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.

Impact of Pharmaceutical Education on Medication Adherence and Its Clinical Efficacy in Patients with Type 2 Diabetes and Systemic Arterial Hypertension.

Purpose: To evaluate the impact of pharmaceutical education on medication adherence in patients with Type 2 Diabetes and Systemic Arterial Hypertension. Patients and Methods: This randomized clinical trial enrolled patients with a diagnosis of Type 2 Diabetes Mellitus and Systemic Arterial Hypertension treated in an internal medicine outpatient clinic of a teaching hospital. One hundred and three patients were randomly assigned to the study groups; 51 to the control group and 52 to the intervention group with a 6 months follow-up. Medication adherence was assessed using the Morisky 8-item medication adherence scale. To improve patient adherence to treatment, a wallet card was provided with an up-to-date list of prescribed medications along with recommendations for follow-up care. Results: One hundred and seventy-nine patients were screened for eligibility, of which 103 (57.5%) participated in the study. The intervention group showed a statistically significant decrease in capillary glucose levels, glycated hemoglobin, systolic and diastolic blood pressure, total cholesterol and triglycerides compared to the control group. The frequencies on medication adherence levels at 3 and 6 months in the control group remained similar to baseline, while in the intervention group the frequency of high adherence increased significantly at 6 months (8.7% to 43.5%). Conclusion: A high percentage of patients are not achieving optimal control of their diabetes. Medication adherence rates were between 45-50% in patients at the baseline of the study, but after receiving education and support from a pharmacist, the intervened group showed a significant increase in their adherence.

Attributional and consequential LCAs of a novel bio-jet fuel from Dutch potato by-products.

To mitigate the climate change impact of aviation, jet fuels from bio-based by-products are considered a promising alternative to conventional jet fuels. Life cycle assessment (LCA) is a commonly applied tool to determine the environmental impacts of bio-jet fuels. This article presents both attributional and consequential LCA models to assess an innovative bio-jet fuel produced from potato by-products in the Netherlands. The two models led to opposite conclusions regarding the overall environmental performance of this bio-jet fuel. The attributional LCA showed that this bio-jet fuel could offer about a 60% GHG emissions reduction compared to conventional jet fuel. In comparison, the consequential LCA estimated either a much lower climate change benefit (5-40%) if the potato by-products taken from the animal feed market are replaced with European animal feed or a 70% increase in GHG emissions if also imported soybean meals are used to replace the feed. Contrasting conclusions were also obtained for photochemical ozone formation. Conversely, the attributional and consequential LCAs agree on acidification, terrestrial eutrophication and depletion of fossil fuels. Although the consequential LCA was affected by higher uncertainties related to the determination of the actual product displaced, it allowed understanding the consequence of additional animal feed production. This process was not included in the system boundaries of the attributional LCA.

Model-driven approach for the production of butyrate from CO2/H2 by a novel co-culture of C. autoethanogenum and C. beijerinckii.

One-carbon (C1) compounds are promising feedstocks for the sustainable production of commodity chemicals. CO2 is a particularly advantageous C1-feedstock since it is an unwanted industrial off-gas that can be converted into valuable products while reducing its atmospheric levels. Acetogens are microorganisms that can grow on CO2/H2 gas mixtures and syngas converting these substrates into ethanol and acetate. Co-cultivation of acetogens with other microbial species that can further process such products, can expand the variety of products to, for example, medium chain fatty acids (MCFA) and longer chain alcohols. Solventogens are microorganisms known to produce MCFA and alcohols via the acetone-butanol-ethanol (ABE) fermentation in which acetate is a key metabolite. Thus, co-cultivation of an acetogen and a solventogen in a consortium provides a potential platform to produce valuable chemicals from CO2. In this study, metabolic modeling was implemented to design a new co-culture of an acetogen and a solventogen to produce butyrate from CO2/H2 mixtures. The model-driven approach suggested the ability of the studied solventogenic species to grow on lactate/glycerol with acetate as co-substrate. This ability was confirmed experimentally by cultivation of Clostridium beijerinckii on these substrates in batch serum bottles and subsequently in pH-controlled bioreactors. Community modeling also suggested that a novel microbial consortium consisting of the acetogen Clostridium autoethanogenum, and the solventogen C. beijerinckii would be feasible and stable. On the basis of this prediction, a co-culture was experimentally established. C. autoethanogenum grew on CO2/H2 producing acetate and traces of ethanol. Acetate was in turn, consumed by C. beijerinckii together with lactate, producing butyrate. These results show that community modeling of metabolism is a valuable tool to guide the design of microbial consortia for the tailored production of chemicals from renewable resources.

