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Sophorolipids (SLs) from Candida batistae has a unique structure that contains ω-hydroxy fatty acids, which can be used as a building block in the polymer and fragrance industries. To improve the production of this industrially important SLs, we optimized the culture medium of C. batistae for the first time. Using an optimized culture medium composed of 50 g/L glucose, 50 g/L rapeseed oil, 5 g/L ammonium nitrate and 5 g/L yeast extract, SLs were produced at a concentration of 24.1 g/L in a flask culture. Sophorolipids production increased by about 19% (28.6 g/L) in a fed-batch fermentation using a 5 L fermentor. Sophorolipids production more increased by about 121% (53.2 g/L), compared with that in a flask culture, in a fed-batch fermentation using a 50 L fermentor, which was about 787% higher than that of the previously reported SLs production (6 g/L). These results indicate that a significant increase in C. batistae-derived SLs production can be achieved by optimization of the culture medium composition and fed-batch fermentation. Finally, we successfully separated and purified the SLs from the culture medium. The improved production of SLs from C. batistae in this study will help facilitate the successful development of applications for the SLs.

In this study, we successfully purified a novel lactose-oxidizing enzyme in Pseudomonas taetrolens for the first time. The purified enzyme was identified as malate:quinone oxidoreductase (MQO, EC 1.1.5.4), which showed the malate-oxidizing activity converting malate into oxaloacetate. We characterized the enzymatic properties of this interesting MQO from P. taetrolens, such as the substrate specificity toward various saccharides and the effects of temperature, pH, and metal ions on the activity and stability of MQO. MQO exhibited unique substrate specificity, as it only oxidized disaccharides with reducing-end glucosyl residues, such as lactose, but not monosaccharides. Using the high oxidizing activity of MQO toward lactose, we successfully produced lactobionic acid (LBA), a valuable organic acid used in the cosmetic, food, and pharmaceutical industries, from lactose in Escherichia coli in which the quinoprotein glucose dehydrogenase gene was inactivated, the LBA nonproducing strain, by heterologously expressing MQO with pyrroloquinoline quinone. At 37 h cultivation in a 300 mL flask culture, the LBA production, yield, and productivity of the recombinant E. coli strain were 23 g/L, 100%, and 0.62 g/L/h, respectively. This study is the first to reveal the lactose-oxidizing activity of MQO, which could be used for producing LBA in heterologous bacteria.

This is the first study on improving lactobionic acid (LBA) production capacity in Pseudomonas taetrolens by genetic engineering. First, quinoprotein glucose dehydrogenase (GDH) was identified as the lactose-oxidizing enzyme of P. taetrolens. Of the two types of GDH genes in P. taetrolens, membrane-bound (GDH1) and soluble (GDH2), only GDH1 showed lactose-oxidizing activity. Next, the genetic tool system for P. taetrolens was developed based on the pDSK519 plasmid for the first time, and GDH1 gene was homologously expressed in P. taetrolens. Recombinant expression of the GDH1 gene enhanced intracellular lactose-oxidizing activity and LBA production of P. taetrolens in flask culture. In batch fermentation of the recombinant P. taetrolens using a 5 L bioreactor, the LBA productivity of the recombinant P. taetrolens was approximately 17% higher (8.70 g/(L h)) than that of the wild type (7.41 g/(L h)). The LBA productivity in this study is the highest ever reported using bacteria as production strains for LBA.

Five genes (mxbDM, mxcQE and mxaB) are responsible for the transcription of methanol oxidation genes in Methylobacterium strains. Among these, MxbDM and MxcQE constitute the two-component system (TCS) regulating methanol metabolism. In this study, we integrated the methanol-sensing domain of MxbD and MxcQ with the EnvZ/OmpR from Escherichia coli. The domain-swapping strategy resulted in chimeric histidine kinases (HK’s) MxbDZ and MxcQZ AM1 containing recombinant E. coli. Real-time quantitative PCR was used to monitor OmpC expression mediated by the chimeric HK and response regulator (RR) OmpR. Further, an ompC promoter based fluorescent biosensor for sensing methanol was developed. GFP fluorescence was studied both qualitatively and quantitatively in response to environmental methanol. GFP measurement also confirmed ompC expression. Maximum fluorescence was observed at 0.05% methanol and 0.01% methanol using MxbDZ and MxcQZ AM1, respectively. Thus the chimeric HK containing E. coli were found to be highly sensitive to methanol, resulting in a rapid response making them an ideal sensor.

