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Clostridium acetobutylicum has been extensively exploited to produce biofuels and solvents and its biofilm could dramatically improve the productivities. However, genetic control of C. acetobutylicum biofilm has not been dissected so far. Here, to identify potential genes controlling C. acetobutylicum biofilm formation, over 40 gene candidates associated with extracellular matrix, cell surface, cell signaling or gene transcription, were tried to be disrupted to examine their individual impact. A total of 25 disruptants were finally obtained over years of attempts, for which biofilm and relevant phenotypes were characterized. Most of these disruptants formed robust biofilm still, or suffered both growth and biofilm defect. Only a strain with a disrupted histidine kinase gene (CA_C2730, designated bfcK in this study) abolished biofilm formation without impairing cell growth or solvent production. Further analysis revealed that bfcK could control flagellar biogenesis and cell motility at protein levels. The bfcK also appeared to repress the phosphorylation of a serine/threonine protein kinase (encoded by CA_C0404) that might negatively regulate biofilm formation. Based on these findings, possible bfcK-mediated mechanisms for biofilm formation were proposed. This is a big step toward understanding the biofilm formation in C. acetobutylicum and will help further engineering of its biofilm-based industrial processes.

Compared with the traditional suspension fermentation, immobilised biofilm fermentation has advantages of strong resistance, high yield and continuous fermentation and is conducive to improving the industrial fermentation methods. The formation of a biofilm on correct supports is critical to immobilised fermentation. In this study, Pichia pastoris was used to understand and control the modification of surface properties of supports and cells. Following deletion of gene PAS_chr2-1_0773, in P. pastoris △0773, the cell-surface negative charge density decreased and hydrophobicity improved, which was conducive to adhesion. Therefore, according to the changing trend of cell surface properties, cotton fibre was used with succinic anhydride (SA) to modify the carrier for changing the surface properties for efficient conduction of immobilised fermentation. In the fermentation process, the adhesion between the modified cotton fibre and yeast was better than that between an unmodified cotton fibre and yeast, and the additive amount increased by ∼25 %. The enzyme activity of the △0773-cotton-SA fermentation system reached 302.7 U/mL, which was 39.6 % higher than that of the original fermentation system. Therefore, this study provides a guide for significant improvement of immobilised fermentation of fungi.

In this study, we found that biofilm formation is a critical factor affecting the activity of Aspergillus niger SJ1 xylanase. Xylanase activity increased 8.8% from 1046.88 to 1147.74 U/mL during A. niger SJ1 immobilized fermentation with biofilm formation. Therefore, we carried out the work of genomic analysis and biofilm-related time-series transcriptome analysis of A. niger SJ1 for better understanding of the ability of A. niger SJ to produce xylanase and biofilm formation. Genome annotation results revealed a complete biofilm polysaccharide component synthesis pathway in A. niger SJ1 and five proteins regarding xylanase synthesis. In addition, results of transcriptome analysis revealed that the genes involved in the synthesis of cell wall polysaccharides and amino acid anabolism were highly expressed in the biofilm. Furthermore, the expression levels of major genes in the gluconeogenesis pathway and mitogen-activated protein kinase pathway were examined.

Biofilm-based fermentation, as a new immobilisation strategy, is beneficial for industrial fermentation due to its excellent environmental resistance, high productivity and continuous fermentation relative to calcium alginate-immobilised fermentation. These two techniques differ mainly regarding cell stages. Here, we describe the cell phenotype of Saccharomyces cerevisiae biofilm-based fermentation and compare cell cycle stages with those during immobilisation in calcium alginate. Most cells in the biofilm-based fermentation adhered to the cotton-fibre carrier of the biofilm and were in the G2/M phase whereas alginate-embedded cells were in the G1/G0 phase. Deletion of the RIM15 gene, which regulates cell cycle progression according to nutritional status, hampered the cell cycle arrest observed in alginate-embedded cells, enhanced biofilm formation and improved fermentation ability. The improved biofilm formation shown by the rim15△ strain could be attributed to an increase in the expression level of the adhesion protein FLO11 and synthesis of trehalose. These findings suggest that the extracellular environment is mainly responsible for the difference between biofilm-based fermentation and alginate-embedded fermentation, and that RIM15 plays an essential role in cell cycle progression. KEY POINTS: • In the biofilm, S. cerevisiae cell populations were mostly in the G2/M phase while alginate-embedded cells were arrested in the G1/G0 phase. • The RIM15 gene partially influenced the cell cycle progression observed during ethanol fermentation. • Biofilm-based cells were actively adsorbed on the physical carrier. • Biofilm immobilisation could maintain cell division activity explaining its fermentation efficiency.

The production of acetone-butanol-ethanol by solventogenic Clostridium using lignocellulosic biomass can be a potential alternative to petroleum-based butanol. However, previous studies on nondetoxified lignocellulose hydrolysate could not provide better results when compared to those in synthetic medium. In this study, we engineered the pentose pathway of Clostridium beijerinckii NCIMB 8052, which was then subjected to adaptive laboratory evolution in the gradient mixture of synthetic medium and pretreated corn stover enzymatic hydrolysate (CSH) prepared according to the National Renewable Energy Laboratory (NREL) standard. The final resultant strain CIBTS1274A produced 20.7 g/L of total solvents in NREL CSH diluted to 6% initial total sugars, supplemented with ammonium acetate. This performance was comparable with that of corn-based butanol. In addition, this strain was successfully used in the scale-up operation using nondetoxified corn stover and corncob hydrolysate at Lignicell Refining Biotechnologies Ltd., which once was the only commercial biobutanol industry in the world.

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

Butanol is an attractive second-generation biofuel as well as an important chemical. Production of butanol from lignocellulosic feedstock via consolidated bioprocessing has attracted increasing int...

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.

