[ Objective] The study was to investigate the volatile components of secondary metabolites from M. alpine producing arachidonic acid and explore the changes in its metabolic pathway. [ Method] The air above M. alpine ...[ Objective] The study was to investigate the volatile components of secondary metabolites from M. alpine producing arachidonic acid and explore the changes in its metabolic pathway. [ Method] The air above M. alpine broth was extracted by solid-phase microextraction（SPME） during the post-exponential phase of growth and analyzed by GC-MS. [Result] 13 compounds were identified, 12 of which were sesquiterpenes with C15H24 formula and accounting for 99.62% of the complete compounds. Thujopsene-（ 12）, α-Guaiene and Aristolene were three most sesquiterpenes accounting for 10.66%, 33.69% and 34.85% of total content respectively. It can be sufficiently certified that sesquiterpene metabolic pathway existing in M. alpine. [ Coclusion] Metabolic flux of sesquiterpene pathway increased to improve its mass accumulation, because one or several key enzyme genes mutation in isoprene or sesquiterpene pathway enhanced their activities during induction of mutation from initial strain.展开更多
Mortierella isabellina was found to accumulate large amounts of unsaturated fatty acids when it was grown in a medium containing butanol(5 g/L) and yeast extract(10 g/L) and cultivated at 25 ℃ for 5 d during which ad...Mortierella isabellina was found to accumulate large amounts of unsaturated fatty acids when it was grown in a medium containing butanol(5 g/L) and yeast extract(10 g/L) and cultivated at 25 ℃ for 5 d during which additional feeding butanol of 2 g/L was fed after being cultivated for 48 h . The resultant mycelial lipids accounted for 40 1% of the dry mycelia, while about 34% of butanol in the medium was converted. The mycelial lipids contained four kinds of unsaturated fatty acids, i.e ., palmitoleic(4 9%), oleic(54 1%), linoleic(10 4%) and linolenic(5 4%) acids. Those accounted for 74 8% of the total fatty acids. The effects of the culture conditions, such as cultivation temperature, initial pH of the medium and additional feeding butanol in the course of cultivation, on the production of mycelial lipids by M. isabellina were studied.展开更多
A developed method was used for the enhancement of arachidonic acid production by M. isabellina. An orthogonal, rotatable and central composite design was applied to determine the optimum conditions for protoplast reg...A developed method was used for the enhancement of arachidonic acid production by M. isabellina. An orthogonal, rotatable and central composite design was applied to determine the optimum conditions for protoplast regeneration mutagenesis. The results showed that a commixture enzyme （cellulase and glusulase） at the concentration of 4%, enzymolysis temperature at 30℃ and enzymolysis time on 7.5 h were the optimal conditions, in which the lethality of M. isabellina spores was 78.4%. After mutagenesis and re-screenings, M. isabellina mutant Y-69 was obtained. GC analysis showed that the yield of arachidonic acid by Y-69 （2.92 g. L-1） was 3.56 times higher than that of the wild-type strain （0.82 g.L-1）. Pass generation tests showed that the properties of Y-69 by mutation were readily inherited.展开更多
AIM： To clone △^12 -fatty acid desaturase gene of Mortierella isabellina and to functionally characterize this gene in vitro and in vivo.METHODS： Reverse transcriptional polymerase chain reaction （RT-PCR） was use...AIM： To clone △^12 -fatty acid desaturase gene of Mortierella isabellina and to functionally characterize this gene in vitro and in vivo.METHODS： Reverse transcriptional polymerase chain reaction （RT-PCR） was used to clone the open reading frame of △^12-fatty acid desaturase gene （D12D） of Mortierella isabellina. Plasmids pEMICL12 and pYMICL12 were constructed with it. pEMICL12 was transformed into Escherichia coli（E.coli） strain BL21 using CaCl2 method for expression after induction with IPTG. pTMICL12 was transformed into Saccharomyces cerevisiae strain IN- VSc1 using lithium acetate method for expression under the induction of galactose. Northern blotting method was used to investigate the effect of temperature on the transcriptional level of this gene in S.cerevisiae strain IN- VSc1.RESULTS： Recombinant plasmids pEMICL12 and pTMICL12 were successfully constructed and transformed into E. coli and S.cerevisiae separately with appropriate method. After induction with IPTG and galactose, it was found that expression of △^12-fatty acid desaturase genes in E.coli and S. cerevisiae under appropriate conditions led to the production of active △^12-fatty acid desaturase, which could convert 17.876% and 17.604% of oleic acid respectively to linoleic acid by GC-MS detection in vitro and in vivo.CONCLUSION： Cloning and expression of M.isabellina D12D gene in E.coli and S.cerevisiae is successfully completed.展开更多
Arachidonic acid （AA or ARA）, an essential to-6 polyunsaturated fatty acid （PUFA）, can be produced by Mortierella isabellina. Mutagenesis on Mortierella isabellina As3.3410 was induced to raise ARA production. The...Arachidonic acid （AA or ARA）, an essential to-6 polyunsaturated fatty acid （PUFA）, can be produced by Mortierella isabellina. Mutagenesis on Mortierella isabellina As3.3410 was induced to raise ARA production. The mutant strain of YZ-124 had the highest ARA of 4.72 g. L-1, which was 5.5 times higher than that of the original strain 3.3410. mRNA expression level of △ 6- desaturase was determined in five different kinds of ARA-producing Mortierella isabellina after cultured for 7 days, and in the mutant strain YZ-124 over a 3-8 day time-course. In addition, the desaturase activity and ARA content were measured at the selected time points. The lowest expression of △6-desaturase was observed in the original strain and the highest expression in the mutant strain YZ-124, which increased with increasing time in culture. Furthermore, a positive correlation was observed between the expression levels of △6-desaturase and ARA content. Based on this, △6-desaturase played a significant role in ARA synthesis pathway in Mortierella isabellina.展开更多