Sporulation in solventogenic and acetogenic clostridia.

The Clostridium genus harbors compelling organisms for biotechnological production processes; while acetogenic clostridia can fix C1-compounds to produce acetate and ethanol, solventogenic clostridia can utilize a wide range of carbon sources to produce commercially valuable carboxylic acids, alcohols, and ketones by fermentation. Despite their potential, the conversion by these bacteria of carbohydrates or C1 compounds to alcohols is not cost-effective enough to result in economically viable processes. Engineering solventogenic clostridia by impairing sporulation is one of the investigated approaches to improve solvent productivity. Sporulation is a cell differentiation process triggered in bacteria in response to exposure to environmental stressors. The generated spores are metabolically inactive but resistant to harsh conditions (UV, chemicals, heat, oxygen). In Firmicutes, sporulation has been mainly studied in bacilli and pathogenic clostridia, and our knowledge of sporulation in solvent-producing or acetogenic clostridia is limited. Still, sporulation is an integral part of the cellular physiology of clostridia; thus, understanding the regulation of sporulation and its connection to solvent production may give clues to improve the performance of solventogenic clostridia. This review aims to provide an overview of the triggers, characteristics, and regulatory mechanism of sporulation in solventogenic clostridia. Those are further compared to the current knowledge on sporulation in the industrially relevant acetogenic clostridia. Finally, the potential applications of spores for process improvement are discussed.Key Points• The regulatory network governing sporulation initiation varies in solventogenic clostridia.• Media composition and cell density are the main triggers of sporulation.• Spores can be used to improve the fermentation process.

Transcriptomic and Phenotypic Analysis of a spoIIE Mutant in Clostridium beijerinckii.

SpoIIE is a phosphatase involved in the activation of the first sigma factor of the forespore, σ F , during sporulation. A ΔspoIIE mutant of Clostridium beijerinckii NCIMB 8052, previously generated by CRISPR-Cas9, did not sporulate but still produced granulose and solvents. Microscopy analysis also showed that the cells of the ΔspoIIE mutant are elongated with the presence of multiple septa. This observation suggests that in C. beijerinckii, SpoIIE is necessary for the completion of the sporulation process, as seen in Bacillus and Clostridium acetobutylicum. Moreover, when grown in reactors, the spoIIE mutant produced higher levels of solvents than the wild type strain. The impact of the spoIIE inactivation on gene transcription was assessed by comparative transcriptome analysis at three time points (4 h, 11 h and 23 h). Approximately 5% of the genes were differentially expressed in the mutant compared to the wild type strain at all time points. Out of those only 12% were known sporulation genes. As expected, the genes belonging to the regulon of the sporulation specific transcription factors (σ F , σ E , σ G , σ K ) were strongly down-regulated in the mutant. Inactivation of spoIIE also caused differential expression of genes involved in various cell processes at each time point. Moreover, at 23 h, genes involved in butanol formation and tolerance, as well as in cell motility, were up-regulated in the mutant. In contrast, several genes involved in cell wall composition, oxidative stress and amino acid transport were down-regulated. These results indicate an intricate interdependence of sporulation and stationary phase cellular events in C. beijerinckii.

Importance of the Use of Oxidative Stress Biomarkers and Inflammatory Profile in Aqueous and Vitreous Humor in Diabetic Retinopathy.

Diabetic retinopathy is one of the leading causes of visual impairment and morbidity worldwide, being the number one cause of blindness in people between 27 and 75 years old. It is estimated that ~191 million people will be diagnosed with this microvascular complication by 2030. Its pathogenesis is due to alterations in the retinal microvasculature as a result of a high concentration of glucose in the blood for a long time which generates numerous molecular changes like oxidative stress. Therefore, this narrative review aims to approach various biomarkers associated with the development of diabetic retinopathy. Focusing on the molecules showing promise as detection tools, among them we consider markers of oxidative stress (TAC, LPO, MDA, 4-HNE, SOD, GPx, and catalase), inflammation (IL-6, IL-1ß, IL-8, IL-10, IL-17A, TNF-α, and MMPs), apoptosis (NF-kB, cyt-c, and caspases), and recently those that have to do with epigenetic modifications, their measurement in different biological matrices obtained from the eye, including importance, obtaining process, handling, and storage of these matrices in order to have the ability to detect the disease in its early stages.

