N-butanol Production Research Articles

Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production.

BackgroundThere is a strong interest in using photosynthetic cyanobacteria as production hosts for biofuels and chemicals. Recent work has shown the benefit of pathway engineering, enzyme tolerance, and co-factor usage for improving yields of fermentation products.ResultsAn n-butanol pathway was inserted into a Synechocystis mutant deficient in polyhydroxybutyrate synthesis. We found that nitrogen starvation increased specific butanol productivity up to threefold, but cessation of cell growth limited total n-butanol titers. Metabolite profiling showed that acetyl-CoA increased twofold during nitrogen starvation. Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions and increased butanol titers from 22 to 37 mg/L at day 8. Flux balance analysis of photoautotrophic metabolism showed that a Calvin–Benson–Bassham-Phosphoketolase pathway had higher theoretical butanol productivity than CBB-Embden–Meyerhof–Parnas and a reduced butanol ATP demand.ConclusionThese results demonstrate that phosphoketolase overexpression and modulation of nitrogen levels are two attractive routes toward increased production of acetyl-CoA derived products in cyanobacteria and could be implemented with complementary metabolic engineering strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0355-9) contains supplementary material, which is available to authorized users.

Economic and environmental assessment of n-butanol production in an integrated first and second generation sugarcane biorefinery: Fermentative versus catalytic routes

n-Butanol produced from renewable resources has attracted increasing interest, mostly for its potential use as liquid biofuel for transportation. Process currently used in the industry (Acetone–Butanol–Ethanol fermentation – ABE) faces major technical challenges, which could be overcome by an alternative production through ethanol catalysis. In this study, both routes are evaluated by means of their financial viabilities and environmental performance assessed through the Virtual Sugarcane Biorefinery methodological framework. Comparative financial analysis of the routes integrated to a first and second generation sugarcane biorefinery shows that, despite the drawbacks, ABE process for fermentation of the pentoses liquor is more attractive than the catalysis of ethanol to n-butanol and co-products. n-Butanol use as fuel demonstrated favorable environmental results for climate change as figures showed over 50% reduction in greenhouse gas emission compared with gasoline.

Modular design of metabolic network for robust production of n-butanol from galactose–glucose mixtures

BackgroundRefactoring microorganisms for efficient production of advanced biofuel such as n-butanol from a mixture of sugars in the cheap feedstock is a prerequisite to achieve economic feasibility in biorefinery. However, production of biofuel from inedible and cheap feedstock is highly challenging due to the slower utilization of biomass-driven sugars, arising from complex assimilation pathway, difficulties in amplification of biosynthetic pathways for heterologous metabolite, and redox imbalance caused by consuming intracellular reducing power to produce quite reduced biofuel. Even with these problems, the microorganisms should show robust production of biofuel to obtain industrial feasibility. Thus, refactoring microorganisms for efficient conversion is highly desirable in biofuel production.ResultsIn this study, we engineered robust Escherichia coli to accomplish high production of n-butanol from galactose–glucose mixtures via the design of modular pathway, an efficient and systematic way, to reconstruct the entire metabolic pathway with many target genes. Three modular pathways designed using the predictable genetic elements were assembled for efficient galactose utilization, n-butanol production, and redox re-balancing to robustly produce n-butanol from a sugar mixture of galactose and glucose. Specifically, the engineered strain showed dramatically increased n-butanol production (3.3-fold increased to 6.2 g/L after 48-h fermentation) compared to the parental strain (1.9 g/L) in galactose-supplemented medium. Moreover, fermentation with mixtures of galactose and glucose at various ratios from 2:1 to 1:2 confirmed that our engineered strain was able to robustly produce n-butanol regardless of sugar composition with simultaneous utilization of galactose and glucose.ConclusionsCollectively, modular pathway engineering of metabolic network can be an effective approach in strain development for optimal biofuel production with cost-effective fermentable sugars. To the best of our knowledge, this study demonstrated the first and highest n-butanol production from galactose in E. coli. Moreover, robust production of n-butanol with sugar mixtures with variable composition would facilitate the economic feasibility of the microbial process using a mixture of sugars from cheap biomass in the near future.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0327-7) contains supplementary material, which is available to authorized users.

Butanol production in S. cerevisiae via a synthetic ABE pathway is enhanced by specific metabolic engineering and butanol resistance.

