高产DHA裂壶藻突变株发酵条件的优化

为了进一步提高裂壶藻突变株的DHA产量，以经过⁶⁰Co-γ射线辐照诱变后所得高产DHA裂壶藻突变株1.6-7-1为研究对象，通过Plackett-Burman实验、最陡爬坡实验和响应面实验对其发酵培养基进行优化，同时通过发酵罐发酵培养研究不同溶氧水平对突变株代谢的影响。结果表明：葡萄糖、C₅H₈NNaO₄和NaCl添加量对该突变株产DHA具有显著影响，其最佳添加量分别为葡萄糖125.46 g/L、C₅H₈NNaO₄ 12.44 g/L、NaCl 4 g/L,在此条件下该突变株DHA产量达6.01 g/L,相较于原始菌株提升了49.88%;在高溶氧水平下，突变株生物量高但油脂产量和DHA产量较低，可能是因为细胞优先用营养物质进行自身的生长繁殖，而过低的溶氧水平会抑制能量代谢，减慢细胞繁殖速度。综上，优化发酵条件可以提高突变株DHA产量。     

化学添加剂对裂壶藻突变株发酵产DHA的影响

为了研究外源化学添加剂对裂壶藻(Schizochytrium limacinum)突变株发酵合成脂肪酸(SFAs、PUFAs和DHA)的影响,采用基于气相色谱-质谱(GC-MS)联用技术分析裂壶藻突变株产油脂肪酸的组成。通过单因素和正交实验设计,优化了乙醇胺(ETA)、萘氧乙酸(BNOA)和水杨酸(SA)三种化学添加剂的最佳添加条件。结果表明,添加150 mg/L的乙醇胺(ETA)、10 mg/L萘氧乙酸(BNOA)和1 mg/L水杨酸(SA),裂壶藻突变株的DHA产量最高,达到6.18 g/L,比对照组提高了12.77%。综上所述,添加适量的化学添加剂可以提高裂壶藻突变株DHA的含量。     

硝态氮对Nannochloropsis oculata生长及合成胞内组分的影响

研究了硝态氮对Nannochloropsis oculata C170细胞生长及胞内组分合成的影响。以硝酸钠为氮源考察不同氮浓度下藻细胞生长、氮消耗及油脂含量变化,并在此基础上分析氮限制处理时油脂、总糖及蛋白等胞内组分合成差异。结果表明:N.oculata C170在42.5mg/L氮浓度时生长最佳,其生物量、比生长速率及产率最高,分别为0.59g/L、0.275d-1和50.98mg/(L·d)。油脂含量与氮浓度呈明显负相关,氮浓度为12.5mg/L时,油脂含量和产率最高,分别为29.17%和11.40mg/(L·d)。而胞内多不饱和脂肪酸比例随着氮浓度的升高显著增加,其中EPA含量由8.06%升高到18.76%。此外藻细胞在限氮培养时,其油脂含量增加至23.07%,为最初含量的2.8倍,同时总糖含量升高了76%左右,叶绿素a和蛋白含量则分别下降了大约29%和24%。高浓度硝态氮有利于N.oculata C170细胞生长及胞内EPA积累,低浓度硝态氮诱导藻细胞油脂积累并与糖类合成、色素和蛋白降解相关联。     

裂壶藻补糖发酵及糖氮代谢对产油的影响

为了探索裂壶藻(Schizochytrium limacinum)突变株生长及产油过程中对碳源和氮源的需求,本文对发酵各阶段的碳源和氮源利用变化情况进行测定,并设定五个在不同时期的补糖实验组进行研究,分别观测各组各时期生物量、油脂产量。结果表明,前96 h是裂壶藻生长的重要时期,生物量大幅度增长,在发酵144~168 h时,油脂产量达到最大,各组经发酵7 d收获总的藻体,对生物量、油脂产量、DHA含量和DHA产量进行综合测定,结果表明,在发酵120 h时进行补糖为最佳时期,且油脂产量及DHA产量相比于对照组分别高出18.80%和22.44%。当氮源耗尽时补糖不仅可以促进油脂的产生,而且对DHA的形成也是有利的。     

