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Utilization of carbon dioxide (CO 2 ) is a huge challenge for global sustainable development. Biological carbon fixation occurs in nature, but the low energy efficiency and slow speed hamper its commercialization. Physical-chemical carbon fixation is efficient, but relies on high energy consumption and often generates unwanted by-products. Combining the advantages of biological, physical and chemical technologies for efficient utilization of CO 2 remains to be an urgent scientific and technological challenge to be addressed. Here, based on the development of Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences in the past decade, we summarize the important progress in the design and construction of functional parts, pathways and systems for artificial bioconversion of carbon dioxide, including the breakthrough on the artificial synthesis of starch from CO 2 . Moreover, we prospect how to further develop the technologies for artificial bioconversion of carbon dioxide. These progress and perspectives provide new insight for achieving the goal of "carbon peaking and carbon neutrality".

Industrial biotechnology offers great promises to the development of a green, low-carbon and sustainable economy. The Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, which was formally established ten years ago, is an important powerhouse in the field of industrial biotechnology. To date, the Institute has developed a scientific and technological innovation chain, covering from basic research in life sciences to industrial application of biotechnology. On the occasion of the Institute's tenth anniversary, we publish this special issue to review the progress and trends of industrial biotechnology from six aspects, namely expert perspectives, development strategy, cutting-edge research, enabling technology, core technology innovation, and industrialization promotion. This will facilitate the construction of a green and low-carbon circular economy system, and promote the high-quality development of bioeconomy.

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.

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.

Unlabelled: The alkaliphilichemicellulolytic bacterium Bacillus sp. N16-5 was isolated from Lake Wudunao in Inner Mongolia. It has a broad substrate spectrum and exhibits a capacity to utilize complex carbohydrates such as galactomannan, xylan and pectin. Previous transcriptional analysis of differential carbohydrate utilization by Bacillus sp. N16-5 has identified a putative gene cluster related to xylan utilization. It contains a putative xylo-oligosaccharide ATP- binding cassette (ABC) transporter encoded by xynEFG gene cluster., Objective: xynE gene is predicted to encode an extracellular solute-binding protein of the ABC transporter. Here, the physiological roles of xynE on the xylan utilization was investigated by gene deletion., Methods: We obtained the xynE deficient strain N16-5 (ΔxynE) through homologous recombination using a temperature sensitive shuttle vector pNNB194. The effects of xynE on xylan utilization by N16-5 were detected by comparing the growth profiles on xylan of the wild type and mutant strains as well as the variation of reducing sugars concentration in the medium during cultivation. We further verified the phenotype by constructing the complementary strain. Moreover, the substrate specificity of XynE was illustrated by the HPLC analysis results of the xylan medium components, which was supplemented with the growth profiles of the wild type strain and N16-5 (ΔxynE) strain on xylose., Results: Compared with the wild type strain, strain N16-5 (ΔxynE) had a delayed exponential phase, obtained a lower maximum optical intensity OD600 value, and presented the accumulation and depletion of reducing sugars during cultivation. The complementary strain retrieved the phenotype of wild type strain, and grown slightly better than it. HPLC analysis showed that N16-5 ( ΔxynE) strain degraded the xylan substrates more slowly than wild type, xylo- oligosaccharides like xylotetraose, xylotriose and xylobiose began to accumulate after 16 h cultivation. Moreover it still maitained a large number of the degradation product xylobiose in the medium after 60 h. When cultured in xylose medium, strain N16-5 (ΔxynE) performed similar growth profile with the wild type strain., Conclusion: XynE played an important role in rapidly and effectively utilizing xylan in Bacillus sp. N16-5 and specifically related with xylo-oligosaccharide uptake.

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.

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.

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.

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.

