Increased Butyrate Production in Clostridium saccharoperbutylacetonicum from Lignocellulose-Derived Sugars.

Butyrate is produced by chemical synthesis based on crude oil, produced by microbial fermentation, or extracted from animal fats (M. Dwidar, J.-Y. Park, R. J. Mitchell, and B.-I. Sang, The Scientific World Journal, 2012:471417, 2012, https://doi.org/10.1100/2012/471417). Butyrate production by anaerobic bacteria is highly favorable since waste or sustainable resources can be used as the substrates. For this purpose, the native hyper-butanol producer Clostridium saccharoperbutylacetonicum N1-4(HMT) was used as a chassis strain due to its broad substrate spectrum. BLASTp analysis of the predicted proteome of C. saccharoperbutylacetonicum N1-4(HMT) resulted in the identification of gene products potentially involved in acetone-butanol-ethanol (ABE) fermentation. Their participation in ABE fermentation was either confirmed or disproven by the parallel production of acids or solvents and the respective transcript levels obtained by transcriptome analysis of this strain. The genes encoding phosphotransacetylase (pta) and butyraldehyde dehydrogenase (bld) were deleted to reduce acetate and alcohol formation. The genes located in the butyryl-CoA synthesis (bcs) operon encoding crotonase, butyryl-CoA dehydrogenase with electron-transferring protein subunits α and ß, and 3-hydroxybutyryl-CoA dehydrogenase were overexpressed to channel the flux further towards butyrate formation. Thereby, the native hyper-butanol producer C. saccharoperbutylacetonicum N1-4(HMT) was converted into the hyper-butyrate producer C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151_BCS_PbgaL]. The transcription pattern following deletion and overexpression was characterized by a second transcriptomic study, revealing partial compensation for the deletion. Furthermore, this strain was characterized in pH-controlled fermentations with either glucose or Excello, a substrate yielded from spruce biomass. Butyrate was the main product, with maximum butyrate concentrations of 11.7 g·L-1 and 14.3 g·L-1, respectively. Minimal amounts of by-products were detected. IMPORTANCE Platform chemicals such as butyrate are usually produced chemically from crude oil, resulting in the carry-over of harmful compounds. The selective production of butyrate using sustainable resources or waste without harmful by-products can be achieved by bacteria such as clostridia. The hyper-butanol producer Clostridium saccharoperbutylacetonicum N1-4(HMT) was converted into a hyper-butyrate producer. Butyrate production with very small amounts of by-products was established with glucose and the sustainable lignocellulosic sugar substrate Excello extracted from spruce biomass by the biorefinery Borregaard (Sarpsborg, Norway).

Production of propionate using metabolically engineered strains of Clostridium saccharoperbutylacetonicum.

The carboxylic acid propionate is a valuable platform chemical with applications in various fields. The biological production of this acid has become of great interest as it can be considered a sustainable alternative to petrochemical synthesis. In this work, Clostridium saccharoperbutylacetonicum was metabolically engineered to produce propionate via the acrylate pathway. In total, the established synthetic pathway comprised eight genes encoding the enzymes catalyzing the conversion of pyruvate to propionate. These included the propionate CoA-transferase, the lactoyl-CoA dehydratase, and the acryloyl-CoA reductase from Anaerotignum neopropionicum as well as a D-lactate dehydrogenase from Leuconostoc mesenteroides subsp. mesenteroides. Due to difficulties in assembling all genes on one plasmid under the control of standard promoters, the PtcdB-tcdR promoter system from Clostridium difficile was integrated into a two-plasmid system carrying the acrylate pathway genes. Several promoters were analyzed for their activity in C. saccharoperbutylacetonicum using the fluorescence-activating and absorption-shifting tag (FAST) as a fluorescent reporter to identify suitable candidates to drive tcdR expression. After selecting the lactose-inducible PbgaL promoter, engineered C. saccharoperbutylacetonicum strains produced 0.7 mM propionate upon induction of gene expression. The low productivity was suspected to be a consequence of a metabolic imbalance leading to acryloyl-CoA accumulation in the cells. To even out the proposed imbalance, the propionate-synthesis operons were rearranged, thereby increasing the propionate concentration by almost four-fold. This study is the first one to report recombinant propionate production using a clostridial host strain that has opened a new path towards bio-based propionate to be improved further in subsequent work. KEY POINTS: • Determination of promoter activities in C. saccharoperbutylacetonicum using FAST. • Implementation of propionate production in C. saccharoperbutylacetonicum. • Elevation of propionate production by 375% to a concentration of 3 mM.

