JBB : Journal of Bioscience and Bioengineering

Journal of Bioscience and Bioengineering vol.126 cover

 



  • Exoelectrogenic response of Pichia fermentans influenced by mediator and reactor design

    Publication date: Available online 11 December 2018

    Source: Journal of Bioscience and Bioengineering

    Author(s): Mamta Pal, Rakesh Kumar Sharma

    Microbial fuel cell is one of the most convenient and cost-effective technology for producing the clean energy. This study explores the exoelectrogenic behavior of Pichia fermentans in a microbial fuel cell. Two different reactor designs (double- and single-chambered) were tested in the presence and in the absence of a mediator (methylene blue). The influence of extracellular polymeric substances in the electricity generation has also been studied. In a double-chambered setup, maximum open circuit voltages were measured as 0.602 and 0.488 V with mediator and without mediator cell, respectively, whereas maximum power densities were measured as 1.23 μWcm−2 and 0.407 μWcm−2, respectively. In addition, maximum open circuit voltages were observed as 0.40 and 0.397 V in a single-chambered fuel cell with and without mediator, respectively. The maximum power density was recorded 1.64 μWcm−2 in the presence of a mediator, whereas the same was found as 0.643 μWcm−2 in the absence of mediator. Thus, these results indicate that P. fermentans has the ability to produce high power density under microaerophilic conditions with mediator in a single-chambered membrane less setup.

  • Cloning, expression, and characterization of novel GH5 endoglucanases from Thermobifida alba AHK119

    Publication date: Available online 2 December 2018

    Source: Journal of Bioscience and Bioengineering

    Author(s): Takeshi Ohta, Hitomi Horie, Aina Matsu-ura, Fusako Kawai

    Thermobifida alba AHK119 exhibits sufficient filter paper-degradation activity in its culture supernatant. AHK119-bMs (1365 bp) and AHK119-E5 (1425 bp), which encode novel GH5 family endoglucanases, were cloned from the genomic DNA of T. alba AHK119. AHK119-bMs and AHK119-E5 consisted of 454 and 474 amino acid residues, respectively, in which the catalytic domain (CD) and carbohydrate-binding module (CBM) were connected by an accessary module (linker region). The amino acid sequences of CD and CBM of AHK119-bMs were most identical to those of endo-β-mannanases (Man5As) from Thermobifidafusca TM51, T. halotolerans YIM90462, and T.cellulosilytica TB100. In contrast, the amino acid sequences of CD and CBM of AHK119-E5 were most identical to those of endo-1,4-β-glucanases (cellulases; Cel5As) from T. fusca and T. halotolerans YIM90462. However, the linker region of both the genes shared low identities with those of Man5As and Cel5As. AHK119-bMs showed broader specificities toward cellulosic substrates than Man5As, whereas AHK119-E5 showed higher activity toward insoluble cellulosic substrates than toward soluble ones, which was conflicting when compared with other Cel5As. In addition, AHK119-bMs and AHK119-E5 showed different requirements for metal ions from those of Man5As and Cel5As, respectively. Therefore, both the enzymes were identified as novel GH5 endoglucanases, and the accessary modules seemed to play important roles in their enzymatic properties.

  • Construction and co-cultivation of two mutant strains harboring key precursor genes to produce prodigiosin

    Publication date: December 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 6

    Author(s): Wei-Chuan Chen, Min-Jun Tsai, Po-Chi Soo, Li-Fen Wang, Shen-Long Tsai, Yu-Kaung Chang, Yu-Hong Wei

    The biosynthesis of prodigiosin (PG) from Serratia marcescens involves the coupling of a bipyrrole, 4-methoxy-2,2′-bipyrrole-5-carboxaldehyde (MBC), with a monopyrrole, 2-methyl-3-n-amyl-pyrrole (MAP), and formation of a linear tripyrrole (PG). We constructed mutant strains in which either the MBC biosynthesis by S. marcescens BMJ816 or the MAP biosynthesis by S. marcescens AMJ817. S. marcescens BMJ816 and AMJ817 confirmed that they lose the ability to synthesize PG when they are cultivated alone. An experiment was also conducted in which cultures of the two mutant strains were grown to the early exponential phase in a semi-defined medium, and one suspension culture was inoculated with the other. This approach yielded 103 mg/L PG. The findings suggest that the addition of precursors may enhance PG production by microorganisms.

