The enhancement of energy supply in syngas-fermenting microorganisms.

After the second industrial revolution, social productivity developed rapidly, and the use of fossil fuels such as coal, oil, and natural gas increased greatly in industrial production. The burning of these fossil fuels releases large amounts of greenhouse gases such as CO2, which has caused greenhouse effects and global warming. This has endangered the planet's ecological balance and brought many species, including animals and plants, to the brink of extinction. Thus, it is crucial to address this problem urgently. One potential solution is the use of syngas fermentation with microbial cell factories. This process can produce chemicals beneficial to humans, such as ethanol as a fuel while consuming large quantities of harmful gases, CO and CO2. However, syngas-fermenting microorganisms often face a metabolic energy deficit, resulting in slow cell growth, metabolic disorders, and low product yields. This problem limits the large-scale industrial application of engineered microorganisms. Therefore, it is imperative to address the energy barriers of these microorganisms. This paper provides an overview of the current research progress in addressing energy barriers in bacteria, including the efficient capture of external energy and the regulation of internal energy metabolic flow. Capturing external energy involves summarizing studies on overexpressing natural photosystems and constructing semiartificial photosynthesis systems using photocatalysts. The regulation of internal energy metabolic flows involves two parts: regulating enzymes and metabolic pathways. Finally, the article discusses current challenges and future perspectives, with a focus on achieving both sustainability and profitability in an economical and energy-efficient manner. These advancements can provide a necessary force for the large-scale industrial application of syngas fermentation microbial cell factories.

Efficient Mycoprotein Production with Low CO2 Emissions through Metabolic Engineering and Fermentation Optimization of Fusarium venenatum.

The global protein shortage is intensifying, and promising means to ensure daily protein supply are desperately needed. The mycoprotein produced by Fusarium venenatum is a good alternative to animal/plant-derived protein. To comprehensively improve the mycoprotein synthesis, a stepwise strategy by blocking the byproduct ethanol synthesis and the gluconeogenesis pathway and by optimizing the fermentation medium was herein employed. Ultimately, compared to the wild-type strain, the synthesis rate, carbon conversion ratio, and protein content of mycoprotein produced from the engineered strain were increased by 57% (0.212 vs 0.135 g/L·h), 62% (0.351 vs 0.217 g/g), and 57% (61.9 vs 39.4%), respectively, accompanied by significant reductions in CO2 emissions. These results provide a referential strategy that could be useful for improving mycoprotein synthesis in other fungi; more importantly, the obtained high-mycoprotein-producing strain has the potential to promote the development of the edible protein industry and compensate for the gap in protein resources.

Anthocyanin and proanthocyanidin from Aronia melanocarpa (Michx.) Ell.: Purification, fractionation, and enzyme inhibition.

Aronia melanocarpa (Michx.) Ell. is a rich source of anthocyanins and proanthocyanidins with confirmed health benefits. Individual cyanidin glucosides (cyanidin 3-galactoside, cyanidin 3-arabinoside, cyanidin 3-xyloside, and cyanidin 3-glucoside) of anthocyanins (calculated by individual cyanin glycoside fractions was 419.9 mg/100 g FW) were isolated by Sephadex LH-20 column and different parts of proanthocyanidins with a different mean degree of polymerization (mDP) were fractionated by the solubility differences in different solvents. The composition of different mDP of proanthocyanidins was as follows: monomers (1.51%), oligomer (mDP of 4.2 ± 0.9, 20.57%), CPP-50 (mDP of 78.9 ± 4.1, 22.17%), CPP-60 (mDP of 66.1 ± 1.2, 27.94%), CPP-70 (mDP of 36.8 ± 3.9, 36.8%), CPP-75 (mDP of 25.2 ± 1.3, 6.14%), CPP-L (mDP of 10.2 ± 2.6, 6.95%), and there were recycling loss of 0.34%. Cyanidin 3-glucoside showed the strongest inhibition effects on α-amylase and lipase and cyanidin 3-arabinoside showed the strongest inhibition effect on α-glucosidase, while cyanidin 3-xyloside has no inhibition effect on the α-amylase, and cyanidin 3-galactoside, cyanidin 3-arabinoside, and cyanidin 3-xyloside have no inhibition effects on lipase. The inhibition effect of proanthocyanidins with different mDP to the enzymes all showed high negative correlations between the mDP and IC50 (half-maximal inhibitory concentration). This study suggests that A. melanocarpa (Michx.) Ell. can have beneficial effects due to inhibition of the digestion enzyme.