Objective: Acetyl-CoA carboxylase (ACC) catalyzes the first step of fatty acid synthesis. In most bacteria, ACC is composed of four subunits encoded by accA, accB, accC, and accD. Of them, accA encodes acetyl-CoA carboxyltransferase alpha-subunit. Our prior work on proteomics of Alkalimonas amylolytica N10 showed that the expression of the Aa-accA has a remarkable response to salt and alkali stress. This research aimed to find out the Aa-accA gene contributing to salt and alkali tolerance., Methods: The Aa-accA was amplified by PCR from A. amylolytica N10 and expressed in E. coli K12 host. The effects of Aa-accA expression on the growth of transgenic strains were examined under different NaCl concentration and pH conditions. Transgenic tobacco BY-2 cells harboring Aa-accA were also generated via Agrobacterium-mediated transformation. The viability of BY-2 cells was determined with FDA staining method after salt and alkali shock., Results: The Aa-accA gene product has 318 amino acids and is homologous to the carboxyl transferase domain of acyl-CoA carboxylases. It showed 76% identity with AccA (acetyl-CoA carboxylase carboxyltransferase subunit alpha) from E. coli. Compared to the wild-type strains, transgenic E. coli K12 strain containing Aa-accA showed remarkable growth superiority when grown in increased NaCl concentrations and pH levels. The final cell density of the transgenic strains was 2.6 and 3.5 times higher than that of the control type when they were cultivated in LB medium containing 6% (W/V) NaCl and at pH 9, respectively. Complementary expression of Aa-accA in an accA-depletion E. coli can recover the tolerance of K12 delta accA to salt and alkali stresses to some extent. Similar to the transgenic E. coli, transgenic tobacco BY-2 cells showed higher percentages of viability compared to the wild BY-2 cells under the salt or alkali stress condition., Conclusion: We found that Aa-accA from A. amylolytica N10 overexpression enhances the tolerance of both transgenic E. coli and tobacco BY-2 cells to NaCl and alkali stresses.

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.

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.

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.

Objective: To isolate and identify an alkaliphilic mannanase producing bacterium, purify and characterize mannanase thereof., Methods: Mannanase-producing alkaliphilic bacterium HMTS15 was isolated by alkaline agar with konjak from water sample of Hamatai Lake in Inner Mongolia, China. The morphological, biochemical and physiological characteristics and 16S rRNA gene were analyzed to identify the taxonomic position of strain HMTS15. Mannanase produced by strain HMTS15 was purified by four steps including (NH4)2SO4 precipitation, cellulose DEAE-sepharose, twice Superdex 200. The enzyme properties including optimal temperature, optimal pH, thermal stability, pH stability, NaCl tolerance, metal ion tolerance, EDTA and SDS tolerance were tested., Results: Strain HMTS15 was Gram-positive rod. Its growth pH ranged from 7.0 to 11.0 and growth temperature ranged from 10 degrees C to 45 degrees C. The G + C content of the DNA was 40 mol%. Phylogenetic analyses based on 16S rRNA gene sequence comparisons indicated that strain HMTS15 was a member of Bacillus. The extracellular mannanase from strain HMTS15 was purified as a single band with molecular weight of about 45 kD on SDS-PAGE. The optimal catalytic activity was showed at 75 degrees C and pH 10. The mananase was stable up to 60 degrees C and retained about 60% residual activity at 65 degrees C for 30 min. The ions Fe2+, Mn2+, Co2+, Zn2+, Ag+, Hg2+ and EDTA inhibited the acitivity of the mannanase., Conclusion: Polyphasic taxonomy revealed that strain HTMS15 was a new member of Bacillus agaradhaerens. The alkaline mannanase produced by strain HMTS15 hold the valuable property in stability at high temperature and broad range of pH.

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.

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.

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.

Objective: To isolate and characterize alkaliphilic bacteria with protease activity., Methods: Protease-producing alkaliphiles were isolated by skim milk agar method. The morphological, biochemical and physiological characteristics, 16S rRNA gene sequence and DNA-DNA hybridization were determined to identify the taxonomic position of strain ZL223. Meanwhile, the enzyme characterization of the protease including optimal temperature and pH range, stability and oxidation resistance were tested by using casein hydrolysis test., Results: A new strain named ZL223 was isolated from Bange Lake in Tibet, China. Cells were Gram-positive, spore-forming, motile rods. Growth pH range was from 7.0 to 10.0, optimum at pH 9.0. Growth temperature range was from 15 degrees C to 45 degrees C, optimum at 37 degrees C. The G + C content of DNA was 40.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain belonged to the genus Bacillus and most closely related to Bacillus pseudofirmus OF4 with 98.6% sequence similarity. DNA-DNA hybridization analysis confirmed the close relationship of strain ZL223 with Bacillus pseudofirmu OF4 (86% relatedness). Strain ZL223 produced extracellular alkaline protease and its maximal enzyme activity was observed at 40 degrees C and pH 12.0., Conclusion: Base on polyphasic taxonomy, strain ZL223 was a new member of Bacillus pseudofirmus. It produced alkaline protease which was worth further research.