  • Pilot-scale bioelectrochemical system for simultaneous nitrogen and carbon removal in urban wastewater treatment plants

    Publication date: December 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 6

    Author(s): María Isabel San-Martín, Raúl Mateos, Begoña Carracedo, Adrián Escapa, Antonio Morán

    This study aims to characterize the performance of a 150 L bioelectrochemical system-based plant, during the simultaneous carbon and nitrogen removal from several waste streams of wastewater treatment plants. The bioelectrochemical system (BES) contained five electrode pairs (operated hydraulically and electrically in parallel) and was fed with either wastewater, centrate (nutrient-rich liquid stream produced during the dewatering of digested biomass), or a mixture of both over 63 days, with a hydraulic retention time of one day. Total organic carbon and total nitrogen removal rates averaged 80% and 70%, respectively, with a specific energy consumption of 0.18 kWh·m−3 (BES + ancillary equipment). This work also underlines the challenges of using BES for nitrogen removal, highlighting the limitations of the current design, and suggesting some strategies for improvement.

  • Novel biobutanol fermentation at a large extractant volume ratio using immobilized Clostridium saccharoperbutylacetonicum N1-4

    Publication date: December 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 6

    Author(s): Rizki Fitria Darmayanti, Yukihiro Tashiro, Takuya Noguchi, Ming Gao, Kenji Sakai, Kenji Sonomoto

    Product inhibition by butanol and acetone is a known drawback in acetone-butanol-ethanol (ABE) fermentation. Extractive fermentation improves butanol production by several ABE-producing Clostridium spp., but only low volume ratios (<4) of extractant to broth (Ve/Vb) have been studied. Here, a novel extractive fermentation process was developed using Clostridium saccharoperbutylacetonicum N1-4 and a large Ve/Vb ratio. A mixture of oleyl alcohol-tributyrin (1:1 (v/v)) yielded high distribution coefficients for both butanol (3.14) and acetone (0.660). Although a fed-batch culture using free cells and the oleyl alcohol-tributyrin mixture at a Ve/Vb ratio of 5 had a lag phase of >24 h, it produced a higher concentration of total butanol (i.e., butanol produced in all the phases per broth volume used) of 24.2 g/L-broth after 96 h compared with 14.4 g/L-broth at a Ve/Vb ratio of 1, resulting in a low butanol concentration in the aqueous phase. The use of cells immobilized with calcium alginate beads shortened the lag phase to <12 h. Butanol production was achieved not only in a 3-phase mode (extractant, beads, and tryptone-yeast extract-acetate (TYA) medium) but also in a 2-phase mode (extractant and beads containing TYA medium, without an aqueous phase) at a Ve/Vb ratio of 5, resulting butanol concentrations of 30.9 g/L-broth and 27.7 g/L-broth, respectively. The 3-phases fed-batch extractive fermentation at a Ve/Vb ratio of 10 showed a better performance compared with published reports: a total butanol concentration of 64.6 g/L-broth and a butanol yield to consumed sugar of 0.378 C-mol/C-mol.

    Graphical abstract

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  • Impact of anaerobic digestion and centrifugation/decanting processes in bacterial communities fractions

    Publication date: December 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 6

    Author(s): Ana Isabel Díaz, Paula Oulego, Sergio Collado, Adriana Laca, José Manuel González, Mario Díaz

    Sewage sludge can be treated by anaerobic processes that frequently are followed by physical separation processes. In this work, a high-throughput sequencing technology, based on variation in the bacterial 16S rRNA gene, has been used to characterise the bacterial populations present in samples taken from different points of an industrial anaerobic digestion process fed with sewage sludge. Relative abundances of phyla and classes throughout the biological process and the subsequent separation steps were determined. Results revealed that the Bacteroidetes, Firmicutes and Proteobacteria phyla were the most representative. However, significant changes in relative abundance were detected along treatments, showing the influence of operational parameters on the distribution of microorganisms throughout the process. After anaerobic digestion, phylum Firmicutes doubled its relative abundance, which seems to indicate that the anaerobic conditions and the nutrients favoured its growth, in contrast to other phyla that almost disappeared. After centrifugation, Proteobacteria went preferentially to the solid phase, in contrast to Firmicutes which was the dominant phylum in the liquid phase. After decanting the liquid phase during 45 h, an important growth of Proteobacteria, Spirochaetes and Tenericutes was detected. At class level, only significantly changes were observed for Proteobacteria classes being α-proteobacteria dominant in the digestate, while γ-proteobacteria was the majority since this point to the final steps. To know the changes on the kind and abundance of microbial populations throughout the anaerobic and separation processes is very important to understand how the facilities design and operation conditions can influence over the efficiencies of next biological treatments.

