Regulating Cardiolipin Biosynthesis for Efficient Production of Colanic Acid in Escherichia coli.

Colanic acid has broad application prospects in the food and healthcare market due to its excellent physical properties and biological activities. In this study, we discovered that colonic acid production in Escherichia coli could be enhanced by regulating cardiolipin biosynthesis. Single deletion of clsA, clsB, or clsC related to cardiolipin biosynthesis in E. coli MG1655 only slightly increased colonic acid production, but double or triple deletion of these three genes in E. coli MG1655 increased colonic acid production up to 2.48-fold. Previously, we have discovered that truncating lipopolysaccharide by deletion of the waaLUZYROBSPGQ gene cluster and enhancing RcsA by deletion of genes lon and hns can increase colonic acid production in E. coli. Therefore, these genes together with clsA, clsB, or/and clsC were deleted in E. coli, and all the resulting mutants showed increased colonic acid production. The best colonic acid production was observed in the mutant WWM16, which is 126-fold higher than in the control MG1655. To further improve colonic acid production, the genes rcsA and rcsD1-466 were overexpressed in WWM16, and the resulting recombinant E. coli WWM16/pWADT could produce 44.9 g/L colonic acid, which is the highest titer reported to date.

Improve L-isoleucine production in Corynebacterium glutamicum WM001 by destructing the biosynthesis of trehalose dicorynomycolate.

Trehalose dicorynomycolates are structurally important constituents of the cell envelope in Corynebacterium glutamicum. The genes treS, treY, otsA, mytA and mytB are necessary for the biosynthesis of trehalose dicorynomycolates. In this study, the effect of biosynthesis of trehalose dicorynomycolates on L-isoleucine production in C. glutamicum has been investigated by deleting the genes treS, treY, otsA, mytA, and mytB in the L-isoleucine producing C. glutamicum WM001. L-isoleucine production was slightly improved in the mutants ΔtreY, ΔotsA, and ΔtreYA, and not improved in the single deletion mutant ΔtreS , but significantly improved in the triple deletion mutant ΔtreSYA. Deletion of mytA or mytB in ΔtreSYA could further improve L-isoleucine production. However, deletion of both mytA and mytB in ΔtreSYA significantly decreased L-isoleucine production. The final L-isoleucine producing C. glutamicum WL001 was constructed by deletion of treS, treY, otsA, and mytB, insertion of lrp, and replacement of the native promoter of ilvA with the L-isoleucine sensitive promoter PbrnFE7. WL001 grew worse than the control WM001, but produced 36.1% more L-isoleucine after 72 h shake flask cultivation than WM001.

The role of trehalose biosynthesis on mycolate composition and L-glutamate production in Corynebacterium glutamicum.

Corynebacterium glutamicum has been widely utilized for the industrial production of various amino acids. Trehalose is a prerequisite for the biosynthesis of mycolates which are structurally important constituents of the cell envelope in C. glutamicum. In this study, C. glutamicum mutant ΔSYA, which is unable to synthesize trehalose was constructed by deleting genes treS, treY and otsA in the three pathways of trehalose biosynthesis. In the fermentation medium, ΔSYA grew as well as the control C. glutamicum ATCC13869, synthesized similar levels of glucose monocorynomycolate, trehalose dicorynomycolate, and phospholipids to ATCC13869, but produced 12.5 times more L-glutamate than ATCC13869. Transcriptional levels of the genes relevant to L-arginine biosynthesis, encoding ODHC and relevant to the biosynthesis of sulfur-containing amino acids were down-regulated in ΔSYA. In minimal medium with different concentrations of glucose, ΔSYA grew worse than ATCC13869 but excreted more L-glutamate. When grown in minimal medium, phospholipids are the major lipid in ΔSYA, while glucose monocorynomycolate, trehalose dicorynomycolate, and phospholipids are the major lipid in ATCC13869. The transcriptional levels of mscCG in ΔSYA was significantly up-regulated when grown in minimal medium.

Free lipid A and full-length lipopolysaccharide coexist in Vibrio parahaemolyticus ATCC33846.

