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To produce maltobionic acid (MBA) from maltose in Escherichia coli, we recombinantly expressed a glucose dehydrogenase gene (gdh1) from Enterobacter cloacae and a pyrroloquinoline quinone (PQQ) synthesis gene cluster (pqqFABCDEMIH) from Pseudomonas taetrolens. Although the recombinant E. coli strain (E. coli [pKK-ECGDH1 + pACYC-PQQ]) successfully produced MBA from maltose, the yield of MBA was rather low, indicating that E. coli has other maltose utilization pathways. Amylomaltase (MalQ) is the first enzyme in the maltose utilization pathway in E. coli. To investigate the potential role of MalQ on MBA production, E. coli malQ was inactivated. The culturing of the recombinant E. coli strain (E. coli ∆malQ [pKK-ECGDH1 + pACYC-PQQ]) in a flask resulted in higher MBA production titer, yield, and productivity (209.3 g/L, 100%, and 1.1 g/L/h, respectively) than those of E. coli [pKK-ECGDH1 + pACYC-PQQ] (162.1 g/L, 77.4%, and 0.5 g/L/h, respectively), indicating that the MalQ inactivation was highly effective in improving the MBA production ability of E. coli. After fermentation using 5-L bioreactor, MBA production titer, yield, and productivity of the recombinant E. coli strain were 209.3 g/L, 100%, and 1.5 g/L/h, respectively, which were 1.3-, 1.3-, 2.3-fold higher than those of E. coli [pKK-ECGDH1 + pACYC-PQQ] (167.3 g/L, 79.9%, and 0.65 g/L/h), respectively. Thus, our results provide an important foundation for efficient MBA production using recombinant E. coli strain.

To reduce food waste (FW) disposal costs, many Koreans now convert FW into residual food dried substances (RFDS) using a house-service food drying machine and then dispose of the RFDS. To recycle RFDS, we tested whether RFDS could be used as a culture nutrient to produce value-added microbial chemicals. As a case study, we attempted to produce lactic acid (LA) by cultivating lactic acid bacteria using RFDS. To prepare the culture medium for LA production, we finely ground the RFDS and dissolved it with CaCO

Pseudomonas taetrolens has previously been shown to convert cellobiose to cellobionic acid (CBA), which can potentially be used in cosmetics, food, and pharmaceutical industries. The cellobiose-oxidizing activity of the P. taetrolens strain, which expressed the homologous quinoprotein glucose dehydrogenase (GDH), was increased by approximately 50.8% compared to the original strain. Whole-cell biocatalyst (WCB) of the genetically modified P. taetrolens strain [pDSK-GDH] was prepared simply by fermentation and washing processes. Reaction conditions for the proper use of WCB, such as reaction temperature, cell density to be added, and cell harvest time for preparing WCB, were investigated. The highest CBA productivity (18.2 g/L/h) was achieved when WCB prepared in the late-exponential phase of cell culture was used at 35 °C with cell density of 10 at OD

Lactobionic acid (LBA) has recently emerged as an important substance in various industries, such as cosmetics, foods, and pharmaceuticals. In this study, we developed a simple, efficient, and high-throughput method for screening LBA-producing microorganisms. First, an agar plate was prepared to isolate LBA-producing microorganisms by utilizing the property of LBA to solubilize colloidal calcium carbonate (CaCO

In this study, we attempted to produce maltobionic acid (MBA) from waste cooked rice (WCR) using maltose as an intermediate. In our previous study, we produced maltose from WCR using a commercial maltogenic amylase (Maltogenase L). However, in the present study, we used wild-type Bacillus subtilis, which inherently produces maltogenic amylase (AmyE), instead of Maltogenase L to produce maltose from WCR. During cultivation of B. subtilis with WCR, maltose was successfully produced by AmyE in the culture medium. To improve maltose production, we constructed a recombinant B. subtilis strain expressing AmyE and used it for maltose production. Following cultivation of the recombinant B. subtilis strain, the maltose production titer (34.6 g/L) increased approximately 3.6-fold that (9.6 g/L) obtained from the cultivation of wild-type B. subtilis. Using Pseudomonas taetrolens, an efficient MBA-producing bacterium, 28.8 g/L of MBA was produced from the prepared maltose (27.6 g/L). The above results indicated that MBA was successfully produced from WCR via a two-step process, which involved the conversion of WCR into maltose by maltogenic amylase-producing B. subtilis and the production of MBA from the WCR-derived maltose by P. taetrolens.