Correlations between contrast-enhanced ultrasound and microvessel density in non-small cell lung cancer: A prospective study.

Background: 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. Methods: 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. Results: 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-1. There was a significant statistical difference in MFE patterns between two pathological subgroups (p < 0.05). Besides, the MVD of "cotton" and "vascular" patterns were significantly higher than that of "dead wood" pattern (both of p < 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, p < 0.001, and r = 0.367, p < 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 p < 0.001). Only the PI was positively correlated with the MVD of lung adenocarcinoma (r = 0.288, p = 0.037). Conclusions: MFE patterns and quantitative parameters of CEUS had good correlation with MVD of NSCLC, especially in squamous cell carcinoma.

Identification of neutral genome integration sites with high expression and high integration efficiency in Fusarium venenatum TB01.

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.

Sustainable production of astaxanthin in microorganisms: the past, present, and future.

Astaxanthin (3,3'-dihydroxy-4,4'-diketo-ß-carotene) is a type of C40 carotenoid with remarkable antioxidant characteristics, showing significant application prospects in many fields. Traditionally, the astaxanthin is mainly obtained from chemical synthesis and natural acquisition, with both approaches having many limitations and not capable of meeting the growing market demand. In order to cope with these challenges, novel techniques, e.g., the innovative cell engineering strategies, have been developed to increase the astaxanthin production. In this review, we first elaborated the biosynthetic pathway of astaxanthin, with the key enzymes and their functions discussed in the metabolic process. Then, we summarized the conventional, non-genetic strategies to promote the production of astaxanthin, including the methods of exogenous additives, mutagenesis, and adaptive evolution. Lastly, we reviewed comprehensively the latest studies on the synthesis of astaxanthin in various recombinant microorganisms based on the concept of microbial cell factory. Furthermore, we have proposed several novel technologies for improving the astaxanthin accumulation in several model species of microorganisms.

Enhanced ultrasound-guided versus non-enhanced ultrasound-guided percutaneous needle biopsy in tissue cellularity of lung malignancies: a propensity score matched study.

Background: Though ultrasound-guided percutaneous lung needle biopsy (US-PLNB) is a first-line small biopsy method for peripheral lung lesions, quality of cellularity in specimens obtained via US-PLNB is uncertain. This study investigated the accuracy, sensitivity, and cellularity of US-PLNB. It examined the ability of contrast-enhanced ultrasound (CEUS) to improve the effectiveness of US-PLNB. Methods: We retrospectively analyzed all data of patients with subpleural lung lesions who underwent US-PLNB. The cellularity of US-PLNB from malignant lesions included the tumor cell number and proportion. The definition of high-quality cellularity (HQC) was concurrently achieving a tumor cell number ≥400 and a proportion ≥20%. The sensitivity, the actual numbers of tumor cell number/proportion, and the rate of HQC were calculated and compared between the CEUS and non-enhanced US groups after propensity score matching (PSM) with subgroup analyses by lesion size (small lesion ≤30 mm and large lesion >30 mm). Results: A total of 345 patients undergoing 345 US-PLNBs were evaluated, with 3.7±1.1 of punctures on average. There were 201 malignant and 144 benign lesions with a mean size of 43.8±24.1 mm. Among the 201 malignant lesions, 124 cases underwent CEUS and 77 underwent non-enhanced US. The quantity of tumor cells, the proportion of tumor cells, and the rate of HQC in 201 cases of US-PLNB from malignant lesions were 2,862.1±2,288.0, 44.6%±24.5%, and 82.1% [95% confidence interval (CI): 76.6% to 87.1%], respectively. The quantity of tumor cells, the proportion of tumor cells, and rate of HQC were significantly higher in the CEUS group than that in the non-enhanced US group, both in the analysis of overall malignant lesions and in large malignant lesions (all P<0.05). Conclusions: The US-PLNB has high sensitivity and thereby obtains HQC samples for subpleural lung malignant lesions. The CEUS helps improve the rate of HQC and tissue cellularity of lung malignancies.

