การทำให้บริสุทธิ์และคุณลักษณะของเอนไซม์ทริปซินและไคโมทริปซินจากเครื่องในปลาทูน่าพันธุ์ครีบเหลือง (Thunnus albacares)และการประยุกต์ใช้

Thesis (M.Sc., Biotechnology)--Prince of Songkla University, 200

Increased Biomass and Polyhydroxybutyrate Production by <i>Synechocystis</i> sp. PCC 6803 Overexpressing <i>RuBisCO</i> Genes

The overexpression of the RuBisCO (rbc) gene has recently become an achievable strategy for increasing cyanobacterial biomass and overcoming the biocompound production restriction. We successfully constructed two rbc-overexpressing Synechocystis sp. PCC 6803 strains (OX), including a strain overexpressing a large subunit of RuBisCO (OXrbcL) and another strain overexpressing all large, chaperone, and small subunits of RuBisCO (OXrbcLXS), resulting in higher and faster growth than wild type under sodium bicarbonate supplementation. This increased biomass of OX strains significantly contributed to the higher polyhydroxybutyrate (PHB) production induced by nutrient-deprived conditions, in particular nitrogen (N) and phosphorus (P). As a result of higher PHB contents in OX strains occurring at days 7 and 9 of nutrient deprivation, this enhancement was apparently made possible by cells preferentially maintaining their internal lipids while accumulating less glycogen. The OXrbcLXS strain, with the highest level of PHB at about 39 %w/dry cell weight (DCW) during 7 days of BG11-NP treatment, contained a lower glycogen level (31.9 %w/DCW) than wild type control (40 %w/DCW). In contrast, the wild type control strain exposed to N- and NP-stresses tended to retain lipid levels and store more glycogen than PHB. In this model, we, for the first time, implemented a RuBisCO-overexpressing cyanobacterial factory for overproducing PHB, destined for biofuel and biomaterial biotechnology

Enhanced productivity of extracellular free fatty acids by gene disruptions of acyl-ACP synthetase and S-layer protein in Synechocystis sp. PCC 6803

Background Based on known metabolic response to excess free fatty acid (FFA) products, cyanobacterium Synechocystis sp. PCC 6803 preferentially both recycles via FFA recycling process and secrets them into medium. Engineered cyanobacteria with well growth and highly secreted FFA capability are considered best resources for biofuel production and sustainable biotechnology. In this study, to achieve the higher FFA secretion goal, we successfully constructs Synechocystis sp. PCC 6803 mutants disrupting genes related to FFA recycling reaction (aas gene encoding acyl-acyl carrier protein synthetase), and surface layer protein (encoded by sll1951). Results Three Synechocystis sp. PCC 6803 engineered strains, including two single mutants lacking aas (KA) and sll1951 (KS), and one double mutant lacking both aas and sll1951 (KAS), significantly secreted FFAs higher than that of wild type (WT). Certain increase of secreted FFAs was noted when cells were exposed to nitrogen-deficient conditions, BG(11)-half N and BG(11)-N conditions, with the exception of strain KS. Under BG(11)-N condition at day 10, strain KAS strikingly secreted FFAs products up to 40%w/DCW or 238.1 mg/L, with trace amounts of PHB. Unexpectedly, strain KS, with S-layer disruption, appeared to have endured longer in BG(11)-N growth medium. This strain KS significantly acclimated to the BG(11)-N environment by accumulating a greater glycogen pool with lower FFA production, whereas strain KA favored higher PHB and intracellular lipid accumulations with moderate FFA secretion. Conclusions Mutations of both aas and sll1951 genes in Synechocystis sp. PCC 6803 significantly improved the productivity of secreted FFAs, especially under nitrogen deprivation

Synechocystis sp. PCC 6803 overexpressing genes involved in CBB cycle and free fatty acid cycling enhances the significant levels of intracellular lipids and secreted free fatty acids