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.

A lactobionic acid (LBA)-producing bacterium isolated from seaside soils was identified as Acinetobacter halotolerans and designated as strain KRICT-1. We determined whether KRICT-1 can produce LBA at high salt concentrations. The KRICT-1 strain grew on a nutrient broth (NB) agar plate with up to 7.0% NaCl, indicating high NaCl tolerance, and 30 °C was the optimum growth temperature for LBA production. We produced LBA using the KRICT-1 strain in NB medium containing various concentrations of NaCl. While Pseudomonas taetrolens, an efficient LBA-producing bacterium, could produce LBA with up to 5.5% NaCl, the KRICT-1 strain could produce LBA at up to 7.0% NaCl and produced more LBA than P. taetrolens with over 5.5% NaCl. We produced LBA using NB medium containing 7.0% NaCl by batch fermentation of the KRICT-1 strain in a 5 L fermenter. The LBA production titer and productivity of the KRICT-1 strain were 32.1 g/L and 0.22 g/L/h, respectively, which were approximately 1.35- and 1.38-fold higher than those (23.7 g/L and 0.16 g/L/h) obtained from flask culture. Additionally, quinoprotein glucose dehydrogenase is an LBA-producing enzyme in A. halotolerans. We demonstrated that the A. halotolerans KRICT-1 strain is appropriate for LBA production at high salt concentrations.

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.

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.

Maltobionic acid (MBA) can be applied to various fields such as food, cosmetics, and pharmaceutical industries. In this study, whole-cell biocatalysis for MBA production was performed using recombinant Pseudomonas taetrolens homologously expressing quinoprotein glucose dehydrogenase (GDH). Various reaction parameters such as temperature, cell density, and cell harvest time, were optimized for improving MBA production. Under the optimized reaction conditions using pure maltose as a substrate, the MBA production titer, yield, and productivity of whole-cell biocatalyst (WCB) were 200 g/L, 95.6%, and 18.18 g/L/h, respectively, which were the highest compared to those reported previously. Productivity, a key factor for industrial MBA production, obtained from whole-cell biocatalysis in this study, was enhanced by approximately 1.9-fold compared to that obtained in our previous work (9.52 g/L/h) using the fermentation method. Additionally, the WCB could be reused up to six times without a significant reduction in MBA productivity, indicating that the WCB is very robust. Although MBA productivity (8.33 g/L/h) obtained from high-maltose corn syrup (HMCS) as a substrate was 45.8% of that using pure maltose, HMCS can be a better substrate for commercial MBA production because its price is only 1.1% of that of pure maltose. The results of this study using a WCB to convert maltose into MBA may support the development of a potential industrial process for more economically effective MBA production in the future.

Polyethylene terephthalate (PET) waste has caused serious environmental pollution. Recently, PET depolymerization by enzymes with PET-depolymerizing activity has received attention as a solution to recycle PET. An engineered variant of leaf-branch compost cutinase (293 amino acid), ICCG (Phe243Ile/Asp238Cys/Ser283Cys/Tyr127Gly), showed excellent depolymerizing activity toward PET at 72 °C, which was the highest depolymerizing activity and thermo-stability ever reported in previous works. However, this enzyme was only produced by heterologous expression in the cytoplasm of Escherichia coli, which requires complex separation and purification steps. To simplify the purification steps of ICCG, we developed a secretory production system using Bacillus subtilis and its 174 types of N-terminal signal peptides. The recombinant strain expressing ICCG with the signal peptide of serine protease secreted the highest amount (9.4 U/mL) of ICCG. We improved the production of ICCG up to 22.6 U/mL (85 μg/mL) by performing batch fermentation of the selected strain in 2 L working volume using a 5-L fermenter, and prepared the crude ICCG solution by concentrating the culture supernatant. The recombinant ICCG successfully depolymerized a PET film with 37% crystallinity at 37 °C and 70 °C. In this study, we developed a secretory production system of the engineered cutinase with PET-depolymerizing activity to obtain high amounts of the enzyme by a relatively simple purification method. This system will contribute to the recycling of PET waste via a more efficient and environmentally friendly method based on enzymes with PET-depolymerizing activity.

