Preface for special issue on industrial biology (2019) / 生物工程学报

Industrial biotechnology promises to make a significant contribution in enabling the sustainable development, and need the solid support from its basic discipline. As the basis of industrial biotechnology, industrial biology is to study the basic laws and mechanisms of biological behavior in industrial environment and to solve the key scientific problems for understanding, designing and constructing the organisms adapted to the application of industrial environment. In order to comprehend the status of industrial biology, we published this special issue to review the progress and trends of industrial biology from the three aspects of industrial protein science, cell science and fermentation science, respectively, for laying the foundation for the development of industrial biotechnology.

Effect of calcium on sporulation of Taiwanofungus camphoratus in submerged fermentation / 生物工程学报

Taiwanofungus camphoratus is a valuable and rare medicinal mushroom with various bioactivities, such as liver protection and anti-cancer. T. camphoratus can produce many arthroconidia at the end of submerged fermentation, but molecular mechanism underlying this submerged conidiation remains unknown. In this study, we found that Ca²⁺ concentration in culture medium significantly affected the arthroconidium production of T. camphoratus. Then, we identified two proteins (CaM and HSP90) involved in Ca²⁺/calmodulin signaling pathway and one protein (AbaA) involved in FluG-mediated conidiation pathway by two-dimensional electrophoresis analyses. Furthermore, we proposed a Ca²⁺/calmodulin- and FluG-mediated signaling pathway by bioinformatics analysis. By real-time quantitative PCR analyses of 23 key genes in the Ca²⁺/calmodulin- and FluG-mediated conidiation pathway, we found that expression levels of 7 genes (crz1, hsp90, flbB, brlA, abaA, wetA and fadA) showed significant responses to Ca²⁺ concentration in fermentation medium. Our research is beneficial for elucidating the underlying mechanism of submerged fermentation conidiation for T. camphoratus.

CT analysis of pulmonary cryptococcosis / 实用放射学杂志

Objective To review and analyse the CT distinguishing features of pulmonary cryptococcosis (PC)for improving the level of image diagnosis of the disease.Methods CT images and clinical details of 19 patients with biopsy or surgery pathology con-firmed PC were reviewed and analysed.Results Nodules or masses were presented in 10 cases,in which most casescases were nod-ules,lesions ranged in diameter from 1 cm to 2.5 cm.Multiple nodules were easy to fuse.Consolidation were presented in 6 cases,with high attenuation and clear border.3 cases were mixed type,with nodule,mass consolidation and ground glass attenuation.The lesions were mostly located in the inferior lobes(47%)and peripheral lung(68%).The features of pleural thickening,air bronchus sign,halo sign and cavitations were observed in 42%,36%,26%,21% of patients respectively.Conclusion CT findings of PC are diversity.The lesions are mostly located in the inferior and peripheral lung.Lesions are mild to moderate enhanced.Pleural thickening,air bronchus sign,halo sign,cavitations will be useful for the diagnosis.

[Construction of expression vectors for alkaliphilic Bacillus sp. N16-5].

OBJECTIVE: To construct an expression vector for alkaliphilic Bacillus sp. N16-5. METHODS: Bacillus subtilis expression vector pHCMC04 was used as a backbone. Its xylose-inducible promoter cassette was replaced by the constitutive promoters P43 (from B. subtilis) and P(EF) (from Bacillus sp. N165), separately, resulting in two expression vectors pABN165P43 and pABN165P(EF). Green fluorescent protein gene gfp was linked to the two vectors as a reporter gene. Fluorescence microscope and multifunctional fluorescent reader were used to test the expression efficiency of the system. RESULTS: Green fluorescence was visualized in Bacillus sp. N16-5 with pABN165P(EF)-gfp or pABN165P43-gfp. Quantitative data analysis revealed that fluorescence was first detected around the 7th hour. The fluorescence intensity increased rapidly from the 7th hour to the 12th hour and reached the maximum at about the 12th hour. CONCLUSION: Two expression vectors for Bacillus sp. N16-5 have been constructed, allowing expression of exogenous protein in alkaliphilic Bacillus sp. N165.