Lactobionic acid (LBA) was produced by fermentation of Pseudomonas taetrolens. First, to increase the production of LBA by P. taetrolens, we controlled the pH of culture medium by CaCO3 addition (30 g/L) and then examined the initial lactose concentration ranging from 50 to 200 g/L and the growth temperature ranging from 20 to 37 °C. Both the LBA production titer (180 g/L) and the productivity (2.5 g/L h) were highest at 200 g/L lactose concentration and 25 °C of cell growth temperature in shake-flask culture. Although the production of LBA (178 g/L) was almost similar during the batch fermentation of P. taetrolens using 5 L bioreactor, the LBA productivity highly increased to 4.9 g/L h. The method using ethanol precipitation and ion-exchange chromatography was developed to recover the pure LBA from the fermentation broth. The optimum volume of ethanol and pH of culture medium for the precipitation of Ca2+ salt form of LBA were six volume of ethanol and pH 6.5, respectively. The cation-exchange resin T42 finally showed the best recovery yield (97.6%) of LBA from the culture supernatant. The production titer (178 g/L) and the productivity (4.9 g/L h) of lactobionic acid in this study were highest among the previous studies ever reported using P. taetrolens as a production strain of LBA.

To produce malic acid from non-oxidative pathway route in Escherichia coli using two key enzymes and synthetic scaffold complex. E. coli was engineered to produce malic acid from glucose by co-localization of two key enzymes phosphoenolpyruvate carboxylase (Ppc) and malate dehydrogenase (MdhA) with synthetic scaffold complex. Scaffold plasmid has produced the maximum concentration of 3.51 g/L malic acid from 10 g/L glucose in 48 h of culture. pH 5.5 and temperature 30°C were optimum for malic acid production without any engineering of competing metabolic pathways. E. coli mutant strains and different concentrations of glucose also tested. When 50 g/L glucose was used as substrate, 20.4 g/L of malic acid was produced.

Although L-Serine has wide applications and great market potential, its application is limited by its high cost and low yields. Microbial factories have many advantages for producing L-serine from renewable carbon resources. To achieve L-serine production with high efficiency, three enzymes (SerB, SerC, and EamA) were physically re-localized by using a scaffold system GBD:SH3:PDZ. Such strategy was highly effective in improving the production of L-serine in Escherichia coli. The highest concentration of L-serine harvested was at 3.8 g/l after 48 h in strain E. coli with the scaffold GBD:SH3:PDZ at ratio of 1:2:1, reaching a high yield of 0.24 mol/mol glucose.

The production of industrial chemicals from renewable biomass resources is a promising solution to overcome the society’s dependence on petroleum and to mitigate the pollution resulting from petroleum processing. Klebsiella pneumoniae is a nutritionally versatile bacterium with numerous native pathways for the production of well-known and industrially important platform chemicals derived from various sugars. Genomic sequence analyses have shown that the K. pneumoniae genome has a high similarity with that of Escherichia coli, the most studied organism, which is used in industrial biotechnology processes for fuel and chemical production. Hence, K. pneumoniae can be considered as a promising platform microorganism that can be metabolically engineered for the high-level production of bio-based chemicals. This review highlights the substrate metabolism and the metabolic engineering strategies developed in K. pneumoniae for the production of bio-based chemicals.

We investigated the effect of cell-surface display of glutamate decarboxylase (GadB) on gamma-aminobutyric acid (GABA) production in recombinant Escherichia coli. We integrated GadB from the hyperthermophilic, anaerobic archaeon Pyrococcus horikoshii to the C-terminus of the E. coli outer membrane protein C (OmpC). After 12 hr of culturing GadB-displaying cells, the GABA concentration in the extracellular medium increased to 3.2 g/l, which is eight times that obtained with cells expressing GadB in the cytosol. To further enhance GABA production, we increased the temperatures of the culture. At 60°C, the obtained GABA concentration was 4.62 g/l after 12 hr of culture, and 5.35 g/l after 24 hr, which corresponds to a yield of 87.7%.

