Effect of hemX gene deletion on heme synthesis in Bacillus amyloliquefaciens / 生物工程学报

Heme, which exists widely in living organisms, is a porphyrin compound with a variety of physiological functions. Bacillus amyloliquefaciens is an important industrial strain with the characteristics of easy cultivation and strong ability for expression and secretion of proteins. In order to screen the optimal starting strain for heme synthesis, the laboratory preserved strains were screened with and without addition of 5-aminolevulinic acid (ALA). There was no significant difference in the heme production of strains BA, BAΔ6 and BAΔ6ΔsigF. However, upon addition of ALA, the heme titer and specific heme production of strain BAΔ6ΔsigF were the highest, reaching 200.77 μmol/L and 615.70 μmol/(L·g DCW), respectively. Subsequently, the hemX gene (encoding the cytochrome assembly protein HemX) of strain BAΔ6ΔsigF was knocked out to explore its role in heme synthesis. It was found that the fermentation broth of the knockout strain turned red, while the growth was not significantly affected. The highest ALA concentration in flask fermentation reached 82.13 mg/L at 12 h, which was slightly higher than that of the control 75.11 mg/L. When ALA was not added, the heme titer and specific heme production were 1.99 times and 1.45 times that of the control, respectively. After adding ALA, the heme titer and specific heme production were 2.08 times and 1.72 times higher than that of the control, respectively. Real-time quantitative fluorescent PCR showed that the expressions of hemA, hemL, hemB, hemC, hemD, and hemQ genes at transcription level were up-regulated. We demonstrated that deletion of hemX gene can improve the production of heme, which may facilitate future development of heme-producing strain.

An efficient marker-free genome editing method for Aspergillus niger / 生物工程学报

Aspergillus niger is an important industrial strain which has been widely used for production of enzymes and organic acids. Genome modification of A. niger is required to further improve its potential for industrial production. CRISPR/Cas9 is a widely used genome editing technique for A. niger, but its application in industrial strains modification is hampered by the need for integration of a selection marker into the genome or low gene editing efficiency. Here we report a highly efficient marker-free genome editing method for A. niger based on CRISPR/Cas9 technique. Firstly, we constructed a co-expression plasmid of sgRNA and Cas9 with a replication initiation region fragment AMA1 (autonomously maintained in Aspergillus) by using 5S rRNA promoter which improved sgRNA expression. Meanwhile, a strain deficient in non-homologous end-joining (NHEJ) was developed by knocking out the kusA gene. Finally, we took advantage of the instability of plasmid containing AMA1 fragment to cure the co-expression plasmid containing sgRNA and Cas9 through passaging on non-selective plate. With this method, the efficiency of gene editing reached 100% when using maker-free donor DNA with a short homologous arm of 20 bp. This method may facilitate investigation of gene functions and construction of cell factories for A. niger.

Effects of deleting peptidoglycan hydrolase genes on the viable cell counts of Bacillus amyloliquefaciens and the yield of alkaline protease / 生物工程学报

In order to explore the effect of peptidoglycan hydrolase on the viable cell counts of Bacillus amyloliquefaciens and the yield of alkaline protease, five peptidoglycan hydrolase genes (lytC, lytD, lytE, lytF and lytG) of B. amyloliquefaciens TCCC111018 were knocked out individually. The viable cell counts of the bacteria and their alkaline protease activities before and after gene deletion were determined. The viable cell counts of the knockout mutants BA ΔlytC and BA ΔlytE achieved 1.67×106 CFU/mL and 1.44×106 CFU/mL respectively after cultivation for 60 h, which were 32.5% and 14.3% higher than that of the control strain BA Δupp. Their alkaline protease activities reached 20 264 U/mL and 17 265 U/mL, respectively, which were 43.1% and 27.3% higher than that of the control strain. The results showed that deleting some of the peptidoglycan hydrolase genes effectively maintained the viable cell counts of bacteria and increased the activity of extracellular enzymes, which may provide a new idea for optimization of the microbial host for production of industrial enzymes.