BackgroundThe fermentation of sugars to alcohols by microbial systems underpins many biofuel initiatives. Short chain alcohols, like n-butanol, isobutanol and isopropanol, offer significant advantages over ethanol in terms of fuel attributes. However, production of ethanol from resistant Saccharomyces cerevisiae strains is significantly less complicated than for these alternative alcohols.ResultsIn this study, we have transplanted an n-butanol synthesis pathway largely from Clostridial sp. to the genome of an S. cerevisiae strain. Production of n-butanol is only observed when additional genetic manipulations are made to restore any redox imbalance and to drive acetyl-CoA production. We have used this butanol production strain to address a key question regarding the sensitivity of cells to short chain alcohols. In the past, we have defined specific point mutations in the translation initiation factor eIF2B based upon phenotypic resistance/sensitivity to high concentrations of exogenously added n-butanol. Here, we show that even during endogenous butanol production, a butanol resistant strain generates more butanol than a butanol sensitive strain.ConclusionThese studies demonstrate that appreciable levels of n-butanol can be achieved in S. cerevisiae but that significant metabolic manipulation is required outside of the pathway converting acetyl-CoA to butanol. Furthermore, this work shows that the regulation of protein synthesis by short chain alcohols in yeast is a critical consideration if higher yields of these alcohols are to be attained.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0281-4) contains supplementary material, which is available to authorized users.

Metabolic engineering of Clostridium tyrobutyricum for n-butanol production from maltose and soluble starch by overexpressing α-glucosidase.

Clostridium tyrobutyricum does not have the enzymes needed for using maltose or starch. Two extracellular α-glucosidases encoded by agluI and agluII from Clostridium acetobutylicum ATCC 824 catalyzing the hydrolysis of α-1,4-glycosidic bonds in maltose and starch from the non-reducing end were cloned and expressed in C. tyrobutyricum (Δack, adhE2), and their effects on n-butanol production from maltose and soluble starch in batch fermentations were studied. Compared to the parental strain grown on glucose, mutants expressing agluI showed robust activity in breaking down maltose and produced more butanol (17.2 vs. 9.5 g/L) with a higher butanol yield (0.20 vs. 0.10 g/g) and productivity (0.29 vs. 0.16 g/L h). The mutant was also able to use soluble starch as substrate, although at a slower rate compared to maltose. Compared to C. acetobutylicum ATCC 824, the mutant produced more butanol from maltose (17.2 vs. 11.2 g/L) and soluble starch (16.2 vs. 8.8 g/L) in batch fermentations. The mutant was stable in batch fermentation without adding antibiotics, achieving a high butanol productivity of 0.40 g/L h. This mutant strain thus can be used in industrial production of n-butanol from maltose and soluble starch.

Investigation of uncertainties associated with the production of n-butanol through ethanol catalysis in sugarcane biorefineries

This study evaluated the viability of n-butanol production integrated within a first and second generation sugarcane biorefinery. The evaluation included a deterministic analysis as well as a stochastic approach, the latter using Monte Carlo simulation. Results were promising for n-butanol production in terms of revenues per tonne of processed sugarcane, but discouraging with respect to internal rate of return (IRR). The uncertainty analysis determined there was high risk involved in producing n-butanol and co-products from ethanol catalysis. It is unlikely that these products and associated production route will be financially attractive in the short term without lower investment costs, supportive public policies and tax incentives coupled with biofuels’ production strategies.

Metabolic engineering of Clostridium tyrobutyricum for n-butanol production: effects of CoA transferase.

The overexpression of CoA transferase (ctfAB), which catalyzes the reaction: acetate/butyrate + acetoacetyl-CoA → acetyl/butyryl-CoA + acetoacetate, was studied for its effects on acid reassimilation and butanol biosynthesis in Clostridium tyrobutyricum (Δack, adhE2). The plasmid pMTL007 was used to co-express adhE2 and ctfAB from Clostridium acetobutylicum ATCC 824. In addition, the sol operon containing ctfAB, adc (acetoacetate decarboxylase), and ald (aldehyde dehydrogenase) was also cloned from Clostridium beijerinckii NCIMB 8052 and expressed in C. tyrobutyricum (Δack, adhE2). Mutants expressing these genes were evaluated for their ability to produce butanol from glucose in batch fermentations at pH5.0 and 6.0. Compared to C. tyrobutyricum (Δack, adhE2) without expressing ctfAB, all mutants with ctfAB overexpression produced more butanol, with butanol yield increased to 0.22 - 0.26g/g (vs. 0.10 - 0.13g/g) and productivity to 0.35g/lh (vs. 0.13g/lh) because of the reduced acetate and butyrate production. The expression of ctfAB also resulted in acetone production from acetoacetate through a non-enzymatic decarboxylation.