Progress in the thermophilic and alkalophilic xylanases / 生物工程学报

Xylanase is the key enzyme to degrade xylan that is a major component of hemicellulose. The enzyme has potential industrial applications in the food, feed, paper and flax degumming industries. The use of xylanases becomes more and more important in the paper industry for bleaching purposes. Xylanases used in the pulp bleaching process should be stable and active at high temperature and alkaline pH. Thermophilic and alkalophilic xylanases could be obtained by screening the wild type xylanases or engineering the mesophilic and neutral enzymes. In this paper, we reviewed recent progress of screening of the thermophilic and alkalophilic xylanases, molecular mechanism of thermal and alkaline adaptation and molecular engineering. Future research prospective was also discussed.

Improving thermal stability of xylanase by introducing aromatic residues at the N-terminus / 生物工程学报

Thermophilic and alkalophilic xylanases have great potential in the pulp bleaching industry. In order to improve the thermal stability of an alkaline family 11 xylanase Xyn11A-LC, aromatic residues were introduced into the N-terminus of the enzyme by rational design. The mutant increased the optimum temperature by 5 degrees C. The wild type had a half-time of 22 min at 65 degrees C and pH 8.0 (Tris-HCl buffer). Under the same condition, the mutant had the half-time of 106 min. CD spectroscopy revealed that the melting temperature (T(m)) values of the wild type and mutant were 55.3 degrees C and 67.9 degrees C, respectively. These results showed that the introduction of aromatic residues could enhance the thermal stability of Xyn11A-LC.

Deficiency of succinic dehydrogenase or succinyl-coA synthetase enhances the production of 5-aminolevulinic acid in recombinant Escherichia coli / 生物工程学报

5-aminolevulinic acid (ALA), a precursor for biosynthesis of pyrrole compounds in living organisms, has been widely used in agriculture and medical photodynamics therapy and is regarded as a promising value-added bio-based chemical. In the previous investigations on ALA production with recombinant Escherichia coli expressing heterogenous C4 pathway gene, LB media supplemented with glucose and ALA precursors succinate and glycine is widely used, leading to high production cost. Succinate participates in ALA biosynthesis in a form of succinyl-CoA. In this study, genes involved in succinyl-CoA consumption, sdhAB (encoding succinic dehydrogenase) or sucCD (encoding succinyl-CoA synthetase) of E. coli MG1655 was knocked out and tested for ALA accumulation. In comparison with the recombinant E. coli strain expressing heterogenous ALA synthetase, the sdhAB- or sucCD-deficient strain accumulate 25.59% and 12.40%, respectively, more ALA in a 5 L fermentor using a defined synthetic medium with glucose as main carbon source and without supplementation of succinate, providing a novel cost-effective approach for industrial production of ALA.

Biosynthesis of adipic acid / 生物工程学报

Adipic acid is a six-carbon dicarboxylic acid, mainly for the production of polymers such as nylon, chemical fiber and engineering plastics. Its annual demand is close to 3 million tons worldwide. Currently, the industrial production of adipic acid is based on the oxidation of aromatics from non-renewable petroleum resources by chemo-catalytic processes. It is heavily polluted and unsustainable, and the possible alternative method for adipic acid production should be developed. In the past years, with the development of synthetic biology and metabolic engineering, green and clean biotechnological methods for adipic acid production attracted more attention. In this study, the research advances of adipic acid and its precursor production are reviewed, followed by addressing the perspective of the possible new pathways for adipic acid production.

Trends in polymer-grade L-lactic acid fermentation by non-food biomass / 生物工程学报

Lactic acid has a wide range of uses in the chemical, pharmaceutical and food industry. With rapid development of poly (lactic acid) industry, the demand for polymer-grade L-lactic acid is continuously increasing. Developing low-cost, non-food-biomass-lactic-acid fermentation process and the fermentation-separation coupled technology are trends to reduce polymer-grade L-lactic acid production cost. This review summarized the most recent advances in low-cost L-lactic acid fermentation based on the use of non-food biomass, followed by addressing the key issue that might be strategically important for future development of polymer-grade L-lactic acid production in industry.