Adjuvant Therapies in Diabetic Retinopathy as an Early Approach to Delay Its Progression: The Importance of Oxidative Stress and Inflammation.

Diabetes mellitus (DM) is a progressive disease induced by a sustained state of chronic hyperglycemia that can lead to several complications targeting highly metabolic cells. Diabetic retinopathy (DR) is a multifactorial microvascular complication of DM, with high prevalence, which can ultimately lead to visual impairment. The genesis of DR involves a complex variety of pathways such as oxidative stress, inflammation, apoptosis, neurodegeneration, angiogenesis, lipid peroxidation, and endoplasmic reticulum (ER) stress, each possessing potential therapeutic biomarkers. A specific treatment has yet to be developed for early stages of DR since no management is given other than glycemic control until the proliferative stage develops, offering a poor visual prognosis to the patient. In this narrative review article, we evaluate different dietary regimens, such as the Mediterranean diet, Dietary Pattern to Stop Hypertension (DASH) and their functional foods, and low-calorie diets (LCDs). Nutraceuticals have also been assessed in DR on account of their antioxidant, anti-inflammatory, and antiangiogenic properties, which may have an important impact on the physiopathology of DR. These nutraceuticals have shown to lower reactive oxygen species (ROS), important inflammatory factors, cytokines, and endothelial damage biomarkers either as monotherapies or combined therapies or concomitantly with established diabetes management or nonconventional adjuvant drugs like topical nonsteroidal anti-inflammatory drugs (NSAIDs).

Adaptation and application of a two-plasmid inducible CRISPR-Cas9 system in Clostridium beijerinckii.

Recent developments in CRISPR technologies have opened new possibilities for improving genome editing tools dedicated to the Clostridium genus. In this study we adapted a two-plasmid tool based on this technology to enable scarless modification of the genome of two reference strains of Clostridium beijerinckii producing an Acetone/Butanol/Ethanol (ABE) or an Isopropanol/Butanol/Ethanol (IBE) mix of solvents. In the NCIMB 8052 ABE-producing strain, inactivation of the SpoIIE sporulation factor encoding gene resulted in sporulation-deficient mutants, and this phenotype was reverted by complementing the mutant strain with a functional spoIIE gene. Furthermore, the fungal cellulase-encoding celA gene was inserted into the C. beijerinckii NCIMB 8052 chromosome, resulting in mutants with endoglucanase activity. A similar two-plasmid approach was next used to edit the genome of the natural IBE-producing strain C. beijerinckii DSM 6423, which has never been genetically engineered before. Firstly, the catB gene conferring thiamphenicol resistance was deleted to make this strain compatible with our dual-plasmid editing system. As a proof of concept, our dual-plasmid system was then used in C. beijerinckii DSM 6423 ΔcatB to remove the endogenous pNF2 plasmid, which led to a sharp increase of transformation efficiencies.

l-Rhamnose Metabolism in Clostridium beijerinckii Strain DSM 6423.

