•Butanol treatment altered the lipid content.•Cells accumulated more C16:0 and less DHA in the presence of butanol.•Butanol treatment triggered squalene and sterol accumulation.•The carotenoid ...content was increased upon butanol treatment.•The antagonism between the formation of DHA and terpenoids was observed.
Schizochytrium is a microalgae-like fungus and is widely used for producing docosahexaenoic acid (DHA). It is also a promising source of squalene and carotenoids. However, few fermentation strategies are available in enhancing squalene and carotenoid content in Schizochytrium. This study showed that butanol addition had multiple effects on Schizochytrium limacinum B4D1. First, butanol addition altered the lipid content of cells. Second, 6g/L of butanol decreased the proportion of DHA by nearly 40%. Third, the squalene content increased 31-fold in the presence of 6g/L butanol. Finally, cells accumulated more carotenoids upon butanol addition. Specifically, when cells were treated with 8g/L butanol, the astaxanthin content increased to 245 times than that of the untreated control. These results are helpful for the commercial exploitation of Schizochytrium in producing squalene and carotenoids.
Methylomonas sp
. ZR1 was an isolated new methanotrophs that could utilize methane and methanol growing fast and synthesizing value added compounds such as lycopene. In this study, the genomic study ...integrated with the comparative transcriptome analysis were taken to understanding the metabolic characteristic of ZR1 grown on methane and methanol at normal and high temperature regime. Complete Embden-Meyerhof-Parnas pathway (EMP), Entner–Doudoroff pathway (ED), Pentose Phosphate Pathway (PP) and Tricarboxy Acid Cycle (TCA) were found to be operated in ZR1. In addition, the energy saving ppi-dependent EMP enzyme, coupled with the complete and efficient central carbon metabolic network might be responsible for its fast growing nature. Transcript level analysis of the central carbon metabolism indicated that formaldehyde metabolism was a key nod that may be in charge of the carbon conversion efficiency (CCE) divergent of ZR1 grown on methanol and methane. Flexible nitrogen and carotene metabolism pattern were also investigated in ZR1. Nitrogenase genes in ZR1 were found to be highly expressed with methane even in the presence of sufficient nitrate. It appears that, higher lycopene production in ZR1 grown on methane might be attributed to the higher proportion of transcript level of C
40
to C
30
metabolic gene. Higher transcript level of exopolysaccharides metabolic gene and stress responding proteins indicated that ZR1 was confronted with severer growth stress with methanol than with methane. Additionally, lower transcript level of the TCA cycle, the dramatic high expression level of the nitric oxide reductase and stress responding protein, revealed the imbalance of the central carbon and nitrogen metabolic status, which would result in the worse growth of ZR1 with methanol at 30 °C.
Immunohistochemical microvessel density (MVD) is an early indicator of angiogenesis and it could be used to evaluate the therapeutic efficacy of non-small cell lung cancer (NSCLC). We sought to ...identify the ability of contrast-enhanced ultrasound (CEUS) in evaluating MVD of subpleural NSCLC.
We prospectively collected CEUS data of NSCLC confirmed by ultrasound-guided transthoracic needle biopsy from October 2019 to February 2021, The MVD of NSCLC counted by CD34-positive vessels of immunohistochemical staining. Microflow enhancement (MFE) of CEUS was divided into "dead wood", "cotton", and "vascular" patterns. Pathology subgroup and MVD between different MFE patterns were analyzed, respectively. The arrival time, time to peak, peak intensity (PI), and area under curve (AUC) derivefrom time-intensity curve of CEUS with MVD in NSCLC and its pathological subgroups (adenocarcinoma and squamous cell carcinoma) were subjected to correlation analysis.
A total of 87 patients were included in this study, consisting of 53 cases of adenocarcinoma and 34 cases of squamous cell carcinoma with a mean MVD of 27.8 ± 12.2 mm
. There was a significant statistical difference in MFE patterns between two pathological subgroups (
< 0.05). Besides, the MVD of "cotton" and "vascular" patterns were significantly higher than that of "dead wood" pattern (both of
< 0.05), whereas there was no significant difference in MVD between "cotton" pattern and "vascular" pattern. PI and AUC of CEUS were positively correlated with the MVD of NSCLC (r = 0.497,
< 0.001, and r = 0.367,
< 0.001, respectively). Besides, PI and AUC of CEUS were positively correlated with the MVD of squamous cell carcinoma (r = 0.802, and r = 0.663, respectively; both of
< 0.001). Only the PI was positively correlated with the MVD of lung adenocarcinoma (r = 0.288,
= 0.037).