Investigation of putative genes for the production of medium-chained acids and alcohols in autotrophic acetogenic bacteria.

Gas fermentation is a technology for producing platform chemicals as well as fuels and one of the most promising alternatives to petrochemicals. Medium-chained acids and alcohols such as hexanoate and hexanol are particularly interesting due to their versatile application. This study elucidated the pathway of chain elongation in native C6 compound-producing acetogens. Essential genes of Clostridium carboxidivorans for synthesis of medium-chained acids and alcohols were identified in order to demonstrate their catalytic activity in the acetogenic model organism Acetobacterium woodii. Two such gene clusters were identified, which are responsible for conversion of acetyl-CoA to butyryl-CoA by reverse ß-oxidation. Using RT-PCR it could be demonstrated that only genes of cluster 1 are expressed constitutively with simultaneous formation of C6 compounds. Based on genes from C. carboxidivorans, a modular hexanoyl-CoA synthesis (hcs) plasmid system was constructed and transferred into A. woodii. With the recombinant A. woodii strains AWO [pPta_hcs1], AWO [pPta_hcs2], AWO [pTet_hcs1], and AWO [pTet_hcs2] butyrate and hexanoate production under heterotrophic (1.22-4.15 mM hexanoate) and autotrophic conditions (0.48-1.56 mM hexanoate) with both hcs clusters could be detected. hcs Cluster 1 from C. carboxidivorans was transferred into the ABE-fermenting strain Clostridium saccharoperbutylacetonicum as well. For further analysis, genes were also cloned into the hcs plasmid system individually. The resulting recombinant C. saccharoperbutylacetonicum strains with just individual genes neither produced hexanoate nor hexanol, but the strains containing the entire gene cluster were capable of chain elongation. A production of 0.8 mM hexanoate and 5.2 mM hexanol in the fermentation with glucose could be observed.

Genome Editing in Clostridium saccharoperbutylacetonicum N1-4 with the CRISPR-Cas9 System.

Clostridium saccharoperbutylacetonicum N1-4 is well known as a hyper-butanol-producing strain. However, the lack of genetic engineering tools hinders further elucidation of its solvent production mechanism and development of more robust strains. In this study, we set out to develop an efficient genome engineering system for this microorganism based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (CRISPR-Cas9) system. First, the functionality of the CRISPR-Cas9 system previously customized for Clostridium beijerinckii was evaluated in C. saccharoperbutylacetonicum by targeting pta and buk, two essential genes for acetate and butyrate production, respectively. pta and buk single and double deletion mutants were successfully obtained based on this system. However, the genome engineering efficiency was rather low (the mutation rate is <20%). Therefore, the efficiency was further optimized by evaluating various promoters for guide RNA (gRNA) expression. With promoter P J23119 , we achieved a mutation rate of 75% for pta deletion without serial subculturing as suggested previously for C. beijerinckii Thus, this developed CRISPR-Cas9 system is highly desirable for efficient genome editing in C. saccharoperbutylacetonicum Batch fermentation results revealed that both the acid and solvent production profiles were altered due to the disruption of acid production pathways; however, neither acetate nor butyrate production was eliminated with the deletion of the corresponding gene. The butanol production, yield, and selectivity were improved in mutants, depending on the fermentation medium. In the pta buk double deletion mutant, the butanol production in P2 medium reached 19.0 g/liter, which is one of the highest levels ever reported from batch fermentations.IMPORTANCE An efficient CRISPR-Cas9 genome engineering system was developed for C. saccharoperbutylacetonicum N1-4. This paves the way for elucidating the solvent production mechanism in this hyper-butanol-producing microorganism and developing strains with desirable butanol-producing features. This tool can be easily adapted for use in closely related microorganisms. As also reported by others, here we demonstrated with solid data that the highly efficient expression of gRNA is the key factor determining the efficiency of CRISPR-Cas9 for genome editing. The protocol developed in this study can provide essential references for other researchers who work in the areas of metabolic engineering and synthetic biology. The developed mutants can be used as excellent starting strains for development of more robust ones for desirable solvent production.