利用伯克霍尔德氏菌脂肪酶富集裂壶藻油脂中的二十二碳六烯酸

目的：研究伯克霍尔德氏菌胞外脂肪酶对裂壶藻油脂中二十二碳六烯酸（docosahexaenoic acid,DHA）的富集作用。方法：用三丁酸甘油酯平板筛选法,筛选产脂肪酶的细菌;以对硝基苯月桂酸酯为底物,对筛选得到的伯克霍尔德氏菌脂肪酶的酶学性质进行研究;用该脂肪酶水解裂壶藻油脂,通过气相色谱法测定酶解后水相和有机相的脂肪酸组成来研究其对裂壶藻油脂DHA的富集作用。结果：从广州的泥土样品中分离获得一株高产胞外脂肪酶的伯克霍尔德氏菌,在产酶培养基中30 ℃、200 r/min的条件下培养40 h后,脂肪酶酶活力达到最大值70 U/mL。脂肪酶粗酶液酶活性的最适温度为45 ℃,最适pH值为8.5。该菌的脂肪酶对裂壶藻油脂中的DHA有一定的富集作用,能够使油脂中DHA占总脂肪酸的质量分数从最初的30%升高到40%。结论：筛选得到一株能够产生选择性富集DHA的脂肪酶的细菌,拓宽了可用于多不饱和脂肪酸分离纯化的脂肪酶的范围。     

光照对杜氏盐藻突变藻株Zea1生长和积累玉米黄素的影响

通过紫外线和亚硝基胍(NTG)诱变得到一株杜氏盐藻高产玉米黄素突变株Zea1,以此突变藻株为实验材料,通过正交试验获得突变藻株生长所需C、N、P的最适浓度分别为15、2.0、0.1mmol/L,积累玉米黄素所需C、N、P的最适浓度分别为15、2.0、0.2mmol/L。分别比较强光照(5000 lx)、正常光照(3000 lx)、低光照(500 lx)对野生型和突变型藻株生长和累积玉米黄素的影响。结果显示：在同等光照条件下,突变型藻株藻细胞数和玉米黄素的积累都略高于野生型藻株;强光照明显有利于突变藻株藻细胞生长和玉米黄素积累,藻细胞数分别为低光照和正常光照条件下的7.08倍和1.29倍,玉米黄素含量分别是低光照和正常光照条件下的4.56倍和1.4倍。     

集胞藻PCC6803中S2P蛋白酶Sll0528参与铵盐胁迫响应的重要性

Sll0528是集胞藻PCC6803中四个二位蛋白酶(site-2-protease, S2P)之一。本研究主要比较了三株集胞藻(编码基因sll0528过表达的藻株OE0528,编码基因sll0528敲除的缺失藻株?sll0528,以及野生型藻株)在不同氯化铵浓度下的生长表型。结果显示,缺失藻株?sll0528对120 mM的氯化铵比野生型更敏感,光合系统损伤严重。在180 mM氯化铵下野生型株的生长受到抑制,而过表达藻株OE0528的生长优于野生型,更耐受高浓度铵盐,其光合系统色素藻蓝蛋白和叶绿素等的损伤程度低于野生型。可见,Sll0528在集胞藻PCC6803适应铵盐胁迫中发挥重要的作用,可能参与了铵盐胁迫的快速响应,并直接或间接介导高铵盐浓度下集胞藻对光合系统的保护或修复机制。本研究首次揭示了S2P蛋白酶微藻具有一定的铵盐胁迫响应作用,相关结果对于深入探究微藻对铵盐的响应机制及提高微藻对铵盐的耐受性和利用率具有重要的意义。     