Utility of high-frequency B-mode and contrast-enhanced ultrasound for the differential diagnosis of benign and malignant pleural diseases: a prospective study.

Background: Pleural disease is a prevalent condition. As precision therapy advances, noninvasive imaging modalities play even more important roles in the evaluation of pleural diseases. This study investigated the diagnostic capabilities of high-frequency B-mode ultrasound (US) and contrast-enhanced US (CEUS) in terms of differentiating between benign and malignant pleural diseases. Methods: Patients with unexplained thickened pleurae were prospectively analyzed via transthoracic US. High-frequency B-mode US was used to derive the pleural thickness, morphology, and echogenicity. We analyzed the high-frequency CEUS data including the enhancement mode and time intensity curve (TIC). The cause of pleural thickening was confirmed by pleural biopsy and follow-up after the biopsy. We analyzed the differences in various ultrasonic features between the malignant and benign groups. Moreover, we plotted receiver operator curves (ROCs) and obtained the area under the curves, sensitivities, and specificities of all significant continuous variables. Multivariate logistic regression was used to assess the combined usefulness of multiple US indicators in terms of predicting malignant pleurae. Results: Thirty malignant and twenty benign thickened pleurae were finally diagnosed via pleural biopsy and at least six months of follow-up. The pleural morphology and enhancement modes significantly differed between the two groups (all P<0.05). The thickness derived from B-mode US and CEUS were significantly thicker in the malignant group (both P<0.05). Arrival time (AT) and the time to peak (TTP) of TIC were significantly shorter in the malignant group, whereas peak intensity and the area under the TIC were significantly higher in the malignant group (all P<0.05). The area under the ROC for pleural thickness derived from B-mode US was 0.819; pleural thickness derived from CEUS was 0.848; AT was 0.804; TTP was 0.750; peak intensity was 0.745; the area under the TIC was 0.743; and the combined various B-mode and CEUS parameter was 0.975. Conclusions: Pleural thickness, morphology, enhancement mode, and the TIC of high-frequency US aided the differentiation of benign from malignant pleural diseases. Combined analysis of US indicators further improved the diagnostic capability.

Diagnostic ability and its influenced factors of ultrasound-guided percutaneous pleural needle biopsy diagnosis for malignant pleural mesothelioma.

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.

Evasion of Cas9 toxicity to develop an efficient genome editing system and its application to increase ethanol yield in Fusarium venenatum TB01.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) system is a powerful genome editing tool that has been successfully established in some filamentous fungi due to its high flexibility and efficiency. However, the potential toxicity of Cas9 restricts the further popularization and application of this system to some degree. The AMA1 element is a self-replicator derived from Aspergillus nidulans, and its derived vectors can be readily lost without selection. In this study, we eliminated Cas9 toxicity to Fusarium venenatum TB01 based on 100% AMA1-based Cas9 expression vector loss. Meanwhile, two available endogenous Pol III promoters (FvU6374 and Fv5SrRNA) used for sgRNA expression of the CRISPR/Cas9 system were excavated. Compared to FvU6374 (40-50%), Fv5SrRNA exhibited higher single-gene editing efficiency (> 85%), and the efficiency of simultaneous editing of the two genes using Fv5SrRNA was over 75%. Based on this system, a butanediol dehydrogenase encoding gene FvBDH was deleted, and the ethanol yield in variants increased by 52% compared with that of the wild-type. The highly efficient CRISPR/Cas9 system developed here lays the technical foundation for advancing the development of F. venenatum TB01 through metabolic engineering, and the obtained FvBDH gene-edited variants have the potential to simultaneously produce mycoprotein and ethanol by further gene modification and fermentation process optimization in the future.Key points• Cas9 toxicity disappeared and DNA-free gene-edited strains obtained after vector loss• Promoter Fv5SrRNA conferred TB01 higher gene editing efficiency than FvU6374•Deletion of the FvBDH gene resulted in a 52% increase in ethanol yield.

