Aurantiochytrium sp., a marine thraustochytrid possesses a remarkable ability to produce lipid rich in polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA). Although gene regulation underlying lipid biosynthesis has been previously reported, proteomic analysis is still limited. In this study, high DHA accumulating strain Aurantiochytrium sp. SW1 has been used as a study model to elucidate the alteration in proteome profile under different cultivation phases i.e. growth, nitrogen-limitation and lipid accumulation. Of the total of 5146 identified proteins, 852 proteins were differentially expressed proteins (DEPs). The largest number of DEPs (488 proteins) was found to be uniquely expressed between lipid accumulating phase and growth phase. Interestingly, there were up-regulated proteins involved in glycolysis, glycerolipid, carotenoid and glutathione metabolism which were preferable metabolic routes towards lipid accumulation and DHA production as well as cellular oxidative defence. Integrated proteomic and transcriptomic data were also conducted to comprehend the gene and protein regulation underlying the lipid and DHA biosynthesis. A significant up-regulation of acetyl-CoA synthetase was observed which suggests alternative route of acetate metabolism for acetyl-CoA producer. This study presents the holistic routes underlying lipid accumulation and DHA production in Aurantiochytrium sp. SW1 and other relevant thraustochytrid.
The present work describes the application of homologous recombination techniques in a wild-type Aspergillus terreus (ATCC 20542) strain to increase the flow of precursors towards the lovastatin biosynthesis pathway. A new strain was generated to overexpress acetyl-CoA carboxylase (ACCase) by replacing the native ACCase promoter with a strong constitutive PadhA promoter from Aspergillus nidulans. Glycerol and a mixture of lactose and glycerol were used independently as the carbon feedstock to determine the degree of response by the A. terreus strains towards the production of acetyl-CoA, and malonyl-CoA. The new strain increased the levels of malonyl-CoA and acetyl-CoA by 240% and 14%, respectively, compared to the wild-type strain. As a result, lovastatin production was increased by 40% and (+)-geodin was decreased by 31% using the new strain. This study shows for the first time that the metabolism of Aspergillus terreus can be manipulated to attain higher levels of precursors and valuable secondary metabolites.
Malonyl-CoA serves as the main building block for the biosynthesis of many important polyketides, as well as fatty acid-derived compounds, such as biofuel. Escherichia coli, Corynebacterium gultamicum, and Saccharomyces cerevisiae have recently been engineered for the biosynthesis of such compounds. However, the developed processes and strains often have insufficient productivity. In the current study, we used enzyme-engineering approach to improve the binding of acetyl-CoA with ACC. We generated different mutations, and the impact was calculated, which reported that three mutations, that is, S343A, T347W, and S350W, significantly improve the substrate binding. Molecular docking investigation revealed an altered binding network compared to the wild type. In mutants, additional interactions stabilize the binding of the inner tail of acetyl-CoA. Using molecular simulation, the stability, compactness, hydrogen bonding, and protein motions were estimated, revealing different dynamic properties owned by the mutants only but not by the wild type. The findings were further validated by using the binding-free energy (BFE) method, which revealed these mutations as favorable substitutions. The total BFE was reported to be -52.66 ± 0.11 kcal/mol for the wild type, -55.87 ± 0.16 kcal/mol for the S343A mutant, -60.52 ± 0.25 kcal/mol for T347W mutant, and -59.64 ± 0.25 kcal/mol for the S350W mutant. This shows that the binding of the substrate is increased due to the induced mutations and strongly corroborates with the docking results. In sum, this study provides information regarding the essential hotspot residues for the substrate binding and can be used for application in industrial processes.
Mixed culture sludge has been widely used as a microbial consortium for biohydrogen production. Simple thermal treatment of sludge is usually required in order to eliminate any H2-consuming bacteria that would reduce H2 production. In this study, thermal treatment of sludge was carried out at various temperatures. Electron flow model was then applied in order to assess community structure in the sludge upon thermal treatment for biohydrogen production. Results show that the dominant electron sink was acetate (150-217 e- meq/mol glucose). The electron equivalent (e- eq) balances were within 0.8-18% for all experiments. Treatment at 100 °C attained the highest H2 yield of 3.44 mol H2/mol glucose from the stoichiometric reaction. As the treatment temperature increased from 80 to 100 °C, the computed acetyl-CoA and reduced form of ferredoxin (Fdred) concentrations increased from 13.01 to 17.34 e- eq (1.63-2.17 mol) and 1.34 to 4.18 e- eq (0.67-2.09 mol), respectively. The NADH2 balance error varied from 3 to 10% and the term e-(Fd↔NADH2) (m) in the NADH2 balance was NADH2 consumption (m = -1). The H2 production was mainly via the Fd:hydrogenase system and this is supported with a good NADH2 balance. Using the modified Gompertz model, the highest maximum H2 production potential was 1194 mL whereas the maximum rate of H2 production was 357 mL/h recorded at 100 °C of treatment.
Lovastatin is widely prescribed to reduce elevated levels of cholesterol and prevent heart-related diseases. Cultivation of Aspergillus terreus (ATCC 20542) with carbohydrates or low-value feedstocks such as glycerol produces lovastatin as a secondary metabolite and (+)-geodin as a by-product. An A. terreus mutant strain was developed (gedCΔ) with a disrupted (+)-geodin biosynthesis pathway. The gedCΔ mutant was created by inserting the antibiotic marker hygromycin B (hyg) within the gedC gene that encodes emodin anthrone polyketide synthase (PKS), a primary gene responsible for initiating (+)-geodin biosynthesis. The effects of emodin anthrone PKS gene disruption on (+)-geodin and lovastatin biosynthesis and the production of the precursors acetyl-CoA and malonyl-CoA were investigated with cultures based on glycerol alone and in combination with lactose. The gedCΔ strain showed improved lovastatin production, particularly when cultivated on the glycerol-lactose mixture, increasing lovastatin production by 80% (113 mg/L) while simultaneously inhibiting (+)-geodin biosynthesis compared to the wild-type strain. This study thus shows that suppression of the (+)-geodin pathway increases lovastatin yield and demonstrates a practical approach of manipulating carbon flux by modulating enzyme activity.
Thiamine is known to be an important compound in human diet and it is a cofactor required for vital metabolic processes such as acetyl-CoA biosynthesis, amino acid biosynthesis, Krebs and Calvin cycle. Besides that, thiamine has been shown to be involved in plant protection against stress. In this study, the level of expression of THIC and THI1/THI4, the genes for the first two enzymes in the thiamine biosynthesis pathway were observed when oil palm (Elaeis guineensis) was subjected to oxidative stress. Primers were designed based on the consensus sequence of thiamine biosynthesis genes obtained from Arabidopsis thaliana, Zea mays, Oryza sativa, and Alnus glutinosa. Oxidative stress were induced with various concentrations of paraquat and samplings were done at various time points post-stress induction. The expression of THIC and THI1/THI4 genes were observed via RT-PCR and qPCR analysis. The expression of THIC was increased 2-fold, while THI1/THI4 gene transcript was increased 4-fold upon induction of oxidative stress. These findings showed that oil palm responded to oxidative stress by over-expressing the genes involved in thiamine biosynthesis. These findings support the suggestion that thiamine may play an important role in plant protection against stress.