Mortierella alpina has been considered as the most effective producer of arachidonic acid(ARA)-rich oil. It was found that several methods could improve the percentage of ARA in total lipids successfully, as they acti...Mortierella alpina has been considered as the most effective producer of arachidonic acid(ARA)-rich oil. It was found that several methods could improve the percentage of ARA in total lipids successfully, as they activated the desaturation system on the endoplasmic reticulum. Additionally, in M. alpina the ARA exists in several forms, such as triacylglycerol(TAG), and diacylglycerol(DAG). These forms are caused by different acyltransferases and they determine the nutrient value of the microbial oil. However, few works revealed detailed fatty acid distribution among lipid classes, which to some extent impeded the accurate regulation in ARA accumulation. Herein, this paper gives information on the accumulation process of main lipid classes and the changes of fatty acid composition in these lipids during ARA accumulation period in M. alpina. The result demonstrates that TAG was the dominant component of the total lipids, and it is the main form for ARA storage. The ARA enrichment stage occurred during 168–192 h when the amount of total lipids maintained steady. Further analysis indicated that the newly formed ARA-TAG might come from the incorporation and modi fication of saturated and monounsaturated fatty acids in other lipid classes. This work could be helpful for further optimization of ARA-rich TAG production.展开更多
The regio- and stereo-selective hydroxylations of two ingenane diterpenoids, 20-deoxyingenol(1) and 13-oxyingenol dodecanoat(2), by the filamentous fungi Mortierella ramanniana and Gibberella fujikuroi were investigat...The regio- and stereo-selective hydroxylations of two ingenane diterpenoids, 20-deoxyingenol(1) and 13-oxyingenol dodecanoat(2), by the filamentous fungi Mortierella ramanniana and Gibberella fujikuroi were investigated in the present study. Four undescribed metabolites(3–6) of substrate 1 and two undescribed metabolites(7 and 8) of substrate 2 were isolated. All the lites were identified as hydroxylated ingenane derivatives by extensive NMR and HR-ESI-MS data analyses. All the biotransformed compounds and the substrates were evaluated for their cytotoxicities against three human cancer cell lines, including human colon cancer Caco-2, breast cancer MCF-7, and adriamycin(ADM)-resistant MCF-7/ADM cell lines. All ingenane alcohols(1, and 3–6) displayed no significant cytotoxic activities. The substrate 13-oxyingenol dodecanoat(2) showed moderate cytotoxicity with IC50 values being 35.59 ± 5.37 μmol·L^(-1)(Caco-2), 24.04 ± 4.70 μmol·L^(-1)(MCF-7), and 22.24 ± 5.19 μmol·L^(-1)(MCF-7/ADM). However, metabolites 7 and 8 displayed no significant cytotoxicity. These results indicated that the hydroxylation at the C^(-1)3 aliphatic acid ester of substrate 2 can significantly reduce the cytotoxic activity.展开更多
Arachidonic acid （ARA or AA）, one of the most important polyunsaturated fatty acids （PUFAs）, has various physiological activities and positive effects on human health. ARA production by Mortierella alpina has beco...Arachidonic acid （ARA or AA）, one of the most important polyunsaturated fatty acids （PUFAs）, has various physiological activities and positive effects on human health. ARA production by Mortierella alpina has become a hot topic in recent years, owing to that it is effective, safe and easy to control. How to improve ARA yield and purification efficiency is important to ARA production in M. alpina. Therefore, in this review, we summarized some methods to improve ARA yield： optimization of culture conditions, mycelium aging technologies and metabolic regulation, and the commonly used methods for ARA isolation and purification, to provide a theoretical basis for ARA production by M. alpina fermentation.展开更多
基金Supported by the National Natural Science Foundation of China(20606018)the National Basic Research Program (also called 973Program)(2007CB707805)the Six Projects Sponsoring Talent Summits of Jiangsu Province~~
文摘[ Objective] The study was to investigate the volatile components of secondary metabolites from M. alpine producing arachidonic acid and explore the changes in its metabolic pathway. [ Method] The air above M. alpine broth was extracted by solid-phase microextraction（SPME） during the post-exponential phase of growth and analyzed by GC-MS. [Result] 13 compounds were identified, 12 of which were sesquiterpenes with C15H24 formula and accounting for 99.62% of the complete compounds. Thujopsene-（ 12）, α-Guaiene and Aristolene were three most sesquiterpenes accounting for 10.66%, 33.69% and 34.85% of total content respectively. It can be sufficiently certified that sesquiterpene metabolic pathway existing in M. alpine. [ Coclusion] Metabolic flux of sesquiterpene pathway increased to improve its mass accumulation, because one or several key enzyme genes mutation in isoprene or sesquiterpene pathway enhanced their activities during induction of mutation from initial strain.