Biochemical characterization and substrate profile of a highly enantioselective carbonyl reductase from Pichia pastoris / 生物工程学报

Carbonyl reductases catalyze carbonyl compounds to chiral alcohols that are important building blocks in fine chemical industry. To study carbonyl reductase from Pichia pastoris GS115 (ppcr), we discovered a new gene (ppcr) encoding an NADPH-dependent carbonyl reductase by genomic data mining. It was amplified by PCR from the genomic DNA, and expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to homogeneity. The optimum temperature was 37 degrees C and the optimum pH of PPCR was 6.0. PPCR was stable below 45 degrees C. The Km and k(cat) value of the enzyme for ethyl 3-methyl-2-oxobutanoate were 9.48 mmol/L and 0.12 s, respectively. The enzyme had broad substrate specificity and high enantioselectivity. It catalyzed the reduction of aldehydes, a-ketoesters, beta-ketoesters and aryl ketones to give the corresponding alcohols with >97% ee with only a few exceptions, showing its application potential in the synthesis of chiral alcohols.

Cloning and characterization of a novel carbonyl reductase for asymmetric reduction of bulky diaryl ketones / 生物工程学报

Asymmetric reduction of bulky diaryl ketones is still one of the challenging tasks in biocatalysis. By genomic data mining, a putative carbonyl reductase gene pascr was found in Pichia pastoris GS115. pascr was cloned and over-expressed in Escherichia coli Rosseta2 (DE3). The recombinant enzyme was purified to homogeneity by Ni-NTA column and its catalytic properties were studied. PasCR strictly used NADPH as cofactor, gel filtration and SDS-PAGE analysis suggested that the native form of PasCR was a dimmer. PasCR exhibited the highest activity at 35 degrees C in phosphate buffer at pH 6.5. The enzyme catalyzed the reduction of some bulky diaryl ketones, such as 4-methylbenzophenone, 2-methylbenzophenone and 4-chlorobenzophenone, especially for 4-methylbenzophenone, the product S--alcohol was obtained with 85% ee.

Crystal structures of D-psicose 3-epimerase from Clostridium cellulolyticum H10 and its complex with ketohexose sugars

D-psicose 3-epimerase (DPEase) is demonstrated to be useful in the bioproduction of D-psicose, a rare hexose sugar, from D-fructose, found plenty in nature. Clostridium cellulolyticum H10 has recently been identified as a DPEase that can epimerize D-fructose to yield D-psicose with a much higher conversion rate when compared with the conventionally used DTEase. In this study, the crystal structure of the C. cellulolyticum DPEase was determined. The enzyme assembles into a tetramer and each subunit shows a (β/α)(8) TIM barrel fold with a Mn(2+) metal ion in the active site. Additional crystal structures of the enzyme in complex with substrates/products (D-psicose, D-fructose, D-tagatose and D-sorbose) were also determined. From the complex structures of C. cellulolyticum DPEase with D-psicose and D-fructose, the enzyme has much more interactions with D-psicose than D-fructose by forming more hydrogen bonds between the substrate and the active site residues. Accordingly, based on these ketohexose-bound complex structures, a C3-O3 proton-exchange mechanism for the conversion between D-psicose and D-fructose is proposed here. These results provide a clear idea for the deprotonation/protonation roles of E150 and E244 in catalysis.

Progress in lignocellulose deconstruction by fungi / 生物工程学报

Inefficient degradation of lignocellulose is one of the main barriers for the utilization of renewable plant biomass for biofuel production. The bottleneck of the biorefinery process is the generation of fermentable sugars from complicated biomass polymers. In nature, the main microbes of lignocelluloses deconstruction are fungi. Therefore, elucidating the mechanism of lignocelluloses degradation by fungi is of critical importance for the commercialization of lignocellulosic biofuels. This review focuses on the progress in lignocelluloses degradation pathways in fungi, especially on the advances made by functional genomics studies.