Inorganic Carbon Assimilation and Electrosynthesis of Platform Chemicals in Bioelectrochemical Systems (BESs) Inoculated with Clostridium saccharoperbutylacetonicum N1-H4.

The need for greener processes to satisfy the demand of platform chemicals together with the possibility of reusing CO2 from human activities has recently encouraged research on the set-up, optimization, and development of bioelectrochemical systems (BESs) for the electrosynthesis of organic compounds from inorganic carbon (CO2, HCO3-). In the present study, we tested the ability of Clostridium saccharoperbutylacetonicum N1-4 (DSMZ 14923) to produce acetate and D-3-hydroxybutyrate from inorganic carbon present in a CO2:N2 gas mix. At the same time, we tested the ability of a Shewanella oneidensis MR1 and Pseudomonas aeruginosa PA1430/CO1 consortium to provide reducing power to sustain carbon assimilation at the cathode. We tested the performance of three different systems with the same layouts, inocula, and media, but with the application of 1.5 V external voltage, of a 1000 Ω external load, and without any connection between the electrodes or external devices (open circuit voltage, OCV). We compared both CO2 assimilation rate and production of metabolites (formate, acetate 3-D-hydroxybutyrate) in our BESs with the values obtained in non-electrogenic control cultures and estimated the energy used by our BESs to assimilate 1 mol of CO2. Our results showed that C. saccharoperbutylacetonicum NT-1 achieved the maximum CO2 assimilation (95.5%) when the microbial fuel cells (MFCs) were connected to the 1000 Ω external resistor, with the Shewanella/Pseudomonas consortium as the only source of electrons. Furthermore, we detected a shift in the metabolism of C. saccharoperbutylacetonicum NT-1 because of its prolonged activity in BESs. Our results open new perspectives for the utilization of BESs in carbon capture and electrosynthesis of platform chemicals.

Modulation of sol mRNA expression by the long non-coding RNA Assolrna in Clostridium saccharoperbutylacetonicum affects solvent formation.

Solvents such as butanol are important platform chemicals and are often produced from petrochemical sources. Production of butanol and other compounds from renewable and sustainable resources can be achieved by solventogenic bacteria, such as the hyper-butanol producer Clostridium saccharoperbutylacetonicum. Its sol operon consists of the genes encoding butyraldehyde dehydrogenase, CoA transferase, and acetoacetate decarboxylase (bld, ctfA, ctfB, adc) and the gene products are involved in butanol and acetone formation. It is important to understand its regulation to further optimize the solvent production. In this study, a new long non-coding antisense transcript complementary to the complete sol operon, now called Assolrna, was identified by transcriptomic analysis and the regulatory mechanism of Assolrna was investigated. For this purpose, the promoter-exchange strain C. saccharoperbutylacetonicum ΔP asr ::P asr ** was constructed. Additionally, Assolrna was expressed plasmid-based under control of the native P asr promoter and the lactose-inducible P bgaL promoter in both the wild type and the promoter-exchange strain. Solvent formation was strongly decreased for all strains based on C. saccharoperbutylacetonicum ΔP asr ::P asr ** and growth could not be restored by plasmid-based complementation of the exchanged promoter. Interestingly, very little sol mRNA expression was detected in the strain C. saccharoperbutylacetonicum ΔP asr ::P asr ** lacking Assolrna expression. Butanol titers were further increased for the overexpression strain C. saccharoperbutylacetonicum [pMTL83151_asr_P bgaL ] compared to the wild type. These results suggest that Assolrna has a positive effect on sol operon expression. Therefore, a possible stabilization mechanism of the sol mRNA by Assolrna under physiological concentrations is proposed.

Establishment of Green- and Red-Fluorescent Reporter Proteins Based on the Fluorescence-Activating and Absorption-Shifting Tag for Use in Acetogenic and Solventogenic Anaerobes.