Improved n-butanol tolerance in Escherichia coli by controlling membrane related functions

As the increasing demand from both chemical and fuel markets, the interest in producing n-butanol using biological route has been rejuvenated to engineer an economical fermentation process, competing with the currently-dominant chemical synthesis. n-Butanol has been traditionally produced from the ABE fermentation of Clostridium acetobutylicum. This system, however, is not economically feasible due to its limited efficiency and the lack of genetic modification tools for further improvements. Alternatively, n-butanol synthesis pathway was successfully transferred into Escherichia coli and rapidly improved to reach a level of production comparable to the native producer. Nevertheless, the toxicity of n-butanol has become a common issue that either approach has to deal with. Previously, we reported our success in improving n-butanol tolerance in E. coli by engineering an Artificial Transcription Factor (ATF) that can modify the expression level of multiple targets simultaneously and improved the n-butanol tolerance of MG1655 strain to 1.5% (vol/vol) n-butanol. However, it was observed that some possible n-butanol tolerance mechanisms did not occurred upon the ATF expression, especially the membrane-related functions such as the homeoviscous adaptation, iron uptaking system, and efflux pump system. In this work, we attempted to enhance the n-butanol tolerance associated with the ATF by combining it with the membrane-related functions in E. coli, including the overexpression of fatty acid synthesis genes, iron-uptaking protein FeoA, and introducing a SrpABC efflux pump from Pseudomonas putida into E. coli. The synergistic effect of this combinatorial approach led to 4, 5, and 9-fold improved growths in the cultures containing 1, 1.5, and 2% (vol/vol) n-butanol, respectively, of an MG1655 knockout strain engineered for n-butanol production, and expanded the tolerance limit to 2% (vol/vol) n-butanol.

Analysis of the molecular response of Pseudomonas putida KT2440 to the next-generation biofuel n-butanol

To increase the efficiency of biocatalysts a thorough understanding of the molecular response of the biocatalyst to precursors, products and environmental conditions applied in bioconversions is essential. Here we performed a comprehensive proteome and phospholipid analysis to characterize the molecular response of the potential biocatalyst Pseudomonas putida KT2440 to the next-generation biofuel n-butanol. Using complementary quantitative proteomics approaches we were able to identify and quantify 1467 proteins, corresponding to 28% of the total KT2440 proteome. 256 proteins were altered in abundance in response to n-butanol. The proteome response entailed an increased abundance of enzymes involved in n-butanol degradation including quinoprotein alcohol dehydrogenases, aldehyde dehydrogenases and enzymes of fatty acid beta oxidation. From these results we were able to construct a pathway for the metabolism of n-butanol in P. putida. The initial oxidation of n-butanol is catalyzed by at least two quinoprotein ethanol dehydrogenases (PedE and PedH). Growth of mutants lacking PedE and PedH on n-butanol was significantly impaired, but not completely inhibited, suggesting that additional alcohol dehydrogenases can at least partially complement their function in KT2440. Furthermore, phospholipid profiling revealed a significantly increased abundance of lyso-phospholipids in response to n-butanol, indicating a rearrangement of the lipid bilayer. Biological significancen-butanol is an important bulk chemical and a promising alternative to gasoline as a transportation fuel. Due to environmental concerns as well as increasing energy prices there is a growing interest in sustainable and cost-effective biotechnological production processes for the production of bulk chemicals and transportation fuels from renewable resources. n-butanol fermentation is well established in Clostridiae, but the efficiency of n-butanol production is mainly limited by its toxicity. Therefore bacterial strains with higher intrinsic tolerance to n-butanol have to be selected as hosts for n-butanol production. Pseudomonas bacteria are metabolically very versatile and exhibit a high intrinsic tolerance to organic solvents making them suitable candidates for bioconversion processes. A prerequisite for a potential production of n-butanol in Pseudomonas bacteria is a thorough understanding of the molecular adaption processes caused by n-butanol and the identification of enzymes involved in n-butanol metabolization. This work describes the impact of n-butanol on the proteome and the phospholipid composition of the reference strain P. putida KT2440. The high proteome coverage of our proteomics survey allowed us to reconstruct the degradation pathway of n-butanol and to monitor the changes in the energy metabolism of KT2440 induced by n-butanol. Key enzymes involved in n-butanol degradation identified in study will be interesting targets for optimization of n-butanol production in Pseudomonads. The present work and the identification of key enzymes involved in butanol metabolism may serve as a fundament to develop new or improve existing strategies for the biotechnological production of the next-generation biofuel n-butanol in Pseudomonads.

