ABSTRACT
A field manipulative experiment was carried out during 2015 and 2016 to examine the changes and influencing factors of root production, turnover rate, and standing crop under different nitrogen (N) addition levels, i.e., 0, 1, 2, 4, 8 and 16 g N·m-2·a-1, in a Tibetan alpine steppe. The results showed that root production and standing crop decreased linearly or exponentially with increasing N addition rates. Compared with control, 16 g N·m-2·a-1 significantly reduced the two-year average root production and standing crop by 43.0% and 45.7%, respectively. Root turnover rate increased first and then decreased along the N addition gradient, with the maximum appearing under 2 and 4 g N·m-2·a-1 treatments for 2015 and 2016, respectively. Results from linear mixed-effects models showed that root starch content was the main factor modulating the N-induced changes in root production and turnover rate, explaining 21.7% and 25.4% of their variations. Root protein content mainly contributed to the variations in standing crop, with an explanation of 20.8% of its variance. Overall, N addition had negative effect on root production and standing crop, and low N promoted while high N inhibited root turnover rate. Root metabolic parameters were the main factors modulating the N-induced changes in root dynamics.
Subject(s)
Nitrogen , Plant Roots , China , TibetABSTRACT
The gene encoding phosphopantetheinyl transferase (PPTase), pfaE, a component of the polyketide synthase (PKS) pathway, is crucial for the production of docosahexaenoic acid (DHA, 22:6ω3), along with the other pfa cluster members pfaA, pfaB, pfaC and pfaD. DHA was produced in Escherichia coli by co-expressing pfaABCD from DHA-producing Colwellia psychrerythraea 34H with one of four pfaE genes from bacteria producing arachidonic acid (ARA, 20:4ω6), eicosapentaenoic acid (EPA, 20:5ω3) or DHA, respectively. Substitution of the pfaE gene from different strain source in E. coli did not influence the function of the PKS pathway producing DHA, although they led to different DHA yields and fatty acid profiles. This result suggested that the pfaE gene could be switchable between these strains for the production of DHA. The DHA production by expressing the reconstituted PKS pathway was also investigated in different E. coli strains, at different temperatures, or with the treatment of cerulenin. The highest DHA production, 2.2 mg of DHA per gram of dry cell weight or 4.1% of total fatty acids, was obtained by co-expressing pfaE(EPA) from the EPA-producing strain Shewanella baltica with pfaABCD in DH5α. Incubation at low temperature (10-15°C) resulted in higher accumulation of DHA compared to higher temperatures. The addition of cerulenin to the medium increased the proportion of DHA and saturated fatty acids, including C12:0, C14:0 and C16:0, at the expense of monounsaturated fatty acids, including C16:1 and C18:1. Supplementation with 1 mg/L cerulenin resulted in the highest DHA yield of 2.4 mg/L upon co-expression of pfaE(DHA) from C. psychrerythraea.
Subject(s)
Alteromonadaceae/enzymology , Bacterial Proteins/metabolism , Docosahexaenoic Acids/biosynthesis , Escherichia coli/metabolism , Polyketide Synthases/metabolism , Transferases (Other Substituted Phosphate Groups)/metabolism , Alteromonadaceae/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Biosynthetic Pathways/genetics , Cerulenin/pharmacology , Escherichia coli/drug effects , Escherichia coli/genetics , Fatty Acid Synthesis Inhibitors/pharmacology , Gene Expression Regulation, Bacterial , Metabolic Engineering/methods , Multigene Family , Polyketide Synthases/genetics , Reverse Transcriptase Polymerase Chain Reaction , Seawater/microbiology , Sequence Homology, Amino Acid , Transferases (Other Substituted Phosphate Groups)/geneticsABSTRACT
BACKGROUND: Colwellia psychrerythraea 34H is a psychrophilic bacterium able to produce docosahexaenoic acid (DHA). Polyketide synthase pathway is assumed to be responsible for DHA production in marine bacteria. RESULTS: Five pfa genes from strain 34H were confirmed to be responsible for DHA formation by heterogeneous expression in Escherichia coli. The complexity of fatty acid profile of this strain was revealed by GC and GC-MS. Treatment of cells with cerulenin resulted in significantly reduced level of C16 monounsaturated fatty acid (C16:1(Δ9t), C16:1(Δ7)). In contrast, the amount of saturated fatty acids (C10:0, C12:0, C14:0), hydroxyl fatty acids (3-OH C10:0 and 3-OH C12:0), as well as C20:4ω3, C20:5ω3 and C22:6ω3 were increased. RNA sequencing (RNA-Seq) revealed the altered gene expression pattern when C. psychrerythraea cells were treated with cerulenin. Genes involved in polyketide synthase pathway and fatty acid biosynthesis pathway were not obviously affected by cerulenin treatment. In contrast, several genes involved in fatty acid degradation or ß-oxidation pathway were dramatically reduced at the transcriptional level. CONCLUSIONS: Genes responsible for DHA formation in C. psychrerythraea was first cloned and characterized. We revealed the complexity of fatty acid profile in this DHA-producing strain. Cerulenin could substantially change the fatty acid composition by affecting the fatty acid degradation at transcriptional level. Acyl-CoA dehydrogenase gene family involved in the first step of ß-oxidation pathway may be important to the selectivity of degraded fatty acids. In addition, inhibition of FabB protein by cerulenin may lead to the accumulation of malonyl-CoA, which is the substrate for DHA formation.