Lactobionic acid (LBA) is a specialty organic acid that is widely employed in the food, cosmetic, and pharmaceutical industries. In the present study, we screened new LBA-producing bacteria from the soil of a poultry farm. Among the 700 bacterial colonies, five that exhibited LBA-producing ability were successfully isolated. Phylogenetic analysis based on 16 S rRNA sequences identified strain 2-15 as an Acinetobacter sp., strains 3-13 and 3-15 as Pseudomonas spp., and strains 7-7 and 7-8 as Psychrobacter spp. The LBA-producing abilities of the five strains were compared in flask culture, whereupon Psychrobacter sp. 7-8 showed the highest LBA titer (203.7 g/L), LBA yield from lactose (97.3%), and LBA productivity (2.83 g/L/h). To our best knowledge, this is the first study showing that Acinetobacter and Psychrobacter spp. can produce LBA from lactose. Our results would help broaden the spectrum of workhorse bacteria available for the industrially important microbial production of LBA. In addition, we improved the LBA-production ability of the three isolated bacteria, namely Acinetobacter sp. 2-15, Pseudomonas spp. strains 3-13 and 3-15, by heterologously expressing quinoprotein glucose dehydrogenase from Pseudomonas taetrolens. In particular, the LBA-production ability of the recombinant Pseudomonas sp. 3-13 were highly improved that the LBA titer and productivity were 19.2- (205.6 vs. 10.7 g/L, respectively) and 17.8-fold (1.07 vs. 0.06 g/L/h, respectively) higher, respectively, than those of the wild-type strain. These values were almost identical to those of the wild-type Psychrobacter sp. 7-8, which showed the highest LBA productivity among the five isolated strains. This result demonstrated that the expression of lactose-oxidizing enzyme in LBA-producing microorganisms was highly effective to enhance their LBA-production ability. Our study presents a practical method to screen for efficient LBA-producing microorganisms and to improve their production ability by genetic engineering for industrial LBA production.

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 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.

Lactobionic acid (LBA), an aldonic acid prepared by oxidation of the free aldehyde group of lactose, has been broadly used in cosmetic, food, and pharmaceutical industries. Although Escherichia coli is unable to produce LBA naturally, a wild-type E. coli strain successfully produced LBA from lactose upon pyrroloquinoline quinone (PQQ) supplementation, indicating that E. coli contains at least one lactose-oxidizing enzyme as an apo-form. By inactivating the candidate genes in the E. coli chromosome, we found that the lactose-oxidizing enzyme of E. coli was the quinoprotein glucose dehydrogenase (GCD). To improve the LBA production ability of the E. coli strain, quinoprotein glucose dehydrogenase (GDH) from Pseudomonas taetrolens was recombinantly expressed and culture conditions such as growth temperature, initial lactose concentration, PQQ concentration, and isopropyl-β-D-1-thiogalactopyranoside induction concentration were optimized. We performed batch fermentation using a 5-L bioreactor under the optimized culture conditions determined in flask culture experiments. After batch fermentation, the LBA production titer, yield, and productivity of the recombinant E. coli strain were 200 g/L, 100 %, and 1.28 g/L/h, respectively. To the best our knowledge, this is the first report to identify the lactose-oxidizing enzyme of E. coli and to produce LBA using a recombinant E. coli strain as the production host. Because E. coli is one of the most easily genetically manipulated bacteria, our result provides the groundwork to further enhance LBA production by metabolic engineering of LBA-producing E. coli.