Performance of Paracoccus pantotrophus MA3 in heterotrophic nitrification-anaerobic denitrification using formic acid as a carbon source.

Excess amount of nitrogen in wastewater has caused serious concerns, such as water eutrophication. Paracoccus pantotrophus MA3, a novel isolated strain of heterotrophic nitrification-anaerobic denitrification bacteria, was evaluated for nitrogen removal using formic acid as the sole carbon source. The results showed that the maximum ammonium removal efficiency was observed under the optimum conditions of 26.25 carbon to nitrogen ratio, 3.39% (v/v) inoculation amount, 34.64 °C temperature, and at 180 rpm shaking speed, respectively. In addition, quantitative real-time PCR technique analysis assured that the gene expression level of formate dehydrogenase, formate tetrahydrofolate ligase, 5,10-methylenetetrahydrofolate dehydrogenase, serine hydroxymethyltransferase, respiratory nitrate reductase beta subunit, L-glutamine synthetase, glutamate dehydrogenase, and glutamate synthase were up-regulated compared to the control group, and combined with nitrogen mass balance analysis to conclude that most of the ammonium was removed by assimilation. A small amount of nitrate and nearly no nitrite were accumulated during heterotrophic nitrification. MA3 exhibited significant denitrification potential under anaerobic conditions with a maximum nitrate removal rate of 4.39 mg/L/h, and the only gas produced was N2. Additionally, 11.50 ± 0.06 mg/L/h of NH4+-N removal rate from biogas slurry was achieved.

Establishment of High-Efficiency Screening System for Gene Deletion in Fusarium venenatum TB01.

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 F. venenatum 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.

From formic acid to single-cell protein: genome-scale revealing the metabolic network of Paracoccus communis MA5.

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 (OD600: 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.

Genome-scale revealing the central metabolic network of the fast growing methanotroph Methylomonas sp. ZR1.

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 C40 to C30 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.

[Simulation of industrial fermentation: current status and future perspectives].

Industrial fermentation is the basic operation unit of industrial biotechnology in large-scale production. Mathematical simulation of microbial cells and their reactors will help deepen the understanding of microorganisms and fermentation processes, and will also provide solutions for the construction of new synthetic organisms. In this paper, the characteristics of industrial fermentation system, the development of mathematical simulation, the classification, characteristics and functions of mathematical models are described in depth, and the development trend of whole fermentation system simulation is prospected.

Effects of Methanol on Carotenoids as Well as Biomass and Fatty Acid Biosynthesis in Schizochytrium limacinum B4D1.

