Enhanced production of itaconic acid from enzymatic hydrolysate of lignocellulosic biomass by recombinant Corynebacteriumglutamicum.

Itaconic acid (IA) is a value-added chemical currently produced by Aspergillus terreus from edible glucose and starch but not from inedible lignocellulosic biomass owing to the high sensitivity to fermentation inhibitors present in the hydrolysate of lignocellulosic biomass. To produce IA from lignocellulosic biomass, a gram-positive bacterium, Corynebacterium glutamicum, with a high tolerance to fermentation inhibitors was metabolically engineered to express a fusion protein composed of cis-aconitate decarboxylase from A. terreus responsible for IA formation from cis-aconitate and a maltose-binding protein (malE) from Escherichia coli. The codon-optimized cadA_malE gene was expressed in C. glutamicum ATCC 13032, and the resulting recombinant strain produced IA from glucose. IA concentration increased 4.7-fold by the deletion of the ldh gene encoding lactate dehydrogenase. With the Δldh strain HKC2029, an 18-fold higher IA production was observed from enzymatic hydrolysate of kraft pulp as a model lignocellulosic biomass than from glucose (6.15 and 0.34 g/L, respectively). The enzymatic hydrolysate of kraft pulp contained various potential fermentation inhibitors involved in furan aldehydes, benzaldehydes, benzoic acids, cinnamic acid derivatives, and aliphatic acid. Whereas cinnamic acid derivatives severely inhibited IA production, furan aldehydes, benzoic acids, and aliphatic acid improved IA production at low concentrations. The present study suggests that lignocellulosic hydrolysate contains various potential fermentation inhibitors; however, some of them can serve as enhancers for microbial fermentation likely due to the changing of redox balance in the cell.

Physiological characteristics of Corynebacterium glutamicum as a cell factory under anaerobic conditions.

Corynebacterium glutamicum, a gram-positive and facultative anaerobic bacterium, is widely used for the industrial production of amino acids, such as L-glutamate and L-lysine. C. glutamicum grows and produces amino acids under aerobic conditions. When restricted under anaerobic conditions, it produces organic acids, such as L-lactate and succinate, through metabolic shift. With the increasing threat of global warming, these organic acids have drawn considerable attention as bio-based plastic monomers. In addition to the organic acids, the anaerobic bioprocess is also used to produce other value-added compounds, including isobutanol, ethanol, 3-methyl-1-butanol, 2,3-butanediol, L-alanine, and L-valine. Therefore, C. glutamicum is now a versatile cell factory for producing a wide variety of useful chemicals under both aerobic and anaerobic conditions. The growth and metabolism of the bacterium depend on the oxygen levels, which modulate the rearrangement of the carbon flux by reprogramming gene expression patterns and intracellular redox states. Anaerobic cell growth and L-lysine production as well as aerobic succinate production have been demonstrated by engineering the metabolic pathways or supplying a terminal electron acceptor instead of oxygen. In this review, we discuss the physiological and metabolic changes in C. glutamicum associated with its application as a cell factory under different oxygen states. Physiological switching in bacteria is initiated with the sensing of oxygen availability. While such a sensor has not been identified in C. glutamicum yet, the molecular mechanism for oxygen sensing in related bacteria is also discussed. KEY POINTS: • C. glutamicum produces a wide variety of useful compounds under anaerobic conditions. • C. glutamicum is a versatile cell factory under both aerobic and anaerobic conditions. • Metabolic fate can be overcome by engineering metabolic pathways.

Advances in metabolic engineering of Corynebacterium glutamicum to produce high-value active ingredients for food, feed, human health, and well-being.

The soil microbe Corynebacterium glutamicum is a leading workhorse in industrial biotechnology and has become famous for its power to synthetise amino acids and a range of bulk chemicals at high titre and yield. The product portfolio of the microbe is continuously expanding. Moreover, metabolically engineered strains of C. glutamicum produce more than 30 high value active ingredients, including signature molecules of raspberry, savoury, and orange flavours, sun blockers, anti-ageing sugars, and polymers for regenerative medicine. Herein, we highlight recent advances in engineering of the microbe into novel cell factories that overproduce these precious molecules from pioneering proofs-of-concept up to industrial productivity.

