylitol concentration and xylitol yield, respectively, as compared to the parental strain, K. marxianus ATCC 36907. According to enzymatic assays for xylose reductase (XR) activities, XR activity from K. marxianus 36907-FMEL1 was around twofold higher than that from the parental strain. Interestingly, the ratios of NADH-linked and NADPH-linked XR activities were highly changed from 1.92 to 1.30 when K. marxianus ATCC 36907 and K. marxianus 36907-FMEL1 were compared. As results of KmXYL1 genes sequencing, it was found that cysteine was substituted to tyrosine at position 36 after strain development which might cause enhanced XR activity from K. marxianus 36907-FMEL1.

Cytoprotective effect of hispidin against palmitate-induced lipotoxicity in C2C12 myotubes.

It is well known that Phellinus linteus, which produces hispidin and its derivatives, possesses antioxidant activities. In this study, we investigated whether hispidin has protective effects on palmitate-induced oxidative stress in C2C12 skeletal muscle cells. Our results showed that palmitate treatment in C2C12 myotubes increased ROS generation and cell death as compared with the control. However, pretreatment of hispidin for 8 h improved the survival of C2C12 myotubes against palmitate-induced oxidative stress via inhibition of intracellular ROS production. Hispidin also inhibited palmitate-induced apoptotic nuclear condensation in C2C12 myotubes. In addition, we found that hispidin can suppress cleavage of caspase-3, expression of Bax, and NF-κB translocation. Therefore, these results suggest that hispidin is capable of protecting C2C12 myotubes against palmitate-induced oxidative stress.

Mechanism of macrophage activation induced by polysaccharide from Cordyceps militaris culture broth.

Mushroom-derived polysaccharides have been shown to stimulate immune responses. Our previous report showed that the novel polysaccharide PLCM isolated from the culture broth of Cordyceps militaris could induce nitric oxide production in the murine macrophage-like cell line RAW264.7. In this study, we show that PLCM enhances immunostimulatory activities such as the release of toxic molecules (nitric oxide and reactive oxygen species), secretion of the cytokine tumor necrosis factor (TNF)-α, and phagocytic uptake in RAW264.7 macrophages. In addition, all the specific inhibitors against the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) (SN50, BAY11-7082, PD98059, SP600125 and SB203580) markedly suppressed the nitric oxide production and phagocytic uptake induced by PLCM. Moreover, antibodies specific to the extracellular domain of Toll-like receptor-2, Toll-like receptor-4 or the macrophage receptor Dectin-1 significantly attenuated PLCM-induced secretion of TNF-α. Our results indicate that the C. militaris polysaccharide activates macrophages through the MAPKs and NF-κB signaling pathways via Toll-like receptor 2, Toll-like receptor 4, and Dectin-1.

Cyanidin-3-glucoside isolated from mulberry fruit protects pancreatic ß-cells against oxidative stress-induced apoptosis.

The extract obtained from berries contains high amounts of anthocyanins, and this extract is used as a phytotherapeutic agent for different types of diseases. In this study, we examined the cytoprotective effects of cyanidin-3-glucoside (C3G) isolated from mulberry fruit against pancreatic ß-cell apoptosis caused by hydrogen peroxide (H2O2)-induced oxidative stress. The MIN6 pancreatic ß-cells were used to investigate the cytoprotective effects of C3G on the oxidative stress-induced apoptosis of cells. Cell viability was examined by MTT assay and lipid peroxidation was assayed by thiobarbituric acid (TBA) reaction. Immunofluorescence staining, flow cytometry and western blot analysis were also used to determine apoptosis and the expression of proteins associated with apoptosis. Our results revealed that H2O2 increased the rate of apoptosis by stimulating various pro-apoptotic processes, such as the generation of intracellular reactive oxygen species (ROS), lipid peroxidation, DNA fragmentation and caspase-3 activation. However, C3G reduced the H2O2-induced cell death in the MIN6N pancreatic ß-cells. In addition, we confirmed that H2O2 activated mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 MAPK. C3G inhibited the phosphorylation of ERK and p38 without inducing the phosphorylation of JNK. Furthermore, C3G regulated the intrinsic apoptotic pathway-associated proteins, such as proteins belonging to the Bcl-2 family, cytochrome c and caspase-3. Taken together, our results suggest that C3G isolated from mulberry fruit has potential for use as a phytotherapeutic agent for the prevention of diabetes by preventing oxidative stress-induced ß-cell apoptosis.

Inonotus obliquus-derived polysaccharide inhibits the migration and invasion of human non-small cell lung carcinoma cells via suppression of MMP-2 and MMP-9.

Polysaccharides isolated from the fruiting body of Inonotus obliquus (PFIO) are known to possess various pharmacological properties including antitumor activity. However, the anti-metastatic effect and its underlying mechanistic signaling pathway involved these polysaccharides in human non-small cell lung carcinoma remain unknown. The present study therefore aimed to determine the anti-metastatic potential and signaling pathways of PFIO in the highly metastatic A549 cells. We found that PFIO suppressed the migration and invasive ability of A549 cells while decreasing the expression levels and activity of matrix metalloproteinase (MMP)-2 and MMP-9. Furthermore, PFIO decreased the phosphorylation levels of mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) as well as the expression level of COX-2, and inhibited the nuclear translocation of nuclear factor κB (NF-κB) in A549 cells. These results suggested that PFIO could suppress the invasion and migration of human lung carcinoma by reducing the expression levels and activity of MMP-2 and MMP-9 via suppression of MAPKs, PI3K/AKT, and NF-κB signaling pathways.