基金Supported by the National Natural Science Foundation of China(No.2 98730 18)
文摘Mortierella isabellina was found to accumulate large amounts of unsaturated fatty acids when it was grown in a medium containing butanol(5 g/L) and yeast extract(10 g/L) and cultivated at 25 ℃ for 5 d during which additional feeding butanol of 2 g/L was fed after being cultivated for 48 h . The resultant mycelial lipids accounted for 40 1% of the dry mycelia, while about 34% of butanol in the medium was converted. The mycelial lipids contained four kinds of unsaturated fatty acids, i.e ., palmitoleic(4 9%), oleic(54 1%), linoleic(10 4%) and linolenic(5 4%) acids. Those accounted for 74 8% of the total fatty acids. The effects of the culture conditions, such as cultivation temperature, initial pH of the medium and additional feeding butanol in the course of cultivation, on the production of mycelial lipids by M. isabellina were studied.
基金Supported by Young Scientifi c Fund of Heilongjiang Province (QC2010093)Project of Daqing Scientifi c Program (sggh2009-029)
文摘A developed method was used for the enhancement of arachidonic acid production by M. isabellina. An orthogonal, rotatable and central composite design was applied to determine the optimum conditions for protoplast regeneration mutagenesis. The results showed that a commixture enzyme （cellulase and glusulase） at the concentration of 4%, enzymolysis temperature at 30℃ and enzymolysis time on 7.5 h were the optimal conditions, in which the lethality of M. isabellina spores was 78.4%. After mutagenesis and re-screenings, M. isabellina mutant Y-69 was obtained. GC analysis showed that the yield of arachidonic acid by Y-69 （2.92 g. L-1） was 3.56 times higher than that of the wild-type strain （0.82 g.L-1）. Pass generation tests showed that the properties of Y-69 by mutation were readily inherited.
基金Supported by the National Natural Science Foundation of China, No. 30200167 Tianjin Natural Science Foundation, No. 013802511
文摘AIM： To clone △^12 -fatty acid desaturase gene of Mortierella isabellina and to functionally characterize this gene in vitro and in vivo.METHODS： Reverse transcriptional polymerase chain reaction （RT-PCR） was used to clone the open reading frame of △^12-fatty acid desaturase gene （D12D） of Mortierella isabellina. Plasmids pEMICL12 and pYMICL12 were constructed with it. pEMICL12 was transformed into Escherichia coli（E.coli） strain BL21 using CaCl2 method for expression after induction with IPTG. pTMICL12 was transformed into Saccharomyces cerevisiae strain IN- VSc1 using lithium acetate method for expression under the induction of galactose. Northern blotting method was used to investigate the effect of temperature on the transcriptional level of this gene in S.cerevisiae strain IN- VSc1.RESULTS： Recombinant plasmids pEMICL12 and pTMICL12 were successfully constructed and transformed into E. coli and S.cerevisiae separately with appropriate method. After induction with IPTG and galactose, it was found that expression of △^12-fatty acid desaturase genes in E.coli and S. cerevisiae under appropriate conditions led to the production of active △^12-fatty acid desaturase, which could convert 17.876% and 17.604% of oleic acid respectively to linoleic acid by GC-MS detection in vitro and in vivo.CONCLUSION： Cloning and expression of M.isabellina D12D gene in E.coli and S.cerevisiae is successfully completed.