Recent progress in production of amino acid-derived chemicals using Corynebacterium glutamicum.

Green chemical production by microbial processes is critical for the development of a sustainable society in the twenty-first century. Among the important industrial microorganisms, the gram-positive bacterium Corynebacterium glutamicum has been utilized for amino acid fermentation, which is one of the largest microbial-based industries. To date, several amino acids, including L-glutamic acid, L-lysine, and L-threonine, have been produced by C. glutamicum. The capability to produce substantial amounts of amino acids has gained immense attention because the amino acids can be used as a precursor to produce other high-value-added chemicals. Recent developments in metabolic engineering and synthetic biology technologies have enabled the extension of metabolic pathways from amino acids. The present review provides an overview of the recent progress in the microbial production of amino acid-derived bio-based monomers such as 1,4-diaminobutane, 1,5-diaminopentane, glutaric acid, 5-aminolevulinic acid, L-pipecolic acid, 4-amino-1-butanol, and 5-aminolevulinic acid, as well as building blocks for healthcare products and pharmaceuticals such as ectoine, L-theanine, and gamma-aminobutyric acid by metabolically engineered C. glutamicum.

Enhanced methane production from cellulose using a two-stage process involving a bioelectrochemical system and a fixed film reactor.

BACKGROUND: It is desirable to improve the anaerobic digestion processes of recalcitrant materials, such as cellulose. Enhancement of methane (CH4) production from organic molecules was previously accomplished through coupling a bioelectrochemical system (BES); however, scaling-up BES-based production is difficult. Here, we developed a two-stage process consisting of a BES using low-cost and low-reactive carbon sheets as the cathode and anode, and a fixed film reactor (FFR) containing conductive material, i.e., carbon fiber textiles (CFTs) (:BES → FFR). By controlling the cathodic current at 2.7 µA/cm2 without abiotic H2 production, the three-electrode BES system was operated to mimic a microbial electrolysis cell. RESULTS: The thermophilic BES (inlet pH: 6.1) and FFR (inlet pH: 7.5) were operated using hydraulic retention times (HRTs) of 2.5 and 4.2 days, respectively, corresponding to a cellulose load of 3555.6 mg-carbon (C)/(L day). The BES → FFR process achieved a higher CH4 yield (37.5%) with 52.8 vol% CH4 in the product gas compared to the non-bioelectrochemical system (NBES) → FFR process, which showed a CH4 yield of 22.1% with 46.8 vol% CH4. The CH4 production rate (67.5 mM/day) obtained with the BER → FFR process was much higher than that obtained using electrochemical methanogenesis (0.27 mM/day). Application of the electrochemical system or CFTs improved the yields of CH4 with the NBES → FFR or BES → non-fixed film reactor process, respectively. Meta 16S rRNA sequencing revealed that putative cellulolytic bacteria (identified as Clostridium species) were present in the BES and NBES, and followed (BES→ and NBES→) FFR. Notably, H2-consuming methanogens, Methanobacterium sp. and Methanosarcina sp., showed increased relative abundances in the suspended fraction and attached fraction of (BES→) FFR, respectively, compared to that of (NBES→) FFR, although these methanogens were observed at trace levels in the BES and NBES. CONCLUSIONS: These results indicate that bioelectrochemical preprocessing at a low current effectively induces interspecies H2 transfer in the FFR with conductive material. Sufficient electrochemical preprocessing was observed using a relatively short HRT. This type of two-stage process, BES → FFR, is useful for stabilization and improvement of the biogas (CH4) production from cellulosic material, and our results imply that the two-stage system developed here may be useful with other recalcitrant materials.

Elevated, non-proliferative temperatures change the profile of fermentation products in Corynebacterium glutamicum.