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

Waste-cooking oil (WCO) is defined as vegetable oil that has been used to fry food at high temperatures. The annual global generation of WCO is 41–67 million tons. Without proper treatment, most WCO is abandoned in sinks and the solid residue of WCO is disposed of in landfills, resulting in serious environmental problems. Recycling and valorizing WCO have received considerable attention to reduce its negative impact on ecosystems. To convert WCO into a high value-added compound, we aimed to produce sophorolipids (SLs) that are industrially important biosurfactants, using WCO as a hydrophobic substrate by the fed-batch fermentation of Starmerella bombicola. The SLs concentration was increased ~3.7-fold compared with flask culture (315.6 vs. 84.8 g/L), which is the highest value ever generated from WCO. To expand the applications of SLs, we prepared methyl hydroxy branched fatty acids (MHBFAs) from SLs, which are important chemicals for various industries yet difficult to produce by chemical methods, using a bio-chemical hybrid approach. We synthesized bio-based plastics using MHBFAs as co-monomers. Compared with the control polymer without MHBFAs, even the incorporation of 1 mol% into polymer chains improved mechanical properties (such as ultimate tensile strength, 1.1-fold increase; toughness, 1.3-fold increase). To the best of our knowledge, this is the first attempt to apply MHBFAs from SLs derived from WCO to building blocks of plastics. Thus, we extended the valorization areas of WCO to one of the world’s largest industries.

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.

Bacterial phospholipase A1 (PLA1) is used in various industrial fields because it can catalyze the hydrolysis, esterification, and transesterification of phospholipids to their functional derivatives. It also has a role in the degumming process of crude plant oils. However, bacterial expression of the foreign PLA1-encoding gene was generally hampered because intracellularly expressed PLA1 is inherently toxic and damages the phospholipid membrane. In this study, we report that secretion-based production of recombinant PlaA, a bacterial PLA1 gene, or co-expression of PlaS, an accessory gene, minimizes this harmful effect. We were able to achieve high-level PlaA production via secretion-based protein production. Here, TliD/TliE/TliF, an ABC transporter complex of Pseudomonas fluorescens SIK-W1, was used to secrete recombinant proteins to the extracellular medium. In order to control the protein expression with induction, a new strain of P. fluorescens, which had the lac operon repressor gene lacI, was constructed and named ZYAI strain. The bacteriotoxic PlaA protein was successfully produced in a bacterial host, with help from ABC transporter-mediated secretion, induction-controlled protein expression, and fermentation. The final protein product is capable of degumming oil efficiently, signifying its application potential.

Pseudomonas taetrolens is one of the most efficient lactobionic acid (LBA)-producing microorganisms. We previously found that it also produced cellobionic acid (CBA) and that quinoprotein glucose dehydrogenase (GDH) from P. taetrolens could convert lactose into LBA. Here, we discovered that GDH could also convert cellobiose into CBA when it was heterologously expressed in Escherichia coli. To enhance the CBA-producing ability of P. taetrolens, we homologously expressed GDH in P. taetrolens, and demonstrated that the intracellular cellobiose-oxidizing activity (1.75 U/μg) and CBA titer (21.4 g/L) of the recombinant P. taetrolens were approximately 54% and 60% higher than those (1.14 U/μg and 13.4 g/L, respectively) of the wild-type P. taetrolens in 12 h flask cultures. We optimized culture conditions of this recombinant strain, such as cellobiose concentration, growth temperature, and cell density of seed culture. Thereafter, we scaled up the CBA production using a 5-L bioreactor under the optimized culture condition determined in flask culture experiments. The CBA production titer, yield, and productivity of the recombinant P. taetrolens strain after batch fermentation were 200 g/L, 95.6%, and 9.52 g/L/h, respectively, which far exceeded those obtained in previous studies. Our study provides important clues to achieve economically feasible production of CBA.

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.

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.

Highlights • Glucose dehydrogenase from Pseudomonas taetrolens could produce maltobionic acid. • The glucose dehydrogenase gene was homologously expressed in P. taetrolens. • Maltose-oxidizing activity and MBA production titer of P. taetrolens was improved. • MBA productivity of the recombinant P. taetrolens was increased to 9.52 g/L/h. • High-level production of MBA from HMCS was achieved by the recombinant P. taetrolens., Maltobionic acid (MBA) has recently emerged as an important material in various industries. Here, we showed that quinoprotein glucose dehydrogenase (GDH) from Pseudomonas taetrolens could convert maltose into MBA by heterologously expressing this enzyme in MBA non-producing Escherichia coli. We homologously expressed GDH in P. taetrolens to improve intracellular maltose-oxidizing activity and MBA production. We optimized culture conditions, then applied these conditions to batch fermentation by recombinant P. taetrolens in a 5-L bioreactor. The MBA production, yield, and productivity of batch fermentation using high-maltose corn syrup (HMCS), an inexpensive maltose source, were 200 g/L, 95.6 %, and 6.67 g/L/h, respectively. Although the MBA productivity from HMCS was 70.1 % of that compared with pure maltose as the substrate, HMCS was a better substrate for commercial MBA production, considering the cost was 1.1 % of that of pure maltose. The present findings provide an economically feasible strategy with which to produce MBA.