基金Supported by the National Natural Science Foundation of China(31171657)Heilongjiang Youth Science Foundation(QC2010093)+1 种基金Heilongjiang Natural Science Foundation(20100481037)Key Projects on Ministry of Education in Heilongjiang(12511Z022)
文摘Arachidonic acid （AA or ARA）, an essential to-6 polyunsaturated fatty acid （PUFA）, can be produced by Mortierella isabellina. Mutagenesis on Mortierella isabellina As3.3410 was induced to raise ARA production. The mutant strain of YZ-124 had the highest ARA of 4.72 g. L-1, which was 5.5 times higher than that of the original strain 3.3410. mRNA expression level of △ 6- desaturase was determined in five different kinds of ARA-producing Mortierella isabellina after cultured for 7 days, and in the mutant strain YZ-124 over a 3-8 day time-course. In addition, the desaturase activity and ARA content were measured at the selected time points. The lowest expression of △6-desaturase was observed in the original strain and the highest expression in the mutant strain YZ-124, which increased with increasing time in culture. Furthermore, a positive correlation was observed between the expression levels of △6-desaturase and ARA content. Based on this, △6-desaturase played a significant role in ARA synthesis pathway in Mortierella isabellina.
基金Supported by the National Science Foundation for Distinguished Young Scholars of China(21225626)the National Basic Research Program of China(2011CBA00800)+3 种基金the National Natural Science Foundation of China(21376002,21476111)the Natural Science Foundation of Jiangsu Province(BK20131405)the National High-Tech R&D Program of China(2014AA021703)the Priority Academic Program Development of Jiangsu Higher Education Institutions
文摘Mortierella alpina has been considered as the most effective producer of arachidonic acid(ARA)-rich oil. It was found that several methods could improve the percentage of ARA in total lipids successfully, as they activated the desaturation system on the endoplasmic reticulum. Additionally, in M. alpina the ARA exists in several forms, such as triacylglycerol(TAG), and diacylglycerol(DAG). These forms are caused by different acyltransferases and they determine the nutrient value of the microbial oil. However, few works revealed detailed fatty acid distribution among lipid classes, which to some extent impeded the accurate regulation in ARA accumulation. Herein, this paper gives information on the accumulation process of main lipid classes and the changes of fatty acid composition in these lipids during ARA accumulation period in M. alpina. The result demonstrates that TAG was the dominant component of the total lipids, and it is the main form for ARA storage. The ARA enrichment stage occurred during 168–192 h when the amount of total lipids maintained steady. Further analysis indicated that the newly formed ARA-TAG might come from the incorporation and modi fication of saturated and monounsaturated fatty acids in other lipid classes. This work could be helpful for further optimization of ARA-rich TAG production.
文摘The regio- and stereo-selective hydroxylations of two ingenane diterpenoids, 20-deoxyingenol(1) and 13-oxyingenol dodecanoat(2), by the filamentous fungi Mortierella ramanniana and Gibberella fujikuroi were investigated in the present study. Four undescribed metabolites(3–6) of substrate 1 and two undescribed metabolites(7 and 8) of substrate 2 were isolated. All the lites were identified as hydroxylated ingenane derivatives by extensive NMR and HR-ESI-MS data analyses. All the biotransformed compounds and the substrates were evaluated for their cytotoxicities against three human cancer cell lines, including human colon cancer Caco-2, breast cancer MCF-7, and adriamycin(ADM)-resistant MCF-7/ADM cell lines. All ingenane alcohols(1, and 3–6) displayed no significant cytotoxic activities. The substrate 13-oxyingenol dodecanoat(2) showed moderate cytotoxicity with IC50 values being 35.59 ± 5.37 μmol·L^(-1)(Caco-2), 24.04 ± 4.70 μmol·L^(-1)(MCF-7), and 22.24 ± 5.19 μmol·L^(-1)(MCF-7/ADM). However, metabolites 7 and 8 displayed no significant cytotoxicity. These results indicated that the hydroxylation at the C^(-1)3 aliphatic acid ester of substrate 2 can significantly reduce the cytotoxic activity.
基金Supported by Natural Science Foundation of Yunnan Province(2016FB030)Young Academic and Technical Talent Program of Yunnan Province(2011CI027)~~
文摘Arachidonic acid （ARA or AA）, one of the most important polyunsaturated fatty acids （PUFAs）, has various physiological activities and positive effects on human health. ARA production by Mortierella alpina has become a hot topic in recent years, owing to that it is effective, safe and easy to control. How to improve ARA yield and purification efficiency is important to ARA production in M. alpina. Therefore, in this review, we summarized some methods to improve ARA yield： optimization of culture conditions, mycelium aging technologies and metabolic regulation, and the commonly used methods for ARA isolation and purification, to provide a theoretical basis for ARA production by M. alpina fermentation.