Although temperature is a crucial factor affecting enzymatic activity on biochemical and biofuel production, the reaction temperature for the generation of these products is usually set at the optimal growth temperature of the host strain, even under non-proliferative conditions. Given that the production of fermentation products only requires a fraction of the cell's metabolic pathways, the optimal temperatures for microbial growth and the fermentative production of a target compound may be different. Here, we investigated the effect of temperature on lactic and succinic acids production, and related enzymatic activities, in wild-type and succinic acid-overproducing strains of Corynebacterium glutamicum. Interestingly, fermentative production of lactic acid increased with the temperature in wild-type: production was 69% higher at 42.5 °C, a temperature that exceeded the upper limit for growth, than that at the optimal growth temperature (30 °C). Conversely, succinic acid production was decreased by 13% under the same conditions in wild-type. The specific activity of phosphoenolpyruvate carboxylase decreased with the increase in temperature. In contrast, the other glycolytic and reductive TCA cycle enzymes demonstrated increased or constant activity as the temperature was increased. When using a succinic acid over-producing strain, succinic acid production was increased by 34% at 42.5 °C. Our findings demonstrate that the profile of fermentation products is dependent upon temperature, which could be caused by the modulation of enzymatic activities. Moreover, we report that elevated temperatures, exceeding the upper limit for cell growth, can be used to increase the production of target compounds in C. glutamicum. KEY POINTS: • Lactate productivity was increased by temperature elevation. • Succinate productivity was increased by temperature elevation when lactate pathway was deleted. • Specific activity of phosphoenolpyruvate carboxylase was decreased by temperature elevation.

Optimal Ratio of Carbon Flux between Glycolysis and the Pentose Phosphate Pathway for Amino Acid Accumulation in Corynebacterium glutamicum.

Glucose is metabolized through central metabolic pathways such as glycolysis and the pentose phosphate pathway (PPP) to synthesize downstream metabolites including amino acids. However, how the split ratio of carbon flux between glycolysis and PPP specifically affects the formation of downstream metabolites remains largely unclear. Here, we conducted a comprehensive metabolomic analysis to investigate the effect of the split ratio between glycolysis and the PPP on the intracellular concentration of amino acids and their derivatives in Corynebacterium glutamicum. The split ratio was varied by exchanging the promoter of a gene encoding glucose 6-phosphate isomerase (PGI). The ratio was correlated with the pgi transcription level and the enzyme activity. Concentrations of threonine and lysine-derivative 1,5-diaminopentane increased with an increase of the split ratio into the PPP. In contrast, concentrations of alanine, leucine, and valine were increased with an increase of the split ratio into glycolysis. These results could provide a new engineering target for improving the production of the amino acids and the derivatives.

Anaerobic glucose consumption is accelerated at non-proliferating elevated temperatures through upregulation of a glucose transporter gene in Corynebacterium glutamicum.

Cell proliferation is achieved through numerous enzyme reactions. Temperature governs the activity of each enzyme, ultimately determining the optimal growth temperature. The synthesis of useful chemicals and fuels utilizes a fraction of available metabolic pathways, primarily central metabolic pathways including glycolysis and the tricarboxylic acid cycle. However, it remains unclear whether the optimal temperature for these pathways is correlated with that for cell proliferation. Here, we found that wild-type Corynebacterium glutamicum displayed increased glycolytic activity under non-growing anaerobic conditions at 42.5 °C, at which cells do not proliferate under aerobic conditions. At this temperature, glucose consumption was not inhibited and increased by 28% compared with that at the optimal growth temperature of 30 °C. Transcriptional analysis revealed that a gene encoding glucose transporter (iolT2) was upregulated by 12.3-fold compared with that at 30 °C, with concomitant upregulation of NCgl2954 encoding the iolT2-regulating transcription factor. Deletion of iolT2 decreased glucose consumption rate at 42.5 °C by 28%. Complementation of iolT2 restored glucose consumption rate, highlighting the involvement of iolT2 in the accelerating glucose consumption at an elevated temperature. This study shows that the optimal temperature for glucose metabolism in C. glutamicum under anaerobic conditions differs greatly from that for cell growth under aerobic conditions, being beyond the upper limit of the growth temperature. This is beneficial for fuel and chemical production not only in terms of increasing productivity but also for saving cooling costs. KEY POINTS: • C. glutamicum accelerated anaerobic glucose consumption at elevated temperature. • The optimal temperature for glucose consumption was above the upper limit for growth. • Gene expression involved in glucose transport was upregulated at elevated temperature. Graphical abstract.