Beta-mannanases (beta-1,4-D-mannanase, EC 3.2.1.78), as a hemicellulose hydrolase, are widely distributed in bacteria, fungi, plants and even animals. They can randomly hydrolyze the beta-1,4-mannosidic linkages in mannan and heteromannan and have great potential in the food/feed, pulp/paper, medicine, oil exploitation and detergent industries. Most beta-mannanases often display a modular organization and usually contain structurally discrete catalytic and non-catalytic modules. Catalytic domains of these enzymes share a (beta/alpha)8-barrel fold, which play important roles in substrate binding and catalysis. Carbohydrate binding modules, as the most common non-catalytic modules, fold as beta-sandwich and facilitate the targeting of these enzymes to polysaccharide. In this review, a brief introduction is given concerning structural characteristics and function of these beta-mannanase modules.

Objective: Examining bacterial diversity in an oil reservoir of Shengli oil field by both culture-independent molecular technique and enrichment method., Methods: The heterotrophic bacteria, hydrocarbon-degrading bacteria and sulfate-reducing bacteria were enriched from S12-4 oil-well samples by the corresponding media. Then the genomic DNAs of the enrichments were extracted and the 16S rRNA gene clone libraries were constructed., Results: The phylogenetic analysis revealed that the bacterial 16S rRNA gene clone libraries of the 3 enrichments were dominated by clones of Thermotoga, Thermaerobacter and Thermotoga, respectively. Sequences of the other co-dominant clones observed only in the enrichments of hydrocarbon-degrading bacteria and sulfate-reducing bacteria were, respectively, associated with Marinobacter and Moorella. The uncultured 16S rRNA gene library was also generated directly from total DNA of S12-4 oil-well samples by bacterial primer set. Sequence analysis of this bacterial library indicated that a large percentage of clones were highly related to the genus Pseudomonas and the dominant species emerging in the enrichment samples had a very low content in the tested oil reservoir. The significant difference of the bacterial composition between the samples obtained from independent-culture method and enrichment method implies that the specialized nutrient may lead to a distinctive selection of dominant organisms., Conclusion: Through culture-dependent and culture-independent methods, we acquired important information on the bacterial diversity of ShengLi oil reservoir. These results may expand our understanding of the microbial diversity of oil reservoir and provide useful information for MEOR(microbial enhancement of oil recovery).

Heat shock protein 60 (HSP60) is one of the important chaperonins, which can assist proper protein folding and configuration,and can prevent denaturation and degradation of proteins as well. It was found that HSP60 in Thermoanaerobacter tengcongensis (T. tengcongensis) was typically temperature-dependent with the highest protein expression at 80 degrees C. To understand the molecular mechanisms of thermophiles in their responding to heat stress, further investigation was conducted to examine the dynamic expression of HSP60 gene induced by higher temperature. The T. tengcongensis cells cultured at optimal temperature (75 degrees C) were transferred to 80 degrees C followed by taking the aliquots at different time intervals. To monitor the expression levels of HSP60 mRNA and protein, these bacterial gene products were analyzed by two-dimensional electrophoresis, MALDI-TOF, HPLC-MS/MS, Western blot and quantitative PCR. The experimental data were calculated statistically and quantitatively, which indicate that the HSP60 protein increases steadily and significantly within 4h period after temperature raised, but its mRNA level is shown as a peak shape change within 1h. Obviously, the expression response of HSP60 protein to heat stress is significantly slower than that of its mRNA expression. Additionally, the expression extent of HSP60 mRNA and protein induced by raised-temperature is very different. In E. coli strains, the expression of HSP60 is usually regulated by the sigma32 factor, which disassociates with DnaK immediately when the environmental temperature elevated. Since the genome of T. tengcongensis contains two sigma32 genes, thus, it is likely that both bacteria share the similar mechanisms which regulate HSP60 transcription responding to the changes of temperature. Interestingly, the mRNA of HSP60 drops down about 70% within 30m responding to heat shock, but goes to the increased expression after then. Although this phenomenon is hard to explain based upon the current theory, it is plausible that the transcription in T. tengcongensis is temporally inhibited due to the culture temperature sharply raised.

With culture-independent approach, microbial DNA was directly extracted from samples of Zabuye saline soda lake. Using the microbial DNA as template, archaeal 16S rDNAs were amplified by PCR. Amplified products were cloned and sequenced. 60 different cloned partial sequences, most of which were related to haloalkaliphilic archaeon, were acquired. In the phylogenetic tree, some clones of Zabuye lake belonged to Genus Natronobacterium, Natrinema, Natronococcus, Natronorubrum, Natronomonas, Halorubrum, Haloterrigena, Halorhabdus in Family Halobacteriaceae. Other clones represent some novel groups. All of them show prolific archaeal diversity of Zabuye lake.