In this study, we tested whether biodiesel could be used as a feedstock for the microbial synthesis of sophorolipid, a high value-added chemical. Sophorolipid was successfully produced from Starmerrella bombicola using biodiesel as a hydrophobic substrate. Fed-batch fermentation was carried out for the high-level production. Compared to flask culture, the sophorolipid production concentration significantly increased from 58.1 ​g/L to 224.2 ​g/L (an approximate increase of 386%) using fed-batch fermentation, which is the highest value ever reported using fatty acid methyl esters and fatty acid ethyl esters as hydrophobic substrates. Different from rapeseed oil-based sophorolipid (ROSL), the biodiesel-based sophorolipid (BDSL) contained a new type of sophorolipid called esterified sophorolipid (approximately 19.8%). The BDSL demonstrated better surface-active properties, lower surface tension (34.2 vs. 35.8 ​mN/m, respectively), and a decreased critical micelle concentration (25.1 vs. 26.3 ​mg/L, respectively) compared to the ROSL. Given these results, the BDSL is expected to be used in various industrial fields where vegetable oil-based sophorolipids, the commercialized forms of sophorolipids, have been used. To our knowledge, this is the first report to describe the conversion of biodiesel for the production of a high value-added chemical.

In this study, we successfully purified a novel lactose-oxidizing enzyme in

This is the first study on improving lactobionic acid (LBA) production capacity in

Lactobionic acid (LBA) has been widely used in the food, pharmaceutical, and cosmetic industries. Pseudomonas taetrolens is an efficient LBA-producing bacterium. To improve the LBA-production ability of P. taetrolens, we modified the strain by genetic engineering. We performed homologous expression of the quinoprotein glucose dehydrogenase gene in P. taetrolens and measured the intracellular lactose-oxidizing activity and LBA production titer. In flask cultures at 12 h of incubation, the intracellular lactose oxidizing activity (0.159 U/g dry weight cell) and LBA production titer (77.2 g/L) of the recombinant P. taetrolens were approximately 118 % and 69 % higher than those (0.073 U/g dry weight cell and 45.8 g/L, respectively) of wild-type P. taetrolens. Using this recombinant strain as a whole-cell biocatalyst (WCB), the effects of reaction parameters, such as reaction temperature, cell density, and cell harvest time, were investigated on LBA production. Under optimized reaction conditions, the LBA production titer, yield, and productivity of WCB were 200 g/L, 95.6 %, and 16.7 g/L/h, respectively. Compared with our previous study, LBA production titer, yield, and productivity, which are key factors for industrial LBA production, were significantly improved by fermentation of wild-type P. taetrolens. Moreover, the reaction for LBA production could be performed up to seven times without a significant reduction in productivity, implying that this WCB was rather robust. Our results suggest that the utilization of whole-cell biocatalysis using recombinant P. taetrolens provides a potential solution to achieve economically feasible production of LBA.

The Two-component Regulatory System (TCS) is the primary mode that bacteria use to continuously sense the environment. A TCS is comprised of a periplasmic sensor Histidine kinase (HK) domain and a cytoplasmic Response regulator (RR) domain. The HK domain phosphorylates the RR domain to activate the effector gene expression. Utilizing a rational approach, the sensor HK was genetically engineered in Escherichia coli to create chimeric HK, by a rewiring or domain swapping strategy. Apart from the wild-type characteristics, chimeric HK imparts novel or the desired characteristics and ability to genetically engineered E. coli for its adaptation and survival. This review focuses on the design, potential applications, and future perspectives of chimeric HKs used as high throughput screening biosensors of various compounds.