An ABC transporter, TliDEF, from Pseudomonas fluorescens SIK W1, mediates the secretion of its cognate lipase, TliA, in a temperature-dependent secretion manner; the TliDEF-mediated secretion of TliA was impossible at the temperatures over 33°C. To isolate a mutant TliDEF capable of secreting TliA at 35°C, the mutagenesis of ABC protein (TliD) was performed. The mutated tliD library where a random point mutation was introduced by error-prone PCR was coexpressed with the wild-type tliE, tliF and tliA in Escherichia coli. Among approximately 10,000 colonies of the tliD library, we selected one colony that formed transparent halo on LB-tributyrin plates at 35°C. At the growth temperature of 35°C, the selected mutant TliD showed 1.75 U/ml of the extracellular lipase activity, while the wild-type TliDEF did not show any detectable lipase activity in the culture supernatant of E. coli. Moreover, the mutant TliD also showed higher level of TliA secretion than the wild-type TliDEF at other culture temperatures, 20°C, 25°C and 30°C. The mutant TliD had a single amino acid change (Ser287Pro) in the predicted transmembrane region in the membrane domain of TliD, implying that the corresponding region of TliD was important for causing the temperature-dependent secretion of TliDEF. These results suggested that the property of ABC transporter could be changed by the change of amino acid in the ABC protein.

To secrete the endo-β-1,4-mannanase of Bacillus subtilis WL7 in Escherichia coli, the ABC exporter (TliDEF) and the C-terminal signal sequences of lipase (TliA) and metalloprotease (PrtA) were used. Six C-terminal fragments of TliA (TliAC1, TliAC2 and TliAC3) and PrtA (PrtAC1, PrtAC2 and PrtAC3) were amplified by PCR, respectively, and were fused to the mature mannanase gene lacking its original N-terminal signal sequence. When coexpressed with TliDEF exporter gene in E. coli, all the fusion mannanases were successfully secreted to the culture supernatant as an enzymatically active form by TliDEF exporter in E. coli. However, the unspecific cell leakage occurred in the fusion mannanases containing TliA-derived signal sequences. Among the fusion mannanases containing the signal sequences of PrtA, the mannanase with the shortest signal sequence fragment (Man-PrtAC3) exhibited the highest secretion level (4.65 mg/L). Moreover, the specific activity, the effect of temperature and pH on activity and stability were almost identical to the wild-type mannanase, indicating that the fused signal sequence of PrtA did not affect the enzymatic properties of mannanase. It was concluded that the efficient secretion of enzymatically active mannanase from Bacillus subtilis WL-7 was achieved by using PrtAC3 as the most adequate signal sequence fragment.

Recently, malic acid has gained attention due to its potential application in food, pharmaceutical, and medical industries. In this study, the synthetic scaffold complex strategy was employed between the two key enzymes pyruvate kinase (PykF) and malic enzyme (SfcA); SH3 ligand was attached to PykF, and the SH3 domain was attached to the C-terminus of ScfA. Synthetic scaffold systems can organize enzymes spatially and temporally to increase the local concentration of intermediates. In a flask culture, the recombinant strain harboring scaffold complex produced a maximum concentration of 5.72 g/L malic acid from 10 g/L glucose. The malic acid production was significantly increased 2.1-fold from the initial culture period. Finally, malic acid production was elevated to 30.2 g in a 5 L bioreactor from recombinant strain XL-1 blue.