Construction and application of a synthetic promoter library for Corynebacterium glutamicum / 生物工程学报

Promoter is an important genetic tool for fine-tuning of gene expression and has been widely used for metabolic engineering. Corynebacterium glutamicum is an important chassis for industrial biotechnology. However, promoter libraries that are applicable to C. glutamicum have been rarely reported, except for a few developed based on synthetic sequences containing random mutations. In this study, we constructed a promoter library based on the native promoter of odhA gene by mutating the -10 region and the bystanders. Using a red fluorescent protein (RFP) as the reporter, 57 promoter mutants were screened by fluorescence imaging technology in a high-throughput manner. These mutants spanned a strength range between 2.4-fold and 19.6-fold improvements of the wild-type promoter. The strongest mutant exhibited a 2.3-fold higher strength than the widely used strong inducible promoter Ptrc. Sequencing of all 57 mutants revealed that 55 mutants share a 1-4 bases shift (4 bases shift for 68% mutants) of the conserved -10 motif "TANNNT" to the 3' end of the promoter, compared to the wild-type promoter. Conserved T or G bases at different positions were observed for strong, moderate, and weak promoter mutants. Finally, five promoter mutants with different strength were employed to fine-tune the expression of γ-glutamyl kinase (ProB) for L-proline biosynthesis. Increased promoter strength led to enhanced L-proline production and the highest L-proline titer of 6.4 g/L was obtained when a promoter mutant with a 9.8-fold higher strength compared to the wild-type promoter was used for ProB expression. The use of stronger promoter variants did not further improve L-proline production. In conclusion, a promoter library was constructed based on a native C. glutamicum promoter PodhA. The new promoter library should be useful for systems metabolic engineering of C. glutamicum. The strategy of mutating native promoter may also guide the construction of promoter libraries for other microorganisms.

Heterogeneous expression of DOPA decarboxylase to improve the production of dopamine in Escherichia coli / 生物工程学报

Dopamine is the precursor of a variety of natural antioxidant compounds. In the body, dopamine acts as a neurotransmitter that regulates a variety of physiological functions of the central nervous system. Thus, dopamine is used for the clinical treatment of various types of shock. Dopamine could be produced by engineered microbes, but with low efficiency. In this study, DOPA decarboxylase gene from Sus scrofa (Ssddc) was cloned into plasmids with different copy numbers, and transformed into a previously developed L-DOPA producing strain Escherichia coli T004. The resulted strain was capable of producing dopamine from glucose directly. To further improve the production of dopamine, a sequence-based homology alignment mining (SHAM) strategy was applied to screen more efficient DOPA decarboxylases, and five DOPA decarboxylase genes were selected from 100 candidates. In shake-flask fermentation, the DOPA decarboxylase gene from Homo sapiens (Hsddc) showed the highest dopamine production (3.33 g/L), while the DOPA decarboxylase gene from Drosophila Melanogaster (Dmddc) showed the least residual L-DOPA concentration (0.02 g/L). In 5 L fed-batch fermentations, production of dopamine by the two engineered strains reached 13.3 g/L and 16.2 g/L, respectively. The residual concentrations of L-DOPA were 0.45 g/L and 0.23 g/L, respectively. Finally, the Ssddc and Dmddc genes were integrated into the genome of E. coli T004 to obtain genetically stable dopamine-producing strains. In 5 L fed-batch fermentation, 17.7 g/L of dopamine was produced, which records the highest titer reported to date.

Advances in gene editing of Corynebacterium glutamate / 生物工程学报

Corynebacterium glutamicum, an important microorganism to produce amino acids and organic acids, has been widely applied in food and medicine fields. Therefore, using editing tools to study the function of unknown genes in C. glutamicum has great significance for systematic development of industrial strain with efficient and novel production capability. Recently, gene editing has been greatly developed. Traditional gene editing based on homologous recombination and gene editing mediated by nuclease are successfully applied in C. glutamicum. Among these, the CRISPR system has been developed to be a main tool used for gene knockout of C. glutamicum due to its advantages of efficiency, simplicity and good target specificity. However, more efficient and reliable knockout system is still urgently demanded, to help develop high-performing strains in industrial application.