In this study, constructed Escherichia coli could efficiently adsorb fenitrothion by displaying a pesticide-binding peptide on it using the anchoring motif OmpC. A codon-optimized, pesticide-binding peptide was attached to the C-terminus of OmpC at loop 7 (993 bp). The efficiency of fenitrothion binding by the monomer peptide was evaluated under different temperatures, pH levels, and fenitrothion concentrations. To enhance fenitrothion adsorption, a dimer of pesticide-binding peptide was also constructed and displayed. Compared with the peptide monomer, the dimer-displaying strain showed superior fenitrothion-binding ability. The performance of the strains was evaluated in artificial polluted soil, and their morphology was analyzed by FE-SEM. The results showed that these two kinds of constructed strains can adsorb fenitrothion in contaminated environments with no cellular activity reduction. ARTICLE INFO.

Bio-based production of industrially important chemicals provides an eco-friendly alternative to current petrochemical-based processes. Because of the limited supply of fossil fuel reserves, various technologies utilizing microbial host strains for the sustainable production of platform chemicals from renewable biomass have been developed. Corynebacterium glutamicum is a non-pathogenic industrial microbial species traditionally used for L-glutamate and L-lysine production. It is a promising species for industrial production of bio-based chemicals because of its flexible metabolism that allows the utilization of a broad spectrum of carbon sources and the production of various amino acids. Classical breeding, systems, synthetic biology, and metabolic engineering approaches have been used to improve its applications, ranging from traditional amino-acid production to modern biorefinery systems for production of value-added platform chemicals. This review describes recent advances in the development of genetic engineering tools and techniques for the establishment and optimization of metabolic pathways for bio-based production of major C2-C6 platform chemicals using recombinant C. glutamicum.

GABA is produced from the decarboxylation of glutamate by glutamate decarboxylase enzyme (GadB). Various hetrologous GadB enzymes has been indentified and introduced in Escherichia coli to improve the productivity of GABA. In this study, cell surface display strategy was employed by displaying Neurospora crassa glutamate decarboxylase (GadB) on the surface of Escherichia coli using OmpC as an anchoring motif. To construct the display system, the GadB was fused to truncated C-terminus of OmpC and expressed in E. coli. The recombinant E. coli displaying GadB has produced 3.03 g/L of GABA from 10 g/L glutamate within 12 h of culture period. When the GadB display system was cultured at high cell density, 100% GABA yeild was achieved from 50 g/L of glutamate. The results suggest that the GadB expression on the E. coli surface is stable and effective on GABA production. The surface display strategy can be applied on other potential enzyme candidates and used in Industry for bulk chemical production.

In a cell-surface display (CSD) system, successful display of a protein or peptide is highly dependent on the anchoring motif and the position of the display in that anchoring motif. In this study, a recombinant bacterial CSD system for manganese (Mn) and cobalt (Co) recovery was developed by employing OmpC as an anchoring motif on three different external loops. A portion of Cap43 protein (TRSRSHTSEG)3 was employed as a manganese and cobalt binding peptide (MCBP), which was fused with OmpC at three different external loops. The fusions were made at the loop 2 [fusion protein-2 (FP2)], loop 6 (FP6), and loop 8 (FP8) of OmpC, respectively. The efficacy of the three recombinant strains in the recovery of Mn and Co was evaluated by varying the concentration of the respective metal. Molecular modeling studies showed that the short trimeric repeats of peptide probably form a secondary structure with OmpC, thereby giving rise to a difference in metal recovery among the three recombinant strains. Among the three recombinant strains, FP6 showed increased metal recovery with both Mn and Co, at 1235.14 (1 mM) and 379.68 (0.2 mM) µmol/g dry cell weight (DCW), respectively.

Gamma-aminobutyric acid is a precursor of nylon-4, which is a promising heat-resistant biopolymer. GABA can be produced from the decarboxylation of glutamate by glutamate decarboxylase. In this study, a synthetic scaffold complex strategy was employed involving the Neurospora crassa glutamate decarboxylase (GadB) and Escherichia coli GABA antiporter (GadC) to improve GABA production. To construct the complex, the SH3 domain was attached to the N. crassa GadB, and the SH3 ligand was attached to the N-terminus, middle, and C-terminus of E. coli GadC. In the C-terminus model, 5.8 g/l of GABA concentration was obtained from 10 g/l glutamate. When a competing pathway engineered strain was used, the final GABA concentration was further increased to 5.94 g/l, which corresponds to 97.5% of GABA yield. With the introduction of the scaffold complex, the GABA productivity increased by 2.9 folds during the initial culture period.

