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Progress and prospect of bio-jet fuels industry in domestic and overseas.

Kai QIAO ; Jie FU ; Feng ZHOU ; Huixia MA

Chinese Journal of Biotechnology.2016;32(10):1309-1321. doi:10.13345/j.cjb.160078

We reviewed the progress of the bio-jet fuels industry in recent years and systematically analyzed the technical routes that have been approved or in the pipeline for approval by ASTM D7566. In addition, we highlighted a novel pathway to produce drop-in fuel by near-critical hydrolysis of waste cooking oils or algal oils followed by catalytic decarboxylation. Also, we introduced the source of oils and fats feedstock and the domestic bio-jet fuel industry status during the 12th Five-Year-Plan period. Based on our own research, we discussed the prospect of the bio-jet fuel industry and future research needs.

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Progress of stress-induced flowering in plants.

Min ZHANG ; Jiaxu ZHU ; Lei WANG ; Miaoyun XU

Chinese Journal of Biotechnology.2016;32(10):1301-1308. doi:10.13345/j.cjb.160012

Plants tend to flower earlier if placed under stress conditions. Those stress factors include drought, high salinity, low temperature, high- or low-intensity light, and ultraviolet light. This phenomenon has been called stress-induced flowering. Stress-induced plant flowering might be helpful for species preservation. Thus, stress-induced flowering might have biological significance and should be considered as important as other plant flowering control strategy. Here, history of stress-induced flowering, metabolic regulation and molecular regulation mechanisms in plants were reviewed. Potential perspective was discussed.

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Effects of light quality on cell growth and psbA promoter of engineered Synechococcus sp. PCC7002.

Yihua SUN ; Chunli ZHANG ; Dingji SHI ; Xiaohui JIA ; Rui JIA ; Peimin HE

Chinese Journal of Biotechnology.2016;32(9):1286-1290. doi:10.13345/j.cjb.160002

Light quality can regulate both psbA genes and vector promoter psbA of the engineered Synechococcus. Through light regulation, we tried to improve yield of the recombinant protein for vp28 gene-expressed Synechococcus sp. PCC7002. To drive photon-capturing efficiently, three limiting factors (irradiance, temperature and pH) were optimized by measuring net photosynthesis. High cell density cultures were performed with variant ratios of white, red and blue light in a 5-L photo-bioreactor. Yields of biomass, expressions of vp28 and transcription levels of psbA were compared. High ratio blue light-induced vp28 transcription had tripled and the relative accumulation of VP28 protein was doubled. The relative expressions of psbAII and psbAIII had positive correlations with higher ratio of blue light, not the red light. With high ratio red light inducing, dry biomass reached 1.5 g/L in three days. Therefore, we speculated that red light accelerated biomass accumulation of the transgenic strain and blue light promoted transcription for PpsbA and psbA. These results provided useful information for mass production of cyanobacteria and its secondary metabolites.
Gene Expression Regulation, Bacterial ; Light ; Photosystem II Protein Complex ; genetics ; Promoter Regions, Genetic ; Synechococcus ; genetics ; growth & development ; radiation effects

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Prokaryotic expression, purification and identification of recombinant human atrial natriuretic peptide.

Chenhui CHEN ; Ziye ZHAO ; Jin XU ; Xuesong CAO ; Shangjing GUO ; Jun LI ; Hao WANG ; Sheng HOU

Chinese Journal of Biotechnology.2016;32(9):1273-1285. doi:10.13345/j.cjb.160018

In order to improve the expression of recombinant human atrial natriuretic peptide (ANP), a new plasmid (pET28a(+)/ANP₃) containing 3 tandem ANP genes with lysine codon as the interval linker, was constructed. Target gene was transformed into Escherichia coli BL21 (DE3) and induced by IPTG, about 60% of the total-cell-protein was the target protein, His₆-ANP₃. After denaturation and refolding, it was digested by Endoproteinase Lys-C and Carboxypeptidase B (CPB) and then purified by a series of purification processes, about 16 mg purified ANP monomer could be obtained from one liter bacteria broth of shaking culture. Ultimately, the purity of protein was above 90% determined by UPLC and Tricine SDS-PAGE, its molecular weight was 3 080 Da according to LC-MS identification and it was proved to be equivalent to the reference product by ELISA. The use of tandem gene expression can provide a new possible model for the expression of other peptide drugs.
Atrial Natriuretic Factor ; biosynthesis ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; metabolism ; Gene Expression ; Humans ; Metalloendopeptidases ; Peptides ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis

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Comparison of MITE transposons mPing in different rice subspecies.