Azo dyes are the most widely applied chemical dyes that have also raised great concerns for environmental contamination and human health issues. There has been a growing interest in discovering bioremediation methods to degrade azo dyes for environmental and economic purposes. Azoreductases are key enzymes evolved in nature capable of degrading azo dyes. The current work reports the identification, expression, and properties of a novel azoreductase (AzoRed2) from Streptomyces sp. S27 which shows an excellent stability against pH change and organic solvents. To overcome the requirements of coenzyme while degrading azo dyes, we introduced a coenzyme regeneration enzyme, Bacillus subtilis glucose 1-dehydrogenase (BsGDH), to construct a recycling system in living cells. The whole-cell biocatalyst containing AzoRed2 and BsGDH was used to degrade a representative azo dye methyl red. The degradation rate of methyl red was up to 99% in 120 min with high substrate concentration (250 μM) and no external coenzyme added. The degradation rate was still 98% in the third batch trial. To sum up, a novel azoreductase with good properties was found, which was applied to construct whole-cell biocatalyst. Both the enzymes and whole-cell biocatalysts are good candidates for the industrial wastewater treatment and environmental restoration.

Clostridium acetobutylicum is an important industrial platform capable of producing a variety of biofuels and bulk chemicals. Biofilm of C. acetobutylicum renders many production advantages and has been long and extensively applied in fermentation. However, molecular and genetic mechanisms underlying the biofilm have been much less studied and remain largely unknown. Here, we review studies to date focusing on C. acetobutylicum biofilms, especially on its physiological and molecular aspects, summarizing the production advantages, cell physiological changes, extracellular matrix components and regulatory genes of the biofilm. This represents the first review dedicated to the biofilm of C. acetobutylicum. Hopefully, it will deepen our understanding toward C. acetobutylicum biofilm and inspire more research to learn and develop more efficient biofilm processes in this industrially important bacterium.

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. Electronic supplementary material The online version of this article (10.1186/s13068-018-1316-4) contains supplementary material, which is available to authorized users.

The sugar alcohols and functional sugars have wide applications in food, pharmaceutical, and chemical industries. However, the smaller quantities of natural occurring sugar alcohols and functional sugars restricted their applications. The enzymatic and whole-cell catalyst production is emerging as the predominant alternatives. The properties of Yarrowia lipolytica make it a promising sugar alcohol and functional sugar producer. However, there are still some issues to be resolved. As there exist reviews about the chemical structures, physicochemical properties, biological functions, applications, and biosynthesis of sugar alcohols and/or functional sugars in Y. lipolytica, this mini review will not only update the recent advances in enzymatic and microbial production of sugar alcohols (erythritol, D-threitol, and xylitol) and functional sugars (isomaltulose, trehalose, fructo-oligosaccharides, and galacto-oligosaccharides) by using recombinant Y. lipolytica but also focus on the studies of gene discovery, pathway engineering, expanding substrate scope, bioprocess engineering, and novel breeding methods to resolve the aforementioned issues.

Light is an important signal source in nature, which regulates the physiological cycle, morphogenetic pathways, and secondary metabolites of fungi. As an external pressure on Aspergillus niger, light signaling transmits stress signals into the cell via the mitogen-activated protein kinase (MAPK) signaling pathway. Studying the effect of light on the biofilm of A. niger will provide a theoretical basis for light in the cultivation of filamentous fungi and industrial applications. Here, the characterization of A. niger biofilm under different light intensities confirmed the effects of light signaling. Our results indicated that A. niger intensely accumulated protective mycelial melanin under light illumination. We also discovered that the RlmA transcription factor in the MAPK signaling pathway is activated by light signaling to promote the synthesis of melanin, chitin, and other exopolysaccharides. However, the importance of melanin to A. niger biofilm is rarely reported; therefore, we knocked out key genes of the melanin biosynthetic pathway-Abr1 and Ayg1 Changes in hydrophobicity and electrostatic forces resulted in the decrease of biofilm caused by the decrease of melanin in mutants.IMPORTANCE As an important industrial filamentous fungus, Aspergillus niger can perceive light. The link between light signaling and A. niger biofilm is worthy of further study since reports are lacking in this area. This study found that light signaling promotes biofilm production in A. niger, wherein melanin plays an important role. It was further discovered that the RlmA transcription factor in the mitogen-activated protein kinase (MAPK) signaling pathway was mediated by light signaling to promote the synthesis of melanin and extracellular polysaccharides. These findings set the stage for light signal regulation of biofilm in filamentous fungi and provide a theoretical basis for the development of a new light-controlled biofilm method to improve biofilm-based industrial fermentation.

Biofilms are a form of microbial community that can be beneficial for industrial fermentation because of their remarkable environmental resistance. However, the mechanism of biofilm formation in Saccharomyces cerevisiae remains to be fully explored, and this may enable improved industrial applications for this organism. Although quorum-sensing (QS) molecules are known to be involved in bacteria biofilm formation, few studies have been undertaken with these in fungi. 2-phenylethanol (2-PE) is considered a QS molecule in S. cerevisiae. Here, we found that exogenous 2-PE could stimulate biofilm formation at low cell concentrations. ARO8p and ARO9p are responsible for the synthesis of 2-PE and were crucial to the formation of biofilm. Deletion of the ARO8 and ARO9 genes reduced the content of 2-PE in the early stage of fermentation, reduced ethanol yield and decreased biofilm formation. The expression of FLOp, which is involved in cell adhesion, and the content of extracellular polysaccharides of mutant strains ΔARO8 and ΔARO9 were also significantly reduced. These findings indicate that the production of 2-PE had a positive effect on biofilm formation in S. cerevisiae, thereby providing further key details for studying the formation of biofilm mechanism in the future. KEY POINTS: • Quorum-sensing molecule 2-PE positively affects biofilm formation in S. cerevisiae. • 2-PE synthetic genes ARO8 and ARO9 deletion reduced extracellular polysaccharide. • ARO8 and ARO9 deletion reduced the gene expression of the FLO family.

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.

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 β-1,3-glucan synthesis gene (FksA) were consistent with the change in chitin and β-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.

