Lignocellulosic ethanol and butanol production by Saccharomyces cerevisiae and Clostridium beijerinckii co-culture using non-detoxified corn stover hydrolysate.

Considering global economic and environmental -benefits, green renewable biofuels such as ethanol and butanol are considered as sustainable alternatives to fossil fuels. Thus, developing a co-culture strategy for ethanol and butanol production by Saccharomyces cerevisiae and Clostridium beijerinckii has emerged as a promising approach for biofuel production from lignocellulosic biomass. This study developed a co-culture of S. cerevisiae and C. beijerinckii for ethanol and butanol production from non-detoxified corn stover hydrolysate. By firstly inoculating 3 % S. cerevisiae and then 7 % C. beijerinckii with 8-10 h time intervals, the optimized co-culture process gave 24.0 g/L ABE (20.8 g/L ethanol and 2.4 g/L butanol), obtaining ABE yield and productivity of 0.421 g/g and 0.55 g/L/h. The demonstrated co-culture strategy made full use of hexose and pentose in hydrolysate and contributed to total yield and efficiency compared to conventional ethanol or ABE fermentation, indicating its great potential for developing economically feasible and sustainable bioalcohols production.

Assessing the Performance of Non-Equilibrium Thermodynamic Integration in Flavodoxin Redox Potential Estimation.

Flavodoxins are enzymes that contain the redox-active flavin mononucleotide (FMN) cofactor and play a crucial role in numerous biological processes, including energy conversion and electron transfer. Since the redox characteristics of flavodoxins are significantly impacted by the molecular environment of the FMN cofactor, the evaluation of the interplay between the redox properties of the flavin cofactor and its molecular surroundings in flavoproteins is a critical area of investigation for both fundamental research and technological advancements, as the electrochemical tuning of flavoproteins is necessary for optimal interaction with redox acceptor or donor molecules. In order to facilitate the rational design of biomolecular devices, it is imperative to have access to computational tools that can accurately predict the redox potential of both natural and artificial flavoproteins. In this study, we have investigated the feasibility of using non-equilibrium thermodynamic integration protocols to reliably predict the redox potential of flavodoxins. Using as a test set the wild-type flavodoxin from Clostridium Beijerinckii and eight experimentally characterized single-point mutants, we have computed their redox potential. Our results show that 75% (6 out of 8) of the calculated reaction free energies are within 1 kcal/mol of the experimental values, and none exceed an error of 2 kcal/mol, confirming that non-equilibrium thermodynamic integration is a trustworthy tool for the quantitative estimation of the redox potential of this biologically and technologically significant class of enzymes.

Multiplex genome engineering in Clostridium beijerinckii NCIMB 8052 using CRISPR-Cas12a.

Clostridium species are re-emerging as biotechnological workhorses for industrial acetone-butanol-ethanol production. This re-emergence is largely due to advances in fermentation technologies but also due to advances in genome engineering and re-programming of the native metabolism. Several genome engineering techniques have been developed including the development of numerous CRISPR-Cas tools. Here, we expanded the CRISPR-Cas toolbox and developed a CRISPR-Cas12a genome engineering tool in Clostridium beijerinckii NCIMB 8052. By controlling the expression of FnCas12a with the xylose-inducible promoter, we achieved efficient (25-100%) single-gene knockout of five C. beijerinckii NCIMB 8052 genes (spo0A, upp, Cbei_1291, Cbei_3238, Cbei_3832). Moreover, we achieved multiplex genome engineering by simultaneously knocking out the spo0A and upp genes in a single step with an efficiency of 18%. Finally, we showed that the spacer sequence and position in the CRISPR array can affect the editing efficiency outcome.

Enzymatic breakdown of biofilm matrix to allow flow cytometry viability analysis of Clostridium beijerinckii cells.