Requirement of de novo synthesis of pyruvate carboxylase in long-term succinic acid production in Corynebacterium glutamicum.

Protein turnover through de novo synthesis is critical for sustainable cellular functions. We previously found that glucose consumption rate in Corynebacterium glutamicum under anaerobic conditions increased at temperature higher than the upper limit of growth temperature. Here, we showed that production of lactic and succinic acids increased at higher temperature for long-term (48 h) anaerobic reaction in metabolically engineered strains. At 42 °C, beyond the upper limit of growth temperature range, biomass-specific lactic acid production rate was 8% higher than that at 30 °C, the optimal growth temperature. In contrast, biomass-specific succinic acid production rate was highest at 36 °C, 28% higher than that at 30 °C, although the production at 42 °C was still 23% higher than that at 30 °C. As enzymes are usually unstable at high temperatures, we investigated whether protein turnover of metabolic enzymes is required for the production of lactic and succinic acids under these conditions. Interestingly, when de novo protein synthesis was inhibited by addition of chloramphenicol, after 6 h, only succinic acid production was inhibited. Because glycolytic enzymes are involved in both lactic and succinic acids synthesis, enzymes in the anaplerotic pathway and the tricarboxylic acid (TCA) cycle leading to succinic acid synthesis were likely to be responsible for its decreased production. Among the five enzymes examined, the specific activity of only pyruvate carboxylase was drastically decreased after 48 h at 42 °C. Thus, the de novo synthesis of pyruvate carboxylase is required for long-term production of succinic acid. Graphical abstract KEY POINTS: • Long-term reaction for organic acids can be improved at temperature beyond ideal growth conditions. • De novo synthesis of pyruvate carboxylase is required for long-term succinic acid production.

Automatic Redirection of Carbon Flux between Glycolysis and Pentose Phosphate Pathway Using an Oxygen-Responsive Metabolic Switch in Corynebacterium glutamicum.

Controlling the carbon flux into a desired pathway is important for improving product yield in metabolic engineering. After entering a cell, glucose is channeled into glycolysis and the pentose phosphate pathway (PPP), which decreases the yield of target products whose synthesis relies on NADPH as a cofactor. Here, we demonstrate redirection of carbon flux into PPP under aerobic conditions in Corynebacterium glutamicum, achieved by replacing the promoter of glucose 6-phosphate isomerase gene (pgi) with an anaerobic-specific promoter of the lactate dehydrogenase gene (ldhA). The promoter replacement increased the split ratio of carbon flux into PPP from 39 to 83% under aerobic conditions. The titer, yield, and production rate of 1,5-diaminopentane, whose synthesis requires NADPH as a cofactor, were increased by 4.6-, 4.4-, and 2.6-fold, respectively. This is the largest improvement in the production of 1,5-diaminopentane or its precursor, lysine, reported to date. After aerobic cell growth, pgi expression was automatically induced under anaerobic conditions, altering the carbon flux from PPP to glycolysis, to produce succinate in a single metabolically engineered strain. Such an automatic redirection of metabolic pathway using an oxygen-responsive switch enables two-stage fermentation for efficient production of two different compounds by a single strain, potentially reducing the production costs and time for practical applications.

Less biomass and intracellular glutamate in anodic biofilms lead to efficient electricity generation by microbial fuel cells.