    Graphical abstract

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  • Accumulation of intracellular S-adenosylmethionine increases the fermentation rate of bottom-fermenting brewer's yeast during high-gravity brewing

    Publication date: December 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 6

    Author(s): Mayu Oomuro, Daisuke Watanabe, Yukiko Sugimoto, Taku Kato, Yasuo Motoyama, Tetsuya Watanabe, Hiroshi Takagi

    High-gravity brewing has been used to reduce costs and energy, as well as to produce new types of beer with high alcohol content. To identify the key metabolic pathways underlying efficient high-gravity brewing, we explored metabolites that were highly accumulated during alcoholic fermentation under high-maltose conditions using bottom-fermenting brewer's yeast, Saccharomyces pastorianus. Based on metabolomic data, we focused on S-adenosylmethionine (SAM), which may be involved in glycolysis and alcoholic fermentation in the closely related yeast species Saccharomyces cerevisiae. Exogenous SAM led to an increase in fermentation rate in both high-maltose synthetic medium and high-gravity wort. Although SAM is composed of methionine and the adenosine moiety of ATP, neither methionine nor adenosine significantly increased the fermentation rate. These results suggest that SAM is specifically associated with the fermentation rate of bottom-fermenting brewer's yeast. Deletion of the adenosine kinase gene ADO1, which leads to an accumulation of SAM in S. cerevisiae cells, elevated the fermentation rate in high-glucose synthetic medium at 15°C; however, this ado1Δ effect became less significant at higher temperatures. Similarly, a SAM-accumulating S. pastorianus mutant strain, with enhanced resistance to the adenosine analog cordycepin, exhibited a higher fermentation rate in both high-maltose synthetic medium and high-gravity wort. Taken together, our study demonstrates that SAM acts as a positive regulator in high-gravity brewing at low temperatures and that cordycepin resistance could serve as a useful indicator for breeding S. pastorianus strains with high fermentation performance.

  • Function of three RuBisCO enzymes under different CO2 conditions in Hydrogenovibrio marinus

    Publication date: December 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 6

    Author(s): Koichi Toyoda, Masaharu Ishii, Hiroyuki Arai

    The obligate chemolithoautotrophic bacterium, Hydrogenovibrio marinus MH-110 has three ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) isoenzymes, designated CbbLS-1, CbbLS-2, and CbbM, which are encoded by the cbbL1S1, cbbL2S2, and cbbM genes, respectively. Functions of these isoenzymes at different CO2 concentrations were investigated using deletion mutants of their genes. Deletion of cbbL1 had no effect on cell growth under any of the test growth conditions. The cbbL2 mutant was unable to grow under lower (≤0.15%) CO2 conditions, though it grew normally under higher (≥2%) CO2 conditions. Growth of the cbbM mutant was retarded under higher CO2 conditions but was not affected by lower CO2 conditions. These results indicate that CbbLS-2 and CbbM specifically function under lower and higher CO2 conditions, respectively. The growth retardation of the cbbL2 and cbbM mutants was not restored by complementation with plasmids carrying the cbbL2S2 and cbbM genes, respectively. The cbbL2S2 and cbbM genes are followed by the carboxysome genes and the cbbQmOm genes, respectively. Co-expression of these downstream genes was probably necessary for the in vivo function of CbbLS-2 and CbbM. CbbLS-1 was upregulated in the cbbL2 and cbbM mutants under the lower and higher CO2 conditions, respectively, indicating that the expression of cbbL1S1 was controlled to compensate the deficiency of the other RuBisCO isoenzymes.