高产油小球藻的低温等离子体诱变育种

以普通小球藻(Chlorella vulgaris)为研究对象进行低温等离子体诱变,测定培养期间小球藻的生物量、总脂含量及脂肪酸组成等指标,旨在筛选获得生长速度快、油脂含量高的小球藻突变株。结果表明:诱变筛选得到3株油脂含量高的藻株,其中诱变株M1的总脂含量、中性脂含量、总脂产率及中性脂产率分别为37. 28%、19. 12%、8. 20 mg/(L·d)和4. 21 mg/(L·d),较野生株分别提高了103. 5%、133. 5%、163. 7%和202. 9%; M1的生物量和蛋白质含量也有一定程度的提高; M1藻细胞油脂中的MUFA和PUFA含量分别为44. 0%和19. 8%,较野生株分别提高了23. 6%和32. 9%,作为生物柴油指标的十八烯酸C18∶1含量也较野生株提高了64. 9%。     

产油脂微藻的分离、鉴定及筛选

微藻油脂是生产生物柴油的重要原料,同时也是生产不饱和脂肪酸的原料来源之一。本文以广州市华南理工大学校内东湖为采样点,筛选鉴定湖中存在的微藻并研究其生长和油脂积累特性,旨在筛选出水体中可能存在的富油微藻以便后续研究分析。研究共筛选出6株绿藻,18Sr DNA鉴定显示6株绿藻中DH1、DH2、DH6均属于小球藻属,DH3、DH4、DH5均属于栅藻属,后分析构建了6株微藻进化发育树。通过藻细胞胞内油脂含量测定,表明DH1、DH2与DH6均能较好地积累油脂,尤其是DH2与DH6两株微藻。培养25 d发现其最大生物量(细胞数)分别达到2.14×10~7个/m L和2.74×10~7个/m L,油脂产量分别达到103.89 mg/L和131.69mg/L,藻细胞油脂百分含量均超过30%,表明该两株微藻是潜在的较为理想的产油微藻,可作为生物柴油制备的备用藻种资源。     

利用海洋微藻生产二十二碳六烯酸的研究进展北大核心CSCD

二十二碳六烯酸(DHA)是一种ω-3系多不饱和脂肪酸,对人体生长发育和健康具有重要作用,但其传统来源并不稳定且会引入鱼腥味,因此积极开发DHA新来源具有重要的研究价值和市场潜力。介绍了DHA的结构、理化性质、功能特性和来源,并从微生物产DHA途径,高产DHA藻种裂壶藻和寇氏隐甲藻的特点、诱变选育种情况、摇瓶培养条件和发酵工艺优化等方面阐述了微生物发酵生产DHA的研究情况。另外,从食用油和乳液两个方面对DHA藻油在食品中的应用情况进行了介绍。目前以裂壶藻和寇氏隐甲藻发酵生产DHA已取得初步成功,可以为现有生产提供理论基础,但有关胞内合成DHA和精准调控实现DHA增产还有待进一步研究。     

A novel mechanism regulates H(2) S and SO(2) production in Saccharomyces cerevisiae

Sulfite (SO(2) ) plays an important role in flavour stability in alcoholic beverages, whereas hydrogen sulfide (H(2) S) has an undesirable aroma. To discover the cellular processes that control SO(2) and H(2) S production, we screened a library of Saccharomyces cerevisiae deletion mutants. Deletion of 12 genes led to increased H(2) S productivity. Ten of these genes are known to be involved in sulfur-containing amino acid metabolism, whereas UBI4 functions in the ubiquitin-proteasome system and SKP2 encodes an F-box-containing protein whose function is unknown. We found that the skp2 mutant accumulated H(2) S and SO(2) , because the adenosylphophosulfate kinase Met14p is a substrate of SCF(Skp2) and more stable in the skp2 mutant than in the wild-type strain. Furthermore, the skp2 mutant grew more slowly than the wild-type strain under nutrient-limited conditions. Metabolome analysis showed that the concentration of intracellular cysteine is lower in the skp2 mutant than in the wild-type strain. The slow growth of the skp2 mutant was due to a lower concentration of intracellular cysteine, because the addition of cysteine suppressed the slow growth. In the skp2 mutant, the cysteine biosynthesis proteins Str2p, Str3p and Str4p are more stable than in the wild-type strain. Moreover, supplementation with methionine, S-adenosylmethionine, S-adenosylhomocysteine and homocysteine also suppressed the slow growth. Overexpression of STR1 or STR4 caused a more severe defect in the skp2 mutant. These results suggest that the balance of methionine and cysteine biosynthesis is important for yeast cell growth. Thus, Skp2p is one of the key components regulating this balance and H(2) S/SO(2) production.     

