Engineering Saccharomyces cerevisiae for improved biofilm formation and ethanol production in continuous fermentation.

BACKGROUND: Biofilm-immobilized continuous fermentation has the potential to enhance cellular environmental tolerance, maintain cell activity and improve production efficiency. RESULTS: In this study, different biofilm-forming genes (FLO5, FLO8 and FLO10) were integrated into the genome of S. cerevisiae for overexpression, while FLO5 and FLO10 gave the best results. The biofilm formation of the engineered strains 1308-FLO5 and 1308-FLO10 was improved by 31.3% and 58.7% compared to that of the WT strain, respectively. The counts of cells adhering onto the biofilm carrier were increased. Compared to free-cell fermentation, the average ethanol production of 1308, 1308-FLO5 and 1308-FLO10 was increased by 17.4%, 20.8% and 19.1% in the biofilm-immobilized continuous fermentation, respectively. Due to good adhering ability, the fermentation broth turbidity of 1308-FLO5 and 1308-FLO10 was decreased by 22.3% and 59.1% in the biofilm-immobilized fermentation, respectively. Subsequently, for biofilm-immobilized fermentation coupled with membrane separation, the engineered strain significantly reduced the pollution of cells onto the membrane and the membrane separation flux was increased by 36.3%. CONCLUSIONS: In conclusion, enhanced biofilm-forming capability of S. cerevisiae could offer multiple benefits in ethanol fermentation.

Biomaterials for chimeric antigen receptor T cell engineering.

Chimeric antigen receptor T (CAR-T) cells have achieved breakthrough efficacies against hematological malignancies, but their unsatisfactory efficacies in solid tumors limit their applications. The prohibitively high prices further restrict their access to broader populations. Novel strategies are urgently needed to address these challenges, and engineering biomaterials can be one promising approach. The established process for manufacturing CAR-T cells involves multiple steps, and biomaterials can help simplify or improve several of them. In this review, we cover recent progress in engineering biomaterials for producing or stimulating CAR-T cells. We focus on the engineering of non-viral gene delivery nanoparticles for transducing CAR into T cells ex vivo/in vitro or in vivo. We also dive into the engineering of nano-/microparticles or implantable scaffolds for local delivery or stimulation of CAR-T cells. These biomaterial-based strategies can potentially change the way CAR-T cells are manufactured, significantly reducing their cost. Modulating the tumor microenvironment with the biomaterials can also considerably enhance the efficacy of CAR-T cells in solid tumors. We pay special attention to progress made in the past five years, and perspectives on future challenges and opportunities are also discussed. STATEMENT OF SIGNIFICANCE: Chimeric antigen receptor T (CAR-T) cell therapies have revolutionized the field of cancer immunotherapy with genetically engineered tumor recognition. They are also promising for treating many other diseases. However, the widespread application of CAR-T cell therapy has been hampered by the high manufacturing cost. Poor penetration of CAR-T cells into solid tissues further restricted their use. While biological strategies have been explored to improve CAR-T cell therapies, such as identifying new cancer targets or integrating smart CARs, biomaterial engineering provides alternative strategies toward better CAR-T cells. In this review, we summarize recent advances in engineering biomaterials for CAR-T cell improvement. Biomaterials ranging from nano-, micro-, and macro-scales have been developed to assist CAR-T cell manufacturing and formulation.

Biofilm-Based Biocatalysis for Galactooligosaccharides Production by the Surface Display of ß-Galactosidase in Pichia pastoris.

Galactooligosaccharides (GOS) are one of the most important functional oligosaccharide prebiotics. The surface display of enzymes was considered one of the most excellent strategies to obtain these products. However, a rough industrial environment would affect the biocatalytic process. The catalytic process could be efficiently improved using biofilm-based fermentation with high resistance and activity. Therefore, the combination of the surface display of ß-galactosidase and biofilm formation in Pichia pastoris was constructed. The results showed that the catalytic conversion rate of GOS was up to 50.3% with the maximum enzyme activity of 5125 U/g by screening the anchorin, and the number of the continuous catalysis batches was up to 23 times. Thus, surface display based on biofilm-immobilized fermentation integrated catalysis and growth was a co-culture system, such that a dynamic equilibrium in the consolidated integrative process was achieved. This study provides the basis for developing biofilm-based surface display methods in P. pastoris during biochemical production processes.