Schizochytrium is a promising source for the production of docosahexaenoic acid and astaxanthin. The effects of different methanol concentrations on astaxanthin, biomass, and production of the lipids, squalene, and total sterol in Schizochytrium limacinum B4D1 were investigated. Astaxanthin began to accumulate when the methanol concentration reached 3.2% and peaked at 5.6% methanol, with a 2,000-fold increase over that in the control. However, under cultivation with 5.6% methanol, the biomass, lipids, squalene, and total sterol decreased to various degrees. Transcriptomic analysis was performed to explore the effects of different methanol concentrations (0%, 3.2%, and 5.6%) on the expression profile of B4D1. Three key signaling pathways were found to play important roles in regulating cell growth and metabolism under cultivation with methanol. Five central carbon metabolism-associated genes were significantly downregulated in response to 5.6% methanol and thus were expected to result in less ATP and NADPH being available for cell growth and synthesis. High methanol conditions significantly downregulated three genes involved in fatty acid and squalene/sterol precursor biosynthesis but significantly upregulated geranylgeranyl diphosphate synthase, lycopene ß-cyclase, and ß-carotene 3-hydroxylase, which are involved in astaxanthin synthesis, thus resulting in an increase in the levels of precursors and the final production of astaxanthin. Additionally, the transcriptional levels of three stress response genes were upregulated. This study investigates gene expression profiles in the astaxanthin producer Schizochytrium when grown under various methanol concentrations. These results broaden current knowledge regarding genetic expression and provide important information for promoting astaxanthin biosynthesis in SchizochytriumIMPORTANCESchizochytrium strains are usually studied as oil-producing strains, but they can also synthesize other secondary metabolites, such as astaxanthin. In this study, methanol was used as an inducer, and we explored its effects on the production of astaxanthin, a highly valuable substance in Schizochytrium Methanol induced Schizochytrium to synthesize large amounts of astaxanthin. Transcriptomic analysis was used to investigate the regulation of signaling and metabolic pathways (mainly relative gene expression) in Schizochytrium grown in the presence of various concentrations of methanol. These results contribute to the understanding of the underlying molecular mechanisms and may aid in the future optimization of Schizochytrium for astaxanthin biosynthesis.

Alanine mother liquor as a nitrogen source for docosahexaenoic acid production by Schizochytrium sp. B4D1

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.

Effects of butanol on high value product production in Schizochytrium limacinum B4D1.

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.

Phospholipase A1-Catalysed Synthesis of Docosahexaenoic Acid-Enriched Phosphatidylcholine in Reverse Micelles System.

Phosphatidylcholine enriched with docosahexaenoic acid (DHA) was successfully produced by phospholipase A1-catalysed acidolysis. Reverse micelles were firstly selected as the reaction system to avoid the process of immobilization and to ensure the efficient catalysis of phospholipase A1. Parameters were optimized for a high incorporation of phosphatidylcholine enriched with DHA (DHA-PC). A response-surface design with four factors, including the water content, enzyme loading, pH and substrate-mass ratio were used to evaluate the influence of the major factors and to predict the optimal reaction conditions. The results indicated that the optimal reaction conditions for the production of DHA-PC were a water content of 0.4%, an enzyme loading of 40%, pH 6.92 and a substrate-mass ratio of 2.13. Under these optimized conditions, 20.90% of DHA content in DHA-PC was obtained.

Regulation of the Docosapentaenoic Acid/Docosahexaenoic Acid Ratio (DPA/DHA Ratio) in Schizochytrium limacinum B4D1.

Docosapentaenoic acid/docosahexaenoic acid ratio (DPA/DHA ratio) in Schizochytrium was relatively stable. But ideally the ratio of DPA/DHA will vary according to the desired end use. This study reports several ways of modulating the DPA/DHA ratio. Incubation times changed the DPA/DHA ratio, and changes in this ratio were associated with the variations in the saturated fatty acid (SFAs) content. Propionic acid sharply increased the SFAs content in lipids, dramatically decreased the even-chain SFAs content, and reduced the DPA/DHA ratio. Pentanoic acid (C5:0) and heptanoic acid (C7:0) had similar effects as propionic acid, whereas butyric acid (C4:0), hexanoic acid (C6:0), and octanoic acid (C8:0) did not change the fatty acid profile and the DPA/DHA ratio. Transcription analyses show that ß-oxidation might be responsible for this phenomenon. Iodoacetamide upregulated polyunsaturated fatty acid (PUFA) synthase genes, reduced the DHA content, and improved the DPA content, causing the DPA/DHA ratio to increase. These results present new insights into the regulation of the DPA/DHA ratio.