AIMS: Flow cytometry (FC) is a good way to enumerate the number of viable cells in suspension but is not adapted to mature biofilm analysis. The aim of this study is to investigate the effect of mechanical treatment coupled with enzymatic hydrolysis of biofilm matrix on FC viability analysis of biofilm cells. METHODS AND RESULTS: Biofilm was grown for 300 h of continuous fermentation on polyurethane foams. Fermentation was stopped, and the biofilm was detached by agitating the foams in PBS buffer with vortex agitation for 2 min. The best enzymatic hydrolysis consisted of sequential use of DNase I and proteinase K incubated for 1 h at 34°C. Biofilm cells detached from polyurethane foams were stained with both propidium iodide (PI) and carboxyfluoresceine diacetate and analyzed by FC. FC analysis performed after vortex agitation revealed the presence of high non-fluorescent events (78.9% ± 3.3%). After enzymatic treatment, a cell population was extracted from background noise and could be observed on FSC-SSC profile. The non-fluorescent events of this cell population decreased drastically to 41.9% ± 6.6%, and the percentage of viable cells was enhanced from 2.6% ± 0.9% to 38.2% ± 4.0% compared to analysis performed after mechanical treatment alone. CONCLUSIONS: Consequently, protease and nuclease activity are essential to hydrolyze extra polymeric substances prior to FC viability analysis in mature biofilm formed by Clostridium beijerinckii.

Avoiding acid crash: From apple pomace hydrolysate to butanol through acetone-butanol-ethanol fermentation in a zero-waste approach.

Apple pomace (AP) is a lignocellulosic residue from the juice and cider industries that can be valorized in a multi-product biorefinery to generate multiple value-added compounds, including biofuels such as butanol. Butanol is produced biologically by acetone-butanol-ethanol (ABE) fermentation using bacteria of the genus Clostridium from sugar-based feedstocks. In this study, AP hydrolysate was used as a substrate for producing butanol by ABE fermentation. Various environmental factors influence the amount of butanol produced, but only under certain conditions the so-called 'acid crash', an undesirable phenomenon characterized by a total arrest of cell growth and solvent production, can be avoided. Operational parameters that may influence the prevention of acid crash occurrence, such as pH, CaCO3 concentration and culture temperature, were optimized in C. beijerinckii CECT 508 cultures applying a Box-Behnken experimental design. The mathematical model of the fermentation found the optimal conditions of pH 7, 6.8 g/L of CaCO3 and 30 °C, and this was validated in an independent experiment carried out at the optimal conditions, reaching 10.75 g/L of butanol. Also, the comparison of butanol production between the supernatant of the AP hydrolysate (10.57 g/L) and the full hydrolysate with solids (11.69 g/L) indicated that it is possible to eliminate the centrifugation step after hydrolysis, which may allow to reduce process costs and the full utilization of apple pomace, aiming a zero-waste approach.

Identification of serine/threonine kinases that regulate metabolism and sporulation in Clostridium beijerinckii.

Serine/threonine protein kinases (STKs) are important for signal transduction and involved in multiple physiological processes, including cell growth, central metabolism, and sporulation in bacteria. However, the role of STKs in solventogenic clostridia remains unclear. Here, we identified and comprehensively investigated six STK candidates in Clostridium beijerinckii. These STKs were classified into four groups with distinct characteristics via analysis of genetic organizations, prediction of protein domains, and multiple sequence alignment. Cbei0566 is a member of the PrkA family with 41% identity to PrkA from Bacillus subtilis, and both Cbei0666 and Cbei0813 are two-component-like STKs. Cbei1151 and Cbei1929 belong to the Hanks family STKs and consist of a cytoplasmic catalytic domain, a transmembrane region, and extracellular sensor domains. In-frame deletion mutants of cbei0566, cbei0666, cbei1929, and cbei2661 displayed similar cell growth with wild type. Both Δcbei0666 and Δcbei2661 improved acetone-butanol-ethanol (ABE) production by 14.3% (19.2 g/L vs. 16.8 g/L), and the sporulation frequencies of Δcbei0566, Δcbei1929, and Δcbei2661 significantly decreased to 35.5%, 55.1% and 44.8%, respectively. The restored phenotypes after genetic complementation demonstrated their direct link to STKs deletion. Remarkably, overexpressing cbei0566 contributed to 41.5% more spore formation and cbei1929 overexpression enhanced ABE production from 19.3 to 24.2 g/L, along with 25% less acids. These results revealed that Cbei0566 and Cbei1929 had prominent regulatory functions. This study expands the current knowledge of the existence and functions of STKs in prokaryotes and highlights the importance of STK-mediated signaling networks in developing superior strains. KEY POINTS: • First reported serine/threonine protein kinases in solventogenic clostridia • Six STKs with distinct properties possessed diverse functions in C. beijerinckii • Cbei1929 and Cbei0566 remarkably regulated solventogenesis and sporulation.