Type I fimbriae subunit fimA enhances Escherichia coli biofilm formation but affects L-threonine carbon distribution.

The biofilm (BF) provides favorable growth conditions to cells, which has been exploited in the field of industrial biotechnology. Based on our previous research works on type I fimbriae for the biosynthesis of L-threonine (LT) in Escherichia coli, in this study, a fimA-overexpressing strain was engineered, which improved BF formation under industrial fermentation conditions. The morphological observation and characterization of BF formation were conducted to verify the function of the subunit FimA. However, it was not suitable for repeated-batch immobilized fermentation as the LT titer was not elevated significantly. The underlying molecular mechanisms of BF formation and the LT carbon flux were explored by transcriptomic analysis. The results showed that fimA regulated E. coli BF formation but affected LT carbon distribution. This study will stimulate thoughts about how the fimbriae gene regulated biofilms and amino acid excretion and will bring some consideration and provide a reference for the development of BF-based biomanufacturing processes in E. coli.

Physiological changes and growth behavior of Corynebacterium glutamicum cells in biofilm.

Biofilm cells are well-known for their increased survival and metabolic capabilities and have been increasingly implemented in industrial and biotechnological processes. Corynebacterium glutamicum is one of the most widely used microorganisms in the fermentation industry. However, C. glutamicum biofilm has been rarely reported and little is known about its cellular basis. Here, the physiological changes and characteristics of C. glutamicum biofilm cells during long-term fermentation were studied for the first time. Results showed that the biofilm cells maintained stable metabolic activity and cell size was enlarged after repeated-batch of fermentation. Cell division was slowed, and chromosome content and cell proliferation efficiency were reduced during long-term fermentation. Compared to free cells, more biofilm cells were stained by the apoptosis indicator dyes Annexin V-FITC and propidium iodide (PI). Overall, these results suggested slow-growing, long-lived cells of C. glutamicum biofilm during fermentation, which could have important industrial implications. This study presents first insights into the physiological changes and growth behavior of C. glutamicum biofilm cell population, which would be valuable for understanding and developing biofilm-based processes.

Nonsterile l-Lysine Fermentation Using Engineered Phosphite-Grown Corynebacterium glutamicum.

Fermentation using Corynebacterium glutamicum is an important method for the industrial production of amino acids. However, conventional fermentation processes using C. glutamicum are susceptible to microbial contamination and therefore require equipment sterilization or antibiotic dosing. To establish a more robust fermentation process, l-lysine-producing C. glutamicum was engineered to efficiently utilize xenobiotic phosphite (Pt) by optimizing the expression of Pt dehydrogenase in the exeR genome locus. This ability provided C. glutamicum with a competitive advantage over common contaminating microbes when grown on media containing Pt as a phosphorus source instead of phosphate. As a result, the engineered strain could produce 41.00 g/L l-lysine under nonsterile conditions during batch fermentation for 60 h, whereas the original strain required 72 h to produce 40.78 g/L l-lysine under sterile conditions. Therefore, the recombinant strain can efficiently produce l-lysine under nonsterilized conditions with unaffected production efficiency. Although this anticontamination strategy has been previously reported for other species, this is the first time it has been demonstrated in C. glutamicum; these findings should aid in the further development of cost-efficient amino acid fermentation processes.

Isolation and characterization of plant growth-promoting rhizobacteria and their effects on the growth of Medicago sativa L. under salinity conditions.

Plant growth-promoting rhizobacteria are a group of free-living bacteria that colonize plant rhizosphere and benefit plant root growth, thereby increasing host plant to cope with salinity induced stress. The aim of this study was to (1) isolate and characterize auxin-producing bacteria showing a high plant growth-promoting (PGP) potential, and (2) evaluate the PGP effects on the growth of Medicago sativa L under salinity stress (130 mM NaCl). Of thirteen isolates, Bacillus megaterium NRCB001 (NRCB001), B. subtilis subsp. subtilis NRCB002 (NRCB002) and B. subtilis NRCB003 (NRCB003) had the ability to produce auxin, which ranged from 47.53 to 154.38 µg ml-1. The three auxin-producing bacterial strains were shown multiple PGP traits, such as producing siderophore and NH3, showing ACC deaminase activity, solubilize phosphate and potassium. Furthermore, NRCB001, NRCB002, and NRCB003 could survive in LB medium containing 1750 mM NaCl. The three auxin-producing with salinity tolerance strains were selected for further analyses. In greenhouse experiments, when inoculated with NRCB001, NRCB002 and NRCB003, dry weight of alfalfa significantly (P < 0.05) increased by 24.1%, 23.1% and 38.5% respectively, compared with those of non-inoculated control seedlings under normal growth condition. When inoculated with NRCB002 and NRCB003, dry weight of alfalfa significantly (P < 0.05) increased by 96.9 and 71.6% respectively, compared with those of non-inoculated control seedlings under 130 mM NaCl condition. Our results indicated that NRCB002 and NRCB003 having PGP traits are promising candidate strains to develop biofertilizers, especially used under salinity stress conditions.