MFE patterns and quantitative parameters of CEUS had good correlation with MVD of NSCLC, especially in squamous cell carcinoma.
With the increase in population growth and environmental pollution, the daily protein supply is facing great challenges. Single-cell protein (SCP) produced by microorganism fermentation is a good ...alternative for substituting plant- and animal-derived proteins. In this study,
Paracoccus communis
MA5 isolated from soil previously demonstrated an excellent ability to synthesize SCP directly from sodium formate. To investigate the central metabolic network of formic acid assimilation and protein synthesis, genome-scale analyses were performed. Genomic analysis showed that complete tetrahydrofolate cycle-, serine cycle-, glycolytic pathway-, tricarboxylic acid (TCA) cycle- and nitrogen metabolism-relevant genes were annotated in the genome. These pathways play key roles in the conversion of formic acid into proteins. Transcriptional analysis showed that sodium formate stress could stimulate the metabolic pathway in response to environmental stress, but weaken the sulfur metabolic pathway to inhibit amino acid synthesis, resulting in a decrease in protein content (30% vs 44%). However, under culture conditions with ammonium sulfate, metabolic pathways associated with protein synthesis were accelerated, causing an increase in protein content (53% vs 44%); while the tetrahydrofolate cycle associated with formic acid assimilation was inhibited, causing a 62.5% decrease in growth rate (OD
600
: 0.21 vs 0.56). These results provide evidence of protein synthesis from sodium formate in strain MA5 at the gene level and lay a theoretical foundation for the optimization of fermentation systems using formic acid as a carbon source.
Graphical Abstract
Thermophilic endoglucanases have become of significant interest for effectively catalyzing the hydrolysis of cellulose. Myceliophthora thermophila is an ideal source of thermophilic enzymes. ...Interestingly, different hosts differently express the same enzymes. In this study, we successfully overexpressed endoglucanase (MtEG5-1) from M. thermophila in the methylotrophic yeast, Pichia pastoris GS115, via electroporation. We found that purified MtEG5-1 exhibited optimum activity levels at pH 5 and 70 °C, with 88% thermal stability after being incubated at 70 °C for 2 h. However, we observed that purified MtEG5-1 had a molecular weight of 55 kDa. The Km and Vmax values of purified MtEG5-1 were approximately 6.11 mg/mL and 91.74 μmol/min/mg at 70 °C (pH 5.0), respectively. Additionally, the optimum NaCl concentration of purified MtEG5-1 was found to be 6 g/L. Furthermore, we observed that the activity of purified MtEG5-1 was significantly enhanced by Mn2+ and was inhibited by K+. These results indicated that MtEG5-1 expressed by P. pastoris GS115 is more heat-tolerant than that expressed by A. niger and P. pastoris X33. These properties of MtEG5-1 make it highly suitable for future academic research and industrial applications.
CRISPR/Cas9-mediated homology-directed recombination is an efficient method to express target genes. Based on the above method, providing ideal neutral integration sites can ensure the reliable, ...stable, and high expression of target genes. In this study, we obtained a fluorescent transformant with neutral integration and high expression of the GFP expression cassette from the constructed GFP expression library and named strain FS. The integration site mapped at 4886 bp upstream of the gene FVRRES_00686 was identified in strain FS based on a Y-shaped adaptor-dependent extension, and the sequence containing 600 bp upstream and downstream of this site was selected as the candidate region for designing sgRNAs (Sites) for CRISPR/Cas9-mediated homology-directed recombination. PCR analysis showed that the integration efficiency of CRISPR/Cas9-mediated integration of target genes in designed sites reached 100%. Further expression stability and applicability analysis revealed that the integration of the target gene into the above designed sites can be stably inherited and expressed and has no negative effect on the growth of F. venenatum TB01. These results indicate the above designed neutral sites have the potential to accelerate the development of F. venenatum TB01 through overexpression of target genes in metabolic engineering.
Display omitted
Genetic engineering is one of the most effective methods to obtain fungus strains with desirable traits. However, in some filamentous fungi, targeted gene deletion transformant screening on primary ...transformation plates is time-consuming and laborious due to a relatively low rate of homologous recombination. A strategy that compensates for the low recombination rate by improving screening efficiency was performed in
TB01. In this study, the visualized gene deletion system that could easily distinguish the fluorescent randomly inserted and nonfluorescent putative deletion transformants using green fluorescence protein (GFP) as the marker and a hand-held lamp as the tool was developed. Compared to direct polymerase chain reaction (PCR) screening, the screening efficiency of gene deletion transformants in this system was increased approximately fourfold. The visualized gene deletion system developed here provides a viable method with convenience, high efficiency, and low cost for reaping gene deletion transformants from species with low recombination rates.