  • Production of fucoxanthin, chrysolaminarin, and eicosapentaenoic acid by Odontella aurita under different nitrogen supply regimes

    Publication date: December 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 6

    Author(s): Song Xia, Baoyan Gao, Jiaqi Fu, Jihai Xiong, Chengwu Zhang

    Microalgae are recognized as promising producers of many bioactive products, but their utility is limited due to high production costs. We subjected the marine diatom Odontella aurita to three nitrogen supply regimes [initial low nitrogen (ILN), initial high nitrogen (IHN), and initial high nitrogen plus supplementary nitrogen (SN)] to investigate the accumulation of three high-value bioactive components: fucoxanthin, chrysolaminarin and eicosapentaenoic acid (EPA). We found that SN conditions maximized fucoxanthin accumulation: a maximum productivity of 6.01 mg L−1 d−1 was obtained, a 4.32-fold and 1.42-fold increase over production in the ILN and IHN groups, respectively. After nitrogen was depleted in the growth medium, chrysolaminarin became the dominant energy storage compound. Chrysolaminarin content rose to 60.33% of dry weight (DW) in the ILN group, and 46.27% of DW in the IHN group. Variations in fatty acid composition across the different nitrogen supply regimes indicated that EPA primarily accumulated in the glycolipids, especially when nitrogen supply was sufficient. The maximum productivity of chrysolaminarin (161.55 mg L−1 d−1) and EPA (9.37 mg L−1 d−1) was observed in the IHN group. However, IHN conditions did not maximize overall content of either compound. Our results demonstrated that O. aurita is potentially useful as a producer of a variety of bioactive products; the compounds produced by this species can be controlled by altering the nitrogen supply.

  • Ergothioneine production using Methylobacterium species, yeast, and fungi

    Publication date: December 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 6

    Author(s): Yoshiko Fujitani, Kabir Md Alamgir, Akio Tani

    Ergothioneine (EGT) is a sulfur-containing, anti-oxidative amino acid derived from histidine. EGT is synthesized in bacteria and fungi but not in animals and plants, and is now recognized as important for human health. Its cost-effective fermentative production has not been elucidated due to the lack of information for productive microorganisms. In this study, we doubled the gene copy for EGT synthesis and deleted the histidine ammonia-lyase gene in a potent EGT-producing methylotrophic bacterium Methylobacterium aquaticum strain 22A, and optimized its culture conditions, resulting in increased EGT production of 7.0 mg EGT/g dry cell weight and 100 μg EGT/5 mL/7 days. In addition, through screening we found EGT-producing eukaryotic strains of Aureobasidium pullulans and Rhodotorula mucilaginosa, which can produce 1.0 and 3.2 mg EGT/g dry cell weight, 70 and 120 μg EGT/5 mL/7 days, respectively. This study proposes practical uses of potent EGT-producing recombinant Methylobacterium species and non-recombinant yeast and fungal strains.

  • Characteristics of hydrogen-producing enrichment cultures from marine sediment using macroalgae Laminaria japonica as a feedstock

    Publication date: December 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 6

    Author(s): Hongyan Liu, Haihua Qin, Hongyu Wang

    This study aimed to investigate the characteristics of hydrogen production by mixed cultures using Laminaria japonica hydrolysates. The hydrolysates of L. japonica were prepared by pretreatment methods, including heat (100°C or 121°C) and acid (HCl or H2SO4) pretreatments. The mixed cultures could produce hydrogen using L. japonica as a substrate, with the highest cumulative hydrogen production of 825 ± 14 mL/L from HCl-pretreated L. japonica. High-throughput sequencing of the 16S rRNA gene revealed that the microbial community in the hydrolysate of HCl-pretreated L. japonica was the most diverse among all the samples, with a Shannon diversity index of 5.253. The mixed culture from HCl-pretreated L. japonica and those from heat-pretreated (100°C and 121°C) L. japonica occupied different regions in a principal component analysis (PCA) plot. The dominant population in the hydrolysate of HCl-pretreated L. japonica was represented by hydrogen-producing bacteria, Clostridium spp. and Bacillus spp. The results suggested that L. japonica was an optimal feedstock for hydrogen production. The acid (HCl) pretreatment method could effectively enhance the hydrogen production from L. japonica.