Three diacylglycerol acyltransferases contribute to oil biosynthesis and normal growth in Yarrowia lipolytica

Diacylglycerol (DAG) acyltransferase catalyses the final and committed step of triacylglycerol biosynthesis. Eukaryotes commonly contain up to three distinct classes of DAG acyltransferases: acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2), and phospholipid:diacylglycerol acyltransferase (PDAT). The non-conventional oleaginous yeast, Yarrowia lipolytica, contains at least one homologue of each class and serves as a good model to understand the role of different DAG acyltransferases in the biosynthesis of oil, a critical cellular component that serves as a storage molecule as well as a buffer for free fatty acids. We used gene disruptions in Y. lipolytica and in vitro enzyme assays to confirm the identity of genes encoding all three DAG acyltransferases and demonstrate that together they account for almost all oil biosynthesis and that all three contribute significantly to its oil biosynthesis. In Y. lipolytica ATCC 20362 strain, the total lipid% dry cell weight (DCW) as a percentage of the wild-type strain in pdat, dgat1, dgat2, dgat1/dgat2 double mutant and dgat1/dgat2/pdat triple mutant was 70%, 57%, 36%, 18% and 13%, respectively.This is the first example of DGAT1 contributing significantly to oil biosynthesis in a microorganism. The triple mutant shows significant growth defect in both increased lag phase and slower growth rate, suggesting that oil biosynthesis contributes to normal growth in this strain.     

Effects of static magnetic fields on growth and membrane lipid composition of Salmonella typhimurium wild-type and dam mutant strains

This study was carried out to explore the adaptive mechanisms of S. typhimurium particularly, the implication of the Dam methyltransferase in the remodelling of membrane lipid composition to overcome magnetic field stress. With this aim, we focused our analyses on the increase in viable numbers and membrane lipid modifications of S. typhimurium wild-type and dam mutant cells exposed for 10h to static magnetic fields (SMF; 200 mT). For the wild-type strain, exposure to SMF induced a significant decrease (p<0.05) of CFU at 6h, followed by an increase between 8 and 10h. Growth of the dam mutant was significantly affected (p<0.05) after 6h and no recovery was observed until 10h, highlighting a different behavior of SMF stressed wild-type and dam mutant strains. SMF significantly affected the phospholipid proportions in the two strains. The most affected were those of the acidic phospholipids, cardiolipins (CL). In the dam strain the phospholipid response to SMF followed a globally similar trend as in the wild-type with however lower effects, leading mainly to an unusual accumulation of CL. This would in part explain the different behavior of the wild-type and the dam strain. Results showed a significant increase of membrane cyclic fatty acids Cyc17 and Cyc19 in the wild-type strain but only the Cyc17 in the dam strain and a meaningful increase of the total unsaturated fatty acids (UFAs) to total saturated fatty acids (SFAs) ratios of the exposed cells compared to controls from 3 to 9h (p<0.05) for both strains. The net increase of the total UFAs to total SFAs ratios seemed to result mainly from the increase of (C18:1) proportion (p<0.05) and to a lower extent from that of (C16:1) (p<0.05). These modifications of cyclic and unsaturated fatty acid proportions constitute an adaptive response to SMF stress in S. typhimurium wild-type and dam mutants to maintain an optimum level of membrane fluidity under SMF.     