BACKGROUND: Using a microbial fuel cell (MFC), we observed that a complex microbial community decomposed starch and transferred electrons to a graphite felt anode to generate current. In spite of the same reactor configuration, inoculum, substrate, temperature, and pH, MFCs produced different current and power density. To understand which factor(s) affected electricity generation, here, we analyzed a complex microbial community in an anodic biofilm and fermentation broth using Illumina MiSeq sequencing and metabolomics. RESULTS: Microbial biomass on the anode was lower in MFCs generating more electricity (0.09-0.16 mg cm-2-anode) than in those generating less electricity (0.60-2.80 mg cm-2-anode), while being equal (3890-4196 mg L-1-broth) in the fermentation broth over the same operational period. Chemical oxygen demand removal and acetate concentration were also similar in fermentation broths. MFCs generating more electricity had relatively more exoelectrogenic bacteria, such as Geobacter sp., but fewer acetate-utilizing Methanosarcina sp. and/or Lactococcus sp. in anodic biofilms. Accordingly, anodic biofilms generating more electricity presented higher levels of most intracellular metabolites related to the tricarboxylic acid cycle and a higher intracellular ATP/ADP ratio, but a lower intracellular NADH/NAD+ ratio. Moreover, the level of intracellular glutamate, an essential metabolite for microbial anabolic reactions, correlated negatively with current density. CONCLUSION: Microbial growth on the anode and intracellular glutamate levels negatively affect electricity generation by MFCs. Reduced formation of anodic biofilm, in which intracellular glutamate concentration is 33.9 µmol g-cell-1 or less, favors the growth of acetate-utilizing Geobacter sp. on the anode and improves current generation.

Metabolic engineering of Corynebacterium glutamicum for hyperproduction of polymer-grade L- and D-lactic acid.

Strain development is critical for microbial production of bio-based chemicals. The stereo-complex form of polylactic acid, a complex of poly-L- and poly-D-lactic acid, is a promising polymer candidate due to its high thermotolerance. Here, we developed Corynebacterium glutamicum strains producing high amounts of L- and D-lactic acid through intensive metabolic engineering. Chromosomal overexpression of genes encoding the glycolytic enzymes, glucokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, triosephosphate isomerase, and enolase, increased L- and D-lactic acid concentration by 146% and 56%, respectively. Chromosomal integration of two genes involved in the Entner-Doudoroff pathway (6-phosphogluconate dehydratase and 2-dehydro-3-deoxyphosphogluconate aldolase), together with a gene encoding glucose-6-phosphate dehydrogenase from Zymomonas mobilis, to bypass the carbon flow from glucose, further increased L- and D-lactic acid concentration by 11% and 44%, respectively. Finally, additional chromosomal overexpression of a gene encoding NADH dehydrogenase to modulate the redox balance resulted in the production of 212 g/L L-lactic acid with a 97.9% yield and 264 g/L D-lactic acid with a 95.0% yield. The optical purity of both L- and D-lactic acid was 99.9%. Because the constructed metabolically engineered strains were devoid of plasmids and antibiotic resistance genes and were cultivated in mineral salts medium, these strains could contribute to the cost-effective production of the stereo-complex form of polylactic acid in practical scale.

Enhanced production of d-lactate from mixed sugars in Corynebacterium glutamicum by overexpression of glycolytic genes encoding phosphofructokinase and triosephosphate isomerase.

The use of mixed sugars containing glucose and xylose in lignocellulosic biomass is desirable for the microbial production of chemicals and fuels. We investigated the effect of individual or simultaneous overexpression of glycolytic genes on d-lactate production from a mixture of glucose and xylose by a recombinant xylose-assimilating Corynebacterium glutamicum strain. The individual overexpression of genes encoding phosphofructokinase (PFK) and triosephosphate isomerase (TPI) increased d-lactate production rate by 71% and 34%, respectively, with corresponding increases (2.4- and 1.8-fold) in the glucose consumption; however, the amount of xylose consumed not altered. d-Lactate yield was also increased by 5.5%, but only in the strain overexpressing the gene encoding PFK. In the parent strain and the strains overexpressing the genes encoding PFK or TPI, a reduction in d-lactate production occurred at approximately 900 mM after 32 h. However, the strain that simultaneously overexpressed the genes encoding PFK and TPI continued to produce d-lactate after 32 h, with the eventual production of 1326 mM after production for 80 h in mineral salts medium. Our findings contribute to the cost-effective, large-scale production of d-lactate from mixed sugars.

Changes in the microbial consortium during dark hydrogen fermentation in a bioelectrochemical system increases methane production during a two-stage process.