Ning ZHANG ; Yanan RUAN ; Shanshan WANG ; Yang LIU ; Chen ZHAO ; Jingjing WANG ; Kaixi WANG ; Yanli WANG ; Hongyan WANG

Chinese Journal of Biotechnology.2016;32(9):1264-1272. doi:10.13345/j.cjb.150554

The mPing family is the first active MITE TE family identified in rice genome. In order to compare the compositions and distributions of mPing family in the genomes of two rice subspecies japonica (cv. Nipponbare) and indica (cv. 93-11), we initially estimated the copy numbers of mPing family in those two subspecies using Southern blot and then confirmed the results by searching homologous copies in each reference genome using Blastn program, which turned out to have 52 and 14 mPing copies in corresponding reference genome, respectively. All mPing members in Nipponbare genome belong to mPing-1, while there are 3 mPing-1 and 11 mPing-2 copies in 93-11 genome. By further investigating the 5-kb flanking sequences of those mPing copies, it was found that 23 and 3 protein-coding genes in Nipponbare and 93-11 genome are residing adjacent to those mPing copies respectively. These results establish the preliminary theoretical foundation for further dissecting the genetic differences of japonica and indica rice in terms of the diversities and distributions of their component mPing.
Animals ; DNA Transposable Elements ; genetics ; Genome, Plant ; Oryza ; classification ; genetics

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Integration and expression of Xa21 in transgenic rice CX8621.

Lifen GAO ; Pengcheng LIU ; Zhihui XIA ; Jiying ZHAO ; Jianan SHI ; Guanghuai JIANG ; Guozhen LIU ; Wenxue ZHAI

Chinese Journal of Biotechnology.2016;32(9):1255-1263. doi:10.13345/j.cjb.150549

Agrobacterium tumefaciens-mediated transformation system has been widely applied. However, the function of target gene is affected by multiple factors. With this system, we obtained a transgenic rice line CX8621 carrying the bacterial blight resistance gene Xa21. In previous work, we have confirmed that it was selectable maker-free and vector backbone-free. And after 16 generations of breeding, it still maintained perfect resistance to bacterial blight disease. On this basis, we analyzed the integration and expression of Xa21 in CX8621 at the present study. First, based on the border sequences of plasmid pBXa21 and Xa21, we designed nested primers and assured the integrity of Xa21 in CX8621. Second, we cloned the flanking sequences and located Xa21 on chromosome 2 using improved Tail-PCR. Then we analyzed the expression pattern of Xa21 in several tissues and at different developmental stages by RT-PCR. The results show that Xa21 can be stably expressed in CX8621, agreeing well with the disease resistance response as reported previously. In addition, we detected the protein levels of XA21 in CX8621 with antibody of natural XA21 protein. Surprisingly, no XA21 protein was detected in the seeds of CX8621. Thus, the integration and expression analysis of Xa21 in CX8621 provided a part of scientific evidences for the safety assessment of genetically modified rice.
Animals ; DNA Primers ; Disease Resistance ; genetics ; Oryza ; genetics ; Plant Proteins ; genetics ; Plants, Genetically Modified ; genetics ; Polymerase Chain Reaction ; Protein-Serine-Threonine Kinases ; genetics ; Seeds

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Thermal stability improvement for phenylalanine hydroxylase by site-directed mutagenesis.

Shuangshuang YE ; Li ZHOU ; Zhemin ZHOU

Chinese Journal of Biotechnology.2016;32(9):1243-1254. doi:10.13345/j.cjb.160022

In proteins of thermophilic bacteria, Gly is tend to be replaced by Ala and Lys is tend to be replaced by Arg to adapt the high temperature. In order to improve the thermal stability of phenylalanine hydroxylase (PAH) from Chromobacterium violaceum, all the Gly on PAH were mutated to Ala and Lys to Arg. Positive mutant enzymes with improved thermal stability were selected, followed by combined mutation and characterization. The results revealed that half-lives of K94R and G221A mutants at 50 °C were 26.2 min and 16.8 min, which were increased by 1.9-times and 0.9-times than the parent enzyme (9.0 min). The residual activity of K94R/G221A mutant was improved to 65.6% after keeping at 50 °C for 1 h, which was 6.6 time higher than the parent enzyme (8.6%). Circular dichroism (CD) spectroscopy revealed that Tm values of the parent enzyme, K94R, G221A and K94R/G221A were 51.5 ℃, 53.8 ℃, 53.1 ℃ and 54.8 ℃, respectively. According to the protein structure simulation, the two mutations were located on flexible loop. In the K94R mutant, the mutated Arg94 on the surface of the enzyme formed an extra hydrogen bond with Ile95, which stabilized the located loop. In the G221A mutant, the mutated Ala221 formed hydrophobic interaction with Leu281, which could stabilize both the loop and flexible area of the C-terminus of G221A. The results not only provided a reference for protein modification on thermal stability, but also laid the foundation for application of phenylalanine hydroxylase in the field of functional foods.
Bacterial Proteins ; biosynthesis ; genetics ; Chromobacterium ; enzymology ; Enzyme Stability ; Hot Temperature ; Kinetics ; Mutagenesis, Site-Directed ; Mutation ; Phenylalanine Hydroxylase ; biosynthesis ; genetics ; Protein Engineering

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Stability enhancement of urethanase from Lysinibacillus fusiformis by site-directed mutagenesis.