Characterization of a novel bidirectional promoter in Bacillus subtilis / 生物工程学报

Based on the transcriptome analysis data of a Bacillus licheniformis, a novel bidirectional promoter was identified from the strain and its transcriptional strength was analyzed. The expression level of a Bacillus clausii derived alkaline protease gene driven by the bidirectional promoter was studied by using the known strong constitutive promoter pShuttle-09 as a control. Three recombinant expression vectors and the corresponding recombinant bacteria were constructed. Under the control of the new promoter pLA and its reverse promoter pLB, the alkaline protease expression level respectively reached 164 U/mL and 111 U/mL. The results indicated that the transcription strength of pLA was significantly higher than that of pShuttle-09 and pLB, and both the pLA and pLB promoters could initiate the expression of the alkaline protease. Thus, it provides a new expression element for the heterogenous genes in Bacillus sp. and a new idea for the co-expression of two genes in one prokaryotic strain.

Improving the production of 4-aminobenzoic in engineered Escherichia coli by combinatorial regulation / 生物工程学报

Para-aminobenzoate (PABA) is an important chemical for organic synthesis and extensively used in pharmaceutical and dye industry. In recent years, PABA has received increasing attention as a potential component of high-strength polymer. In Escherichia coli, three genes of pabA, pabB and pabC are responsible for PABA production from chorismate in folate synthetic pathway. However, E. coli does not accumulate or accumulates very few amounts of PABA under normal growth condition. In this study, the tyrosine-producing E. coli TYR002 constructed previously was used as the starting strain for developing PABA-producing strain. First, the activity of bifunctional chorismate mutase/prephenate dehydrogenase TyrA in E. coli TYR002 was weakened to reduce the production of tyrosine. Then, three different constitutive promoters were used to regulate the expression of pabA, pabB and pabC in recombinant plasmid which was transformed into E. coli for improving PABA production. The shake-flask fermentation showed that the different combination of constitutive promoters significantly affected the production of PABA, and the highest shake-flask fermentation titer was 0.67 g/L. After further condition optimization, the engineered E. coli produced 6.4 g/L PABA under 5 L fed-batch fermentation. This study could be a good reference for improving microbial production of PABA.

Advances in hydroxylation of hydrophobic amino acid / 生物工程学报

Hydroxy amino acids, constituents of chiral pharmaceutical intermediates or precursors, have a variety of unique functions in the research fields of biotechnology and molecular biology, i.e. antifungal, antibacterial, antiviral and anticancer properties. Biosynthesis of hydroxy amino acids is preferred because of its high specificity and selectivity. The hydroxylation of hydrophobic amino acids is catalyzed by hydroxylase, which belongs to the mononuclear non-heme Fe(Ⅱ)/α-ketoglutarate-dependent dioxygenases (Fe/αKGDs). Fe/αKGDs utilize an (Fe(Ⅳ)=O) intermediate to activate diverse oxidative transformations with key biological roles in the process of catalytic reaction. Here, we review the physiological properties and synthesis of hydroxy amino acids, especially for the 4-HIL and hydroxyproline. The catalytic mechanism of Fe/αKGDs is elucidated, and the applications of hydroxy amino acids in industrial engineering are also discussed.

Construction and optimization of microbial cell factories for producing cis, cis-muconic acid / 生物工程学报

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.

Progress in biosythesis of diaminopentane / 生物工程学报

Air pollution and global warming are increasingly deteriorating. Large amounts of polyamides derived from fossil fuel sources are consumed around the world. Cadaverine is an important building monomer block of bio-based polyamides, thus biotechnological processes for these polymers possess enormous ecological and economical potential. Currently, the engineered strains for biological production of cadaverine are Corynebacterium glutamicum and Escherichia coli. We review here the latest research progress of biosynthesis of cadaverine including metabolism of cadaverine in microorganisms, key enzymes and transport proteins in cadaverine synthesis pathway, optimum pathways and cadaverine yields.