BACKGROUND: Bioelectrochemical systems (BESs) are an innovative technology developed to influence conventional anaerobic digestion. We examined the feasibility of applying a BES to dark hydrogen fermentation and its effects on a two-stage fermentation process comprising hydrogen and methane production. The BES used low-cost, low-reactivity carbon sheets as the cathode and anode, and the cathodic potential was controlled at - 1.0 V (vs. Ag/AgCl) with a potentiostat. The operation used 10 g/L glucose as the major carbon source. RESULTS: The electric current density was low throughout (0.30-0.88 A/m2 per electrode corresponding to 0.5-1.5 mM/day of hydrogen production) and water electrolysis was prevented. At a hydraulic retention time of 2 days with a substrate pH of 6.5, the BES decreased gas production (hydrogen and carbon dioxide contents: 52.1 and 47.1%, respectively), compared to the non-bioelectrochemical system (NBES), although they had similar gas compositions. In addition, a methane fermenter (MF) was applied after the BES, which increased gas production (methane and carbon dioxide contents: 85.1 and 14.9%, respectively) compared to the case when the MF was applied after the NBES. Meta 16S rRNA sequencing revealed that the BES accelerated the growth of Ruminococcus sp. and Veillonellaceae sp. and decreased Clostridium sp. and Thermoanaerobacterium sp., resulting in increased propionate and ethanol generation and decreased butyrate generation; however, unknowingly, acetate generation was increased in the BES. CONCLUSIONS: The altered redox potential in the BES likely transformed the structure of the microbial consortium and metabolic pattern to increase methane production and decrease carbon dioxide production in the two-stage process. This study showed the utility of the BES to act on the microbial consortium, resulting in improved gas production from carbohydrate compounds.

Metabolic engineering of Corynebacterium glutamicum for production of sunscreen shinorine.

Ultraviolet-absorbing chemicals are useful in cosmetics and skin care to prevent UV-induced skin damage. We demonstrate here that heterologous production of shinorine, which shows broad absorption maxima in the UV-A and UV-B region. A shinorine producing Corynebacterium glutamicum strain was constructed by expressing four genes from Actinosynnema mirum DSM 43827, which are responsible for the biosynthesis of shinorine from sedoheptulose-7-phosphate in the pentose phosphate pathway. Deletion of transaldolase encoding gene improved shinorine production by 5.2-fold. Among the other genes in pentose phosphate pathway, overexpression of 6-phosphogluconate dehydrogenase encoding gene further increased shinorine production by 60% (19.1 mg/L). The genetic engineering of the pentose phosphate pathway in C. glutamicum improved shinorine production by 8.3-fold in total, and could be applied to produce the other chemicals derived from sedoheptulose-7-phosphate.

Application of microalgae hydrolysate as a fermentation medium for microbial production of 2-pyrone 4,6-dicarboxylic acid.

Actual biomass of microalgae was tested as a fermentation substrate for microbial production of 2-pyrone 4,6-dicarboxylic acid (PDC). Acid-hydrolyzed green microalgae Chlorella emersonii (algae hydrolysate) was diluted to adjust the glucose concentration to 2 g/L and supplemented with the nutrients of Luria-Bertani (LB) medium (tryptone 10 g/L and yeast extract 5 g/L). When the algae hydrolysate was used as a fermentation source for recombinant Escherichia coli producing PDC, 0.43 g/L PDC was produced with a yield of 20.1% (mol PDC/mol glucose), whereas 0.19 g/L PDC was produced with a yield of 8.6% when LB medium supplemented with glucose was used. To evaluate the potential of algae hydrolysate alone as a fermentation medium for E. coli growth and PDC production, the nutrients of LB medium were reduced from the algae hydrolysate medium. Interestingly, 0.17 g/L PDC was produced even without additional nutrient, which was comparable to the case using pure glucose medium with nutrients of LB medium. When using a high concentration of hydrolysate without additional nutrients, 1.22 g/L PDC was produced after a 24-h cultivation with the yield of 16.1%. Overall, C. emersonii has high potential as cost-effective fermentation substrate for the microbial production of PDC.

Lignocellulose nanofibers prepared by ionic liquid pretreatment and subsequent mechanical nanofibrillation of bagasse powder: Application to esterified bagasse/polypropylene composites.