Preparation of a Copper Polyphosphate Kinase Hybrid Nanoflower and Its Application in ADP Regeneration from AMP.

In this research article, we reported a self-assembly approach to prepare a copper polyphosphate kinase 2 hybrid nanoflower and established a cofactor ADP regeneration system from AMP using the nanoflower. First, the structure of the hybrid nanoflower was confirmed by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy, which indicated the successful loading of the enzyme in the hybrid nanoflower. Moreover, compared to the free enzyme, the hybrid nanoflower exhibited a better performance in ADP production and possessed wider catalytic pH and temperature ranges as well as improved storage stability. The hybrid nanoflower also exhibited well reusability, preserving 71.7% of initial activity after being used for ten cycles. In addition, the phosphorylation of glucose was conducted by utilizing ADP-dependent glucokinase coupled with the ADP regeneration system, in which the hybrid nanoflower was used for regenerating ADP from AMP. It was observed that the ADP regeneration system operated effectively at a very small amount of AMP. Thus, the hybrid nanoflower had great application potential in industrial catalytic processes that were coupled with ADP-dependent enzymes.

Effects of Spo0A on Clostridium acetobutylicum with an emphasis on biofilm formation.

Clostridium acetobutylicum is a well-known strain for biofuel production. In previous work, it was found that this strain formed biofilm readily during fermentation processes. Biofilm formation could protect cells and enhance productivities under environmental stresses in our previous work. To explore the molecular mechanism of biofilm formation, Spo0A of C. acetobutylicum was selected to investigate its influences on biofilm formation and other physiological performances. When spo0A gene was disrupted, the spo0A mutant could hardly form biofilm. The aggregation and adhesion abilities of the spo0A mutant as well as its swarming motility were dramatically reduced compared to those of wild type strain. Sporulation was also negatively influenced by spo0A disruption, and solvent production was almost undetectable in the spo0A mutant fermentation. Furthermore, proteomic differences between wild type strain and the spo0A mutant were consistent with physiological performances. This is the first study confirming a genetic clue to C. acetobutylicum biofilm and will be valuable for biofilm optimization through genetic engineering in the future.

Calcineurin signaling pathway influences Aspergillus niger biofilm formation by affecting hydrophobicity and cell wall integrity.

BACKGROUND: Biofilms, as a kind of fixed-cell community, can greatly improve industrial fermentation efficiency in immobilized fermentation, but the regulation process is still unclear, which restricts their application. Ca2+ was reported to be a key factor affecting biofilm formation. However, the effect of Ca2+ on biofilm structure and microbiology was yet only studied in bacteria. How Ca2+-mediated calcineurin signaling pathway (CSP) alters biofilm formation in bacteria and fungi has rarely been reported. On this basis, we investigated the regulation of CSP on the formation of biofilm in Aspergillus niger. RESULTS: Deletion of the key genes MidA, CchA, CrzA or CnaA in the CSP lowered the Ca2+ concentration in the mycelium to a different extent, inhibited the formation of A. niger biofilm, reduced the hydrophobicity and adhesion of spores, destroyed the cell wall integrity of hyphae, and reduced the flocculation ability of hyphae. qRT-PCR results showed that the expression of spore hydrophobic protein RodA, galactosaminogalactan (GAG) biosynthesis genes (uge3, uge5, agd3, gtb3), and α-1,3-glucan biosynthesis genes (ags1, ags3) in the ∆MidA, ∆CchA, ∆CrzA, ∆CnaA strains were significantly down-regulated compared with those of the wild type (WT). In addition, the transcription levels of the chitin synthesis gene (chsB, chsD) and ß-1,3-glucan synthesis gene (FksA) were consistent with the change in chitin and ß-1,3-glucan contents in mutant strains. CONCLUSION: These results indicated that CSP affected the hydrophobicity and adhesion of spores, the integrity of mycelial cell walls and flocculation by affecting Ca2+ levels in mycelium, which in turn affected biofilm formation. This work provides a possible explanation for how CSP changes the formation of A. niger biofilm, and reveals a pathway for controlling biofilm formation in industrial immobilized fermentation.