Transcriptomic studies of solventogenic clostridia, Clostridium acetobutylicum and Clostridium beijerinckii.

Solventogenic clostridia are not a strictly defined group within the genus Clostridium but its representatives share some common features, i.e. they are anaerobic, non-pathogenic, non-toxinogenic and endospore forming bacteria. Their main metabolite is typically 1-butanol but depending on species and culture conditions, they can form other metabolites such as acetone, isopropanol, ethanol, butyric, lactic and acetic acids, and hydrogen. Although these organisms were previously used for the industrial production of solvents, they later fell into disuse, being replaced by more efficient chemical production. A return to a more biological production of solvents therefore requires a thorough understanding of clostridial metabolism. Transcriptome analysis, which reflects the involvement of individual genes in all cellular processes within a population, at any given (sampling) moment, is a valuable tool for gaining a deeper insight into clostridial life. In this review, we describe techniques to study transcription, summarize the evolution of these techniques and compare methods for data processing and visualization of solventogenic clostridia, particularly the species Clostridium acetobutylicum and Clostridium beijerinckii. Individual approaches for evaluating transcriptomic data are compared and their contributions to advancements in the field are assessed. Moreover, utilization of transcriptomic data for reconstruction of computational clostridial metabolic models is considered and particular models are described. Transcriptional changes in glucose transport, central carbon metabolism, the sporulation cycle, butanol and butyrate stress responses, the influence of lignocellulose-derived inhibitors on growth and solvent production, and other respective topics, are addressed and common trends are highlighted.

Viable strategies for enhancing acetone-butanol-ethanol production from non-detoxified switchgrass hydrolysates.

A process engineering strategy was investigated towards developing a viable scheme for effective conversion of hydrothermolysis pretreated non-detoxified switchgrass hydrolysates (SH) to acetone butanol ethanol (ABE) using a metabolically engineered strain of Clostridium beijerinckii NCIMB 8052, C. beijerinckii_AKR. The engineered strain was modified by homologous integration into the chromosome and constitutive expression of Cbei_3974, which encodes an aldo-keto reductase. Intermittent feeding strategy was employed in which fermentation was initiated with 30% of the SH and the remaining 70% SH was added when the optical density (OD600nm) of C. beijerinckii attained 0.5. The ABE (14.9 g/L) produced from non-detoxified SH by the inhibitor-tolerant C. beijerinckii_AKR was comparable to the P2-glucose control medium (14.7 g/L). Using intermittent feeding, wildtype and C. beijerinckii_AKR produced similar amounts of ABE (about 17.5 g/L). This shows that intermittent feeding strategy and C. beijerinckii_AKR enhanced ABE fermentation and eliminated the need for SH detoxification prior to fermentation.

Kinetic model of Clostridium beijerinckii's Acetone-Butanol-Ethanol fermentation considering metabolically diverse cell types.

Clostridium beijerinckii population branches into metabolically diverse cell types in batch cultures. Here, we present a new kinetic model of C. beijerinckii's Acetone-Butanol-Ethanol fermentation that considers three cell types: producers of acids (acidogenic), consumer of acids and producers of solvents (solventogenic), and spores cells. The model accurately recapitulates batch culture data. Also, the model estimates cell type-specific kinetic parameters, which can be helpful to improve the operation of the ABE fermentation and give a framework to study acidogenic and solventogenic metabolic pathways. To exemplify the latter, we used a constraint-based model to study how the ABE pathways are used among acidogenic and solventogenic cell types. We found that among both cell types, glycolytic production of ATP and consumption of NAD+ varies widely during the fermentation, with their maximum production/consumption rates happening when acidogenic and solventogenic growth rates were at their highest. However, acidogenic cells use the ABE pathway to contribute with an extra 12.5% of the total production of ATP, whereas solventogenic cell types use the ABE pathway to supply more than 75% of the demand for NAD+, alternating between the production of lactate and butyrate, being both coupled to the production of NAD+.

Comparative genome analysis of Clostridium beijerinckii strains isolated from pit mud of Chinese strong flavor baijiu ecosystem.