硫酸二乙酯化学诱变选育高核糖核酸酿酒酵母及培养基组成优化

该研究以酿酒酵母（Saccharomyces cerevisiae）BY23为出发菌株,采用硫酸二乙酯（DES）对其进行化学诱变,筛选出一株生长性能好、胞内核糖核酸（RNA）含量高的突变株BY23-195,并以胞内RNA含量为评价指标,通过单因素及正交试验对其糖蜜培养基成分进行优化。结果表明：突变株BY23-195生长性能较好,在酵母浸出粉胨葡萄糖（YEPD）培养基中,RNA含量较出发菌株BY23提高了18.85%。最优糖蜜培养基组分为糖蜜（糖度调至12 °Bx）、酵母浸粉5%、硫酸铵0.05%、磷酸二氢钾0.05%、硫酸亚铁0.05%、硫酸锌0.10%。在此最优培养基组成下,突变株BY23-195胞内其RNA含量达到16.01%,较优化前（13.66%）提高了17.20%。     

The putative monocarboxylate permeases of the yeast Saccharomyces cerevisiae do not transport monocarboxylic acids across the plasma membrane

We have characterized the monocarboxylate permease family of Saccharomyces cerevisiae comprising five proteins. We could not find any evidence that the monocarboxylate transporter-homologous (Mch) proteins of S. cerevisiae are involved in the uptake or secretion of monocarboxylates such as lactate, pyruvate or acetate across the plasma membrane. A yeast mutant strain deleted for all five MCH genes exhibited no growth defects on monocarboxylic acids as the sole carbon and energy sources. Moreover, the uptake and secretion rates of monocarboxylic acids were indistinguishable from the wild-type strain. Additional deletion of the JEN1 lactate transporter gene completely blocked uptake of lactate and pyruvate. However, uptake of acetate was not even affected after the additional deletion of the gene YHL008c, which had been proposed to code for an acetate transporter. The mch1-5 mutant strain showed strongly reduced biomass yields in aerobic glucose-limited chemostat cultures, pointing to the involvement of Mch transporters in mitochondrial metabolism. Indeed, intracellular localization studies indicated that at least some of the Mch proteins reside in intracellular membranes. However, pyruvate uptake into isolated mitochondria was not affected in the mch1-5 mutant strain. It is concluded that the yeast monocarboxylate transporter-homologous proteins perform other functions than do their mammalian counterparts.     

不同极性酸浆提取液对体外诱导的非酒精性脂肪肝细胞的影响

探讨不同极性酸浆提取液对体外诱导的非酒精性脂肪肝(NAFLD)细胞的影响及其可能的作用机制。用不同极性有机溶剂对酸浆进行萃取,采用MTT法筛选医用脂肪乳注射液使用浓度、酸浆提取液作用浓度,光学显微镜观察油红O染色情况,全自动生化仪检测甘油三酯(TG)、谷丙转氨酶(ALT)、谷草转氨酶(AST)和谷氨酰转肽酶(GGT)水平。与正常肝细胞株HL7702相比,模型组油红O染色后细胞浆内有大量被染成橘红色的脂滴,细胞内TG、ALT、AST和GGT水平显著升高(p0.01)。与模型组相比,不同酸浆提取物治疗12 h组细胞浆内橘红色的脂滴数量明显减少,TG、ALT、AST和GGT水平显著降低(p0.01)。在体外成功建立非酒精性脂肪肝的细胞模型,不同极性酸浆提取液处理能减轻NAFLD细胞中TG蓄积和ALT、AST和GGT的泄漏量。     

Studies of yeast Kluyveromyces lactis mutations conferring super-secretion of recombinant proteins