Macroalgae (or seaweeds) are considered potential biomass feedstocks for the production of renewable fuels and chemicals. Their sugar composition is different from that of lignocellulosic biomasses, and in green species, including Ulva lactuca, the major sugars are l-rhamnose and d-glucose. C. beijerinckii DSM 6423 utilized these sugars in a U. lactuca hydrolysate to produce acetic acid, butyric acid, isopropanol, butanol, and ethanol (IBE), and 1,2-propanediol. d-Glucose was almost completely consumed in diluted hydrolysates, while l-rhamnose or d-xylose was only partially utilized. In this study, the metabolism of l-rhamnose by C. beijerinckii DSM 6423 was investigated to improve its utilization from natural resources. Fermentations on d-glucose, l-rhamnose, and a mixture of d-glucose and l-rhamnose were performed. On l-rhamnose, the cultures showed low growth and sugar consumption and produced 1,2-propanediol, propionic acid, and n-propanol in addition to acetic and butyric acids, whereas on d-glucose, IBE was the major product. On a d-glucose-l-rhamnose mixture, both sugars were converted simultaneously and l-rhamnose consumption was higher, leading to high levels of 1,2-propanediol (78.4 mM), in addition to 59.4 mM butanol and 31.9 mM isopropanol. Genome and transcriptomics analysis of d-glucose- and l-rhamnose-grown cells revealed the presence and transcription of genes involved in l-rhamnose utilization and in bacterial microcompartment (BMC) formation. These data provide useful insights into the metabolic pathways involved in l-rhamnose utilization and the effects on the general metabolism (glycolysis, early sporulation, and stress response) induced by growth on l-rhamnose.IMPORTANCE A prerequisite for a successful biobased economy is the efficient conversion of biomass resources into useful products, such as biofuels and bulk and specialty chemicals. In contrast to other industrial microorganisms, natural solvent-producing clostridia utilize a wide range of sugars, including C5, C6, and deoxy-sugars, for production of long-chain alcohols (butanol and 2,3-butanediol), isopropanol, acetone, n-propanol, and organic acids. Butanol production by clostridia from first-generation sugars is already a commercial process, but for the expansion and diversification of the acetone, butanol, and ethanol (ABE)/IBE process to other substrates, more knowledge is needed on the regulation and physiology of fermentation of sugar mixtures. Green macroalgae, produced in aquaculture systems, harvested from the sea or from tides, can be processed into hydrolysates containing mixtures of d-glucose and l-rhamnose, which can be fermented. The knowledge generated in this study will contribute to the development of more efficient processes for macroalga fermentation and of mixed-sugar fermentation in general.

Correction to: Genome and transcriptome of the natural isopropanol producer Clostridium beijerinckii DSM6423.

Following the publication of this article [1], the authors noticed that Figs. 2, 3 and 4 were in the incorrect order and thus had incorrect captions.

Genome and transcriptome of the natural isopropanol producer Clostridium beijerinckii DSM6423.

BACKGROUND: There is a worldwide interest for sustainable and environmentally-friendly ways to produce fuels and chemicals from renewable resources. Among them, the production of acetone, butanol and ethanol (ABE) or Isopropanol, Butanol and Ethanol (IBE) by anaerobic fermentation has already a long industrial history. Isopropanol has recently received a specific interest and the best studied natural isopropanol producer is C. beijerinckii DSM 6423 (NRRL B-593). This strain metabolizes sugars into a mix of IBE with only low concentrations of ethanol produced (< 1 g/L). However, despite its relative ancient discovery, few genomic details have been described for this strain. Research efforts including omics and genetic engineering approaches are therefore needed to enable the use of C. beijerinckii as a microbial cell factory for production of isopropanol. RESULTS: The complete genome sequence and a first transcriptome analysis of C. beijerinckii DSM 6423 are described in this manuscript. The combination of MiSeq and de novo PacBio sequencing revealed a 6.38 Mbp chromosome containing 6254 genomic objects. Three Mobile Genetic Elements (MGE) were also detected: a linear double stranded DNA bacteriophage (ϕ6423) and two plasmids (pNF1 and pNF2) highlighting the genomic complexity of this strain. A first RNA-seq transcriptomic study was then performed on 3 independent glucose fermentations. Clustering analysis allowed us to detect some key gene clusters involved in the main life cycle steps (acidogenesis, solvantogenesis and sporulation) and differentially regulated among the fermentation. These putative clusters included some putative metabolic operons comparable to those found in other reference strains such as C. beijerinckii NCIMB 8052 or C. acetobutylicum ATCC 824. Interestingly, only one gene was encoding for an alcohol dehydrogenase converting acetone into isopropanol, suggesting a single genomic event occurred on this strain to produce isopropanol. CONCLUSIONS: We present the full genome sequence of Clostridium beijerinckii DSM 6423, providing a complete genetic background of this strain. This offer a great opportunity for the development of dedicated genetic tools currently lacking for this strain. Moreover, a first RNA-seq analysis allow us to better understand the global metabolism of this natural isopropanol producer, opening the door to future targeted engineering approaches.

A two-plasmid inducible CRISPR/Cas9 genome editing tool for Clostridium acetobutylicum.

