All lactose-oxidizing enzymes of Pseudomonas taetrolens, a highly efficient lactobionic acid-producing microorganism, are pyrroloquinoline quinone-dependent enzymes.

In previous and present studies, four enzymes (GCD1, GCD3, GCD4, and MQO1) have been found to act as lactose-oxidizing enzymes of Pseudomonas taetrolens. To investigate whether the four enzymes were the only lactose-oxidizing enzymes of P. taetrolens, we performed the inactivation of gcd1, gcd3, gcd4, and mqo1 genes in P. taetrolens. Compared to the wild-type strain, the lactobionic acid (LBA)-producing ability of P. taetrolens ∆gcd1 ∆gcd3 ∆gcd4 ∆mqo1 was only slightly decreased, implying that P. taetrolens possesses more lactose-oxidizing enzymes. Interestingly, the four lactose-oxidizing enzymes were all pyrroloquinoline quinone (PQQ)-dependent. To identify other unidentified lactose-oxidizing enzymes of P. taetrolens, we prevented the synthesis of PQQ in P. taetrolens by inactivating the genes related to PQQ synthesis such as pqqC, pqqD, and pqqE. Surprisingly, all three knocked-out strains were unable to convert lactose to LBA, indicating that all lactose-oxidizing enzymes in P. taetrolens were inactivated by eliminating PQQ synthesis. In addition, external PQQ supplementation restored the LBA production ability of P. taetrolens ∆pqqC, comparable to the wild-type strain. These results indicate that all lactose-oxidizing enzymes in P. taetrolens are PQQ-dependent.

Efficient production of cellobionic acid using whole-cell biocatalyst of genetically modified Pseudomonas taetrolens.

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 OD600nm. Under these conditions, 200 g/L of cellobiose was all converted to CBA in 11 h, and the WCB of P. taetrolens [pDSK-GDH] maintained the maximum catalytic activity during at least six cycles without a significant decline in the productivity. Our results suggest that the manufacture of WCB based on genetically engineered P. taetrolens and its optimized use could be further developed as an economically viable option for the large-scale production of CBA.

Whole-cell biocatalysis using genetically modified Pseudomonas taetrolens for efficient production of maltobionic acid from pure maltose and high-maltose corn syrup.

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.

Efficient Production of Lactobionic Acid Using Escherichia coli Capable of Synthesizing Pyrroloquinoline Quinone.

Lactobionic acid (LBA) is an emerging chemical that has been widely utilized in food, cosmetic, and pharmaceutical industries. We sought to produce LBA using Escherichia coli. LBA can be produced from lactose in E. coli, which is innately unable to produce LBA, by coexpressing a heterologous quinoprotein glucose dehydrogenase (GDH) and a pyrroloquinoline quinone (PQQ) synthesis gene cluster. Using a recombinant E. coli strain, we successfully produced LBA without additional supplementation of PQQ, and changing the type of heterologous GDH improved the LBA production titer and productivity. To further enhance LBA production, culture conditions, such as growth temperature and isopropyl-ß-d-1-thiogalactopyranoside concentration, were optimized. Using optimized culture conditions, batch fermentation of the recombinant E. coli strain was performed using a 5 L bioreactor. After fermentation, this strain produced an LBA titer of 209.3 g/L, a yield of 100%, and a productivity of 1.45 g/L/h. To our best knowledge, this is the first study to produce LBA using heterologous GDH in an E. coli strain without any additional cofactors. Our results provide a simple method to produce LBA from lactose in a naturally non-LBA-producing bacterium and lay the groundwork for highly efficient LBA production in E. coli, which is one of the most versatile metabolite-producing bacterial hosts.

Efficient isolation of new lactobionic acid-producing microorganisms from environmental samples by colloidal calcium carbonate agar plate-based screening.

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 (CaCO3), resulting in the formation of a clear halo around colonies on a nutrient broth agar plate containing CaCO3. Subsequently, LBA production from the isolated microorganisms was confirmed using high-performance liquid chromatography (HPLC). Approximately 560 colonies from soil samples in Ulsan, Korea were screened and a clear halo was observed around three colonies on the prepared LBA-screening agar plate. The culture supernatants of these three colonies were analyzed by HPLC and it was found that these strains could produce LBA from lactose. Phylogenetic analysis by comparing their 16S rRNA nucleotide sequences revealed that these strains were Pseudomonas spp. and Alcaligenes faecalis. This is the first report highlighting that A. faecalis can produce LBA. As per the aforementioned results, the LBA-screening method that we devised here is highly effective for isolating and identifying new LBA-producing microorganisms.

Secretory production of an engineered cutinase in Bacillus subtilis for efficient biocatalytic depolymerization of polyethylene terephthalate.