Clostridium beijerinckii is a well-known anaerobic solventogenic bacterium which inhabits a wide range of different niches. Previously, we isolated five butyrate-producing C. beijerinckii strains from pit mud (PM) of strong-flavor baijiu (SFB) ecosystems. Genome annotation of the five strains showed that they could assimilate various carbon sources as well as ammonium to produce acetate, butyrate, lactate, hydrogen, and esters but did not produce the undesirable flavors isopropanol and acetone, making them useful for further exploration in SFB production. Our analysis of the genomes of an additional 233 C. beijerinckii strains revealed an open pangenome based on current sampling and will likely change with additional genomes. The core genome, accessory genome, and strain-specific genes comprised 1567, 8851, and 2154 genes, respectively. A total of 298 genes were found only in the five C. beijerinckii strains from PM, among which only 77 genes were assigned to Clusters of Orthologous Genes categories. In addition, 15 transposase and 12 phage integrase families were found in all five C. beijerinckii strains from PM. Between 18 and 21 genome islands were predicted for the five C. beijerinckii genomes. The existence of a large number of mobile genetic elements indicated that the genomes of the five C. beijerinckii strains evolved with the loss or insertion of DNA fragments in the PM of SFB ecosystems. This study presents a genomic framework of C. beijerinckii strains from PM that could be used for genetic diversification studies and further exploration of these strains.

Production of butanol from distillers' grain waste by a new aerotolerant strain of Clostridium beijerinckii LY-5.

A new aerotolerant strain of Clostridium beijerinckii LY-5 was isolated from the pit mud of the Chinese Baijiu-making process for butanol production. Plackett-Burman design and artificial neural network were used to optimize the fermentation medium and a total of 13.54 ± 0.22 g/L butanol and 19.91 ± 0.52 g/L ABE were attained under aerotolerant condition. Moreover, distillers' grain waste (DGW), the main by-product in the Baijiu production process, was utilized as potential substrate for butanol production. DGW was hydrolyzed by α-amylase and glucoamylase and then fermented after a detoxifying process of overliming. Butanol and ABE concentrations were 9.02 ± 0.18 and 9.57 ± 0.19 g/L with the yield of 0.21 and 0.23 g/g sugar, respectively. The higher ratio of butanol to ABE might be caused by the inhibitors in DGW medium affecting the metabolic pathways of C. beijerinckii LY-5 and approximately 1.48 ± 0.04 g/L isopropanol was found at the end of fermentation. This work highlights the feasibility of using DGW as a promising feedstock for butanol production by a new aerotolerant strain of C. beijerinckii LY-5, with benefit to the environment.

A neutral red mediated electro-fermentation system of Clostridium beijerinckii for effective co-production of butanol and hydrogen.

To enhance the co-production of butanol and hydrogen by the acetone-butanol-ethanol (ABE) fermentation of Clostridium beijerinckii, a novel cathodic electro-fermentation (CEF) system was constructed with neutral red (NR) as electron mediator. With the mediation of NR, production of butanol and hydrogen from glucose in the CEF system achieved 5.49 ± 0.28 g/L and 3.74 ± 0.16 L/L, 569.5% and 325.0% higher than that in the open circuit (OC) system, respectively. The butanol and hydrogen yield of 0.30 ± 0.02 g/g and 206.53 ± 8.20 mL/g was 172.7% and 71.4% higher than that in the OC system, respectively. The effective co-production of butanol and hydrogen in the NR-mediated CEF system was attributed to the cooperation of the introduced polarized electrode and the additional NR. With the control of the polarized electrode, a feasible ORP was available for the effective hydrogen production. And the additional NR had induced more carbon source and electrons to the synthesis of butanol.

Ideal conditions of microwave-assisted acid pretreatment of sugarcane straw allow fermentative butyric acid production without detoxification step.

Sugarcane straw (SCS) was pretreated with dilute sulfuric acid assisted by microwave to magnify fermentable sugars and to minimize the concentration of inhibitors in the hydrolysates. The optimum conditions for maximum recovery of sugars were 162 °C and 0.6% (w/v) H2SO4. The low level of inhibitors, such as acetate (2.9 g/L) and total phenolics (1.4 g/L), in the SCS slurry from the pretreatment stage allowed the enzymatic hydrolysis and fermentation steps to occur without detoxification. Besides consuming the total sugar content (31.0 g/L), Clostridium beijerinckii Br21 was able to use acetate from the SCS hydrolysate, to give butyric acid at high conversion factor (0.49 g of butyric acid /g of sugar). The optimized pretreatment conditions spared acid, time, and the detoxification stage, making bio-butyric acid production from SCS extremely attractive.