CRISPR/Cas-based genetic engineering has revolutionised molecular biology in both eukaryotes and prokaryotes. Several tools dedicated to the genomic transformation of the Clostridium genus of Gram-positive bacteria have been described in the literature; however, the integration of large DNA fragments still remains relatively limited. In this study, a CRISPR/Cas9 genome editing tool using a two-plasmid strategy was developed for the solventogenic strain Clostridium acetobutylicum ATCC 824. Codon-optimised cas9 from Streptococcus pyogenes was placed under the control of an anhydrotetracycline-inducible promoter on one plasmid, while the gRNA expression cassettes and editing templates were located on a second plasmid. Through the sequential introduction of these vectors into the cell, we achieved highly accurate genome modifications, including nucleotide substitution, gene deletion and cassette insertion up to 3.6kb. To demonstrate its potential, this genome editing tool was used to generate a marker-free mutant of ATCC 824 that produced an isopropanol-butanol-ethanol mixture. Whole-genome sequencing confirmed that no off-target modifications were present in the mutants. Such a tool is a prerequisite for efficient metabolic engineering in this solventogenic strain and provides an alternative editing strategy that might be applicable to other Clostridium strains.

Biorefinery of the green seaweed Ulva lactuca to produce animal feed, chemicals and biofuels.

The growing world population demands an increase in animal protein production. Seaweed may be a valuable source of protein for animal feed. However, a biorefinery approach aimed at cascading valorisation of both protein and non-protein seaweed constituents is required to realise an economically feasible value chain. In this study, such a biorefinery approach is presented for the green seaweed Ulva lactuca containing 225 g protein (N × 4.6) kg-1 dry matter (DM). The sugars in the biomass were solubilised by hot water treatment followed by enzymatic hydrolysis and centrifugation resulting in a sugar-rich hydrolysate (38.8 g L-1 sugars) containing glucose, rhamnose and xylose, and a protein-enriched (343 g kg-1 in DM) extracted fraction. This extracted fraction was characterised for use in animal feed, as compared to U. lactuca biomass. Based on the content of essential amino acids and the in vitro N (85 %) and organic matter (90 %) digestibility, the extracted fraction seems a promising protein source in diets for monogastric animals with improved characteristics as compared to the intact U. lactuca. The gas production test indicated a moderate rumen fermentation of U. lactuca and the extracted fraction, about similar to that of alfalfa. Reduction of the high content of minerals and trace elements may be required to allow a high inclusion level of U. lactuca products in animal diets. The hydrolysate was used successfully for the production of acetone, butanol, ethanol and 1,2-propanediol by clostridial fermentation, and the rhamnose fermentation pattern was studied.

"In situ" removal of isopropanol, butanol and ethanol from fermentation broth by gas stripping.

In this study, the removal of IBE from aqueous solutions by gas stripping has been characterized. The effect of one or more components in the solution on the kinetics of the separation has been studied, both at 37°C and at 70°C. Gas stripping has been applied to batch, repeated batch and continuous cultures of Clostridium beijerinckii grown on a glucose/xylose mixed sugar substrate mimicking lignocellulosic hydrolysates, with the aim of finding optimal conditions for a stable IBE-producing culture with high productivity. An innovative repeated-batch process has been demonstrated in which the gas-stripping is performed at 70°C, resulting in a prolonged stable IBE culture.

Production of acetone, butanol, and ethanol from biomass of the green seaweed Ulva lactuca.

Green seaweed Ulva lactuca harvested from the North Sea near Zeeland (The Netherlands) was characterized as feedstock for acetone, ethanol and ethanol fermentation. Solubilization of over 90% of sugars was achieved by hot-water treatment followed by hydrolysis using commercial cellulases. A hydrolysate was used for the production of acetone, butanol and ethanol (ABE) by Clostridium acetobutylicum and Clostridium beijerinckii. Hydrolysate-based media were fermentable without nutrient supplementation. C. beijerinckii utilized all sugars in the hydrolysate and produced ABE at high yields (0.35 g ABE/g sugar consumed), while C. acetobutylicum produced mostly organic acids (acetic and butyric acids). These results demonstrate the great potential of U. lactuca as feedstock for fermentation. Interestingly, in control cultures of C. beijerinckii on rhamnose and glucose, 1,2 propanediol was the main fermentation product (9.7 g/L).