The integrative aspect on carbon fixation and lipid production is firstly implemented in cyanobacterium Synechocystis sp. PCC 6803 using metabolic engineering approach. Genes related to Calvin-Benson-Bassham (CBB) cycle including rbcLXS and glpD and free fatty acid recycling including aas encoding acyl-ACP synthetase were practically manipulated in single, double and triple overexpressions via single homologous recombination. The significantly increased growth rate and intracellular pigment contents were evident in glpD-overexpressing (OG) strain among all strains studied under normal growth condition. The triple aas_glpD_rbcLXS-overexpressing (OAGR) strain notably gave the highest contents of both intracellular lipids and extracellular free fatty acids (FFAs) of about 35.9 and 9.6% w/DCW, respectively, when compared to other strains at day 5 of cultivation. However, the highest intracellular lipid titer and production rate were observed in OA strain at day 5 (228.7mg/L and 45.7mg/L/day, respectively) and OG strain at day 10 (358.3mg/L and 35.8mg/L/day, respectively) due to their higher growth. For fatty acid (FA) compositions, the main saturated fatty acid of palmitic acid (C16:0) was dominantly found in both intracellular lipid and secreted FFAs fractions. Notably, intracellular FA proportion of myristic acid (C14:0) was induced in all engineered strains whereas the increase of stearic acid (C18:0) composition was found in extracellular FFAs fraction. Altogether, these overexpressing strains efficiently produced higher lipid production via homeostasis balance on both its lipid synthesis and FFAs secretion

Improved lipid production and component of mycosporine-like amino acids by co-overexpression of amt1 and aroB genes in Synechocystis sp. PCC6803

Implementing homologous overexpression of the amt1 (A) and aroB (B) genes involved in ammonium transporter and the synthesis of mycosporine-like amino acids (MAAs) and aromatic amino acids, respectively, we created three engineered Synechocystis sp. PCC6803 strains, including Ox-A, Ox-B, and Ox-AB, to study the utilization of carbon and nitrogen in cyanobacteria for the production of valuable products. With respect to amt1 overexpression, the Ox-A and Ox-AB strains had a greater growth rate under (NH4)(2)SO4 supplemented condition. Both the higher level of intracellular accumulation of lipids in Ox-A and Ox-AB as well as the increased secretion of free fatty acids from the Ox-A strain were impacted by the late-log phase of cell growth. It is noteworthy that among all strains, the Ox-B strain undoubtedly spotted a substantial accumulation of glycogen as a consequence of aroB overexpression. Additionally, the ammonium condition drove the potent antioxidant activity in Ox strains with a late-log phase, particularly in the Ox-B and Ox-AB strains. This was probably related to the altered MAA component inside the cells. The higher proportion of P4-fraction was induced by the ammonium condition in both Ox-B and Ox-AB, while the noted increase of the P1 component was found in the Ox-A strain

Overexpressing Carotenoid Biosynthetic Genes in Synechocystis sp. PCC 6803 Improved Intracellular Pigments and Antioxidant Activity, Which Can Decrease the Viability and Proliferation of Lung Cancer Cells In Vitro

In the antioxidant system in cyanobacteria, non-enzymatic antioxidants, such as carotenoids, are considered good candidates for coping with oxidative stress, particularly light stress, and pharmaceutical therapeutic applications. A significant amount of carotenoid accumulation has been recently improved by genetic engineering. In this study, to achieve higher carotenoid production with higher antioxidant activity, we successfully constructed five Synechocystis sp. PCC 6803 strains overexpressing (OX) native genes related to the carotenoids biosynthetic pathway, including OX_CrtB, OX_CrtP, OX_CrtQ, OX_CrtO, and OX_CrtR. All of the engineered strains maintained a significant quantity of myxoxanthophyll, while increasing zeaxanthin and echinenone accumulation. In addition, higher components of zeaxanthin and echinenone were noted in all OX strains, ranging from 14 to 19% and from 17 to 22%, respectively. It is worth noting that the enhanced echinenone component responded to low light conditions, while the increased beta-carotene component contributed to a high light stress response. According to the higher antioxidant activity of all OX strains, the carotenoid extracts presented lower IC50 in lung cancer cell lines H460 and A549, with values less than 157 and 139 mu g/mL, respectively, when compared with those of WTc, particularly OX_CrtR and OX_CrtQ. A higher proportion of zeaxanthin and beta-carotene in OX_CrtR and OX_CrtQ, respectively, may considerably contribute to the ability to treat lung cancer cells with antiproliferative and cytotoxic effects

Overexpression of lipA or glpD_RuBisCO in the Synechocystis sp. PCC 6803 Mutant Lacking the Aas Gene Enhances Free Fatty-Acid Secretion and Intracellular Lipid Accumulation