  • SKIK-zipbody-alkaline phosphatase, a novel antibody fusion protein expressed in Escherichia coli cytoplasm

    Publication date: December 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 6

    Author(s): Panwad Ritthisan, Teruyo Ojima-Kato, Jasmina Damnjanović, Takaaki Kojima, Hideo Nakano

    Antibody-enzyme fusion proteins have been used for various immunological detection techniques, such as ELISA, Western blotting and so on. The use of genetically-fused antibody-enzyme complexes has advantages over conventional chemical conjugation methods, as they require no complex chemical reactions and allow for the strict control of the number of enzymes fused with antibodies, resulting in a more stable performance of the bifunctional protein. Here, we describe efficient cytoplasmic soluble expression of an antigen-binding fragment (Fab) fused with Escherichia coli alkaline phosphatase (AP), N-terminal Ser-Lys-Ile-Lys (SKIK) tag that can improve the synthesis of the tagged protein, as well as leucine zipper (LZ) to enhance the association of the light chain and the heavy chain of Fab. Our results demonstrated that the SKIK-Fab-LZ-AP fusion was well expressed in E. coli oxidative cytoplasm in soluble form having both antigen-binding and AP activity, and was purified to homogeneity by two step column chromatography, suggesting that the combination of the SKIK tag and AP fusion can greatly increase the productivity and solubility of the Fab-enzyme fusion in an E. coli cytoplasmic expression system.

  • Evaluation of β-galactosidase from Lactobacillus acidophilus as biocatalyst for galacto-oligosaccharides synthesis: Product structural characterization and enzyme immobilization

    Publication date: December 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 6

    Author(s): Milica Carević, Maja Vukašinović-Sekulić, Marija Ćorović, Hélène Rogniaux, David Ropartz, Dušan Veličković, Dejan Bezbradica

    β-Galactosidase is an important industrial enzyme that catalyzes reaction of lactose hydrolysis and recently more interesting reaction of transgalactosylation, yielding a highly valuable group of prebiotic compounds named galacto-oligosaccharides (GOS). In this paper, parameters for achieving high yields of tailor-made GOS using crude β-galactosidase obtained from Lactobacillus acidophilus ATCC 4356, probiotic bacteria regarded as safe for human consumption, were optimized. At the same time, detailed structural elucidation of obtained GOS was conducted, and it was concluded that β-galactosidase from L. acidophilus shows a particular specificity towards the formation of β-(1→6) glycosidic bonds. In order to develop more stable and economically cost-effective preparation, crude enzyme was successfully immobilized on a methacrylic polymer carrier Lifetech ECR8409, leading to its simultaneous 2-fold purification. This immobilized preparation showed unchanged specificity towards the transgalactosylation reaction, thus yielding 86 g/l GOS under the previously optimized conditions (lactose concentration 400 g/l in 0.1 M sodium phosphate buffer, pH 6.8 and temperature 50°C).

    Graphical abstract

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  • Expression and characterization of a pectin methylesterase from Aspergillus niger ZJ5 and its application in fruit processing

    Publication date: December 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 6

    Author(s): Zhiwei Zhang, Junshuai Dong, Deqing Zhang, Jiaojiao Wang, Xing Qin, Bo Liu, Xinxin Xu, Wei Zhang, Yuhong Zhang

    A pectin methylesterase gene, pme-zj5a, from Aspergillus niger ZJ5 was cloned and high-level expressed in Pichia pastoris. The highest PME activity was 71.11 U/ml after induction with methanol for 20 h at 30 °C. The molecular mass of purified PME-ZJ5A was estimated to be 37 kDa by SDS-PAGE, and its Km, Vmax and kcat values of PME-ZJ5A were determined to be 3.27 mg/ml, 5.36 μmol/min/mg, and 22.33 s−1 with pectin. Purified recombinant PME-ZJ5A exhibited optimal activity at pH 3.8 and 45 °C. It retained more than 60% of its maximum activity at 10 °C. Moreover, recombinant PME-ZJ5A can increase the transmittance of pineapple juice by 60.8%, and increase the firmness of pineapple cubes nearly double when combined with CaCl2, which showed good potential in fruit processing.

  • Overexpression of PkINO1 improves ethanol resistance of Pichia kudriavzevii N77-4 isolated from the Korean traditional fermentation starter nuruk

    Publication date: December 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 6

    Author(s): Minetaka Sugiyama, Seong Yeol Baek, Shohei Takashima, Natsumi Miyashita, Kei Ishida, Jiyoung Mun, Soo-Hwan Yeo