The efficiency of current methods for industrial production of citric acid is limited. To achieve continuous citric acid production with enhanced yield and reduced cost, immobilized fermentation was employed in an Aspergillus niger 831 repeated fed-batch fermentation system. We developed a new type of material (PAF201), which was used as a carrier for the novel adsorption immobilization system. Hydrophobicity, pore size and concentration of carriers were researched in A. niger immobilization. The efficiency of the A. niger immobilization process was analyzed by scanning electron microscopy. Then eight-cycle repeated fed-batch cultures for citric acid production were carried out over 600 h, which showed stable production with maximum citric acid concentrations and productivity levels of 162.7 g/L and 2.26 g L−1 h−1, respectively. Compared with some other literatures about citric acid yield, PAF201 immobilization system is 11.3% higher than previous results. These results indicated that use of the new adsorption immobilization system could greatly improve citric acid productivity in repeated fed-batch fermentation. Moreover, these results could provide a guideline for A.niger or other filamentous fungi immobilization in industry.

ρ-coumaric acid with strong antibacterial activity is produced during the pretreatment and hydrolysis of lignocellulosic biomass to monomeric sugars. The cell growth and metabolism of C. beijerinckii NCIMB 8052 is significantly inhibited by ρ-coumaric acid. In this study, adaptation of C. beijerinckii NCIMB 8052 to ρ-coumaric acid was remarkably enhanced after disruption of the intracellular electron transport chain, and the antibacterial activity assay showed a 2-fold increase in cell viability in the presence of 0.5 g/L ρ-coumaric acid 24 h after inoculation. Additionally, electricity generation in the wild-type and recombinant strains was measured using microbial fuel cell devices, and the intracellular levels of co-factor NAD(P)H were determined by the enzyme cycling method to show disturbed intracellular electron transfer after disruption of gene Cbei_2996. Furthermore, the metabolism of ρ-coumaric acid by C. beijerinckii was analyzed. These data indicated that gene Cbei_2996 plays a significant role in regulating ρ-coumaric acid tolerance in C. beijerinckii; the metabolism of ρ-coumaric acid mainly coupled with co-factor NADH is catalyzed by reductase. The strategy used in this study provides a potential approach for producing dominant microorganisms with high inhibitor tolerance for butanol production using renewable lignocellulosic materials.

Arthrobacter sp. CGMCC 3584 is able to produce high yields of extracellular cyclic adenosine monophosphate (cAMP), which plays a vital role in the field of treatment of disease and animal food, during aerobic fermentation. However, the molecular basis of cAMP production in Arthrobacter species is rarely explored. Here, for the first time, we report the comparative transcriptomic and proteomic study of Arthrobacter cells to elucidate the higher productivity of cAMP under high oxygen supply. We finally obtained 14.1% and 19.3% of the Arthrobacter genome genes which were up-regulated and down-regulated notably, respectively, with high oxygen supply, and identified 54 differently expressed proteins. Our results revealed that high oxygen supply had two major effects on metabolism: inhibition of glycolysis, pyruvate metabolism, nitrogen metabolism, and amino acid metabolism (histidine, branched-chain amino acids and glutamate metabolism); enhancement of the tricarboxylic acid cycle and purine metabolism. We also found that regulation of adenylate cyclase and phosphodiesterase was not significant under high oxygen supply, suggesting efficient cAMP export might be important in cAMP production. These findings may contribute to further understanding of capacities of Arthrobacter species and would be highly useful in genetic regulation for desirable production.

The increasing demand for biocatalysts in synthesizing enantiomerically pure chiral alcohols results from the outstanding characteristics of enzymes in reaction, economic, ecological issues. Many carbonyl reductases for producing chiral alcohols have been reported but there is still a lack of good catalytic efficacies. Herein, five carbonyl reductases from different Streptomyces were discovered by the strategy of genome mining. These reductases were overexpressed, and we chose SgCR for further study as it owned better enzyme activity. This protein was purified to apparent homogeneity, and its amino acid sequence was analyzed in comparison with that of the reported SDRs. The biocatalytic properties of SgCR were investigated, and this enzyme was confirmed to have the ability to convert various prochiral ketones into highly optically active alcohols. SgCR exhibited the highest activity towards ethyl 4-chloro-3-oxobutanoate (COBE) and the corresponding product ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) was obtained with high yield and excellent e.e. value by optimizing the biphasic system. Eventually, using isopropanol as the co-substrate for NADH recycling in the substrate-coupled reaction, the yield and enantioselectivity of (S)-CHBE were obtained at the values of 90% and 99%, respectively. These results indicate that SgCR is a promising boicatalyst for the synthesis of chiral alcohols in industry.

Redox cofactors play an important role in biosynthetic and catabolic reactions and the transfer of energy for the cell. Therefore, studying the relationship between cofactor perturbation and metabolism is a useful approach to improve the yield of target products. To study RNA accumulation and metabolism when intracellular cofactor balance was impaired, the water-forming NADH oxidase (NoxE) from Lactococcus lactis and membrane-bound transhydrogenase (PntAB) from Escherichia coli were expressed in Candidatropicalis no. 121, respectively. Expression of noxE significantly decreased the intracellular NADH/NAD+ ratio, but the NADPH/NADP+ ratio did not differ significantly. PntAB increased the intracellular NADH pool, while the NADPH/NADP+ ratio decreased. The perturbation of the cofactors caused a large redistribution of metabolic fluxes. The biomass and RNA content decreased by 11.0% and 10.6% in pAUR-noxE strain, respectively, while the RNA content increased by 5.5% and the biomass showed no signification difference in pAUR-pntAB strain. Expression of noxE and pntAB led to decreases and increases in the ATP concentration and yield of RNA, respectively, which also indicated that ATP plays an important role in the RNA biosynthesis.

Butanol is an important industrial chemical and an attractive transportation fuel. However, the deficiency of reducing equivalents NAD(P)H in butanol fermentation results in a large quantity of oxidation products, which is a major problem limiting the atom economy and economic viability of bio-butanol processes. Here, we integrated the butanol fermentation process with a NADH-generating, acetoin biosynthesis process to improve the butanol production. By overexpressing the α-acetolactate decarboxylase gene alsD from Bacillus subtilis in Clostridium acetobutylicum , acetoin yield was significantly increased at the cost of acetone. After optimization of fermentation conditions, butanol (12.9 g/L), acetoin (6.5 g/L), and ethanol (1.9 g/L) were generated by the recombinant strain, with acetone no more than 1.8 g/L. Thus, both mass yield and product value were greatly improved. This study demonstrates that reducing power compensation is effective to improve the atom economy of butanol fermentation, and provides a novel approach to improve the economic viability of bio-butanol production.