Knockout of pde gene in Arthrobacter sp. CGMCC 3584 and transcriptomic analysis of its effects on cAMP production.

Arthrobacter sp. CGMCC 3584 is used for the industrial production of cyclic adenosine monophosphate (cAMP). However, because of the paucity of genetic engineering tools for genetic manipulation on Arthrobacter species, only a few metabolically engineered Arthrobacter have been constructed and investigated. In this study, for the first time, we constructed an arpde knockout mutant of Arthrobacter without any antibiotic resistance marker by a PCR-targeting-based homologous recombination method. Our results revealed that the deletion of arpde had little effect on biomass production and improved cAMP production by 31.1%. Furthermore, we compared the transcriptomes of the arpde knockout strain and the wild strain, aiming to understand the capacities of cAMP production due to arpde inactivation at the molecular level. Comparative transcriptomic analysis revealed that arpde inactivation had two major effects on metabolism: inhibition of glycolysis, PP pathway, and amino acid metabolism (phenylalanine, tryptophan, branched-chain amino acids, and glutamate metabolism); promotion of the purine metabolism and carbon flux from the precursor 5'-phosphoribosyl 1-pyrophosphate, which benefited cAMP production.

pH-Neutralization, Redox-Balanced Process with Coupled Formate Dehydrogenase and Glucose Dehydrogenase Supports Efficient Xylitol Production in Pure Water.

Enzymatic production of xylitol is a promising alternative to the chemical hydrogenation process. However, it encounters problems that are largely due to protein susceptibility to environmental factors. In this study, to develop a robust, practical enzymatic process for xylitol production, a coupled enzyme system consisting of formate dehydrogenase (FDH), glucose dehydrogenase (GDH), and xylose reductase (XR) was constructed, wherein the alkaline product produced by FDH and the acidic product produced by GDH could neutralize each other during cofactor regeneration. After optimization of conditions, a pH-neutralization, redox-balanced process was developed that could be carried out in pure water requiring no pH regulation. As a result, a xylitol production of 273.6 g/L that is much higher than those yet reported was obtained from 2 M xylose in 24 h, with a relatively high productivity of 11.4 g/(L h). The strategy demonstrated here can be adapted for the production of other NADH-consuming products.

Immobilization of a polyphosphate kinase 2 by coordinative self-assembly of his-tagged units with metal-organic frameworks and its application in ATP regeneration from AMP.

Self-assembly of the functional units onto the surface of nanoparticles is a powerful approach to generate functional nanosystems. In this work, we first expressed a recombinant class III polyphosphate kinase 2 (ArPPK2) with his-tag. It is able to synthesize ATP from AMP by a single enzyme, simplifying two-step reaction of ATP regeneration from AMP. Then we chose the Fe-based metal-organic frameworks (MOF)s as carriers to produce the enzyme-MOF biocomposite, based on the interaction between the his-tags and coordinatively unsaturated metal sites present on the external surface of MOFs by a self-assembly process. It was found that ArPPK2@MIL-101-NH2@Fe3O4-COOH exhibited better reusability than other candidates during cycle analysis, preserving 70.1% of initial activity after reusing thirteen times, and also retained high storage stability. The optimum pH of the enzyme-MOF biocomposite was increased from 8.0 to 9.0 and the optimum temperature was increased from 30℃ to 45℃. Compared to free ArPPK2, the enzyme-MOF biocomposite showed increased thermal and pH stability. In addition, we successfully constructed an ATP regeneration system from AMP using the enzyme-MOF biocomposite, coupled with amide bond formation catalyzed by the adenylation domain of tyrocidine synthetase A (TycA-A). The immobilized ArPPK2 will provide a promising route for ATP regeneration from AMP in industrial processes. And the generation of the enzyme-MOF biocomposite by the self-assembly approach can be extended to efficiently immobilize other recombinant his-tagged enzymes.