Escherichia coli was engineered to sense methanol by employing a chimeric two-component system (TCS) strategy. A chimeric MxaY/EnvZ (MxaYZ) TCS was constructed by fusing the Paracoccus denitrificans MxaY with the E. coli EnvZ. Real-time quantitative PCR analysis and GFP-based fluorescence analysis showed maximum transcription of ompC and the fluorescence at 0.01% of methanol, respectively. These results suggested that E. coli was successfully engineered to sense methanol by the introduction of chimeric MxaYZ. By using this strategy, various chimeric TCS-based bacterial biosensors can be constructed and used for the development of biochemical-producing recombinant microorganisms.

Escherichia coli does not have the methanol sensing apparatus, was engineered to sense methanol by employing chimeric two-component system (TCS) strategy. A chimeric FlhS/EnvZ (FlhSZ) chimeric histidine kinase (HK) was constructed by fusing the sensing domain of Paracoccus denitrificans FlhS with the catalytic domain of E. coli EnvZ. The constructed chimeric TCS FlhSZ/OmpR could sense methanol by the expression of ompC and gfp gene regulated by ompC promoter. Real-time quantitative PCR analysis and GFP-based fluorescence analysis showed the dynamic response of the chimeric TCS to methanol. The expression of ompC and the gfp fluorescence was maximum at 0.01 and 0.5% of methanol, respectively. These results suggested that E. coli was successfully engineered to sense methanol by the introduction of chimeric HK FlhSZ. This strategy can be employed for the construction of several chimeric TCS based bacterial biosensors for the development of biochemical producing recombinant microorganisms.

Bisphenol-A (BPA)-sensing and adsorption recombinant Escherichia coli were constructed based on the recA promoter. Quantitative, real-time PCR was carried out to analyze the expression profile of the ompC gene in response to various concentrations of BPA. The results show that this system senses BPA from 50 ppb to a maximum concentration of 40 ppm. The adsorption system based on the cell surface display fused with a BPA binding peptide can absorb 354.0 μmol/g cell DCW at 180 ppm of BPA. The strains used in the study show high selectivity towards BPA adsorption and sensing, and this novel system could thus be employed for sensing and adsorption in BPA contaminated environments, such as in thermal paper wastewater industry and other BPA leached water.

Background Posterior reversible encephalopathy syndrome (PRES) is a neurological complication caused by cerebral hyperperfusion. Case Report A 46-year-old male presented with decreased mental status, left facial palsy, and left-sided weakness after video-assisted thoracoscopic surgery for a solitary pulmonary nodule. During the surgery, phenylephrine was infused intravenously for general anesthesia-induced hypotension. High signal intensity at the right parietooccipital lobe was noted on fluid-attenuated inversion recovering imaging and diffusion-weighted imaging. His neurological symptoms improved two days after initial presentation. Follow-up diffusion-weighted imaging showed resolution of the brain lesions 10 days after the surgery. Conclusions We report a patient who presented with PRES after administration of phenylephrine during resection of a solitary pulmonary nodule. PRES should be considered for patients presented with acute neurologic symptoms following surgical procedures.

Methylobacterium organophilum XX is a type II facultative methylotroph that can grow on methanol. In M. organophilum XX, the MxcQ/MxcE two-component system (TCS) is involved in methanol metabolism. EnvZ/OmpR in E. coli TCS was exploited to develop a methanol biosensor by engaging the MxcQ/MxcE TCS system. The MxcQZ/OmpR methanol sensing chimeric TCS was constructed by integrating the sensing domain of M. organophilum MxcQ with the transmitter domain of E. coli EnvZ. The response regulator of the chimeric TCS system is OmpR, which regulates the expression of the ompC and gfp. The expression of ompC was monitored by real-time quantitative PCR analysis. The expression of gfp also confirmed the expression of the ompC. The maximum expression of ompC and gfp occurred with 0.05% of methanol, and the expression started to decline with further increases in methanol concentration. This system delivers rapid detection of methanol in the environment.