In this study, the heterogeneous expression of the formate dehydrogenase gene for nicotinamide adenine dinucleotide (NADH) regeneration is successfully achieved in Clostridium ljungdahlii. The specific activity of formate dehydrogenase is dependent on cell growth, reaching its highest value (3.16 U mg−1 for the engineered strain, 1.59 U mg−1 for the parent strain) when cells entered the log phase. The NADH concentration in engineered C. ljungdahlii (12.64 μm per g DCW) increased 4.3 fold compared with the parent strain (2.93 μm per g DCW). The genetically engineered bacterium C. ljungdahlii is employed in a microbial fuel cell to gauge the potential for bioelectricity generation improvement. The voltage of a sodium formate fed microbial fuel cell with C. ljungdahlii is enhanced to 290 mV ± 10, which is 3.8 times the parent strain (76 ± 8 mV). The higher NADH pool in engineered C. ljungdahlii can facilitate electron transfer in the system, which contributes to an increase in maximum power density from 15 mW m−2 to 35 mW m−2. On the basis of these results, the manipulation of intracellular cofactors is shown to be an efficient approach to improve bioelectricity generation in the microbial fuel cell, and indicates the great potential of metabolic engineering for improvements in bioelectricity.

Biofilm-based immobilization of solventogenic Clostridia has been extensively exploited to overcome traditional bottlenecks in biobutanol production like solvent toxicity and low productivities. However, the molecular basis of solventogenic Clostridia biofilm is rarely explored. Here, for the first time, we report DNA array-based study of Clostridium acetobutylicum biofilm cells to elucidate the transcriptional modulation. Results showed that 16.2% of the C. acetobutylicum genome genes within the biofilm cells were differentially expressed, with most genes being up-regulated. The most dramatic changes occurred with amino acid biosynthesis, with sulfur uptake and cysteine biosynthesis being the most up-regulated and histidine biosynthesis being the most down-regulated in the biofilm cells. It was demonstrated that C. acetobutylicum biofilm cells increased metabolic activities probably by up-regulating iron and sulfur uptake and Fe-S cluster biosynthesis genes as well as glycolysis genes. Furthermore, genes involved in sporulation, granulose formation, extracellular polymer degradation, pentose catabolisms, and various other processes were also notably regulated, indicating that the biofilm mode of growth rendered the cells a distinct phenotype. This study provides valuable insights into the transcriptional regulation in C. acetobutylicum biofilm cells and should be highly useful for understanding and developing the biofilm-based processes.

Saccharomyces cerevisiae immobilization is commonly used for efficient ethanol fuel production in industry due to the relatively higher ethanol stress resistance of S. cerevisiae in biofilms relative to planktonic cells. The mechanisms of biofilm formation and stress resistance, however, remain ambiguous. By analyzing biofilm and planktonic cell transcriptomes, this study observed that MIG1 (encoding a transcription factor) expression in cells increases during the biofilm formation process. To identify the role of MIG1 in yeast biofilm formation and the ethanol resistance of these cells, MIG1 was deleted and complemented in S. cerevisiae 1308. Results showed the MIG1 deletion mutant strain demonstrated weaker biofilm formation ability both on fibers and plastic than the wild-type and these could be restored by expressing MIG1 in deletion mutant. To verify the ability of MIG1 to regulate the expression of FLO genes, which encode adhesions responsible for yeast biofilm formation, FLO gene transcription levels were measured via qRT-PCR. Relative to wild-type S. cerevisiae, the adhesion genes FLO1, 5, and 9 which also demonstrate increased expression in the transcriptome of yeast cells during biofilm formation, but not FLO11, were down-regulated in the MIG1 mutant strain. Additionally, the MIG1 mutant lost a majority of its flocculation ability, which depended on cell-cell adhesions and its slightly invasive growth ability, dependent on cell-substrate adhesion. Deleting FLO1, 5, and 9 decreased biofilm formation on plastics, suggesting these FLO genes contribute to the biofilm formation process alongside FLO11. Moreover, the ethanol tolerance of yeast decreased in the MIG1 deletion mutant as well as the FLO11 deletion mutant, resulting in reduced biofilm formation during fermentation. It remains possible that in the later period of fermentation, when ethanol has accumulated, an over-expression of the FLO1, 5, and 9 genes regulated by MIG1 would enhanced cell-cell adhesions and thus protect cells in the outer layer of biofilms from ethanol, a function primarily dependent on cell-cell adhesions. This work offers a possible explanation for how biofilm formation is regulated during the immobilized fermentation process, and can enhance environmental tolerance in industrial production.

Redox homeostasis is essential to the maintenance of cell metabolism. Changes in the redox state cause global metabolic and transcriptional changes. Our previous study indicated that the overexpression of NADH oxidase in Saccharomyces cerevisiae led to increased glucose consumption and ethanol production. Gene expression related to thiamine synthesis and osmotolerance as well as HAP4 expression was increased in response to redox change caused by the overexpression of NADH oxidase. To identify detailed relationships among cofactor levels, thiamine synthesis, expression of HAP4, and osmotolerance, and to determine whether these changes are interdependent, THI4 and HAP4 were overexpressed in S. cerevisiae BY4741. The glucose consumption rate of THI4-overexpressing strain (thi4-OE) was the highest, followed by HAP4-overexpressing strain (hap4-OE) > NADH oxidase-overexpressing strain (nox-OE) > control strain (con), while strain hap4-OE showed the highest concentration of ethanol after 26 h of fermentation. Reduced glycerol production and increased osmotolerance were observed in thi4-OE and hap4-OE, as well as in nox-OE. HAP4 globally regulated thiamine synthesis, biomass synthesis, respiration, and osmotolerance of cells, which conferred the recombinant strain hap4-OE with faster glucose metabolism and enhanced stress resistance. Moreover, overexpression of HAP4 might extend the life span of cells under caloric restriction by lowering the NADH level. Although overexpression of THI4 and HAP4 induced various similar changes at both the metabolic and the transcriptional level, the regulatory effect of THI4 was more limited than that of HAP4, and was restricted to the growth phase of cells. Our findings are expected to benefit the bio-ethanol industry.