Competitive adsorption of vanillin and syringaldehyde on a macro-mesopore polymeric resin: modeling.

Vanillin and syringaldehyde are widely used as flavoring and fragrance agents in the food products. The potential of a macro-mesoporous adsorption resin was assessed for separation of these binary mixtures. This work focuses on modeling of the competitive adsorption behaviors and exploration of the adsorption mechanism. The characterization results showed the resin had a large BET surface area and specific pore structure with hydrophobic properties. By analysis of the physicochemical properties of the solutes and the resin, the separation mechanism was mainly contributed by hydrophobic effect. Subsequently, the competitive Langmuir isotherm model was used to fit the competitive adsorption isotherms. The pore diffusion coefficient was obtained by macropore diffusion model. Afterwards, a mathematical model was established to predict the breakthrough curves of the binary mixture at various operating conditions. The data and model presented are valuable for design and simulation of the continuous chromatographic separation process.

Surface functionalization of graphene oxide by amino acids for Thermomyces lanuginosus lipase adsorption.

Graphene oxide (GO) with oxygen containing functional groups can be selectively modified by small biomolecules to achieve heterogeneous surface properties. To achieve a hyper-enzymatic activity, the surface functionality of GO should be tailored to the orientation adsorption of the Thermomyces lanuginosus (TL) lipase, and the active center can be covered by a relatively hydrophobic helical lid for protection. In this work, amino acids were used to interact with GO through reduction reaction, hydrophobic forces, electrostatic forces, or hydrogen bonding to alter the surface hydrophobicity and charge density. Characterization of the structure and surface properties confirmed that the GO samples decorated with phenylalanine (Phe) and glutamic acid (Glu) exhibited superior hydrophobicity than other modifications, whereas tryptophan (Trp) and cysteine (Cys) provided weaker reduction effects on GO. Moreover, the zeta potential of the samples modified by amino acids of lysine (Lys) and arginine (Arg) is higher than other modified samples. The adsorption amount of lipase on Glu-GO reached 172 mg/g and the relative enzymatic activity reached up to 200%. The thermodynamic data and the Freundlich isotherm model fitting showed that the lipase adsorption process on modified samples was spontaneous, endothermic and entropy increase.

Co-localization of glucose oxidase and catalase enabled by a self-assembly approach: Matching between molecular dimensions and hierarchical pore sizes.

To achieve efficient one-step production of gluconic acid, cascade reactions of glucose oxidase (GOD) and catalase (CAT) have been advocated in the biocatalysis system. In this work, the methodology of co-immobilization of GOD and CAT was investigated in details for obtaining improved enzyme loading and activity. The maximum adsorption capability of GOD and CAT was 24.18 and 14.33 mg·g-1, respectively. The matching between dimensions of enzymes and hierarchical pore sizes of carriers are critical to the success of immobilization process. The simultaneous self-assembly on glutaraldehyde cross-linked mesoporous carriers exhibited favorable properties in comparison with sequential immobilization of GOD and CAT. The conversion of glucose under adequate air by co-localized GOD&CAT sustained the activity more than 90% after repeated utilization in the production of sodium gluconate and gluconic acid, suggesting that the co-immobilized GOD&CAT could be a promising catalyst for gluconate and gluconic acid production in some chemical and food industries.

Computation-aided rational design of a halophilic choline kinase for cytidine diphosphate choline production in high-salt condition.

Biocatalysis has become the main approach to produce cytidine diphosphate choline (CDP-choline), which has been applied for treatment of acute craniocerebral injury and consciousness after brain surgery. However, salt accumulates with the production and inhibits enzyme activity, and eventually reduces yield and product accumulation rate. Our work provided a possible solution to this problem by applying a computational designed halophilic choline kinase. The halotolerant CKI (choline kinase) was designed following a unique strategy considering the most variable residue positions on the protein surface among target enzymes from different sources. The basic and neutral surface residues were replaced with acidic ones. This approach was enlightened by features of natural halophilic enzymes. Mutants in the work represented higher catalytic activities and IC50 (inhibit activity by 50%) at high salt concentrations (over 1200 mM). Furthermore, when the mutant was used in fed-batch production, the CDP-choline accumulation rate doubled comparing with process using wild-type CKI at acetate concentration of over 700 mM. The maximum titer was 151 ± 3.2 mM, the productivity was 5.8 ± 0.1 mM·L-1 h-1, and molar yield to CMP and utilization efficiency of energy were 85.3 and 63.5%. The idea of computational design in our work can also be applied to modify other enzymes in industry, and sheds light on alleviating effect of salt accumulation during industrial manufacturing process.