Lipid A plays an important role in the pathogenicity and antimicrobial resistance of Vibrio parahaemolyticus, but little is known about the structure and biosynthesis of lipid A in V. parahaemolyticus. In this study, lipid A species were either directly extracted or obtained by the acid hydrolysis of lipopolysaccharide from V. parahaemolyticus ATCC33846 cells and analyzed by thin-layer chromatography and high-performance liquid chromatography-tandem mass spectrometry. Several lipid A species in V. parahaemolyticus cells were characterized, and two of these species were not connected to polysaccharides. One free lipid A species has the similar structure as the hexa-acylated lipid A in Escherichia coli, and the other is a hepta-acylated lipid A with an additional secondary C16:0 acyl chain. Three lipid A species were isolated by the acid hydrolysis of lipopolysaccharide: the 1st one has the similar structure as the hexa-acylated lipid A in E. coli, the 2nd one is a hepta-acylated lipid A with an additional secondary C16:0 acyl chain and a secondary 2-OH C12:0 acyl chain, and the 3rd one is equal to the 2nd species with a phosphoethanolamine modification. These results are important for understanding the biosynthesis of lipid A in V. parahaemolyticus.

Characterization of ampicillin-resistant genes in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is strongly resistant to ampicillin (AMP). In this study, AMP-resistant genes in V. parahaemolyticus ATCC33846 were characterized. Transcriptomic analysis of V. parahaemolyticus exposed to AMP revealed 4608 differentially transcribed genes, including 670 significantly up-regulated genes and 655 significantly down-regulated genes. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, significantly modulated genes in ATCC33846 under AMP stimulation were observed in the following categories: microbial metabolism in diverse environments, metabolic pathways, bacterial secretion system, citrate cycle, biofilm formation, oxidative phosphorylation, ribosome, citrate cycle, pyruvate metabolism, carbon metabolism, nitrogen metabolism, fatty acid metabolism and tryptophan metabolism. The genes VPA0510, VPA0252, VPA0699, VPA0768, VPA0320, VP0636, VPA1096, VPA0947 and VP1775 were significantly up-regulated at the similar level to blaA in V. parahaemolyticus under AMP stimulation, and their overexpression in V. parahaemolyticus could increase its resistance to AMP. These results indicate that AMP has a global influence on V. parahaemolyticus cells. The findings would provide new insights into the resistant mechanism of V. parahaemolyticus to AMP, which would be helpful for developing novel drugs for treating V. parahaemolyticus infection.

Global metabolic regulation in Vibrio parahaemolyticus under polymyxin B stimulation.

Vibrio parahaemolyticus is responsible for infection diseases of people who consume the contaminated seafood, but its metabolic regulation profile in response to colistin, the last treatment option for multidrug-resistant Gram-negative bacteria, remains unclear. In this study, the metabolic regulation profile of V. parahaemolyticus ATCC33846 under polymyxin B stimulation has been investigated. V. parahaemolyticus exposed to polymyxin B resulted in 4597 differentially transcribed genes, including 673 significantly up-regulated genes and 569 significantly down-regulated genes. In V. parahaemolyticus under polymyxin B stimulation, the cellular antioxidant systems to prevent bacteria from oxidant stress was activated, the synthesis of some nonessential macromolecules was reduced, and the assembly and modification of lipopolysaccharide and peptidoglycan to resist the attack from other antibiotics were promoted. These findings provide new insights into polymyxin B-related stress response in V. parahaemolyticus which should be useful for developing novel drugs for infection.

Correlation between knee anatomical angles and anterior cruciate ligament injury in males.