Xiaohui LIU ; Fang FANG ; Xiaole XIA ; Guocheng DU ; Jian CHEN

Chinese Journal of Biotechnology.2016;32(9):1233-1242. doi:10.13345/j.cjb.150527

Ethyl carbamate as a potential carcinogen commonly exists in traditional fermented foods and beverages. Enzymatic removal of ethyl carbamate from fermented foods and beverages is an efficient and safe method. In this study, we mutated urethanase from Lysinibacillus fusiformis SC02 on the Q328 site through computer aided design approaches. The half-life of resulting mutants Q328C and Q328V was detected to be 7.46 and 1.96 folds higher than that of the original enzyme, and Q328R presented better thermal-tolerance than the original urethanase when incubated at high temperature. The tolerance of Q328C to ethanol and acid also increased when compared with that of the original enzyme. The stability and tolerance to acid and ethanol of urethanase could be improved by modification on its Q328 site.
Amidohydrolases ; biosynthesis ; genetics ; Bacillaceae ; enzymology ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; Computer-Aided Design ; Enzyme Stability ; Ethanol ; Mutagenesis, Site-Directed ; Protein Engineering

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Fitness analysis between the L-sorbosone dehydrogenase modules and Ketogulonigenium vulgare chassis.

Si CHEN ; Nan JIA ; Mingzhu DING ; Yingjin YUAN

Chinese Journal of Biotechnology.2016;32(9):1224-1232. doi:10.13345/j.cjb.150523

Ketogulonigenium vulgare is an acid-producing strain in the process of two-step vitamin C fermentation. L-sorbosone dehydrogenase (SNDH) is one of the key enzymes during the biosynthesis of 2-keto-L-gulonic acid (2-KGA), the precursor of vitamin C. However, the catalytic mechanism of SNDH is unclear. According to the whole genome sequencing of K. vulgare, two genes encoding sorbosone dehydrogenases, one derived from the chromosome (named as sndhg) and one from plasmid (named as sndhp), were introduced into an industrial strain K. vulgare. The overexpression of gene sndhg had hardly effect on 2-KGA production, and the overexpression of gene sndhp produced an obvious byproduct in the fermentation broth. Combinational expression of sndhg/sndhp with pqqA (obtaining sndhg-pqqA and sndhp-pqqA modules) in K. vulgare resulted in the similar fermentation phenotype to two previous strains. After serial sub-cultivation of co-cultured Bacillus endophyticus with each engineered K. vulgare for 50 d, the conversion rate of 2-KGA increased by 15.4%, 179%, 0.65% and 125% compared with that of the parental K. vulgare with B. endophyticus. This study shows that adaptive evolution of microbial consortium is an effective strategy to increase the fitness between functional modules and chassis, thus quickly getting better strains for production of 2-KGA.
Aldehyde Oxidoreductases ; genetics ; metabolism ; Ascorbic Acid ; Bacillus ; Bacterial Proteins ; genetics ; metabolism ; Coculture Techniques ; Fermentation ; Industrial Microbiology ; Microorganisms, Genetically-Modified ; Rhodobacteraceae ; enzymology ; genetics ; Sugar Acids ; metabolism

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Construction and optimization of microbial cell factories for producing cis, cis-muconic acid.

Guotian SONG ; Xiaolong JIANG ; Wujiu CHEN ; Yanfeng PENG ; Fuping LU ; Qinhong WANG

Chinese Journal of Biotechnology.2016;32(9):1212-1223. doi:10.13345/j.cjb.150541

cis, cis-muconic acid (MA) is an important platform chemical. Now, majority of reported engineered strains are genetically instable, the exogenous genes are expressed under the control of expensive inducer and the components of their fermentation medium are complex, thus large-scale microbial production of MA is limited due to the lack of suitable strains. Hence, it is still necessary to construct novel high-performance strain that is genetically stable, no induction and grows in simple inorganic fermentation medium. In this study, after 3 exogenous genes (aroZ, aroY, catA) for biosynthesis of MA were integrated into previously constructed 3-hydroshikimate producing Escherichia coli WJ060 strain and combinatorially regulated with 3 constitutive promoters with different strengths, 27 engineered strains were constructed. The best engineered strain, E. coli MA30 could produce 1.7 g/L MA in the simple inorganic fermentation medium without induction. To further enhance the production capacity of MA, the mutant library of E. coli MA30 was constructed by genome replication engineering and screened via high-throughput assay. After two-round screening, the new strain, E. coli MA30-G2 with improved production of MA was obtained, and the titer of MA increased more than 8%. Under the condition of 5 L fed-batch fermentation, E. coli MA30-G2 could produce about 11.5 g/L MA. Combinatorial regulation and high-throughput screening provide important reference to microbial production of other bio-based chemicals.
Escherichia coli ; metabolism ; Fermentation ; Industrial Microbiology ; Metabolic Engineering ; Microorganisms, Genetically-Modified ; Promoter Regions, Genetic ; Sorbic Acid ; analogs & derivatives ; metabolism

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