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.

Isolation of new lactobionic acid-producing microorganisms and improvement of their production ability by heterologous expression of glucose dehydrogenase from Pseudomonas taetrolens.

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.

Identification of a lactose-oxidizing enzyme in Escherichia coli and improvement of lactobionic acid production by recombinant expression of a quinoprotein glucose dehydrogenase from Pseudomonas taetrolens.

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-ß-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.

Enhancement of sophorolipids production in Candida batistae, an unexplored sophorolipids producer, by fed-batch fermentation.

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.

Valorization of waste-cooking oil into sophorolipids and application of their methyl hydroxyl branched fatty acid derivatives to produce engineering bioplastics.

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.

High-level production of maltobionic acid from high-maltose corn syrup by genetically engineered Pseudomonas 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.

Purification and Characterization of a Malate:Quinone Oxidoreductase from Pseudomonas taetrolens Capable of Producing Valuable Lactobionic Acid.

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.

Enhancement of Lactobionic Acid Productivity by Homologous Expression of Quinoprotein Glucose Dehydrogenase in Pseudomonas taetrolens.

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.

Efficient production of lactobionic acid using genetically engineered Pseudomonas taetrolens as a whole-cell biocatalyst.

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.

Mycoplasma hyorhinis is a potential pathogen of porcine respiratory disease complex that aggravates pneumonia caused by porcine reproductive and respiratory syndrome virus.

The porcine respiratory disease complex (PRDC) caused by numerous bacterial and viral agents has a great impact on pig industry worldwide. Although Mycoplasma hyorhinis (Mhr) has been frequently isolated from lung lesions from pigs with PRDC, the pathological importance of Mhr may have been underestimated. In this study, 383 serum samples obtained from seven herds with a history of PRDC were tested for specific antibodies to Mhr, Mycoplasma hyopneumoniae (Mhp), and porcine reproductive and respiratory syndrome virus (PRRSV). Seropositive rates of PRRSV were significantly correlated with those of Mhr (correlation coefficient, 0.862; P-value, 0.013), but not with those of Mhp (correlation coefficient, -0.555; P-value, 0.196). In vivo experiments demonstrated that pigs co-infected with Mhr and PRRSV induced more severe lung lesions than pigs infected with Mhr or PRRSV alone. These findings suggest that Mhr is closely associated with pneumonia caused by PRRSV and provide important information on Mhr pathogenesis within PRDC. Therefore, effective PRDC control strategies should also consider the potential impact of Mhr in the pathogenesis of PRDC.

Production of biodiesel from carbon sources of macroalgae, Laminaria japonica.

As aquatic biomass which is called "the third generation biomass", Laminaria japonica (also known as Saccharina japonica) consists of mannitol and alginate which are the main polysaccharides of algal carbohydrates. In this study, oleaginous yeast (Cryptococcus curvatus) was used to produce lipid from carbon sources derived from Laminaria japonica. Volatile fatty acids (VFAs) were produced by fermentation of alginate extracted from L. japonica. Thereafter, mannitol was mixed with VFAs to culture the oleaginous yeast. The highest lipid content was 48.30%. The composition of the fatty acids was similar to vegetable oils. This is the first confirmation of the feasibility of using macroalgae as a carbon source for biodiesel production.

Efficacy of a commercial live attenuated Lawsonia intracellularis vaccine in a large scale field trial in Korea.

PURPOSE: Porcine proliferative enteropathy (PPE) is known as one of the most important risk factors causing economic losses in swine industry worldwide. This study was conducted to evaluate the efficacy of a commercial oral attenuated Lawsonia intracellularis vaccine (Enterisol Ileitis) against PPE under a commercial pig farm condition in Korea. MATERIALS AND METHODS: Thirty two-day-old 672 piglets were randomly allocated into vaccinated and control groups. All piglets in the vaccinated group were inoculated with a commercial attenuated L. intracellularis vaccine as following the manufacturer's instruction. Body weights of all pigs in both groups were measured on the vaccination day and 6, 14, and 20 weeks post vaccination and an average daily weight gain (ADWG) was calculated. Health status was observed biweekly during the whole trial. RESULTS: The vaccinated group showed significantly higher body weight (p<0.05) and ADWG (p<0.05) than those of the control group. The vaccinated group had significantly reduced impairments in activity, growth, defecation frequency, and stool hardness (p<0.05). Additional health benefits and improved weight gain by the vaccination produced a 4.2:1 return of investment, and the higher gross margin was $4.80 per pig. CONCLUSION: Our finding suggests that the L. intracellularis vaccine program has effects on the substantial health and economic benefits in the Korean swine industry.