Available molecular and genetic tools for the genetic manipulation of Arthrobacter species are limited until now. In gene engineering, a continuous set of promoters with various strengths are of importance for fine-tuning gene expression in metabolic optimization and control analysis. Here, for the first time, we constructed a promoter trap system using green fluorescence protein (GFP) as a reporter, for screening and characterizing functional Arthrobacter promoters. Twenty-three Arthrobacter transformants of various GFP fluorescence strengths were isolated and characterized through the analysis of DNA sequences. Among the 23 putative promoters, 2 were selected for deletion analysis of promoter elements. As a result, the deletion of the upstream of the putative promoter P8 and P13 caused a 43.8% decrease and a 29.1% increase in the fluorescence signals, respectively. Finally, we obtained the strongest promoter P13-3 which was 4.4 times more potent than the promoter of 6-hydroxyl-D-nicotine oxidase gene which was previously reported in Arthrobacter nicotinovorans, and the obtained promoter was used to improve the production of cyclic adenosine monophosphate in Arthrobacter sp. CGMCC 3584. The screening strategy together with obtained promoters in this study would contribute to the future engineering of Arthrobacter species.

A mutant strain of Clostridium beijerinckii, with high tolerance to ferulic acid, was generated using atmospheric pressure glow discharge and high-throughput screening of C. beijerinckii NCIMB 8052. The mutant strain M11 produced 7.24 g/L of butanol when grown in P2 medium containing 30 g/L of glucose and 0.5 g/L of ferulic acid, which is comparable to the production from non-ferulic acid cultures (8.11 g/L of butanol). When 0.8 g/L of ferulic acid was introduced into the P2 medium, C. beijerinckii M11 grew well and produced 4.91 g/L of butanol. Both cell growth and butanol production of C. beijerinckii M11 were seriously inhibited when 0.9 g/L of ferulic acid was added into the P2 medium. Furthermore, C. beijerinckii M11 could produce 6.13 g/L of butanol using non-detoxified hemicellulosic hydrolysate from diluted sulfuric acid-treated corn fiber (SAHHC) as the carbon source. These results demonstrate that C. beijerinckii M11 has a high ferulic acid tolerance and is able to use non-detoxified SAHHC for butanol production.

Biobutanol is a potential fuel substitute and has been receiving increased attention in recent years. However, the economics of biobutanol production have been hampered by a number of bottlenecks such as high cost of raw material and low yield of solvent. Co-production of value-added products is a possible way to improve the economics of biobutanol production. Here, we present metabolic engineering strategies to substitute the major by-product acetone for a value-added product acetoin during butanol fermentation. By overexpressing the α-acetolactate decarboxylase gene alsD in Clostridium acetobutylicum B3, the acetoin yield was markedly increased while acetone formation was reduced. Subsequent disruption of adc gene effectively abolished acetone formation and further increased acetoin yield. After optimization of fermentation conditions, the alsD-overexpressing adc mutant generated butanol (13.8g/L), acetoin (4.3g/L), and ethanol (3.9g/L), but no acetone. Thus, acetone was completely substituted for acetoin, and both mass yield and product value were improved. This study provides valuable insights into the regulation of acetoin synthesis and should be highly useful for the development of acetoin-derived products like 2,3-butanediol and 2-butanol in C. acetobutylicum.

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 (

Clostridium beijerinckii 4693:int with high ferulic acid (FA) tolerance was engineered and characterized in our lab. In this study, the minimum inhibition concentrations of FA against C. beijerinckii NCIMB 8052 (wild-type) and 4693:int were 1.0 and 1.5g/l, respectively; cell viability was 18.5% and 106.7%, respectively, in the presence of 0.5g/l FA. A comparative transcriptome analysis was carried out at two different growth stages to evaluate sensitivity to FA. Genes that were differentially expressed included those related to redox and associated cofactors, riboflavin metabolism, two-component system, glycolysis and butanoate metabolism, and DNA replication as well as those encoding ATP-binding cassette transporters. Cbei_2134 and Cbei_2135 encoding alkyl hydroperoxide reductases are thought to be involved in antibacterial and adaptation mechanisms in C. beijerinckii in the presence of FA.

The objective of this research was to understand how the initial glucose concentration influences adenosine (AR) production and metabolic flux shift on the cultivation of Bacillus subtilis CGMCC 4484. Experiments confirmed that initial glucose concentration affects cell growth, AR production and metabolites, significantly. The flux distribution at the key nodes of glucose-6-phosphate (G6P), pyruvate (PYR) and acetyl coenzyme-A (AcCoA) could be affected by changing the glucose concentration. Based on kinetic analysis of specific rates, the low-glucose concentration was better for both cell growth and AR production during the first 12 h. However, the high-glucose concentration was more favorable for AR formation after 18 h. Furthermore, different simplified feeding strategies were designed to achieve higher AR accumulation. The final AR concentration of 15.60 g L(-1) was achieved when an optimized constant-feeding strategy was used, which was 21.02 % higher than batch fermentation. This was the first time to investigate the regulation of the glucose metabolism of AR-producing B. subtilis.

Pretreated cotton towels were used as carriers to immobilize Clostridium acetobutylicum CGMCC 5234 cells for butanol or ABE production from glucose and xylose. Results showed that cell immobilization was a promising method to increase butanol concentration, yield and productivity regardless of the sugar sources compared with cell suspension. In this study, a high butanol concentration of 10.02 g/L with a yield of 0.20 g/g was obtained from 60 g/L xylose with 9.9 g/L residual xylose using immobilized cells compared with 8.48 g/L butanol and a yield of 0.141 g/g with 20.2 g/L residual xylose from 60 g/L xylose using suspended cells. In mixed-sugar fermentation (30 g/L glucose plus 30 g/L xylose), the immobilized cultures produced 11.1 g/L butanol with a yield of 0.190 g/g, which were 28.3% higher than with suspended cells (8.65 g/L) during which 30 g/L glucose was utilized completely using both immobilized and suspended cells while 3.46 and 13.1 g/L xylose maintained untilized for immobilized and suspended cells, respectively. Based on the results, we speculated that immobilized cells showed enhanced tolerance to butanol toxicity and the cultures preferred glucose to xylose during ABE fermentation. Moreover, the cultures showed obvious difference when grown between high initial concentrations of glucose and those of xylose. Repeated-batch fermentations from glucose with immobilized cells showed better long-term stability than from xylose. At last, the morphologies of free and immobilized cells adsorbed on pretreated cotton towels during the growth cycle were examined by SEM.