Serratia proteamaculans metalloprotease (SPP) was successfully secreted by a heterologous ABC protein exporter, the Pseudomonas fluorescens TliDEF, in recombinant host strains. Escherichia coli and P. fluorescens cells containing the SPP-encoding gene showed the extracellular protease activity only when the TliDEF-encoding gene cluster was coexpressed. Recombinant P. fluorescens produced an approximately 34.8-fold higher amount of extracellular SPP than did E. coli. The use of a more nutrient-rich medium and controlled dissolved oxygen conditions was effective in increasing SPP secretion in P. fluorescens batch fermentation (an 8.7-fold increase from 41.8 U/mL to 365.2 U/mL). Therefore, SPP, which could not be secreted without an ABC protein exporter, was produced in large quantities by applying the heterologous TliDEF exporter in P. fluorescens. The results also suggest that the use of the ABC protein exporter in P. fluorescens could be an efficient production platform for an industrially promising type I secretion pathway-dependent enzyme.

The industrially-important thermostable lipase, TliA, was extracellularly produced in the recombinant Pseudomonas fluorescens by the homologous expression of TliA and its cognate ABC protein exporter, TliDEF. To increase the secretory production of TliA, we optimized the growth temperature and the culture medium of P. fluorescens. The total amount and the specific productivity of lipase was highest at 25 °C of cell growth temperature, although maximal cell growth was observed at 30 °C. Using the culture medium composed of 20 g dextrin l(-1), 40 g Tween 80 l(-1) and 30 g peptone l(-1), TliA was produced at a level of 2,200 U ml(-1) in a flask culture. The TliA production increased about 3.8-fold (8,450 U ml(-1)) in batch fermentation using a 2.5 l fermentor, which was about 7.7-fold higher than that of previously reported TliA production.

The gene encoding lipase B from Candida antarctica (CalB) was expressed in Pichia pastoris after it was synthesized by the recursive PCR and cloned into the Pichia expression plasmid, pPICZαA. The CalB was successfully secreted in the recombinant P. pastoris strain X-33 with an apparent molecular weight of 34 kDa. For 140 h flask culture, the dry cell weight and the extracellular lipase activity reached at 5.4 g/l and 57.9 U/l toward p-nitrophenyl palmitate, respectively. When we performed the fed-batch fermentation using a methanol feeding strategy for 110 h, the dry cell weight and the extracellular lipase activity were increased to 135.7 g/l and 11,900 U/l; the CalB protein concentration was 1.18 g/l of culture supernatant. The characteristics of CalB recovered from the P. pastoris culture were compared with the commercial form of CalB produced in Aspergillus oryzae. The kinetic constants and specific activity, the effects of activity and stability on temperature and pH, the glycosylation extent, the degree of immobilization on macroporous resin and the yield of esterification reaction between oleic acid and n-butanol were almost identical to each other. Therefore, we successfully proved that the Pichia-based expression system for CalB in this study was industrially promising compared with one of the most efficient production systems.

The enzymatic transesterifications of fatty acid methyl esters (FAME) with hydroxyethyl methacrylate (HEMA) were carried out using the Candida antarctica lipase B immobilized within a porous polymethacrylate resin. The enzymatic activity in the transesterification reaction of FAME with HEMA depended on the polarity of the solvent and the highest yield was obtained in toluene (non-polar). The molar ratio of 1:4 (for methyl laurate:HEMA) and 1:2 (for methyl oleate:HEMA) was most favorable for the transesterification yield. The reaction condition (at 60 °C/24 h), and the enzyme concentration of 5% (w/w) for methyl laurate with HEMA, 2% (w/w) for methyl oleate with HEMA resulted in the highest final yield. Under these conditions, the maximum yields for the transesterification of methyl laurate with HEMA, methyl oleate with HEMA were 97 ± 5.4% and 91 ± 4.7%, respectively. After ten batches of transesterification of FAME with HEMA, enzyme activity was retained at the level of 88 ± 2.6% and 76 ± 2.3%, respectively, compared with their initial activity. Also, alkyl methacrylate/styrene copolymers were synthesized by radical polymerization of HEMA-LMA (or HEMA-OMA) and styrene. The prepared copolymers have average molecular weights from 2.6 × 104 to 5.5 × 104. Especially, the poly(styrene-co-alkyl methacrylate)s (PStmHAMAn) led to a reduction in the pour point in ultra low sulfur diesel (ULSD) treated with 200–1,000 ppm of poly(styrene-co-alkyl methacrylate). Diesel fuel containing 1,000 ppm of the copolymer (PSt2HLMA8) showed a 15 ± 1.25 °C reduction in its pour point.