INTRODUCTION: To determine the correlation between anatomical angles of knee joint and anterior cruciate ligament (ACL) injury, and evaluate the effects of these angles on identifying people prone to ACL injury in males. MATERIALS AND METHODS: From January 2013 to October 2017, male patients with and without non-contact ACL injury were included in the case and control groups, respectively. Anatomical angles on the sagittal and coronal magnetic resonance (MR) images of these patients were measured by senior radiologist and orthopaedic surgeon. The parameters contained medial tibial slope (MTS), lateral tibial slope (LTS), medial-lateral plateau slope (MLPS), femoral axis-Blumensaat line angle (FABA), anterior tibia slope (ATS), anterior tibial-Blumensaat line angle (ATBA). The Student's-t test or rank sum test was used to compare the independent samples between different groups. Binary logistic regression analysis was used to analyse the effects on identifying people apt to suffer an ACL injury of these angles. RESULTS: A total of 150 male patients were included in the study. There were 72 patients in the case group and 78 patients in the control group. The MTS, LTS and ATBA in the case group were significantly greater than those in the control group (P = 0.021, P < 0.001, P = 0.046). The FABA of the case group was significantly smaller than that of the control group (P = 0.006). There was no significant difference in MLPS and ATS between the two groups. The area under the curve (AUC) of LTS was 0.762, the best anatomical angle for identifying people prone to ACL injury. Combining these anatomical angles can improve the accuracy (AUC = 0.800). CONCLUSION: The male ACL injury was associated with MTS, LTS, ATBA and FABA of the knee. The LTS might be more suitable for predicting ACL injury. Analysis of these angles alone or in combination could help identify the people apt to suffer ACL damage.

Construction and Application of an Escherichia coli Strain Lacking 62 Genes Responsible for the Biosynthesis of Enterobacterial Common Antigen and Flagella.

The biosynthesis of the enterobacterial common antigen and flagella in Escherichia coli consumes lots of substrates and energy. In this study, 12 genes responsible for the biosynthesis of the enterobacterial common antigen were deleted in E. coli MG1655, resulting in WQM021. WQM021 grew better than MG1655 in both rich LB medium and minimum M9 medium. Compared with MG1655, WQM021 showed higher membrane permeability and higher production efficiency for recombinant proteins, polyhydroxyalkanoate, and l-threonine. Transcriptome analysis revealed that genes relevant to glucose consumption, glycolysis, and flagellar synthesis were significantly upregulated in WQM021. Therefore, 50 genes responsible for flagellar biosynthesis were further deleted in WQM021, resulting in WQM022. WQM022 grew better and could synthesize more polyhydroxyalkanoate and l-threonine than WQM021. The results demonstrate that the productivity of E. coli can be efficiently improved when the enterobacterial common antigen and flagella are eliminated. This strategy has guiding significance in the optimization of other industrial products and microorganisms.

Predictive effects of the intercondylar notch morphology on anterior cruciate ligament injury in males: A magnetic resonance imaging analysis.

The effects of the intercondylar notch morphology on predicting anterior crucaite ligament (ACL) injury in males were unknown. We aimed to determine the risk factors of the intercondylar notch on ACL injury, and evaluate the predictive effects of the morphological parameters on ACL injury in males. Sixty-one patients with ACL injury and seventy-eight patients with intact ACLs were assigned to the case group and control group respectively. The notch width (NW), bicondylar width, notch width index (NWI), notch height (NH), notch cross-sectional area (CSA), notch angle (NA) and notch shape were obtained from the magnetic resonance images of male patients. Comparisons were performed between the case and control groups. Logistic regression model and the receiver operating characteristic curve were used to assess the predictive effects of these parameters on ACL injury. The NW, NWI, NH, CSA and NA in the case group were significantly smaller than those in the control group on the coronal magnetic resonance images. The NW and NWI were significantly smaller, while no significant differences of the NH and CSA were found between the 2 groups on the axial images. There was no significant difference in the notch shape between the 2 groups. The maximum value of area under the curve calculated by combining all relevant morphological parameters was 0.966. The ACL injury in males was associated with NW, NH, NWI, CSA, and NA. These were good indicators for predicting ACL injury in males.

Development of a Novel Gene Expression System for Secretory Production of Heterologous Proteins via the General Secretory (Sec) Pathway in Corynebacterium Glutamicum.