Based on thermal asymmetric interlaced polymerase chain reaction, the arpde gene encoding a cyclic nucleotide-specific phosphodiesterase was cloned from Arthrobacter sp. CGMCC 3584 for the first time. The 930-bp region encoded a 309-amino-acid protein with a molecular weight of 33.6 kDa. The recombinant ArPDE was able to hydrolyze 3',5'-cAMP, 3',5'-cGMP, and 2',3'-cAMP. The K m values of ArPDE for 3',5'-cAMP and 3',5'-cGMP were 6.82 and 12.82 mM, respectively. ArPDE was thermostable and displayed optimal activity at 45 °C and pH 7.5. The enzyme did not require any metal cofactors, although its activity was stimulated by 2 mM Co(2+) and inhibited by Zn(2+). Nucleotides, reducing agents, and sulfhydryl reagents had different inhibitory effects on the activity of ArPDE. NaF, the actual compound used to improve the industrial yield of cAMP, exhibited 62 % inhibitions at concentrations of 10 mM.

Redox homeostasis is fundamental to the maintenance of metabolism. Redox imbalance can cause oxidative stress, which affects metabolism and growth. Water-forming NADH oxidase regulates the redox balance by oxidizing cytosolic NADH to NAD(+), which relieves cytosolic NADH accumulation through rapid glucose consumption in Saccharomyces cerevisiae, thus decreasing the production of the by product glycerol in industrial ethanol production. Here, we studied the effects of overexpression of a water-forming NADH oxidase from Lactococcus lactis on the stress response of S. cerevisiae in aerobic batch fermentation, and we constructed an interaction network of transcriptional regulation and metabolic networks to study the effects of and mechanisms underlying NADH oxidase regulation. The oxidase-overexpressing strain (NOX) showed increased glucose consumption, growth, and ethanol production, while glycerol production was remarkably lower. Glucose was exhausted by NOX at 26 h, while 18.92 ± 0.94 g/L residual glucose was left in the fermentation broth of the control strain (CON) at this time point. At 29.5 h, the ethanol concentration for NOX peaked at 35.25 ± 1.76 g/L, which was 14.37% higher than that for CON (30.82 ± 1.54 g/L). Gene expression involved in the synthesis of thiamine, which is associated with stress responses in various organisms, was increased in NOX. The transcription factor HAP4 was significantly upregulated in NOX at the late-exponential phase, indicating a diauxic shift in response to starvation. The apoptosis-inducing factor Nuc1 was downregulated while the transcription factor Sok2, which regulates the production of the small signaling molecule ammonia, was upregulated at the late-exponential phase, benefiting young cells on the rim. Reactive oxygen species production was decreased by 10% in NOX, supporting a decrease in apoptosis. The HOG pathway was not activated, although the osmotic stress was truly higher, indicating improved osmotolerance. Thus, the NADH oxidase can regulate the metabolism during aerobic fermentation in S. cerevisiae, thereby protecting cells against several stresses. Our findings indicate its suitability for use in industrial processes.

Low D-ribose production by B. subtilis was resulted from plenty of acid formation as responsible for carbon overflow. An approach of co-feeding glucose and sodium citrate is developed here and proved to be useful for D-ribose production. This strategy is critical because it affects the cell concentration, the productivity of D-ribose and, especially, the formation of by-products such as acetoin, lactate, and acetate. D-ribose production was increased by 59.6% from 71.06 to 113.41 g l−1 without acid formation via co-feeding of 2.22 g l−1 h−1 glucose and 0.036 g l−1 h−1 sodium citrate to a 60 g l−1 glucose reaction system. Actually, the cell density was also enhanced from 11.51 to 13.84 g l−1. These parameters revealed the importance of optimization and modeling of the D-ribose production process. Not only could zero acid formation was achieved over a wide range of co-feeding rate by reducing glycolytic flux drastically, but also the cell density and D-ribose yield were elevated by increasing the hexose monophosphate pathway flux. This article is protected by copyright. All rights reserved.

Summary Immobilized fermentation has several advantages over traditional suspended fermentation, including simple and continuous operation, improved fermentation performance and reduced cost. Carrier is the most adjustable element among three elements of immobilized fermentation, including carrier, bacteria and environment. In this study, we characterized carrier roughness and surface properties of four types of natural fibres, including linen, cotton, bamboo fibre and silk, to assess their effects on cell immobilization, fermentation performance and stability. Linen with higher specific surface area and roughness could adsorb more bacteria during immobilized fermentation, thereby improving fermentation performance; thus, linen was selected as a suitable carrier and was applied for acetone–butanol–ethanol (ABE) fermentation. To further improve fermentation performance, we also found that microbes of Clostridium acetobutylicum were negatively charged surfaces during fermentation. Therefore, we then modified linen with polyetherimide (PEI) and steric acid (SA) to increase surface positive charge and improve surface property. During ABE fermentation, the adhesion between modified linen and bacteria was increased, adsorption was increased about twofold compared with that of unmodified linen, and butanol productivity was increased 8.16% and 6.80% with PEI‐ and SA‐modified linen as carriers respectively.

A mutant strain of Clostridium beijerinckii NCIMB 8052, C. beijerinckii M11, which exhibited ferulic acid tolerance up to 0.9g/L, was generated using atmospheric pressure glow discharge and high-throughput screening. Comparative genomic analysis revealed that this strain harbored a mutation of the Cbei_4693 gene, which encodes a hypothetical protein suspected to be an NADPH-dependent FMN reductase. After disrupting the Cbei_4693 gene in C. beijerinckii NCIMB 8052 using the ClosTron group II intron-based gene inactivation system, we obtained the Cbei_4693 gene inactivated mutant strain, C. beijerinckii 4693::int. Compared with C. beijerinckii NCIMB 8052, 6.23g/L of butanol was produced in P2 medium containing 0.5g/L of ferulic acid by 4693::int, and the ferulic acid tolerance was also significantly increased up to 0.8g/L. These data showed, for the first time, that the Cbei_4693 gene plays an important role in regulating ferulic acid tolerance in ABE fermentation by C. beijerinckii.