The type I secretion system-dependent lipase, TliA, of Pseudomonas fluorescens was successfully produced in quantity in Serratia marcescens by coexpressing its cognate ABC transporter, TliDEF. Compared with P. fluorescens and Escherichia coli, S. marcescens showed an outstanding capacity for the secretory production of TliA, which was done with the expression vectors available for use in E. coli, and no growth phase-dependency, which was unlike the typical feature of TOSS-mediated protein secretion. Among the S. marcescens tested, the highest amount of TliA (approximately 2600 units ml(-1)) was achieved by S. marcescens KCTC 2798 containing the expression plasmid pTliDEFA-223. Our results also suggest that strains of Serratia will provide a valuable opportunity for producing other extracellular TOSS-dependent proteins effectively as well as the TliDEF-dependent TliA in this study.

Gene-expression cassettes for the construction of recombinant Clostridium beijerinckii were developed as potential tools for metabolic engineering of C. beijerinckii. Gene expression cassettes containing ColE1 origin and pAMB origin along with the erythromycin resistance gene were constructed, in which promoters from Escherichia coli, Lactococcus lactis, Ralstonia eutropha, C. acetobutylicum, and C. beijerinckii are examined as potential promoters in C. beijerinckii. Zymogram analysis of the cell extracts and comparison of lipase activities of the recombinant C. beijerinckii strains expressing Pseudomonas fluorescens tliA gene suggested that the tliA gene was functionally expressed by all the examined promoters with different expression level. Also, recombinant C. beijerinckii expressing C. beijerinckii secondary alcohol dehydrogenase by the constructed expression cassettes successfully produced 2-propanol from glucose. The best promoter for TliA expression was the R. eutropha phaP promoter while that for 2-propanol production was the putative C. beijerinckii pta promoter. Gene expression cassettes developed in this study may be useful tools for the construction of recombinant C. beijerinckii strains as host strains for the valuable chemicals and fuels from renewable resources.

Three Escherichia coli-Clostridia shuttle vectors, pKBA411-MCS, pKBE411-MCS, and pKBM411-MCS, which contain p15A, ColE1, and pMB1 origins for replication in E. coli, respectively, along with the pAMB origin for replication in C. beijerinckii, were constructed and examined for their transformation efficiencies into Clostridium beijerinckii NCIMB8052. The transformation condition of pKBM411-MCS, which was optimized by varying resistance, buffer composition, and DNA concentration, was further employed for the transformation of the other plasmids, pKBA411-MCS and pKBE411-MCS into C. beijerinckii. It was found out that transformation efficiency is highly dependent on the origin of replication. The highest transformation efficiency of 7.44 × 10(3) colony-forming units per microgram of DNA was obtained at 5.0 kV cm(-1) field strength, 200 Ω resistance, 270 mM sucrose concentration, 150 ng μg(-1), and 3.0 μg DNA using pKBM411-MCS having pMB1 and pAMB origins of replication. The application of the newly constructed vector system was also investigated by introducing the putative alcohol dehydrogenase gene of C. beijerinckii.

Heterologous ABC protein exporters, the apparatus of type I secretion pathway in Gram-negative bacteria, were used for extracellular production of Pseudomonas fluorescens lipase (TliA) in recombinant Escherichia coli. The effect of the expression of different ABC protein exporter gene clusters (P. fluorescens tliDEF, Pseudomonas aeruginosa aprDEF, Erwinia chrysanthemi prtDEF, and Serratia marcescens lipBCD genes) was examined on the secretion of TliA at growth temperatures of 20, 25, 30 and 35 °C. TliA secretion in recombinant E. coli XL10-Gold varied depending upon type of ABC protein exporter and culture temperature. E. coli expressing S. marcescens lipBCD genes showed the highest secretion level of TliA (122.8 U ml(-1)) when cultured at 25 °C. Thus, optimized culture conditions for efficient extracellular production of lipase in recombinant E. coli can be designed by changing the type of ABC protein exporter and the growth temperature.