Anaerobic bacteria are promising biocatalysts to produce industrially relevant products from nonfood feedstocks. Several anaerobes are genetically accessible, and various molecular tools for metabolic engineering are available. Still, the use of bright fluorescent reporters, which are commonly used in molecular biological approaches is limited under anaerobic conditions. Therefore, the establishment of different anaerobic fluorescent reporter proteins is of great interest. Here, we present the establishment of the green- and red-fluorescent reporter proteins greenFAST and redFAST for use in different solventogenic and acetogenic bacteria. Green fluorescence of greenFAST was bright in Clostridium saccharoperbutylacetonicum, Clostridium acetobutylicum, Acetobacterium woodii, and Eubacterium limosum, while only C. saccharoperbutylacetonicum showed bright red fluorescence when producing redFAST. We used both reporter proteins in C. saccharoperbutylacetonicum for multicolor approaches. These include the investigation of the co-culture dynamics of metabolically engineered strains. Moreover, we established a tightly regulated inducible two-plasmid system and used greenFAST and redFAST to track the coexistence and interaction of both plasmids under anaerobic conditions in C. saccharoperbutylacetonicum. The establishment of greenFAST and redFAST as fluorescent reporters opens the door for further multicolor approaches to investigate cell dynamics, gene expression, or protein localization under anaerobic conditions.

Effect of changes in continuous carboxylate feeding on the specific production rate of butanol using Clostridium saccharoperbutylacetonicum.

Substrate variability in multi-feedstock biorefineries has implications for the stability of downstream bioprocesses. Here, we studied potential effects of fluctuating feed rates and pH on substrate uptake and butanol production by Clostridium saccharoperbutylacetonicum during continuous co-feeding with butyric and acetic acid. Monitoring the fermentation extensively and at high frequency, enabled us to perform irregular fraction experimental designs. The total acid feed rate and the ratio of butyric acid to acetic acid in the feed were found to be significant factors in their uptake by the culture. Furthermore, to maximize the specific butanol production rate, glucose may not be limited and butyric acid should be supplied at a rate of 7.5 mmol L-1 h-1. Surprisingly, pH played a role only indirectly, in its effect on process stability. Obtained results facilitate the control of feed rates based on physiological descriptors, which will be a critical factor in the establishment of multi-feedstock biorefineries.

Development of Clostridium saccharoperbutylacetonicum as a Whole Cell Biocatalyst for Production of Chirally Pure (R)-1,3-Butanediol.

Chirally pure (R)-1,3-butanediol ((R)-1,3-BDO) is a valuable intermediate for the production of fragrances, pheromones, insecticides and antibiotics. Biotechnological production results in superior enantiomeric excess over chemical production and is therefore the preferred production route. In this study (R)-1,3-BDO was produced in the industrially important whole cell biocatalyst Clostridium saccharoperbutylacetonicum through expression of the enantio-specific phaB gene from Cupriavidus necator. The heterologous pathway was optimised in three ways: at the transcriptional level choosing strongly expressed promoters and comparing plasmid borne with chromosomal gene expression, at the translational level by optimising the codon usage of the gene to fit the inherent codon adaptation index of C. saccharoperbutylacetonicum, and at the enzyme level by introducing point mutations which led to increased enzymatic activity. The resulting whole cell catalyst produced up to 20 mM (1.8 g/l) (R)-1,3-BDO in non-optimised batch fermentation which is a promising starting position for economical production of this chiral chemical.

Kinetic modeling of butyric acid effects on butanol fermentation by Clostridium saccharoperbutylacetonicum.

A kinetic model of acetone-butanol-ethanol (ABE) fermentation taking into account butyric acid effects was developed and implemented in COPASI. The model was validated by comparing the simulation results with experimental data in batch cultures of Clostridium saccharoperbutylacetonicum under various concentrations of initial glucose (97.1 to 152.6 mM) and butyric acid (90.7 to 153.2 mM). The modeling results suggested that increasing the conversion rates from butyryl-CoA (BCoA) to butanol, from butyrate to BCoA, or from pyruvate to lactate would increase butanol synthesis. Similarly, reducing glucose uptake rate or the reaction rates from pyruvate to acetyl CoA (ACoA), from acetoacetyl CoA (AACoA) to BCoA, or from BCoA to butyrate would improve butanol production. Overall, the kinetic model developed can accurately predict the dynamic behavior of metabolites in ABE fermentation with butyric acid addition, which may subsequently be used to identify genetic manipulation strategies for higher bio-butanol production.