    The yeast Pichia kudriavzevii N77-4 was isolated from the Korean traditional fermentation starter nuruk. In this study, fermentation performance and stress resistance ability of N77-4 was analyzed. N77-4 displayed superior thermotolerance (up to 44°C) in addition to enhanced acetic acid resistance compared to Saccharomyces cerevisiae. Moreover, N77-4 produced 7.4 g/L of ethanol with an overall production yield of 0.37 g/g glucose in 20 g/L glucose medium. However, in 250 g/L glucose medium the growth of N77-4 slowed down when the concentration of ethanol reached 14 g/L or more and ethanol production yield also decreased to 0.30 g/g glucose. An ethanol sensitivity test indicated that N77-4 was sensitive to the presence of 1% ethanol, which was not the case for S. cerevisiae. Furthermore, N77-4 displayed a severe growth defect in the presence of 6% ethanol. Because inositol biosynthesis is critical for ethanol resistance, expression levels of the PkINO1 encoding a key enzyme for inositol biosynthesis was analyzed under ethanol stress conditions. We found that ethanol stress clearly repressed PkINO1 expression in a dose-dependent manner and overexpression of PkINO1 improved the growth of N77-4 by 19% in the presence of 6% ethanol. Furthermore, inositol supplementation also enhanced the growth by 13% under 6% ethanol condition. These findings indicate that preventing downregulation in PkINO1 expression caused by ethanol stress improves ethanol resistance and enhances the utility of P. kudriavzevii N77-4 in brewing and fermentation biotechnology.

  • Identification of the gene PtMAT1 encoding acetyltransferase from the diastereomer type of mannosylerythritol lipid-B producer Pseudozyma tsukubaensis

    Publication date: December 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 6

    Author(s): Azusa Saika, Yu Utashima, Hideaki Koike, Shuhei Yamamoto, Takahide Kishimoto, Tokuma Fukuoka, Tomotake Morita

    Mannosylerythritol lipids (MELs) are biosurfactants produced from feedstocks by basidiomycetous yeasts. MELs exhibit different properties depending on their structures, such as the degree of acetylation or acylation and the chirality of the mannosylerythritol moiety. Pseudozyma tsukubaensis produces a diastereomer type of MEL-B (mono-acetylated MEL); therefore, deletion of an acetyltransferase could yield a diastereomer type of MEL-D (deacetylated MEL), which has only been produced in in vitro reactions of lipase using MEL-B as a substrate. Here, we deleted the gene PtMAT1 in P. tsukubaensis 1E5 encoding an acetyltransferase related to MEL biosynthesis via targeted gene deletion and generated a producer of the diastereomer type of MEL-D. The uracil auxotrophic mutant of P. tsukubaensis 1E5 (PtURA5-mutant) was used as a host strain for gene deletion. The gene PtMAT1 was replaced with a PtURA5 cassette by homologous recombination using uracil auxotrophy as a selectable marker. According to thin-layer chromatography and nuclear magnetic resonation spectroscopy, we identified the strain ΔPtMAT1 as a producer of the diastereomer type of MEL-D instead of MEL-B.

  • Genomic characterization of methylotrophy of Oharaeibacter diazotrophicus strain SM30T

    Publication date: December 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 6

    Author(s): Haoxin Lv, Akio Tani

    Oharaeibacter diazotrophicus strain SM30T, isolated from rice rhizosphere, is an aerobic, facultative lanthanide (Ln3+)-utilizing methylotroph and diazotroph that belongs to the Methylocystaceae family. In this research, the complete genome sequence of strain SM30T was determined, and its methylotrophy modules were characterized. The genome consists of one chromosome and two plasmids, comprising a total of 5,004,097 bp, and the GC content was 71.6 mol%. A total of 4497 CDSs, 67 tRNA, and 9 rRNA were encoded. Typical alpha-proteobacterial methylotrophy genes were found: pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH) (mxaF and xoxF1-4), methylotrophy regulatory proteins (mxbDM and mxcQE), PQQ synthesis, H4F pathway, H4MPT pathway, formate oxidation, serine cycle, and ethylmalonyl-CoA pathway. SDS-PAGE and subsequent LC–MS analysis, and qPCR analysis revealed that MxaF and XoxF1 were the dominant MDH in the absence or presence of lanthanum (La3+), respectively. The growth of MDH gene-deletion mutants on alcohols and qPCR results indicated that mxaF and xoxF1 are also involved in ethanol and propanol oxidation, xoxF2 participates in methanol oxidation in the presence of La3+, while xoxF3 was associated with methanol and ethanol oxidation in the absence of La3+, implying that XoxF3 is a calcium (Ca2+)-binding XoxF. Four Ln3+ such as La3+, cerium (Ce3+), praseodymium (Pr3+), and neodymium (Nd3+) served as cofactors for XoxF1 by supporting ΔmxaF growth on methanol. Some heavier lanthanides inhibited growth of SM30 on methanol. This study contributes to the understanding of the function of various XoxF-type MDHs and their roles in methylotrophs.