16β-hydroxylation of 4-androstene-3,17-dione by Aspergillus niger / 生物工程学报

In order to discover the steroid biotransformation ability of filamentous fungus Aspergillus niger TCCC41650, we studied the fermentation of 4-androstene-3,17-dione with A. niger TCCC41650. The transformation product was purified, crystallized and determined as 16β-hydroxy-androst-4-ene-3,17-dione by X-ray single crystal diffraction method. The best fermentation condition was found to be pH 6.0, ethanol amount 2% with a substrate concentration of 1 per thousand, the transformation rate is 85.81% after 72 h. Based on the best of our knowledge, 16β-hydroxylation rarely occurs in microbial transformations of steroid. This study laid the foundation for the research of 16β-hydroxylation steroids

High-efficiency L-lactate production from glycerol by metabolically engineered Escherichia coli / 生物工程学报

High-efficient conversion of glycerol to L-lactate is beneficial for the development of both oil hydrolysis industry and biodegradable materials manufacturing industry. In order to construct an L-lactate producer, we first cloned a coding region of gene BcoaLDH encoding an L-lactate dehydrogenase from Bacillus coagulans CICIM B1821 and the promoter sequence (P(ldhA)) of the D-lactate dehydrogenase (LdhA) from Escherichia coli CICIM B0013. Then we assembled these two DNA fragments in vitro and yielded an expression cassette, P(ldhA)-BcoaLDH. Then, the cassette was chromosomally integrated into an ldhA mutant strain, Escherichia coli CICIM B0013-080C, by replacing lldD encoding an FMN-dependent L-lactate dehydrogenase. An L-lactate higher-producer strain, designated as E. coli B0013-090B, possessing genotype of lldD::P(ldhA)-BcoaLDH, deltaack-pta deltapps deltapflB deltadld deltapoxB deltaadhE deltafrdA and deltaldhA, was generated. Under the optimal condition, 132.4 g/L L-lactate was accumulated by B0013-090B with the lactate productivity of 4.90 g/Lh and the yield of 93.7% in 27 h from glycerol. The optical purity of L-lactate in broth is above 99.95%.

Temperature-switched high-efficiency D-lactate production from glycerol / 生物工程学报

Glycerol from oil hydrolysis industry is being considered as one of the abundent raw materials for fermentation industry. In present study, the aerobic and anaerobic metabolism and growth properties on glycerol by Esherichia coli CICIM B0013-070, a D-lactate over-producing strain constructed previously, at different temperatures were investigated, followed by a novel fermentation process, named temperature-switched process, was established for D-lactate production from glycerol. Under the optimal condition, lactate yield was increased from 64.0% to 82.6%. Subsequently, the yield of D-lactate from glycerol was reached up to 88.9% while a thermo-inducible promoter was used to regulate D-lactate dehydrogenase transcription.

Effects of space flight on protein content and electrophoresis in Glycyrrhiza uralensis / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the space environment on the role of licorice mutagenesis analysis of proteins.</p><p><b>METHOD</b>Licorice (Glycyrrhiza uralensis) seeds were carried by a recoverable satellite for 18 days (the average radiation dose in the flight recovery module was 0.102 m x d(-1), the orbit semidiameter 350 km, gravity 10(-6)). After return, The satellite-flown seeds and the unflown seeds (ground control) were planted in the fields of experimental farm. The leaves of each group were used for studying the effects of space flight on CAT, SOD activity, the protein content and electrophoresis.</p><p><b>RESULT</b>After the space flight, CAT, SOD activity of licorice increased in varying degrees, the difference was significant (P<0.05), two types of enzyme activity of sample from Ordos were higher than that from Hangjinqi. The protein content of licorice increased in a certain extent, the difference was significant (P<0.05), while protein electrophoresis also showed differences, weak new bands appeared.</p><p><b>CONCLUSION</b>These results indicated that spaceflight has effect on protein of licorice, these changes may be used as a tool for accelerating the progress in G. uralensis breeding.</p>

Research progress on mutation by spaceflight in medicinal plants breeding / 中国中药杂志

Space breeding in medicinal plants is special characteristics in China. Compared with other plants, in spite of a relatively small number, Medicinal plants have more obvious characteristics and advantages. Research on medicinal plants has also been carried into all aspects, such as biological traits, physiology and biochemistry, genomics, as well as differences in chemical composition, and chemical composition analysis is also involved. However, compared with other plants, especially crops and vegetables, biological research is an obvious deficiency, that is mainly reflected in the insufficient genetics and breeding researches, the stability of genetic traits from generation to generation were not followed up and in-depth study in breeding areas was not carried out. If medicinal plants resources from space with the genetic stability good quality were selected, it would address the problem of lack of resources and ease the pressure on wild resources of medicinal plants. It would at the same time play an important role in promoting the development of medicinal botany space breeding and the implementation of modernization of traditional Chinese medicine.