Enhancing the tolerance of Clostridium saccharoperbutylacetonicum to lignocellulosic-biomass-derived inhibitors for efficient biobutanol production by overexpressing efflux pumps genes from Pseudomonas putida.

Furan aldehydes and phenolic compounds generated during biomass pretreatment can inhibit fermentation for biofuel production. Efflux pumps actively transport small molecules out of cells, thus sustaining normal microbial metabolism. Pseudomonas putida has outstanding tolerance to butanol and other small molecules, and we hypothesize that its efflux pump could play essential roles for such robustness. Here, we overexpressed efflux pump genes from P. putida to enhance tolerance of hyper-butanol producing Clostridium saccharoperbutylacetonicum to fermentation inhibitors. Interestingly, overexpression of the whole unit resulted in decreased tolerance, while overexpression of the subunit (srpB) alone exerted significant enhanced robustness of the strain. Compared to the control, the engineered strain had enhanced capability to grow in media containing 17% more furfural or 50% more ferulic acid, and produced ~14 g/L butanol (comparable to fermentation under regular conditions without inhibitors). This study provided valuable reference for boosting microbial robustness towards efficient biofuel production from lignocellulosic materials.

Genome sequence analysis of the temperate bacteriophage TBP2 of the solvent producer Clostridium saccharoperbutylacetonicum N1-4 (HMT, ATCC 27021).

The genus Clostridium consists of a diverse group of pathogenic and non-pathogenic bacteria. The non-pathogenic clostridia contain several solventogenic members of industrial importance, such as Clostridium acetobutylicum or C. beijerinckii. In the process of acetone-butanol-ethanol (ABE) fermentation, these strains are used in large scale fermentation plants since almost 100 years. Soon after establishment of the first plants, the fermentation processes suffered from different bacteriophage infections worldwide. A limited set of studies addressing bacteriophages in solventogenic clostridia have been conducted since then. In this study, we present the genome sequence of the temperate bacteriophage TBP2 of the solventogenic strain C. saccharoperbutylacetonicum N1-4 (HMT) that is used for ABE fermentation. The phage genome consists of 38 039 bp and includes 48 open reading frames. Sequence analysis indicates that the genome encloses random parts of the bacterial genome in addition to its own DNA. It represents the first fully sequenced genome of a temperate bacteriophage infecting solventogenic clostridia.

RRNPP-type quorum-sensing systems regulate solvent formation, sporulation and cell motility in Clostridium saccharoperbutylacetonicum.

BACKGROUND: Clostridium saccharoperbutylacetonicum N1-4 (HMT) is a strictly anaerobic, spore-forming Gram-positive bacterium capable of hyper-butanol production through the well-known acetone-butanol-ethanol fermentation process. Recently, five putative RRNPP-type QSSs (here designated as QSS1 to QSS5) were predicted in this bacterial strain, each of which comprises a putative RRNPP-type regulator (QssR1 to QssR5) and a cognate signaling peptide precursor (QssP1 to QssP5). In addition, both proteins are encoded by the same operon. The functions of these multiple RRNPP-type QSSs are unknown. RESULTS: To elucidate the function of multiple RRNPP-type QSSs as related to cell metabolism and solvent production in N1-4 (HMT), we constructed qssR-deficient mutants ΔR1, ΔR2, ΔR3 and ΔR5 through gene deletion using CRISPR-Cas9 and N1-4-dcas9-R4 (with the QssR4 expression suppressed using CRISPR-dCas9). We also constructed complementation strains by overexpressing the corresponding regulator gene. Based on systematic characterization, results indicate that QSS1, QSS2, QSS3, and QSS5 positively regulate the sol operon expression and thus solvent production, but they likely negatively regulate cell motility. Consequently, QSS4 might not directly regulate solvent production, but positively affect cell migration. In addition, QSS3 and QSS5 appear to positively regulate sporulation efficiency. CONCLUSIONS: Our study provides the first insights into the roles of multiple RRNPP-type QSSs of C. saccharoperbutylacetonicum for the regulation of solvent production, cell motility, and sporulation. Results of this study expand our knowledge of how multiple paralogous QSSs are involved in the regulation of essential bacterial metabolism pathways.

Enhancement of acid re-assimilation and biosolvent production in Clostridium saccharoperbutylacetonicum through metabolic engineering for efficient biofuel production from lignocellulosic biomass.