  • Performance and dynamic characteristics of microbial communities in multi-stage anaerobic reactors treating gibberellin wastewater

    Publication date: Available online 1 December 2018

    Source: Journal of Bioscience and Bioengineering

    Author(s): Erming Ouyang, Yao Lu, Jiating Ouyang, Lele Wang, Xiaohui Wang

    To treat gibberellin (GA) wastewater, a full-scale, multi-stage combined contact process was developed. This whole process employs three anaerobic reactors followed by micro-aerobic, anoxic/aeration and biological oxidation treatment. Pollutant removal results showed that the combined process could remove more than 98% of the chemical oxygen demand (COD), NH3N, and SO42− pollutants because of the different microbial communities in each reactor. 16S rRNA gene sequencing was used to examine the microbial communities in the internal circulation (IC) and in the two up flow anaerobic sludge blanket (UASB) reactors, as well as to investigate the effect of sampling elevation on the microbial community. The results showed that Firmicutes and Euryarchaeota were the most dominant phyla at the bacterial and archaeal levels, respectively. High levels of Synergistaceae_uncultured were detected in IC and UASB1. Chloroflexi_uncultured was the dominant genus of bacterial communities within UASB2, and Methanosaeta was the dominant genus of archaeal communities. Principal coordinates analysis (PCoA) revealed variations among the microbial communities in 9 samples, and Venn analysis showed different operational taxonomic units (OTUs) among samples collected at various elevations within the three anaerobic reactors. However, partial Mantel tests indicated no significant correlation between the microbial community structure and elevation in the three anaerobic reactors.

  • Combinational biosynthesis and characterization of fusion proteins with tandem repeats of allophycocyanin holo-α subunits, and their application as bright fluorescent labels for immunofluorescence assay

    Publication date: December 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 6

    Author(s): Huaxin Chen, Peng Jiang

    Fusion protein of streptavidin and allophycocyanin holo-α subunit (ApcA) is fluorescent and is able to bind biotin. This fusion protein (SLA) can be used as fluorescent label in immunofluorescence assay. In this study, one or two repeats of ApcA were fused to the protein SLA, with the aim to improve its brightness. The fusion proteins SLA2 (two repeats of ApcA) and SLA3 (three repeats of ApcA), together with lyase (cpcS) and phycoerythrobilin synthesizing enzymes (Ho1 and PebS), were co-expressed in Escherichia coli. These fusion proteins were purified by affinity chromatography. While SLA2 and SLA3 shared similar absorbance spectra, fluorescence spectra and biotin-binding activities with SLA, their brightness were much higher than that of SLA. When used as fluorescent labels in immunofluorescence assay, SLA2 and SLA3 showed higher detection sensitivity than SLA. These results suggested that SLA2 and SLA3 were the preferable fluorescent labels in immunofluorescence assays.

  • NADPH supply for poly(3-hydroxybutyrate) synthesis concomitant with enzymatic oxidation of phosphite

    Publication date: December 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 6

    Author(s): Yuki Miyahara, Mino Oota, Takeharu Tsuge

    Acetoacetyl-CoA reductase (PhaB), involved in poly(3-hydroxybutyrate) [P(3HB)] biosynthesis, requires the coenzyme NADPH as a reducing agent. In this study, the effect of NADPH supply on P(3HB) production was investigated in vitro and in vivo using a phosphite dehydrogenase double mutant (PtxDEAAR), which catalyzes oxidation of phosphite to phosphate with the generation of NADH and NADPH. In an in vitro assay using purified enzymes, P(3HB) polymerization was observed only when phosphite and PtxDEAAR were present, confirming that NADPH was supplied to PhaB. In an in vivo assay using Escherichia coli as a production host for P(3HB), the presence of phosphite and PtxDEAAR did not influence the yield of P(3HB) under normal growth conditions. However, P(3HB) yield increased 3.2-fold in non-growing E. coli cells compared to the control, suggesting that PtxDEAAR-mediated NADPH generation is coupled with P(3HB) biosynthesis. This study confirmed the use of PtxDEAAR for supplying NADPH during P(3HB) synthesis in vitro and in vivo.