A safety cap protects hydrogenase from oxygen attack.

[FeFe]-hydrogenases are efficient H2-catalysts, yet upon contact with dioxygen their catalytic cofactor (H-cluster) is irreversibly inactivated. Here, we combine X-ray crystallography, rational protein design, direct electrochemistry, and Fourier-transform infrared spectroscopy to describe a protein morphing mechanism that controls the reversible transition between the catalytic Hox-state and the inactive but oxygen-resistant Hinact-state in [FeFe]-hydrogenase CbA5H of Clostridium beijerinckii. The X-ray structure of air-exposed CbA5H reveals that a conserved cysteine residue in the local environment of the active site (H-cluster) directly coordinates the substrate-binding site, providing a safety cap that prevents O2-binding and consequently, cofactor degradation. This protection mechanism depends on three non-conserved amino acids situated approximately 13 Å away from the H-cluster, demonstrating that the 1st coordination sphere chemistry of the H-cluster can be remote-controlled by distant residues.

A comparative phenotypic and genomic analysis of Clostridium beijerinckii mutant with enhanced solvent production.

The acetone-butanol-ethanol (ABE) fermentation by solventogenic clostridia has a long history of industrial butanol production. The Clostridium beijerinckii mutant BA101 has been widely studied for ABE fermentation owing to its enhanced butanol production capacity. Here, we characterized the BA101 mutant under controlled environmental conditions in parallel with the parental strain C. beijerinckii NCIMB 8052. To investigate the correlation between phenotype and genotype, we carried out the genome sequencing of BA101. Through comparative genomic analysis, several mutations in the genes encoding transcriptional regulator, sensor kinase, and phosphatase were identified in the BA101 genome as well as other sibling mutants. Among them, the SNP in the Cbei_3078 gene encoding PAS/PAC sensor hybrid histidine kinase was unique to the BA101 strain. The identified mutations relevant to the observed physiological behaviors of BA101 could be potential genetic targets for rational engineering of solventogenic clostridia toward desired phenotypes.

Changes in efflux pump activity of Clostridium beijerinckii throughout ABE fermentation.

Pumping toxic substances through a cytoplasmic membrane by protein transporters known as efflux pumps represents one bacterial mechanism involved in the stress response to the presence of toxic compounds. The active efflux might also take part in exporting low-molecular-weight alcohols produced by intrinsic cell metabolism; in the case of solventogenic clostridia, predominantly acetone, butanol and ethanol (ABE). However, little is known about this active efflux, even though some evidence exists that membrane pumps might be involved in solvent tolerance. In this study, we investigated changes in overall active efflux during ABE fermentation, employing a flow cytometric protocol adjusted for Clostridia and using ethidium bromide (EB) as a fluorescence marker for quantification of direct efflux. A fluctuation in efflux during the course of standard ABE fermentation was observed, with a maximum reached during late acidogenesis, a high efflux rate during early and mid-solventogenesis and an apparent decrease in EB efflux rate in late solventogenesis. The fluctuation in efflux activity was in accordance with transcriptomic data obtained for various membrane exporters in a former study. Surprisingly, under altered cultivation conditions, when solvent production was attenuated, and extended acidogenesis was promoted, stable low efflux activity was reached after an initial peak that appeared in the stage comparable to standard ABE fermentation. This study confirmed that efflux pump activity is not constant during ABE fermentation and suggests that undisturbed solvent production might be a trigger for activation of pumps involved in solvent efflux. KEY POINTS: • Flow cytometric assay for efflux quantification in Clostridia was established. • Efflux rate peaked in late acidogenesis and in early solventogenesis. • Impaired solventogenesis led to an overall decrease in efflux.

Deeper below the surface-transcriptional changes in selected genes of Clostridium beijerinckii in response to butanol shock.

The main bottleneck in the return of industrial butanol production from renewable feedstock through acetone-butanol-ethanol (ABE) fermentation by clostridia, such as Clostridium beijerinckii, is the low final butanol concentration. The problem is caused by the high toxicity of butanol to the production cells, and therefore, understanding the mechanisms by which clostridia react to butanol shock is of key importance. Detailed analyses of transcriptome data that were obtained after butanol shock and their comparison with data from standard ABE fermentation have resulted in new findings, while confirmed expected population responses. Although butanol shock resulted in upregulation of heat shock protein genes, their regulation is different than was assumed based on standard ABE fermentation transcriptome data. While glucose uptake, glycolysis, and acidogenesis genes were downregulated after butanol shock, solventogenesis genes were upregulated. Cyclopropanation of fatty acids and formation of plasmalogens seem to be significant processes involved in cell membrane stabilization in the presence of butanol. Surprisingly, one of the three identified Agr quorum-sensing system genes was upregulated. Upregulation of several putative butanol efflux pumps was described after butanol addition and a large putative polyketide gene cluster was found, the transcription of which seemed to depend on the concentration of butanol.