Alanine mother liquor, a type of industrial waste from alanine fermentation, was used as a nitrogen source to produce docosahexaenoic acid (DHA) by Schizochytrium sp. B4D1. The results indicated that ...yeast extract could trigger the utilization of the alanine mother liquor. Additionally, the alanine can be quenched during the culture, which aids in DHA accumulation. The medium components were optimized via response surface methodology as follows: 99.98-g/L glucose, 0.05-g/L yeast extract and a 183.17 dilution factor of the alanine mother liquid (v/v, with an alanine content of 0.72 g/L) and 17.98% inoculum concentration (v/v). Finally, in a 50-mL shake-flask fermentation, the DHA yield was 2.29 g/L.
How to cite: Xu J, Zhu Y, Li H, et al. Alanine mother liquor as a nitrogen source for docosahexaenoic acid production by Schizochytrium sp. B4D1. Electron J Biotechnol 2018;35. https://doi.org/10.1016/j.ejbt.2018.06.002ç
Background
Malignant pleural mesothelioma (MPM) is a highly invasive malignant tumor. Ultrasound guidance has the advantages of real-time, convenience and nonradiative. We sought to identify ...diagnostic value and its influenced factors of ultrasound-guided percutaneous pleural needle biopsy (US-PPNB) for MPM.
Methods
Patients who underwent US-PPNB between March 2014 and March 2020 and were finally diagnosed with MPM were retrospectively analyzed. We retrospectively analyzed the US-PPNBs pathological results of all patients clinically confirmed as MPM, and divided US-PPNBs into correctly and incorrectly diagnosed groups. Patient, thoracic, and biopsy variables that affected diagnostic accuracy were assessed. All variables significant on univariate analyses were subjected to multivariate logistic regression to identify significant predictors of diagnostic accuracy. We derived cutoffs for all significant continuous variables and used the Mantel–Haenszel test to determine whether the diagnostic accuracy of US-PPNB for MPM increased with pleural thickness.
Results
In total, 49 patients with clinically confirmed MPM underwent US-PPNB; 37 diagnoses were correct and 12 were incorrect (accuracy = 75.5%). The pleura was significantly thicker in the correctly diagnosed group (
p
< 0.001). The pleural thickness cutoff was 4.15 mm and diagnostic accuracy increased with pleural thickness grade (
p
for trend <0.05). The diagnostic accuracy was significantly higher when 16-G rather than 18-G biopsy needles were used (
p
< 0.05). Multivariate logistic regression showed that pleural thickness (odds ratio: 17.2, 95% confidence interval: 2.8–104.1,
p
= 0.002) and needle size (odds ratio: 6.8, 95% confidence interval: 1.0–44.5,
p
= 0.044) independently predicted diagnostic accuracy.
Conclusion
US-PPNB afforded high MPM diagnostic accuracy, and pleural thickness and needle size significantly impacted accuracy.
The aim of the study was to evaluate the potential of acetate as a carbon source by examining the fermentation performance of Schizochytrium limacinum B4D1 and its effects on metabolism using ...physiology and transcriptomics studies. The results demonstrated that, while the metabolic rate of acetate in S. limacinum B4D1 was lower than glucose, it increased the content of docosahexaenoic acid (DHA) in the total fatty acids. Transcriptomic analysis indicated that the use of acetate as a carbon source significantly upregulated the acetyl-CoA synthetase gene, which increased the source of acetyl-CoA for fatty acid biosynthesis. Moreover, the upregulation of polyunsaturated fatty acid enzymes promoted the polyketide synthase (PKS) pathway of fatty acid synthesis, resulting in an increase in DHA content. Meanwhile, the upregulation of genes such as long-chain acyl-CoA synthetase and 3-hydroxyacyl-CoA dehydrogenase enhanced the β-oxidation process of saturated fatty acids in mitochondria, while the downregulation of acyl-CoA oxidase and catalase genes resulted the β-oxidation process of polyunsaturated fatty acids in peroxisomes was inhibited, leading to an increased proportion of DHA in the total fatty acids. Thus, these changes in gene expression contributed to the increased proportion of DHA in the total fatty acids.
•The fermentation performance of sodium acetate and glucose was compared.•Sodium acetate increased the content of DHA in the total fatty acids.•The effects of different carbon sources on DHA synthesis was studied by transcriptome analysis.•The upregulation of acetyl-CoA synthetase gene increased the source of acetyl-CoA for fatty acid biosynthesis.•The polyketide synthase pathway and the β-oxidation process were key metabolic pathways.
PDF