In the clostridial acetone-butanol-ethanol (ABE) fermentation, the intermediate acetate and butyrate are re-assimilated for solvent production. Here, key genes in ABE pathways in Clostridium saccharoperbutylacetonicum N1-4 were overexpressed to enhance acid re-assimilation and solvent production. With the overexpression of sol operon, acid re-assimilation was enhanced, and ABE production was increased by 20%, with ethanol production increased by six times but almost no increase in butanol production. To further drive carbon flux for C4 metabolites and ultimate butanol production, key genes including hbd, thl, crt and bcd in butanol production pathway were further overexpressed. Compared to the control, butanol, acetone and total ABE production in the new strain was increased by 8%, 18%, and 12.4%, respectively. Finally, simultaneous saccharification and fermentation was carried out using acetate-pretreated switchgrass. 15.4 g/L total ABE (with a yield of 0.31 g/g) was produced in both engineered strains, which was significantly higher than the control.

CRISPR-Cas, a highly effective tool for genome editing in Clostridium saccharoperbutylacetonicum N1-4(HMT).

The solventogenic clostridia have long been known for their ability to convert sugars from complex feedstocks into commercially important solvents. Although the acetone-butanol-ethanol process fell out of favour decades ago, renewed interest in sustainability and 'green' chemistry has re-established our appetite for reviving technologies such as these, albeit with 21st century improvements. As CRISPR-Cas genome editing tools are being developed and applied to the solventogenic clostridia, their industrial potential is growing. Through integration of new pathways, the beneficial traits and historical track record of clostridial fermentation can be exploited to generate a much wider range of industrially relevant products. Here we show the application of genome editing using the endogenous CRISPR-Cas mechanism of Clostridium saccharoperbutylacetonicum N1-4(HMT), to generate a deletion, SNP and to integrate new DNA into the genome. These technological advancements pave the way for application of clostridial species to the production of an array of products.

Enhancement of sucrose metabolism in Clostridium saccharoperbutylacetonicum N1-4 through metabolic engineering for improved acetone-butanol-ethanol (ABE) fermentation.

This work investigated sucrose metabolism in C. saccharoperbutylacetonicum. Inactivation of sucrose catabolism operon resulted in 28.9% decrease in sucrose consumption and 44.1% decrease in ABE production with sucrose as sole carbon source. Interestingly, a large amount of colloid-like polysaccharides were generated in the mutant, which might be due to inefficient intracellular sucrose metabolism. Deletion of transcriptional repressor gene successfully alleviated CCR and enhanced ABE production by 24.7%. Additional overexpression of endogenous sucrose pathway further elevated sucrose consumption and enhanced ABE production by 17.2%, 45.7%, or 22.5% compared to wild type with sucrose, mixed sugars or sugarcane juice as substrate, respectively. The engineered strain could be a robust platform for efficient biofuel production from inexpensive sucrose-based carbon sources.

Novel biobutanol fermentation at a large extractant volume ratio using immobilized Clostridium saccharoperbutylacetonicum N1-4.

Product inhibition by butanol and acetone is a known drawback in acetone-butanol-ethanol (ABE) fermentation. Extractive fermentation improves butanol production by several ABE-producing Clostridium spp., but only low volume ratios (<4) of extractant to broth (Ve/Vb) have been studied. Here, a novel extractive fermentation process was developed using Clostridium saccharoperbutylacetonicum N1-4 and a large Ve/Vb ratio. A mixture of oleyl alcohol-tributyrin (1:1 (v/v)) yielded high distribution coefficients for both butanol (3.14) and acetone (0.660). Although a fed-batch culture using free cells and the oleyl alcohol-tributyrin mixture at a Ve/Vb ratio of 5 had a lag phase of >24 h, it produced a higher concentration of total butanol (i.e., butanol produced in all the phases per broth volume used) of 24.2 g/L-broth after 96 h compared with 14.4 g/L-broth at a Ve/Vb ratio of 1, resulting in a low butanol concentration in the aqueous phase. The use of cells immobilized with calcium alginate beads shortened the lag phase to <12 h. Butanol production was achieved not only in a 3-phase mode (extractant, beads, and tryptone-yeast extract-acetate (TYA) medium) but also in a 2-phase mode (extractant and beads containing TYA medium, without an aqueous phase) at a Ve/Vb ratio of 5, resulting butanol concentrations of 30.9 g/L-broth and 27.7 g/L-broth, respectively. The 3-phases fed-batch extractive fermentation at a Ve/Vb ratio of 10 showed a better performance compared with published reports: a total butanol concentration of 64.6 g/L-broth and a butanol yield to consumed sugar of 0.378 C-mol/C-mol.

Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance.

Ribosome engineering, originally applied to Streptomyces lividans, has been widely utilized for strain improvement, especially for the activation of bacterial secondary metabolism. This study assessed ribosome engineering technology to modulate primary metabolism, taking butanol production as a representative example. The introduction into Clostridium saccharoperbutylacetonicum of mutations conferring resistance to butanol (ButR) and of the str mutation (SmR; a mutation in the rpsL gene encoding ribosomal protein S12), conferring high-level resistance to streptomycin, increased butanol production 1.6-fold, to 16.5 g butanol/L. Real-time qPCR analysis demonstrated that the genes involved in butanol metabolism by C. saccharoperbutylacetonicum were activated at the transcriptional level in the drug-resistant mutants, providing a mechanism for the higher yields of butanol by the mutants. Moreover, the activity of enzymes butyraldehyde dehydrogenase (AdhE) and butanol dehydrogenases (BdhAB), the key enzymes involved in butanol synthesis, was both markedly increased in the ButR SmR mutant, reflecting the significant up-regulation of adhE and bdhA at transcriptional level in this mutant strain. These results demonstrate the efficacy of ribosome engineering for the production of not only secondary metabolites but of industrially important primary metabolites. The possible ways to overcome the reduced growth rate and/or fitness cost caused by the mutation were also discussed.

Mono- and co-substrate utilization kinetics using mono- and co-culture of Clostridium beijerinckii and Clostridium saccharoperbutylacetonicum.

The effect of co-culturing C. beijerinckii and C. saccharoperbutylacetonicum for H2 production using mono- and co-substrates of glucose, starch, and cellulose was assessed. Monod kinetic parameters (K, maximum specific substrate utilization rate; and Ks, half-saturation constant) of the C. beijerinckii, C. saccharoperbutylacetonicum, and the co-culture were determined. Co-cultures utilizing glucose competed for the substrate, but showed enhancement for utilizing starch. The maximum values for K on glucose and starch were 0.48g substrate/gVSS.h achieved by C. saccharoperbutylacetonicum mono-culture and 0.39g substrate/gVSS.h achieved by the co-culture, respectively. The average Ks for all mono- and co-culture experiments was 0.93±0.03g/L. Acetate, butyrate, and propionate were the main fermentation products for all experiments. Maximum H2 production yields on glucose (2.69mol/molglucose) and starch (1.07mol/molhexose) were achieved by C. beijerinckii and C. saccharoperbutylacetonicum mono-cultures, respectively; however, neither culture was able to degrade cellulose as a mono-substrate.

Enhancement of solvent production by overexpressing key genes of the acetone-butanol-ethanol fermentation pathway in Clostridium saccharoperbutylacetonicum N1-4.

Clostridium saccharoperbutylacetonicum N1-4 is well known as a hyper-butanol producing strain. However, little information is available concerning its butanol production mechanism and the development of more robust strains. In this study, key biosynthetic genes (either endogenous or exogenous) including the sol operon (bld-ctfA-ctfB-adc), adhE1, adhE1D485G, thl, thlA1V5A, thlAV5A and the expression cassette EC (thl-hbd-crt-bcd) were overexpressed in C. saccharoperbutylacetonicum N1-4 to evaluate their potential in enhancement of butanol production. The overexpression of sol operon increased ethanol production by 400%. The overexpression of adhE1 and adhED485G resulted in a 5.6- and 4.9-fold higher ethanol production, respectively, producing final acetone-butanol-ethanol (ABE) titers (30.6 and 30.1gL-1) of among the highest as ever reported for solventogenic clostridia. The most significant increase of butanol production (by 13.7%) and selectivity (73.7%) was achieved by the overexpression of EC. These results provides a solid foundation and essential references for the further development of more robust strains.