BACKGROUND: Corynebacterium glutamicum (C. glutamicum) is a potential host for the secretory production of the heterologous proteins. However, to this date few secretion-type gene expression systems in C. glutamicum have been developed, which limit applications of C. glutamicum in a secretory production of the heterologous proteins. OBJECTIVES: In this study, a novel and efficient general secretory (Sec) pathway-dependent type gene expression system for the production of heterologous proteins was developed in C. glutamicum. MATERIALS AND METHODS: The synthesized cloning/expression cassette C was assembled into the basic E. coli-C. glutamicum shuttle vector pAU2, generating the Sec-dependent type gene expression vector pAU5. Subsequently, the applicability of the C. glutamicum/pAU5 system was tested using the α-amylase AmyE from Bacillus subtilis as a reporter protein. RESULTS: The vector pAU5 was successfully constructed. The SDS-PAGE experiment showed the AmyE protein band could be observed in the original culture supernatant of the 14067/pAU5-amyE. The Western blotting experiment showed that the AmyE polypeptide could be detected in the culture supernatant of the 14067/pAU5-amyE, not in the cell lysate of 14067/pAU5-amyE. The α-amylase specific activity of the culture supernatant of 14067/pAU5-amyE was 103.24±7.14 U.mg-1 protein, while no α-amylase activity was detected in the cell homogenate supernatant of 14067/pAU5-amyE. These results demonstrate that the recombinant AmyE was efficiently expressed and completely secreted into the extracellular environmentin an active form in C. glutamicum/pAU5 system. CONCLUSIONS: A novel efficient Sec-dependent type gene expression vector pAU5 was constructed in the C. glutamicum. The vector pAU5 employs the strong promoter tac-M for controlling a constitutive transcription of the target gene, the consensus ribosome binding site (RBS) sequence of C. glutamicum to ensure protein translation, and the efficient Sec-type cgR_2070 signal sequence to mediate protein secretion in the C. glutamicum. The C. glutamicum/pAU5 system is an efficient expression system for the secretory production of the heterologous proteins.

Development of a novel vector for cloning and expressing extremely toxic genes in Escherichia coli

Background: Escherichia coli has been widely used as a host to clone and express heterologous genes. However, there are few vectors available for cloning and expressing extremely toxic genes, which limits further basic and applied research on extremely toxic proteins. Results: In this study, a novel vector pAU10 was constructed in E. coli. pAU10 utilizes the combination of the efficient but highly repressible T7-lacO promoter/operator and the strong rrnBT2 transcriptional terminator upstream of the T7 promoter to strictly control unwanted transcription of the extremely toxic gene; in addition, the trp promoter/operator is oriented opposite to the T7 promoter to control the production of the antisense RNA that may block the translation of leaky mRNA. Without the supplementation of IPTG and L-tryptophan in the culture medium, transcription of the extremely toxic gene by the T7 promoter is highly repressed, and the trp promoter produces the antisense RNA, which strictly prevents unwanted expression of the extremely toxic protein in E. coli. With the supplementation of IPTG and L-tryptophan, the T7 promoter efficiently transcribes the extremely toxic gene, and the trp promoter does not produce the antisense RNA, ensuring efficient expression of the extremely toxic protein in E. coli. Tight regulation and efficiency of expression of an extremely toxic gene cloned in the vector pAU10 were confirmed by cloning and expressing the restriction endonuclease-encoding gene bamHI without its corresponding methylase gene in E. coli JM109(DE3). Conclusion: pAU10 is a good vector used for cloning and expressing extremely toxic genes in E. coli.

Development of a genetically engineered Escherichia coli strain for plasmid transformation in Corynebacterium glutamicum.

Gene disruption and replacement in Corynebacterium glutamicum is dependent upon a high transformation efficiency. The cglIR-cgIIR restriction system is a major barrier to introduction of foreign DNA into Corynebacterium glutamicum cells. To improve the transformation efficiency of C. glutamicum, the cglIM gene encoding methyltransferase in the cglIR-cglIIR-cglIM restriction-modification system of C. glutamicum ATCC 13032 was chromosomally integrated and expressed in Escherichia coli, resulting in an engineered strain E. coli AU1. The electro-transformation experiments of C. glutamicum ATCC 13032 with the E. coli-C. glutamicum shuttle plasmid pAU4 showed that the transformation efficiency of C. glutamicum with pAU4 DNA extracted from E. coli TG1/pAU4 was 1.80±0.21×102cfu/µg plasmid DNA, while using pAU4 DNA extracted from E. coli AU1/pAU4, the transformation efficiency reached up to 5.22±0.33×106cfu/µg plasmid DNA. The results demonstrated that E. coli AU1 is able to confer the cglIM-specific DNA methylation pattern to its resident plasmid, which makes the plasmid resistant to the cglIR-cglIIR restriction and efficiently transferred into C. glutamicum. E. coli AU1 is a useful intermediate host for efficient transformation of C. glutamicum.