In the present study, we examined the efficacy of choline acetate (ChOAc, a cholinium ionic liquid))-assisted pretreatment of bagasse powder for subsequent mechanical nanofibrillation to produce lignocellulose nanofibers. Bagasse sample with ChOAc pretreatment and subsequent nanofibrillation (ChOAc/NF-bagasse) was prepared and compared to untreated control bagasse sample (control bagasse), bagasse sample with nanofibrillation only (NF-bagasse) and with ChOAc pretreatment only (ChOAc-bagasse). The specific surface area was 0.83m2/g, 3.1m2/g, 6.3m2/g, and 32m2/g for the control bagasse, ChOAc-bagasse, NF-bagasse, and the ChOAc/NF-bagasse, respectively. Esterified bagasse/polypropylene composites were prepared using the bagasse samples. ChOAc/NF-bagasse exhibited the best dispersion in the composites. The tensile toughness of the composites was 0.52J/cm3, 0.73J/cm3, 0.92J/cm3, and 1.29J/cm3 for the composites prepared using control bagasse, ChOAc-bagasse, NF-bagasse, and ChOAc/NF-bagasse, respectively. Therefore, ChOAc pretreatment and subsequent nanofibrillation of bagasse powder resulted in enhanced tensile toughness of esterified bagasse/polypropylene composites.

Design of Wall-Destructive but Membrane-Compatible Solvents.

We report an extremely biocompatible solvent for plant cell walls based on a polar liquid zwitterion that dissolves cellulose, the most recalcitrant component of the plant cell walls. The polar liquid zwitterion does not affect the viability and activity of Escherichia coli, even at high concentrations. We demonstrate conversion of cell walls to ethanol via a starch-like process, namely successive dissolution, hydrolysis and fermentation in the same reaction pot.

1,5-Diaminopentane production from xylooligosaccharides using metabolically engineered Corynebacterium glutamicum displaying beta-xylosidase on the cell surface.

Xylooligosaccharide-assimilating Corynebacterium glutamicum strains were constructed using metabolic engineering and cell surface display techniques. First, C. glutamicum was metabolically engineered to create lysine-producing strains. Beta-xylosidase BSU17580 derived from Bacillus subtilis was then expressed on the C. glutamicum cell surface using PorH anchor protein, and enzymes involved in the xylose assimilation pathway were also expressed. Metabolic engineering had no effect on the activity of beta-xylosidase. The engineered strains efficiently consumed xylooligosaccharides and produced 12.4mM of lysine from 11.9g/L of xylooligosaccharides as the carbon source. Finally, co-expression of lysine decarboxylase enabled production of 11.6mM of 1,5-diaminopentane (cadaverine) from 13g/L of consumed xylooligosaccharides.

Sucrose purification and repeated ethanol production from sugars remaining in sweet sorghum juice subjected to a membrane separation process.

The juice from sweet sorghum cultivar SIL-05 (harvested at physiological maturity) was extracted, and the component sucrose and reducing sugars (such as glucose and fructose) were subjected to a membrane separation process to purify the sucrose for subsequent sugar refining and to obtain a feedstock for repeated bioethanol production. Nanofiltration (NF) of an ultrafiltration (UF) permeate using an NTR-7450 membrane (Nitto Denko Corporation, Osaka, Japan) concentrated the juice and produced a sucrose-rich fraction (143.2 g L-1 sucrose, 8.5 g L-1 glucose, and 4.5 g L-1 fructose). In addition, the above NF permeate was concentrated using an ESNA3 NF membrane to provide concentrated permeated sugars (227.9 g L-1) and capture various amino acids in the juice, enabling subsequent ethanol fermentation without the addition of an exogenous nitrogen source. Sequential batch fermentation using the ESNA3 membrane concentrate provided an ethanol titer and theoretical ethanol yield of 102.5-109.5 g L-1 and 84.4-89.6%, respectively, throughout the five-cycle batch fermentation by Saccharomyces cerevisiae BY4741. Our results demonstrate that a membrane process using UF and two types of NF membranes has the potential to allow sucrose purification and repeated bioethanol production.