Simultaneous production of isopropanol, butanol, ethanol and 2,3-butanediol by Clostridium acetobutylicum ATCC 824 engineered strains.

Isopropanol represents a widely-used commercial alcohol which is currently produced from petroleum. In nature, isopropanol is excreted by some strains of Clostridium beijerinckii, simultaneously with butanol and ethanol during the isopropanol butanol ethanol (IBE) fermentation. In order to increase isopropanol production, the gene encoding the secondary-alcohol dehydrogenase enzyme from C. beijerinckii NRRL B593 (adh) which catalyzes the reduction of acetone to isopropanol, was cloned into the acetone, butanol and ethanol (ABE)-producing strain C. acetobutylicum ATCC 824. The transformants showed high capacity for conversion of acetone into isopropanol (> 95%). To increase isopropanol production levels in ATCC 824, polycistronic transcription units containing, in addition to the adh gene, homologous genes of the acetoacetate decarboxylase (adc), and/or the acetoacetyl-CoA:acetate/butyrate:CoA transferase subunits A and B (ctfA and ctfB) were constructed and introduced into the wild-type strain. Combined overexpression of the ctfA and ctfB genes resulted in enhanced solvent production. In non-pH-controlled batch cultures, the total solvents excreted by the transformant overexpressing the adh, ctfA, ctfB and adc genes were 24.4 g/L IBE (including 8.8 g/L isopropanol), while the control strain harbouring an empty plasmid produced only 20.2 g/L ABE (including 7.6 g/L acetone). The overexpression of the adc gene had limited effect on IBE production. Interestingly, all transformants with the adh gene converted acetoin (a minor fermentation product) into 2,3-butanediol, highlighting the wide metabolic versatility of solvent-producing Clostridia.

Disruption of the acetate kinase (ack) gene of Clostridium acetobutylicum results in delayed acetate production.

In microorganisms, the enzyme acetate kinase (AK) catalyses the formation of ATP from ADP by de-phosphorylation of acetyl phosphate into acetic acid. A mutant strain of Clostridium acetobutylicum lacking acetate kinase activity is expected to have reduced acetate and acetone production compared to the wild type. In this work, a C. acetobutylicum mutant strain with a selectively disrupted ack gene, encoding AK, was constructed and genetically and physiologically characterized. The ack (-) strain showed a reduction in acetate kinase activity of more than 97% compared to the wild type. The fermentation profiles of the ack (-) and wild-type strain were compared using two different fermentation media, CGM and CM1. The latter contains acetate and has a higher iron and magnesium content than CGM. In general, fermentations by the mutant strain showed a clear shift in the timing of peak acetate production relative to butyrate and had increased acid uptake after the onset of solvent formation. Specifically, in acetate containing CM1 medium, acetate production was reduced by more than 80% compared to the wild type under the same conditions, but both strains produced similar final amounts of solvents. Fermentations in CGM showed similar peak acetate and butyrate levels, but increased acetoin (60%), ethanol (63%) and butanol (16%) production and reduced lactate (-50%) formation by the mutant compared to the wild type. These findings are in agreement with the proposed regulatory function of butyryl phosphate as opposed to acetyl phosphate in the metabolic switch of solventogenic clostridia.

D-2,3-butanediol production due to heterologous expression of an acetoin reductase in Clostridium acetobutylicum.

Acetoin reductase (ACR) catalyzes the conversion of acetoin to 2,3-butanediol. Under certain conditions, Clostridium acetobutylicum ATCC 824 (and strains derived from it) generates both d- and l-stereoisomers of acetoin, but because of the absence of an ACR enzyme, it does not produce 2,3-butanediol. A gene encoding ACR from Clostridium beijerinckii NCIMB 8052 was functionally expressed in C. acetobutylicum under the control of two strong promoters, the constitutive thl promoter and the late exponential adc promoter. Both ACR-overproducing strains were grown in batch cultures, during which 89 to 90% of the natively produced acetoin was converted to 20 to 22 mM d-2,3-butanediol. The addition of a racemic mixture of acetoin led to the production of both d-2,3-butanediol and meso-2,3-butanediol. A metabolic network that is in agreement with the experimental data is proposed. Native 2,3-butanediol production is a first step toward a potential homofermentative 2-butanol-producing strain of C. acetobutylicum.