Efficient Xylitol Production from Cornstalk Hydrolysate Using Engineered Escherichia coli Whole Cells.

Economic transformation of lignocellulose hydrolysate into valued-added products is of particular importance for energy and environmental issues. In this study, xylose reductase and glucose dehydrogenase were cloned into plasmid pETDuet-1 and then simultaneously expressed in Escherichia coli BL21(DE3), which was used as whole-cell catalyst for the first time to convert xylose into xylitol coupled with gluconate production. When tested with reconstituted xylose and glucose solution, 0.1 g/mL cells could convert 1 M xylose and 1 M glucose completely and produced 145.81 g/L xylitol with a yield of 0.97 (g/g) and 184.85 g/L gluconic acid with a yield of 1.03 (g/g) in 24 h. Subsequently, the engineered cells were applied in real cornstalk hydrolysate, which generated 30.88 g/L xylitol and 50.89 g/L gluconic acid. The cells were used without penetration treatment, and CaCO3 was used to effectively regulate the pH during the production, which further saved costs.

Clostridium acetobutylicum grows vegetatively in a biofilm rich in heteropolysaccharides and cytoplasmic proteins.

BACKGROUND: Biofilms are cell communities wherein cells are embedded in a self-produced extracellular polymeric substances (EPS). The biofilm of Clostridium acetobutylicum confers the cells superior phenotypes and has been extensively exploited to produce a variety of liquid biofuels and bulk chemicals. However, little has been known about the physiology of C. acetobutylicum in biofilm as well as the composition and biosynthesis of the EPS. Thus, this study is focused on revealing the cell physiology and EPS composition of C. acetobutylicum biofilm. RESULTS: Here, we revealed a novel lifestyle of C. acetobutylicum in biofilm: elimination of sporulation and vegetative growth. Extracellular polymeric substances and wire-like structures were also observed in the biofilm. Furthermore, for the first time, the biofilm polysaccharides and proteins were isolated and characterized. The biofilm contained three heteropolysaccharides. The major fraction consisted of predominantly glucose, mannose and aminoglucose. Also, a great variety of proteins including many non-classically secreted proteins moonlighting as adhesins were found considerably present in the biofilm, with GroEL, a S-layer protein and rubrerythrin being the most abundant ones. CONCLUSIONS: This study evidenced that vegetative C. acetobutylicum cells rather than commonly assumed spore-forming cells were essentially the solvent-forming cells. The abundant non-classically secreted moonlighting proteins might be important for the biofilm formation. This study provides the first physiological and molecular insights into C. acetobutylicum biofilm which should be valuable for understanding and development of the biofilm-based processes.

Towards acetone-uncoupled biofuels production in solventogenic Clostridium through reducing power conservation.

Microbial production of butanol by solventogenic Clostridium has long been complicated with the formation of acetone as an unwanted product, which causes poor product yields and creates a most important problem concerning substrate transformation. Intensive attempts concentrate on carbon conversion pathways to eliminate acetone, but have actually achieved little so far. Here, we believe microbial product distribution can largely depend on how the cell plays its energetic cofactors in central metabolism, and demonstrate that by introducing a synthetic 2,3-butanediol synthesis pathway in Clostridium acetobutylicum as an NADH-compensating module to readjust the reducing power at a systems level, the production of acetone can be selectively and efficiently eliminated (< 0.3 g/L). H2 evolution was reduced by 78%, and the total alcohol yield was strikingly increased by 19% to 0.44 g/g glucose, much higher than those yet reported for butanol fermentation. These findings highlight that it is the loss of reducing power rather than typically manipulated solventogenesis genes that dominates acetone formation. Further study revealed that the NADH-module triggered apparent regulation of pathways involved in electron transfer and reducing power conservation. The study also suggested the key to conservation of intracellular reducing power might essentially lie in the intermediate processes in central metabolism that are related to redox partners, butyrate or C4 branches, and possibly NADH and NADPH specificity. This study represents the first effective redox-based configuration of C. acetobutylicum and provides valuable understandings for redox engineering of native Clostridium species towards advanced production of biofuels and alcohols.