Most reductases which belong to the short chain dehydrogenase/reductase (SDR) superfamily require NAD (P) H for activity. Addition of this cofactor was still necessary for the production of ethyl (S)-4-chloro-3-hydroxybutanoate by Escherichia coli even when a cofactor regeneration system was constructed by co-expressing carbonyl reductase from Pichia stipitis (PsCRI) and glucose dehydrogenase from Bacillus megaterium (BmGDH). In an attempt to reduce dependence on the expensive cofactor, compounds directly or indirectly involved in NADP synthesis were added to the medium. Only glutamine and xylose enhanced the content of intracellular NADP (H) and the concentration of product. The concentration and yield of (S)-CHBE reached 730 mM and 48.7%, with 30 g/L of glutamine and 40 g/L of xylose, a 2.6-fold increase over the control without the addition of the two compounds.

Experiments confirmed dissolved oxygen (DO) definitely affects cyclic adenosine monophosphate (cAMP) production by Arthrobacter A302. Production of cAMP by batch fermentation was investigated under various DO conditions. A two-stage DO control strategy was proposed to achieve optimal production of cAMP based on the kinetic analysis: the DO level was controlled at 40% during the first 18 h and then switched to 30%. Relatively high cAMP production (9.9 g L(-1)) was achieved by applying this strategy. The cAMP productivity (0.14 g L(-1) h(-1)) was also successfully improved by 85.1, 59.3, 15.1 and 28.0%, compared to cases in which DO was uncontrolled or DO levels were held at 20, 30 and 40%, respectively. This is the first report of the use of a two-stage DO control strategy in cAMP production, and it was verified to be an effective method for enhancing the cAMP yield via this strain.

The cya gene encoding adenylate cyclase was cloned from Arthrobacter sp. CGMCC 3584 by thermal asymmetric interlaced PCR for the first time. It exhibited an open reading frame containing 1,125 bp and encoding 374 amino acids. Amino acid sequence analysis showed that this enzyme was a class III adenylate cyclase. Expression of the cya gene was carried out in Escherichia coli Rosetta, and purification was performed via Ni2+-NTA agarose gel column. SDS-PAGE indicated that the molecular mass of the recombinant adenylate cyclase was 45 kDa. The V max and K m were determined to be 5.06 μmol/min/mg and 7.56 mM, respectively. The optimum pH and temperature were 8.0 and 35 °C. Several divalent metal ions were found to activate the enzyme to different extents, and the maximal specific activity reached 3.04 μmol/min/mg when 50 mM Mg2+ was added. This was the first report of the cloning of an adenylate cyclase gene from Arthrobacter sp.

A whole-cell biocatalytic process for uridine 5'-monophosphate (UMP) production from orotic acid by Saccharomyces cerevisiae was developed. To rationally redistribute the metabolic flux between glycolysis and pentose phosphate pathway, statistical methods were employed first to find out the critical factors in the process. NaH(2)PO(4), MgCl(2) and pH were found to be the important factors affecting UMP production significantly. The levels of these three factors required for the maximum production of UMP were determined: NaH(2)PO(4) 22.1 g/L; MgCl(2) 2.55 g/L; pH 8.15. An enhancement of UMP production from 6.12 to 8.13 g/L was achieved. A significant redistribution of metabolic fluxes was observed and the underlying mechanism was discussed.

The experiments were based on multivariate statistical concepts, and response surface methodology (RSM) was applied to optimize the fermentation medium for the production of ribonucleic acid (RNA) by Candida tropicalis no. 121. The process involved the individual adjustment and optimization of various medium components at shake flask level. The two-level Plackett-Burman (PB) design was used to screen the medium components, which significantly influenced RNA production. Among seven variables, the concentrations of molasses, ZnSO4, and H3PO4 were found to be the important factors that significantly affected RNA production (confidence levels above 95%). These factors were further optimized using a central composite design (CCD) and RSM. The optimum values for the critical components were as follows: molasses 47.21 g/L: ZnSO4 0.048 g/L; H3PO4 1.19 g/L. Under optimal conditions, RNA production was 2.56 g/L, which was in excellent agreement with the predicted value (2.561 g/L), and led to a 2.1-fold increase compare with that using the original medium in RNA production.

A glucose utilizing strain, Arthrobacter A302 was used for cyclic adenosine monophosphate (cAMP) production in batch modes. The non-structured model in a 5 l stirred tank bioreactor for understanding, controlling, and optimizing the fermentation process was proposed using the logistic equation for microbial growth, the Luedeking-Piret equation for product formation and Luedeking-Piret-like equation for substrate uptake, respectively. The production of cAMP was a mixed-growth-associated pattern. Based on model prediction, a comparison of calculated value using the parameters evaluated above with another experimental data in 30 l bioreactor was used to test the model. The results predicted from the model were in good agreement with the experimental observations in 30 l bioreactor, which demonstrated that the model might be useful for the development and optimization of production of cAMP in industrial scale. Based on estimated kinetic parameters, three different fed-batch modes, constant rate and intermittent (once and repeated), were adopted in order to obtain more cAMP accumulation. Furthermore, the final production of cAMP reached 11.24 g l−1 after 72 h incubation using three stages feeding strategy. In particular, the cAMP productivity (0.156 g l−1 h−1) was successfully improved by 22.83, 11.43 and 9.86%, respectively, compared with the modes of the batch, constant rate fed-batch and intermittent fed-batch once.

