PMC:4301621 / 22531-28945 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4301621","sourcedb":"PMC","sourceid":"4301621","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4301621","text":"Regulation of PUFA biosynthesis and metabolism based on GSMM\nAcetyl-CoA is the essential two-carbon donor molecule for fatty acid synthesis. In model iCY1106, there were ten pathways that could produce acetyl-CoA, including amino acid degradation (from aspartate, isoleucine, leucine, and lysine), fatty acid beta-oxidation, and other acetyl-CoA generation processes (phosphoenolpyruvate [PEP], malate, L-lactate, acetate, and citrate) (Figure 4). However, only the pathway from PEP to pyruvate could generate sufficient flux for synthesis of PUFAs on the fermentation medium. During the growth stage, the flux of acetyl-CoA generated by pyruvate was 1.22 mmol gDW−1 h−1, while this value was 1.56 mmol gDW−1 h−1 during the product synthesis stage. This corresponds to a 27.87% increase, which would ensure acetyl-CoA was available for ARA production due to the enhanced activity of the pyruvate dehydrogenase complex (EC: 1.2.4.1, 2.3.1.12, 1.8.1.4) [37]. Although aspartate, isoleucine, leucine, and lysine could all be used to generate acetyl-CoA for ARA production. Asparate has four carbon, while isoleucine, leucine, and lysine all contains six carbon. When the maximum uptake rate of asparate and the other amino acids were set at 0.15 mmol gDW−1 h−1 and 0.1 mmol gDW−1 h−1, respectively. FBA showed only that leucine and lysine could increase ARA production by 14.06% and 13.28%.\nFigure 4 The sources of acetyl-CoA in M . alpina . (cit: citrate, ac: acetate, llac: l-lactate, mal: malate, pep: phosphoenolpyruvate, pyr: pyruvate, accoa: acrtyl-CoA, asp: l-aspartate, bala: beta-alanine, oppa: 3-oxopropanoate, ile: l-isoleucine, omval: (s)-3-nethyl-2-oxopentanoate, mbcoa: 2-methylbutanoyl-CoA, mcrocoa: 2-methylcrotonoyl-CoA, metbycoa: (2S,3S)-3-Hydroxy-2-methylbutanoyl-CoA, mcecoa: 2-Methylacetoacetyl-CoA, lys: l-lysine, sacp: l-saccharopine, amasa: l-2-aminoadipate 6-semialdehyde, ama: l-2-aminoadipate, aka: 2-oxoadipate, gltcoa: glutaryl-CoA, c4dcoa: crotonoyl-coa, c4hcoa: (S)-3-Hydroxybutanoyl-CoA, aaccoa: acetoacetyl-CoA, leu: l-leucine, 4mop: 4-methyl-2-oxopentanoate, ivacoa: 3-isovaleryl-coa, mcrcoa: 3-nethylcrotonyl-CoA, mgcoa: trans-3-methylglutaconyl-CoA, hmgcoa: (s)-3-hydroxy-3-methylglutaryl-coa).\nIn model iCY1106, acetyl-CoA can be consumed by amino acid synthesis, amino sugar metabolism and fatty acid synthesis as well as ARA production (Additional file 5b). During the synthesis of six amino acids (threonine, methionine, lysine, cysteine, ornithine, and leucine), acetyl-CoA was required. FBA results showed that during the growth stage, 96.5% of the acetyl-CoA flux was used to synthesize fatty acids and 2.58% was used for amino acid synthesis. In contrast, during the product synthesis stage, the flux of acetyl-CoA used for fatty acid synthesis accounted for 99.52%, which indicates that amino acid synthesis was inhibited during ARA production. Limiting the nitrogen source can be an effective strategy to control amino acids biosynthesis [38], in which the lipid yield may be increased despite decreases in mycelia concentration.\nDuring the growth stages, the flux of acetyl-CoA used to synthesize malonyl-CoA, catalyzed by acetyl-CoA carboxylase (ACC, EC 6.4.1.2), was 0.79 mmol gDW−1 h−1. In contrast during the ARA production stage, the corresponding flux was 1.43 mmol gDW−1 h−1 (81.0%). ACC catalyzes the first committal step of the fatty acid biosynthetic pathway, and should be overexpressed to maximize ARA production [39]. Glutamate has been shown to activate ACC, and adding glutamate to the culture medium led to an increase in total lipid (21.81%) and ARA yield (66.07%) [40]. As the M. alpina growth rate in batch culture ranged from 0.06 h−1 to 0.19 h-1 [1,7,8,41,42], the glutamate uptake rates used for the in silico analysis should be set between 0 and 1.8 mmol gDW−1 h−1. When the glutamate uptake rate was increased, ARA production increased from 0.128 mmol gDW−1 h−1 to 0.355 mmol gDW−1 h−1 (Additional file 5c).