Effects of space flight on glycyrrhizic acid-related gene mutation in Glycyrrhiza uralensis / 中国中药杂志

<p><b>OBJECTIVE</b>To substantiate the effects of spaceflight on the glycyrrhizic acid-related gene mutation in Glycyrrhiza uralensis.</p><p><b>METHOD</b>Licorice (G. uralensis) seeds were carried by a recoverable satellite for 18 days (the average radiation dose in the flight recovery module was 0. 102 m x d(-1), the orbit semidiameter 350 km, gravity 10(-6)). After returned to the earth, the satellite-flown seeds and the un-flown seeds (ground control) were planted in the fields of experimental farm. The leaves of each group were used for studying the effects of space flight on the glycyrrhizic acid-related gene mutation including ITS sequence and beta-amyrine synthase gene.</p><p><b>RESULT</b>The ITS sequence of glycyrrhizic acid related gene showed no changes after spaceflight. While beta-amyrine synthase gene had some different points after spaceflight and the different points could get the expression.</p><p><b>CONCLUSION</b>The results indicated that spaceflight induce genetic variation in G. uralensis and spaceflight could also have effects on glycyrrhizic acid-related gene mutation in G. uralensis. It may need to further research how the spaceflight induced the mutation of the glycyrrhizic acid related gene. The results suggested that recoverable satellite-flown condition could bring inheritable mutagenic effects on G. uralensis seeds and maybe used as a tool for accelerating the progress in G. uralensis breeding.</p>

Expression of ksdD Gene Encoding 3-ketosteroid-△ 1-dehydrogenase from Arthrobacter simplex in Bacillus subtilis and Analysis of Steroid Transformation / 中国生物工程杂志

To improve 3-ketosteroid-△1-dehydrogenase (KSDH) activity and the transformation level for androst-4-ene-3,17-dione,3-ketosteroid-△1 -dehydrogenase gene (ksdD) from Arthrobacter simplex was cloned into plasmid pWB980 and expressed in B. subtilis WB600 under the control of promoter P43. The molecular weight of expressed enzyme was about 55kDa by SDS-PAGE analysis. The activitities assayed by spectrophotometrical method of intracellular and extracellular soluble enzyme were 110 ± 0.5mU and 15 ± 0.6mU per milligram of protein respectively. The transformation rate of androst-4-ene-3,17-dione by the B. subtilis recombinant cells was 45.3%. Compared with Arthrobacter simplex, the enzyme activity of KSDH expressed in B. subtilis was improved about 30 fold, and the transformation level of androst-4-ene-3,17-dione by the B.subtilis recombinant cells was improved about 10 fold. The recombinant B. subtilis cells used in biotransformation of steroids provided a new way for steroid medicines production.

Physiological features and genetic polymorphism of a new breed licorice Wuxin No.1 / 中草药

Objective A new breed of licorice seeds(Wuxin No.1) was selected from wild licorice,Glycyrrhiza uralensis.Methods Its germination rate,content of soluble protein,and peroxidase(POD) activities were investigated and compared with the wild licorice seeds(control group).The leaves of Wuxin No.1 were collected and its genetic polymorphism was analyzed by inter-simple sequence repeat technique(ISSR).Results The results suggested that the seed vitality in Wuxin No.1 was higher than that in the control group.The change of soluble protein and POD activity also demonstrated that the seeds in Wuxin No.1 have the higher vigor.ISSR Analysis showed that among 22 random primers used in this experiment,six primers generated different DNA band types,which meant that there was genetic polymorphism in Wuxin No.1.Conclusion All these changes indicate that Wuxin No.1 is a prospective domestication species of licorice and may be cultivated widely in the future.

PRELIMINARY STUDIES ON LIVE YEAST CELL DERIVATIVE / 微生物学通报

Live Yeast Cell Derivative (LYCD) was based on a living cells response to a controlled injury, which stimulated it to produce protective substance to increase cellular respiration and wound healing The experiment suggested that LYCD had the ability to improve cellular respiration, and this ability became strongest after the cell was treated with H 2O 2 for 15min, while the quantity of reduced glutathione (GSH) in LYCD reached the highest at 30min By contrast, almost the same biological activity of LYCD was observed under different stress conditions