Attempts were made with success to produce uridine 5′-monophosphate (UMP) from orotic acid by a recombinant Saccharomyces cerevisiae strain pYX212-URA5/BJX12, using the whole cell biocatalytic process. URA5 and URA3 genes, encoding orotate phosphoribosytransferase (OPRTase) and orotidine monophosphate decarboxylase (ODCase), respectively were successfully overexpressed in the industrial yeast strain. As a result, S .cerevisae pYX212-URA5/BJX12 exhibited the highest biocatalytic ability, in contrast with the original industrial yeast strain and S. cerevisae pYX212/BJX12 that overexpressed ODCase only. It indicated that the first step of UMP production from orotic acid is a rate-limiting step. Effects of cultivation for the recombinant strain and biocatalytic reaction conditions on UMP production were also investigated. Cultivating the cells in malt extract medium for 36 h in the exponential phase of growth is in favor of converting orotic acid to UMP. To acquire a higher UMP yield, the conditions of the whole cell biocatalytic reaction were optimized and up to 3.8 g l−1 UMP was produced in 24 h consequently. The yield was fivefold higher than the original UMP yield from the industrial yeast. In addition, the accumulation of 2.6 g l−1 UDP (uridne 5′-diphosphate) in the process demonstrated the possibility for further genetic manipulation: deleting the UMPK (Uridylate Kinase, catalyzing UMP–UDP).

The enzymatic hydrolysate of spent yeast cells was evaluated as a nitrogen source for succinic acid production by Actinobacillus succinogenes NJ113, using corn fiber hydrolysate as a carbon source. When spent yeast cell hydrolysate was used directly as a nitrogen source, a maximum succinic acid concentration of 35.5 g/l was obtained from a glucose concentration of 50 g/l, with a glucose utilization of 95.2%. Supplementation with individual vitamins showed that biotin was the most likely factor to be limiting for succinic acid production with spent yeast cell hydrolysate. After supplementing spent yeast cell hydrolysate and 90 g/l of glucose with 150 μg/l of biotin, cell growth increased 32.5%, glucose utilization increased 37.6%, and succinic acid concentration was enhanced 49.0%. As a result, when biotin-supplemented spent yeast cell hydrolysate was used with corn fiber hydrolysate, a succinic acid yield of 67.7% was obtained from 70.3 g/l of total sugar concentration, with a productivity of 0.63 g/(l h). Our results suggest that biotin-supplemented spent yeast cell hydrolysate may be an alternative nitrogen source for the efficient production of succinic acid by A. succinogenes NJ113, using renewable resources.

A novel NADH-dependent dehydrogenases/reductases (SDRs) superfamily reductase (PsCRII) was isolated from Pichia stipitis. It produced ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE] in greater than 99% enantiomeric excess. This enzyme was purified to homogeneity by ammonium sulfate precipitation followed by Q-Sepharose chromatography. Compared to similar known reductases producing (S)-CHBE, PsCR II was more suitable for production since the purified PsCRII preferred the inexpensive cofactor NADH to NADPH as the electron donor. Furthermore, the Km of PsCRII for ethyl 4-chloro-3-oxobutanoate (COBE) was 3.3 mM, and the corresponding Vmax was 224 μmol/mg protein/min. The catalytic efficiency is the highest value ever reported for NADH-dependent reductases from yeasts that produce CHBE with high enantioselectivity. In addition, this enzyme exhibited broad substrate specificity for several β-keto esters using NADH as the coenzyme. The properties of PsCRII with those of other carbonyl reductases from yeasts were also compared in this study.

Escherichia coli AFP111, a pflB, ldhA, ptsG triple mutant of E. coli W1485, can be recovered for additional succinate production in fresh medium after two-stage fermentation (an aerobic growth stage followed by an anaerobic production stage). However, the specific productivity is lower than that of two-stage fermentation. In this study, three strategies were compared for reusing the cells. It was found when cells were aerobically cultivated at the end of two-stage fermentation without supplementing any carbon source, metabolites (mainly succinate and acetate) could be consumed. As a result, enzyme activities involved in the reductive arm of tricarboxylic acid cycle and the glyoxylate shunt were enhanced, yielding a succinate specific productivity above g⁻¹(DCW)h⁻¹ and a mass yield above 0.90 g g⁻¹ in the subsequent anaerobic fermentation. In addition, the intracellular NADH of cells subjected to aerobic cultivation with metabolites increased by more than 3.6 times and the ratio of NADH to NAD+ increased from 0.4 to 1.3, which were both favorable for driving the TCA branch to succinate.

Statistical experimental designs were used to optimize the composition of culture media for the production of D-ribose by Bacillus subtilis. A fractional factorial design 2 (5-2) was used to determine medium components that sig- nificantly affected D-ribose production. The concentrations of glucose and (NH4)2SO4 were the significant factors. Cen- tral composite design and response surface methodology were then used to estimate the quadratic response surface and determine the factor levels for maximum production of D-ribose. Finally, the optimal medium composition was obtained (g/L): glucose, 172.75; (NH4) 2SO4, 13.2; yeast powder, 4; corn steep liquor, 8 and MnSO4, 0.5. This optimization strategy increased D-ribose production from 73.21 g/L to 88.57 g/L, an increase of 22% compared with the original conditions. The D-ribose production yield to glucose concentration was also enhanced from 0.37 g/g to 0.52 g/g. Con- firmatory experiments were also performed to demonstrate the accuracy of the model. Under the optimal medium using ammonia to control pH in a 5 L fermenter, the D-ribose yield was increased to 95.28 g/L after 3 days of cultivation at 37 o C.

Biocatalysis of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl ( S )-4-chloro-3-hydroxybutanoate [( S )-CHBE] was carried out using Escherichia coli co-expressing a carbonyl reductase gene from Pichia stipitis and a glucose dehydrogenase gene from Bacillus megaterium . An efficient polycistronic plasmid with a high-level of enzyme co-expression was constructed by changing the order of the genes, altering the Shine–Dalgarno (SD) regions, and aligned spacing (AS) between the SD sequence and the translation initiation codon. The optimal SD sequence was 5-TAAGGAGG-3, and the optimal AS distance was eight nucleotides. Asymmetric reduction of COBE to ( S )-CHBE with more than 99% enantiomeric excess was demonstrated by transformants, using a water/ethyl caprylate system. The recombinant cells produced 1260 mM product in the organic phase, and the total turnover number, defined as moles ( S )-CHBE formed per mole NADP + , was 12,600, which was more than 10-fold higher than in aqueous systems.