\nDuring PUFA biosynthesis, NADPH is a necessary cofactor for de novo fatty acid synthesis involving desaturation and elongation. In model iCY1106, there were 172 reactions involving NADPH (Additional file 6). With ARA production fixed at 0.059 mmol gDW−1 h−1 (μ = 0.06 h−1) and 0.128 mmol gDW−1 h−1 (μ = 0.03 h−1), the flux of NADPH reactions were investigated. In total, 53 NADPH reactions exhibited flux changes of \u003e 10−6 mmol gDW−1 h−1 (Figure 5).\nFigure 5 Changes of reactions involving NADPH at different ARA levels. (Different colors represent different changes on NADPH flux. Flux obviously changed reactions are listed. R2: D-glucose 6-phosphate + NADP ⬄ 6-phospho-D-glucono-1,5-lactone + NADPH + H, R3: 6-phospho-D-gluconate + NADP - \u003e D-ribulose 5-phosphate + CO2 + NADPH + H, R4: (S)-malate + NADP - \u003e pyruvate + CO2 + NADPH, R5: L-glutamate 5-semialdehyde + NAD P+ H2O - \u003e L-glutamate + NADPH + H, R6: 1-pyrroline-5-carboxylate + NADPH + H ⬄ L-proline + NADP, R16: Nitrite + 3 NADPH + 5 H - \u003e Ammonia + 3 NADP + 2 H2O, R51: N-acetyl-L-glutamyl 5-phosphate + NADPH + H ⬄ N-acetyl-L-glutamate 5-semialdehyde + NADP + Phosphate, R53: (S)-3-hydroxy-3-methylglutaryl-CoA + 2 NADPH + 2 H ⬄ (R)-mevalonate + CoA + 2 NADP).\nFurther research showed that only five reactions (R2, R3, R4, R5, and R6) involving accumulation of NADPH were significantly altered. When ARA production was increased, three reactions (R4, R5, and R6) underwent flux increases, which corresponded to enhanced enzyme activities due to up-regulation of the genes encoding the enzymes. Of these, malic enzyme (ME, EC 1.1.1.40), which catalyzes the conversion of malate to pyruvate (R4), is considered a key enzyme in lipogenesis in M. alpina. ME expression and enzyme activity are enhanced during ARA production [43], and the minimization of metabolic adjustment (MOMA) method was used to investigate the role of this enzyme. With the biomass and ARA flux maximized by ME deletion using MOMA, 410 (22.11%) reactions showed flux changes (Additional file 6) compared with the wild type model. The ARA exchange reaction flux was lowered from 0.128 mmol gDW−1 h−1 to 0.079 mmol gDW−1 h−1, a decrease of 38.28%. Additionally, all reactions involving NADPH consumption reactions other than R13, R16, R51, and R53 were associated with lipid metabolism. These results indicated that the increase in ARA production was directly correlated with the NADPH consumption rate.","divisions":[{"label":"title","span":{"begin":0,"end":60}},{"label":"p","span":{"begin":61,"end":2228}},{"label":"figure","span":{"begin":1388,"end":2228}},{"label":"label","span":{"begin":1388,"end":1396}},{"label":"caption","span":{"begin":1397,"end":2228}},{"label":"p","span":{"begin":1397,"end":2228}},{"label":"p","span":{"begin":2229,"end":3073}},{"label":"p","span":{"begin":3074,"end":3976}},{"label":"p","span":{"begin":3977,"end":5204}},{"label":"figure","span":{"begin":4427,"end":5204}},{"label":"label","span":{"begin":4427,"end":4435}},{"label":"caption","span":{"begin":4436,"end":5204}},{"label":"p","span":{"begin":4436,"end":5204}}],"tracks":[{"project":"2_test","denotations":[{"id":"25582171-167775-14905497","span":{"begin":952,"end":954},"obj":"167775"},{"id":"25582171-16233009-14905498","span":{"begin":2983,"end":2985},"obj":"16233009"},{"id":"25582171-10893421-14905499","span":{"begin":3471,"end":3473},"obj":"10893421"},{"id":"25582171-12618051-14905500","span":{"begin":3628,"end":3630},"obj":"12618051"},{"id":"25582171-17439233-14905501","span":{"begin":3713,"end":3714},"obj":"17439233"},{"id":"25582171-10463157-14905502","span":{"begin":5765,"end":5767},"obj":"10463157"}],"attributes":[{"subj":"25582171-167775-14905497","pred":"source","obj":"2_test"},{"subj":"25582171-16233009-14905498","pred":"source","obj":"2_test"},{"subj":"25582171-10893421-14905499","pred":"source","obj":"2_test"},{"subj":"25582171-12618051-14905500","pred":"source","obj":"2_test"},{"subj":"25582171-17439233-14905501","pred":"source","obj":"2_test"},{"subj":"25582171-10463157-14905502","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#bc93ec","default":true}]}]}}