Gamma-aminobutyric acid (GABA) is a precursor to pyrrolidone, a monomer used for the production of a biodegradable polymer known as nylon-4. GABA is also widely used in the medical industry to treat conditions such as high blood pressure, anxiety and depression. Generally, GABA is produced from glutamate by the enzyme glutamate decarboxylase (GadB). In this study, a synthetic scaffold complex was introduced between Pyrococcus horikoshii GadB and the GABA antiporter (GadC) from E. coli. P. horikoshii GadB was attached to the N-terminus, C-terminus and middle of E. coli GadC via scaffolding. Among the three scaffold complexes evaluated, the N-terminus scaffold model produced 5.93 g/L of GABA from 10 g/L monosodium glutamate (MSG). When the gabT mutant E. coli XBT strain was used, the highest GABA concentration of 5.96 g/L was obtained, which is 97.8% of GABA yield. In addition to GABA concentration, GABA productivity was increased 3.5 fold via the synthetic scaffold complex.

Gamma-aminobutyric acid (GABA) is a non-protein amino acid, which is an important inhibitor of neurotransmission in the human brain. GABA is also used as the precursor of biopolymer Nylon-4 production. In this study, the carbon flux from the tricarboxylic acid cycle was directed to the GABA shunt pathway for the production of GABA from glucose. The GABA shunt enzymes succinate-semialdehyde dehydrogenase (GabD) and GABA aminotransferase (GabT) were co-localized along with the GABA transporter (GadC) by using a synthetic scaffold complex. The co-localized enzyme scaffold complex produced 0.71 g/l of GABA from 10 g/l of glucose. Inactivation of competing metabolic pathways in mutant E. coli strains XBM1 and XBM6 increased GABA production 13% to reach 0.80 g/l GABA by the enzymes co-localized and expressed in the mutant strains. The recombinant E. coli system developed in this study demonstrated the possibility of the pathway of the GABA shunt as a novel GABA production pathway.

Nylon 4 is a biodegradable polymer which can be produced from the monomer of pyrrolidone. Gammaaminobutyric acid (GABA) is a precursor of pyrrolidone used for the production of bioplastics. In this study, Escherichia coli were engineered to produce gammaaminobutyric acid from glucose via an alternative novel pathway by the introduction of synthetic scaffolds. The GABA pathway constructed contained succinate dehydrogenase, succinate-semialdehyde dehydrogenase and GABA aminotransferase to redirect the Krebs cycle flux to GABA production. By introduction of a synthetic scaffold, production of 0.64 g/L GABA was achieved at 30°C and pH 6.5. Final GABA concentration was increased by 11.3% via the inactivation of competing pathways, and higher initial glucose concentration led to the enhanced final GABA concentration of 1.01 g/L.

The expression of anti-freeze proteins (AFPs) on the Escherichia coli using the cell surface display (CSD) strategy was investigated to determine the feasibility of the method as a potential application of kinetic hydrate inhibitors. Monomers to hexamers of type II and III AFPs were displayed on the surfaces of bacterial cells. The kinetic inhibition performance of the AFPs on the synthetic natural gas hydrate formation was tested. The displayed AFPs exhibited higher KHI effectiveness with the increase in the number of peptides attached to the cells. In addition, best KHI performance of the AFPs could be achieved with higher dosages of the cultures when the concentration of the inducer is optimized, regardless of AFP type. Only a small portion of AFPs in the cell cultures exhibited considerable KHI performance, which implies that the CSD could be an effective strategy, reducing the cost for the production and purification of the AFPs.

Previous studies constructed a chimeric MalKZ two-component system to sense environmental malate. In this study, we used a positive feedback loop to accelerate and amplify the output signal indicating malate concentration. The positive feedback loop was constructed by cloning ompR gene, which encodes ompC and induces OmpR protein; ompC promoter was used to control the process. The transcriptional expression profile showed that the expression level of ompC gene increased about two-fold after the positive feedback loop was introduced. When GFP was used as a reporter protein, a 71% increase in fluorescence level was observed. The results indicate that the signal transduction kinetics of MalKZ can be engineered by introducing the positive feedback loop.