Engineering Escherichia coli Cell Factories for n-Butanol Production.

The production of n-butanol, as a widely applied solvent and potential fuel, is attracting much attention. The fermentative production of butanol coupled with the production of acetone and ethanol by Clostridium (ABE fermentation) was once one of the oldest biotechnological processes, ranking second in scale behind ethanol fermentation. However, there remain problems with butanol production by Clostridium, especially the difficulty in genetically manipulating clostridial strains. In recent years, many efforts have been made to produce butanol using non-native strains. Until now, the most advanced effort was the engineering of the user-friendly and widely studied Escherichia coli for butanol production. This paper reviews the current progress and problems relating to butanol production by engineered E. coli in terms of prediction using mathematical models, pathway construction, novel enzyme replacement, butanol toxicity, and tolerance engineering strategies.

Metabolic process engineering of Clostridium tyrobutyricum Δack-adhE2 for enhanced n-butanol production from glucose: effects of methyl viologen on NADH availability, flux distribution, and fermentation kinetics.

Butanol biosynthesis through aldehyde/alcohol dehydrogenase (adhE2) is usually limited by NADH availability, resulting in low butanol titer, yield, and productivity. To alleviate this limitation and improve n-butanol production by Clostridium tyrobutyricum Δack-adhE2 overexpressing adhE2, the NADH availability was increased by using methyl viologen (MV) as an artificial electron carrier to divert electrons from ferredoxin normally used for H2 production. In the batch fermentation with the addition of 500 μM MV, H2 , acetate, and butyrate production was reduced by more than 80-90%, while butanol production increased more than 40% to 14.5 g/L. Metabolic flux analysis revealed that butanol production increased in the fermentation with MV because of increased NADH availability as a result of reduced H2 production. Furthermore, continuous butanol production of ∼55 g/L with a high yield of ∼0.33 g/g glucose and extremely low ethanol, acetate, and butyrate production was obtained in fed-batch fermentation with gas stripping for in situ butanol recovery. This study demonstrated a stable and reliable process for high-yield and high-titer n-butanol production by metabolically engineered C. tyrobutyricum by applying MV as an electron carrier to increase butanol biosynthesis.

Potential production platform of n-butanol in Escherichia coli

We proposed a potential production platform of n-butanol in Escherichia coli. First, a butyrate-conversion strain was developed by removal of undesired genes and recruiting endogenous atoDA and Clostridium adhE2. Consequently, this E. coli strain grown on the M9 mineral salt with yeast extract (M9Y) was shown to produce 6.2g/L n-butanol from supplemented butyrate at 36h. The molar conversion yield of n-butanol on butyrate reaches 92%. Moreover, the production platform was advanced by additional inclusion of a butyrate-producing strain. This strain was equipped with a pathway comprising atoDA and heterologous genes for the synthesis of butyrate. Without butyrate, the butyrate-conversion and the butyrate-producing strains were co-cultured in M9Y medium and produced 5.5g/L n-butanol from glucose at 24h. The production yield on glucose accounts for 69% of the theoretical yield. Overall, it indicates a promise of the developed platform for n-butanol production in E. coli.