Sugarcane bagasse hydrolysates as feedstock to produce the isopropanol-butanol-ethanol fuel mixture: Effect of lactic acid derived from microbial contamination on Clostridium beijerinckii DSM 6423.

Enzymatic hydrolysis of lignocellulose under industrial conditions is prone to contamination by lactic acid bacteria, and in this study, a cellulose hydrolysate produced from dilute-acid pretreatedsugarcane bagasse contained 13 g/L lactic acid and was used for IBE production by Clostridium beijerinckii DSM 6423. In fermentation of the cellulose hydrolysate supplemented with sugarcane molasses for nutrients and buffering of the medium (40 g/L total sugar), 92% of the lactic acid was consumed, and the butanol yield was as high as 0.28 (7.9 g/L butanol), suggesting that lactic acid was preferentially metabolized to butanol. When the hydrolysate was mixed with a detoxified bagasse hemicellulose hydrolysate and supplemented with molasses (35 g/L total sugar), the culture was able to exhaust glucose and utilized sucrose (by 38%), xylose (31%), and lactic acid (70%). Overall, this study shows that C. beijerinckii DSM 6423 can co-ferment first- and second-generation sugars while consuming lactic acid.

Orange Bagasse Pellets as a Carbon Source for Biobutanol Production.

Due to the environmental concerns, the conversion of lignocellulosic waste can be the key to produce bioproducts and biofuels such as butanol. This study aimed to present and evaluate orange bagasse pellets (OBP) as a carbon source to produce butan-1-ol production via ABE fermentation using Clostridium beijerinckii. These bagasse pellets were characterized, holocellulose (18.99%), alfacellulose (5.37%), hemicellulose (13.62%), lignin (6.16%), pectin (7.21%), protein (3.14%), and was tested under three different pretreatments, which were the following: (a) ultrasound, (b) autohydrolysis, and (c) acid-diluted hydrolysis followed by enzymatic hydrolysis to verify an amount of fermentable total reducing sugars. ANOVA was used and pretreatments followed by enzymatic hydrolysis do not enhance a significant amount of available sugars compared to raw bagasse. The ABE fermentation was carried out in batch reactors at 37 °C under agitation of 160 rpm and anaerobic conditions, using OBP without treatment followed by enzymatic hydrolysis. Using a non-mutant microorganism, the fermentation achieved butyric acid yields of 3762.68 mg L-1 for control and 2488.82 mg L-1 for OBP and the butanol production was 63.86 mg L-1 and 196.80 mg L-1 for OBP and the control (glucose) assay, respectively. The results of this solvent's production have shown that OBP has the potential for ABE fermentation and a promising feedstock.

Metabolic and Process Engineering of Clostridium beijerinckii for Butyl Acetate Production in One Step.

n-Butyl acetate is an important food additive commonly produced via concentrated sulfuric acid catalysis or immobilized lipase catalysis of butanol and acetic acid. Compared with chemical methods, an enzymatic approach is more environmentally friendly; however, it incurs a higher cost due to lipase production. In vivo biosynthesis via metabolic engineering offers an alternative to produce n-butyl acetate. This alternative combines substrate production (butanol and acetyl-coenzyme A (acetyl-CoA)), alcohol acyltransferase expression, and esterification reaction in one reactor. The alcohol acyltransferase gene ATF1 from Saccharomyces cerevisiae was introduced into Clostridium beijerinckii NCIMB 8052, enabling it to directly produce n-butyl acetate from glucose without lipase addition. Extractants were compared and adapted to realize glucose fermentation with in situ n-butyl acetate extraction. Finally, 5.57 g/L of butyl acetate was produced from 38.2 g/L of glucose within 48 h, which is 665-fold higher than that reported previously. This demonstrated the potential of such a metabolic approach to produce n-butyl acetate from biomass.