Gamma-aminobutyric acid (GABA) is an important bio-product, which is used in pharmaceutical formulations, nutritional supplements, and biopolymer monomer. The traditional GABA process involves the decarboxylation of glutamate. However, the direct production of GABA from glucose is a more efficient process. To construct the recombinant strains of Escherichia coli, a novel synthetic scaffold was introduced. By carrying out the co-localization of glutamate synthase, glutamate decarboxylase, and GABA transporter, we redirected the TCA cycle flux to GABA pathway. The genetically engineered E. coli strain produced 1.08 g/L of GABA from 10 g/L of initial glucose. Thus, with the introduction of a synthetic scaffold, we increased GABA production by 2.2-fold. The final GABA concentration was increased by 21.8 % by inactivating competing pathways.

Biochemical conversion of biomass into biofuels, biochemicals, and biopolymers has attracted much interest throughout the world in terms of biorefineries. Lignocellulosic biomass is one of the most plentifully available biomass resources on the earth. It is composed of three main biopolymers - cellulose, hemicelluloses, and lignin, all of which are cross-linked to each other to resist degradation by enzymes and microorganisms resulting in so-called biomass recalcitrance. The biorefinery process typically consists of three steps: pretreatment, hydrolysis, and fermentation. Energy and cost efficiency of biorefinery is predominantly dependent on how to produce inexpensive sugars from complex cell wall component of lignocellulosic biomass by overcoming biomass recalcitrance. There have been tremendous efforts to develop effective biomass pretreatment technologies for obtaining the highest yield of fermentable sugars from biomass feedstocks at the lowest cost. The present review discusses various pretreatment technologies to understand how to effectively break down biomass into fermentable sugars that are eventually used for microbial fermentation to produce biomass-based fuels, chemicals, and polymers.

A positive feedback loop was introduced to modify the dynamic behavior of fumarate sensing DcuSZ chimera TCS. To construct the positive feedback loop, the ompR gene was cloned downstream of the ompC promoter. The ompC promoter induced the expression of OmpR, which in turn induced the expression of the ompC promoter. Through the introduction of this positive feedback loop, the transcriptional expression levels of ompC increased 2.6-fold. When GFP was used as a reporter protein, a 64% increase in fluorescence level was observed. These results suggest that sensitivity of the TCS based fumarate sensing system can be engineered through the introduction of a positive feedback loop.

Escherichia coli were engineered for the direct production of gamma-aminobutyric acid from glucose by introduction of synthetic protein scaffold. In this study, three enzymes consisting GABA pathway (isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase) were connected via synthetic protein scaffold. By introduction of scaffold, 0.92g/L of GABA was produced from 10g/L of glucose while no GABA was produced in wild type E. coli. The optimum pH and temperature for GABA production were 4.5 and 30°C, respectively. When competing metabolic network was inactivated by knockout mutation, maximum GABA concentration of 1.3g/L was obtained from 10g/L glucose. The recombinant E. coli strain which produces GABA directly from glucose was successfully constructed by introduction of protein scaffold.

In an attempt to create an acidic amino acid-sensing Escherichia coli, a chimeric sensor kinase (SK)-based biosensor was constructed using Pseudomonas putida AauS. AauS is a sensor kinase that ultimately controls expression of the aau gene through its cognate response regulator AauR, and is found only in P. putida KT2440. The AauZ chimera SK was constructed by integration of the sensing domain of AauS with the catalytic domain of EnvZ to control the expression of the ompC gene in response to acidic amino acids. Real-time quantitative PCR and GFP fluorescence studies showed increased ompC gene expression and GFP fluorescence as the concentration of acidic amino acids increased. These data suggest that AauS-based recombinant E. coli can be used as a bacterial biosensor of acidic amino acids. By employing the chimeric SK strategy, various bacteria biosensors for use in the development of biochemical-producing recombinant microorganisms can be constructed.