Design and construction of acetyl-CoA overproducing Saccharomyces cerevisiae strains

Saccharomyces cerevisiae has increasingly been engineered as a cell factory for efficient and economic production of fuels and chemicals from renewable resources. Notably, a wide variety of industrially important products are derived from the same precursor metabolite, acetyl-CoA. However, the limited supply of acetyl-CoA in the cytosol, where biosynthesis generally happens, often leads to low titer and yield of the desired products in yeast. In the present work, combined strategies of disrupting competing pathways and introducing heterologous biosynthetic pathways were carried out to increase acetyl-CoA levels by using the CoA-dependent n-butanol production as a reporter. By inactivating ADH1 and ADH4 for ethanol formation and GPD1 and GPD2 for glycerol production, the glycolytic flux was redirected towards acetyl-CoA, resulting in 4-fold improvement in n-butanol production. Subsequent introduction of heterologous acetyl-CoA biosynthetic pathways, including pyruvate dehydrogenase (PDH), ATP-dependent citrate lyase (ACL), and PDH-bypass, further increased n-butanol production. Recombinant PDHs localized in the cytosol (cytoPDHs) were found to be the most efficient, which increased n-butanol production by additional 3 fold. In total, n-butanol titer and acetyl-CoA concentration were increased more than 12 fold and 3 fold, respectively. By combining the most effective and complementary acetyl-CoA pathways, more than 100mg/L n-butanol could be produced using high cell density fermentation, which represents the highest titer ever reported in yeast using the clostridial CoA-dependent pathway.

Enhancement of n-butanol production by in situ butanol removal using permeating-heating-gas stripping in acetone-butanol-ethanol fermentation.

Butanol recovery from acetone-butanol-ethanol (ABE) fed-batch fermentation using permeating-heating-gas was determined in this study. Fermentation was performed with Clostridium acetobutylicum B3 in a fibrous bed bioreactor and permeating-heating-gas stripping was used to eliminate substrate and product inhibition, which normally restrict ABE production and sugar utilization to below 20 g/L and 60 g/L, respectively. In batch fermentation (without permeating-heating-gas stripping), C. acetobutylicum B3 utilized 60 g/L glucose and produced 19.9 g/L ABE and 12 g/L butanol, while in the integrated process 290 g/L glucose was utilized and 106.27 g/L ABE and 66.09 g/L butanol were produced. The intermittent gas stripping process generated a highly concentrated condensate containing approximately 15% (w/v) butanol, 4% (w/v) acetone, a small amount of ethanol (<1%), and almost no acids, resulting in a highly concentrated butanol solution [∼ 70% (w/v)] after phase separation. Butanol removal by permeating-heating-gas stripping has potential for commercial ABE production.

Stable high-titer n-butanol production from sucrose and sugarcane juice by Clostridium acetobutylicum JB200 in repeated batch fermentations.

The production of n-butanol, a widely used industrial chemical and promising transportation fuel, from abundant, low-cost substrates, such as sugarcane juice, in acetone-butanol-ethanol (ABE) fermentation was studied with Clostridium acetobutylicum JB200, a mutant with high butanol tolerance and capable of producing high-titer (>20 g/L) n-butanol from glucose. Although JB200 is a favorable host for industrial bio-butanol production, its fermentation performance with sucrose and sugarcane juice as substrates has not been well studied. In this study, the long-term n-butanol production from sucrose by JB200 was evaluated with cells immobilized in a fibrous-bed bioreactor (FBB), showing stable performance with high titer (16-20 g/L), yield (∼ 0.21 g/g sucrose) and productivity (∼ 0.32 g/Lh) for 16 consecutive batches over 800 h. Sugarcane thick juice as low-cost substrate was then tested in 3 consecutive batches, which gave similar n-butanol production, demonstrating that JB200 is a robust and promising strain for industrial ABE fermentation.

Solvent screening methodology for in situ ABE extractive fermentation

Solvent screening for in situ liquid extraction of products from acetone-butanol-ethanol (ABE) fermentation was carried out, taking into account biological parameters (biocompatibility, bioavailability, and product yield) and extraction performance (partition coefficient and selectivity) determined in real fermentation broth. On the basis of different solvent characteristics obtained from literature, 16 compounds from different chemical families were selected and experimentally evaluated for their extraction capabilities in a real ABE fermentation broth system. From these compounds, nine potential solvents were also tested for their biocompatibility towards Clostridium acetobutylicum. Moreover, bioavailability and differences in substrate consumption and total n-butanol production with respect to solvent-free fermentations were quantified for each biocompatible solvent. Product yield was enhanced in the presence of organic solvents having higher affinity for butanol and butyric acid. Applying this methodology, it was found that the Guerbet alcohol 2-butyl-1-octanol presented the best extracting characteristics (the highest partition coefficient (6.76) and the third highest selectivity (644)), the highest butanol yield (27.4%), and maintained biocompatibility with C. acetobutylicum.