Synergistic systems for biodegradations of lignocellulose in microorganisms: a review / 生物工程学报

Lignocellulose is the most abundant natural biomass. Bioconversion of lignocelluloses becomes a bottleneck for biorefinery, because of its complex structures and heterogeneous composition. Besides screening or engineering approach for single free enzymes with improved properties, an alternative approach is to study synergistic pattern with hydrolysis systems or mimic natural cellulosome for better performance in cellulolytic substrate degradation. Besides, bacterial co-cultures provide another synergistic cellulolytic system. Engineered strains with modified metabolic network could facilitate consolidated bioprocess by increasing yields as well as reducing costs.

Cell factories for biorefinery: core of the technology for biomanufacture / 生物工程学报

The background of developing cell factories for biorefinery was reviewed. Seventeen papers published in this special issue, covering the molecular mechanism of sugar utilization, genome-scale metabolic and regulative networks, the construction technologies, and the optimization of cell factories for biorefinery, were introduced.

Application value of prospective electrocardiogram-gating for 64-slice CT coronary angiography / 中国医学影像技术

Objective To compare radiation dose and image quality between prospective and retrospective ECG gating CT coronary angiography (CTCA) with 64-slice CT scanner. Methods Sixty patients with suspected coronary artery disease were selected, and randomly devided into two groups. Thirty patients in research group (average body-mass index [BMI]:[25.30±3.15] kg/m~2, heart rate ≤65 beats per minute [bpm]) underwent CTCA using prospective ECG-gating (120 kV, mA tailored to weight), while the rest 30 patients in control group (average BMI:[25.40±3.00] kg/m~2, heart rate ≤65 bpm) underwent CTCA using retrospective ECG-gating (120 kV, 650 mA). The average effective dose (ED) and standardizing radiation dose (ED_(Sd)) which was standardized by 12 cm length were respectively calculated, and statistical analysis was then conducted, meanwhile image quality was evaluated. Results The average ED of research group and control group was (5.97±1.04) mSv and (26.25±2.36) mSv, respectively (t_(A,B)=43.13, P<0.05). The ED_(Sd) of two groups was (5.49±0.45) mSv and (23.77±1.64) mSv (t_(Sd)=59.00, P<0.05). ED_(Sd) decreased by 76.62% in research group compared with that of control group. No significant difference of image quality was found between two groups (χ~2=4.26, P=0.235). Conclusion Prospective ECG-gating CTCA can effectively reduce radiation dose without compromising image quality.

Nanoarchaeal 16S rRNA gene sequences are widely dispersed in hyperthermophilic and mesophilic halophilic environments.

The Nanoarchaeota, proposed as the fourth sub-division of the Archaea in 2002, are known from a single isolate, Nanoarchaeum equitans, which exists in a symbiotic association with the hyperthermophilic Crenarchaeote, Ignicoccus. N. equitans fails to amplify with standard archaeal 16S PCR primers and can only be amplified using specifically designed primers. We have designed a new set of universal archaeal primers that amplify the 16S rRNA gene of all four archaeal sub-divisions, and present two new sets of Nanoarchaeota-specific primers based on all known nanoarchaeal 16S rRNA gene sequences. These primers can be used to detect N. equitans and have generated nanoarchaeal amplicons from community DNA extracted from Chinese, New Zealand, Chilean and Tibetan hydrothermal sites. Sequence analysis indicates that these environments harbour novel nanoarchaeal phylotypes, which, however, do not cluster into clear phylogeographical clades. Mesophilic hypersaline environments from Inner Mongolia and South Africa were analysed using the nanoarchaeal-specific primers and found to contain a number of nanoarchaeal phylotypes. These results suggest that nanoarchaeotes are not strictly hyperthermophilic organisms, are not restricted to hyperthermophilic hosts and may be found in a large range of environmental conditions.

STUDY ON THE TECHNOLOGY OF ALKALINE ?-MANNANASE FERMENTATION / 微生物学通报

The fermentation of ?-mannanase from an alkaliphilic Bacillus sp. has been studied in 16L tank. The optimum ventilation quantity and agitation speed were 1:1 vvm and 500r/min respectively. The fermentation cycle was 40 h and the highest ?- mannanase activity was 300 u/ml.