Cordyceps militaris extract protects human dermal fibroblasts against oxidative stress-induced apoptosis and premature senescence.

Oxidative stress induced by reactive oxygen species (ROS) is the major cause of degenerative disorders including aging and disease. In this study, we investigated whether Cordyceps militaris extract (CME) has in vitro protective effects on hydrogen peroxide-induced oxidative stress in human dermal fibroblasts (HDFs). Our results showed that the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of CME was increased in a dose-dependent manner. We found that hydrogen peroxide treatment in HDFs increased ROS generation and cell death as compared with the control. However, CME improved the survival of HDFs against hydrogen peroxide-induced oxidative stress via inhibition of intracellular ROS production. CME treatment inhibited hydrogen peroxide-induced apoptotic cell death and apoptotic nuclear condensation in HDFs. In addition, CME prevented hydrogen peroxide-induced SA-ß-gal-positive cells suggesting CME could inhibit oxidative stress-induced premature senescence. Therefore, these results suggest that CME might have protective effects against oxidative stress-induced premature senescence via scavenging ROS.

Mulberry fruit extract protects pancreatic ß-cells against hydrogen peroxide-induced apoptosis via antioxidative activity.

Among the many environmental stresses, excessive production of reactive oxygen species (ROS) and the ensuring oxidative stress are known to cause significant cellular damage. This has clinical implications in the onset of type 1 diabetes, which is triggered by the destruction of pancreatic ß-cells and is associated with oxidative stress. In this study, we investigated the protective and antioxidative effects of mulberry extract (ME) in insulin-producing pancreatic ß-cells. We found that ME protects pancreatic ß-cells against hydrogen peroxide (H2O2)-induced oxidative stress and the associated apoptotic cell death. ME treatment significantly reduced the levels of H2O2-induced 2-diphenyl-1-picrylhydrazyl (DPPH) radicals, and lipid peroxidation and intracellular ROS accumulation. In addition, ME inhibited DNA condensation and/or fragmentation induced by H2O2. These results suggest that ME protects pancreatic ß-cells against hydrogen peroxide-induced oxidative stress.

Overcoming inefficient cellobiose fermentation by cellobiose phosphorylase in the presence of xylose.

BACKGROUND: Cellobiose and xylose co-fermentation holds promise for efficiently producing biofuels from plant biomass. Cellobiose phosphorylase (CBP), an intracellular enzyme generally found in anaerobic bacteria, cleaves cellobiose to glucose and glucose-1-phosphate, providing energetic advantages under the anaerobic conditions required for large-scale biofuel production. However, the efficiency of CBP to cleave cellobiose in the presence of xylose is unknown. This study investigated the effect of xylose on anaerobic CBP-mediated cellobiose fermentation by Saccharomyces cerevisiae. RESULTS: Yeast capable of fermenting cellobiose by the CBP pathway consumed cellobiose and produced ethanol at rates 61% and 42% slower, respectively, in the presence of xylose than in its absence. The system generated significant amounts of the byproduct 4-O-ß-d-glucopyranosyl-d-xylose (GX), produced by CBP from glucose-1-phosphate and xylose. In vitro competition assays identified xylose as a mixed-inhibitor for cellobiose phosphorylase activity. The negative effects of xylose were effectively relieved by efficient cellobiose and xylose co-utilization. GX was also shown to be a substrate for cleavage by an intracellular ß-glucosidase. CONCLUSIONS: Xylose exerted negative impacts on CBP-mediated cellobiose fermentation by acting as a substrate for GX byproduct formation and a mixed-inhibitor for cellobiose phosphorylase activity. Future efforts will require efficient xylose utilization, GX cleavage by a ß-glucosidase, and/or a CBP with improved substrate specificity to overcome the negative impacts of xylose on CBP in cellobiose and xylose co-fermentation.

A biosynthetic pathway for hexanoic acid production in Kluyveromyces marxianus.

Hexanoic acid can be used for diverse industrial applications and is a precursor for fine chemistry. Although some natural microorganisms have been screened and evolved to produce hexanoic acid, the construction of an engineered biosynthetic pathway for producing hexanoic acid in yeast has not been reported. Here we constructed hexanoic acid pathways in Kluyveromyces marxianus by integrating 5 combinations of seven genes (AtoB, BktB, Crt, Hbd, MCT1, Ter, and TES1), by which random chromosomal sites of the strain are overwritten by the new genes from bacteria and yeast. One recombinant strain, H4A, which contained AtoB, BktB, Crt, Hbd, and Ter, produced 154mg/L of hexanoic acid from galactose as the sole substrate. However, the hexanoic acid produced by the H4A strain was re-assimilated during the fermentation due to the reverse activity of AtoB, which condenses two acetyl-CoAs into a single acetoacetyl-CoA. This product instability could be overcome by the replacement of AtoB with a malonyl CoA-acyl carrier protein transacylase (MCT1) from Saccharomyces cerevisiae. Our results suggest that Mct1 provides a slow but stable acetyl-CoA chain elongation pathway, whereas the AtoB-mediated route is fast but unstable. In conclusion, hexanoic acid was produced for the first time in yeast by the construction of chain elongation pathways comprising 5-7 genes in K. marxianus.