Techno-economics of carbon preserving butanol production using a combined fermentative and catalytic approach

This paper presents a novel process for n-butanol production which combines a fermentation consuming carbon dioxide (succinic acid fermentation) with subsequent catalytic reduction steps to add hydrogen to form butanol. Process simulations in Aspen Plus have been the basis for the techno-economic analyses performed. The overall economy for the novel process cannot be justified, as production of succinic acid by fermentation is too costly. Though, succinic acid price is expected to drop drastically in a near future. By fully integrating the succinic acid fermentation with the catalytic conversion the need for costly recovery operations could be reduced. The hybrid process would need 22% less raw material than the butanol fermentation at a succinic acid fermentation yield of 0.7g/g substrate. Additionally, a carbon dioxide fixation of up to 13ktonnes could be achieved at a plant with an annual butanol production of 10ktonnes.

Homeoviscous response of Clostridium pasteurianum to butanol toxicity during glycerol fermentation

Clostridium pasteurianum ATCC 6013 achieves high n-butanol production when glycerol is used as the sole carbon source. In this study, the homeoviscous membrane response of C. pasteurianum ATCC 6013 has been examined through n-butanol challenge experiments. Homeoviscous response is a critical aspect of n-butanol tolerance and has not been examined in detail for C. pasteurianum. Lipid membrane compositions were examined for glycerol fermentations with n-butanol production, and during cell growth in the absence of n-butanol production, using gas chromatography–mass spectrometry (GC–MS) and proton nuclear magnetic resonance (1H-NMR). Membrane stabilization due to homeoviscous response was further examined by surface pressure–area (π–A) analysis of membrane extract monolayers. C. pasteurianum was found to exert a homeoviscous response that was comprised of an increase lipid tail length and a decrease in the percentage of unsaturated fatty acids with increasing n-butanol challenge. This led to a more rigid or stable membrane that counteracted n-butanol fluidization. This is the first report on the changes in the membrane lipid composition during n-butanol production by C. pasteurianum ATCC 6013, which is a versatile microorganism that has the potential to be engineered as an industrial n-butanol producer using crude glycerol.

Improved n-butanol production by a non-acetone producing Clostridium pasteurianum DSMZ 525 in mixed substrate fermentation

The kinetics of growth, acid and solvent production in batch culture of Clostridium pasteurianum DSMZ 525 were examined in mixed or mono-substrate fermentations. In pH-uncontrolled batch cultures, the addition of butyric acid or glucose significantly enhanced n-butanol production and the ratio of butanol/1,3-propanediol. In pH-controlled batch culture at pH = 6, butyric acid addition had a negative effect on growth and did not lead to a higher n-butanol productivity. On the other hand, mixed substrate fermentation using glucose and glycerol enhanced the growth and acid production significantly. Glucose limitation in the mixed substrate fermentation led to the reduction or inhibition of the glycerol consumption by the growing bacteria. Therefore, for the optimal growth and n-butanol production by C. pasteurianum, a limitation of either substrate should be avoided. Under optimized batch conditions, n-butanol concentration and maximum productivity achieved were 21 g/L, and 0.96 g/L × h, respectively. In comparison, mixed substrate fermentation using biomass hydrolysate and glycerol gave a n-butanol concentration of 17 g/L with a maximum productivity of 1.1 g/L × h. In terms of productivity and final n-butanol concentration, the results demonstrated that C. pasteurianum DSMZ 525 is well suitable for n-butanol production from mixed substrates of biomass hydrolysate and glycerol and represents an alternative promising production strain.

Metabolic engineering of Thermoanaerobacterium saccharolyticum for n-butanol production

The thermophilic anaerobe Thermoanaerobacterium saccharolyticum JW/SL-YS485 was investigated as a host for n-butanol production. A systematic approach was taken to demonstrate functionality of heterologous components of the clostridial n-butanol pathway via gene expression and enzymatic activity assays in this organism. Subsequently, integration of the entire pathway in the wild-type strain resulted in n-butanol production of 0.85g/L from 10g/L xylose, corresponding to 21% of the theoretical maximum yield. We were unable to integrate the n-butanol pathway in strains lacking the ability to produce acetate, despite the theoretical overall redox neutrality of n-butanol formation. However, integration of the n-butanol pathway in lactate deficient strains resulted in n-butanol production of 1.05g/L from 10g/L xylose, corresponding to 26% of the theoretical maximum.