Although engineered cyanobacteria for the production of lipids and fatty acids (FAs) are intelligently used as sustainable biofuel resources, intracellularly overproduced FAs disturb cellular homeostasis and eventually generate lethal toxicity. In order to improve their production by enhancing FFAs secretion into a medium, we constructed three engineered Synechocystis 6803 strains including KA (a mutant lacking the aas gene), KAOL (KA overexpressing lipA, encoding lipase A in membrane lipid hydrolysis), and KAOGR (KA overexpressing quadruple glpD/rbcLXS, related to the CBB cycle). Certain contents of intracellular lipids and secreted FFAs of all engineered strains were higher than those of the wild type. Remarkably, the KAOL strain attained the highest level of secreted FFAs by about 21.9%w/DCW at day 5 of normal BG11 cultivation, with a higher growth rate and shorter doubling time. TEM images provided crucial evidence on the morphological changes of the KAOL strain, which accumulated abundant droplets on regions of thylakoid membranes throughout the cell when compared with wild type. On the other hand, BG11-N condition significantly induced contents of both intracellular lipids and secreted FFAs of the KAOL strain up to 37.2 and 24.5%w/DCW, respectively, within 5 days. Then, for the first time, we shone a spotlight onto the overexpression of lipA in the aas mutant of Synechocystis as another potential strategy to achieve higher FFAs secretion with sustainable growth

Improved lipid production via fatty acid biosynthesis and free fatty acid recycling in engineered Synechocystis sp. PCC 6803

Background Cyanobacteria are potential sources for third generation biofuels. Their capacity for biofuel production has been widely improved using metabolically engineered strains. In this study, we employed metabolic engineering design with target genes involved in selected processes including the fatty acid synthesis (a cassette of accD, accA, accC and accB encoding acetyl-CoA carboxylase, ACC), phospholipid hydrolysis (lipA encoding lipase A), alkane synthesis (aar encoding acyl-ACP reductase, AAR), and recycling of free fatty acid (FFA) (aas encoding acyl-acyl carrier protein synthetase, AAS) in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Results To enhance lipid production, engineered strains were successfully obtained including an aas-overexpressing strain (OXAas), an aas-overexpressing strain with aar knockout (OXAas/KOAar), and an accDACB-overexpressing strain with lipA knockout (OXAccDACB/KOLipA). All engineered strains grew slightly slower than wild-type (WT), as well as with reduced levels of intracellular pigment levels of chlorophyll a and carotenoids. A higher lipid content was noted in all the engineered strains compared to WT cells, especially in OXAas, with maximal content and production rate of 34.5% w/DCW and 41.4mg/L/day, respectively, during growth phase at day 4. The OXAccDACB/KOLipA strain, with an impediment of phospholipid hydrolysis to FFA, also showed a similarly high content of total lipid of about 32.5% w/DCW but a lower production rate of 31.5mg/L/day due to a reduced cell growth. The knockout interruptions generated, upon a downstream flow from intermediate fatty acyl-ACP, an induced unsaturated lipid production as observed in OXAas/KOAar and OXAccDACB/KOLipA strains with 5.4% and 3.1% w/DCW, respectively. Conclusions Among the three metabolically engineered Synechocystis strains, the OXAas with enhanced free fatty acid recycling had the highest efficiency to increase lipid production

Co-overexpression of native phospholipid-biosynthetic genes plsX and plsC enhances lipid production in Synechocystis sp PCC 6803

The overexpression of native plsX and plsC genes involving in fatty acid/phospholipid synthesis first timely-reported the significantly enhanced lipid contents in Synechocystis sp. PCC 6803. Growth rate, intracellular pigment contents including chlorophyll a and carotenoids, and oxygen evolution rate of all overexpressing (OX) strains were normally similar as wild type. For fatty acid compositions, saturated fatty acid, in particular palmitic acid (16:0) was dominantly increased in OX strains whereas slight increases of unsaturated fatty acids were observed, specifically linoleic acid (18:2) and alpha-linolenic acid (18:3). The plsC/plsX-overexpressing (OX + XC) strain produced high lipid content of about 24.3% w/dcw under normal condition and was further enhanced up to 39.1% w/dcw by acetate induction. This OX + XC engineered strain was capable of decreasing phaA transcript level which related to poly-3-hydroxybutyrate (PHB) synthesis under acetate treatment. Moreover, the expression level of gene transcripts revealed that the plsX-and plsC/plsX-overexpression strains had also increased accA transcript amounts which involved in the irreversible carboxylation of acetyl-CoA to malonyl-CoA. Altogether, these overexpressing strains significantly augmented higher lipid contents when compared to wild type by partly overcoming the limitation of lipid production