We have isolated mutants responsible for a super-secretion phenotype in Kluyveromyces lactis using the gene coding for a Bacillus amyloliquefaciens alpha-amylase as a marker for secretion. These mutations defined two groups, dominant and recessive. The recessive mutant strain, which secreted the heterologous protein in five-fold excess compared to the wild-type strain, was used for the cloning of genes, restraining the super-secreting phenotype. In screening for genes affecting super-secreting phenotype, we found that multiple copies of 10 different independently isolated DNA sequences suppressed the super-secreting phenotype. The first among the genes characterized, named KlSEL1 ('secretion lowering') showed homology to Saccharomyces cerevisiae ORF YML013w. The KlSEL1 gene is predicted to encode a polypeptide of 620 amino acid residues containing a putative transmembrane domain and UBX domain, characteristic for the ubiquitin-regulatory proteins. We demonstrated that the disruption of the SEL1 orthologues in K. lactis and S. cerevisiae conferred the super-secreting phenotype. SEL1 isolated from S. cerevisiae suppressed the super-secretion phenotype in K. lactis klsel1 strain, likewise homologous KlSEL1. No other phenotypic features for strains lacking the SEL1 gene were noticed except for the S. cerevisiae mutant growth being notably slower than in a wt strain. No growth changes were observed in the K. lactis klsel1 mutant. The set of genes (suppressors of over-secreting phenotype) could be attractive for further analysis of gene functions, super-secreting mechanisms and construction of new strains. This collection could be useful for the expedient construction of reduced yeast genomes, optimized for heterologous protein secretion.     

单核细胞性李斯特菌基因dal的敲除及其生物学特性初步分析

在单核细胞性李斯特菌(Listeria monocytogenes)野生株EGDe act A及inl B双基因缺失株(EGDeΔact AΔinl B)的基础上,利用同源重组的方法进一步构建了缺失营养基因dal的菌株(EGDe Δact AΔinl BΔdal),并对该缺失菌株生长状态、毒力基因表达水平、生物被膜的形成量及细胞侵袭等方面作进一步分析。结果显示,37℃摇床培养6 h后,缺失株的菌浓度显著低于EGDe Δact AΔinl B(P0.001),培养基中补充D-丙氨酸的缺失株生长速率与亲本株相比无显著差异;实时荧光定量聚合酶链式反应结果显示,缺失株的sig B基因表达水平变化最明显(P0.01),约下调90%;缺失株生物被膜形成量显著增加(P0.05),培养基补充D-丙氨酸后缺失株生物被膜的生成量与亲本株相比无差异;对Coca-2细胞的侵袭无影响,表明该基因对细菌生长能力及生物被膜形成具有重要的调控作用,并不影响菌株对细胞的侵袭力。此缺失株的构建为进一步研究基因dal的功能提供了理论支持。     

Hyaluronic acid production by recombinant Streptococcus thermophilus

Generally recognized as safe, Streptococcus thermophilus was transformed using a plasmid expressing endogenous hyaluronic acid (HA) synthase genes. A single expression of hyaluronic acid synthase (hasA), uridine diphosphate-glucose dehydrogenase gene (hasB), or pyrophosphorylase gene (glmU) and double expression of hasA and hasB were attempted. A streptococcus-Escherichia coli shuttle vector, pBE31, was successfully transfected in S. thermophilus. The single expression of hasA or hasB allowed S. thermophilus to produce about 0.5-1.0 g/l HA. The strains coexpressing of hasA and hasB showed a markedly increased HA production (1.2g/l) which was six-fold increase compared with the wild-type strain. The maximum cell concentration and specific growth rate of each recombinant strain were lower than those of the wild-type strain; however, the specific production rate was more than 100-fold higher. Galactose concentration decreased in the coexpressing strain after depletion of lactose. The bacterial metabolism would be altered in order to achieve a higher production by changing the intracellular metabolism. The average molecular weight of HA (1.0 × 10(6) Da) was not affected by the expression of hasA and hasB. HA produced from recombinant strain could be an alternative material for medical, cosmetic and food utilization instead of HA from conventional pathogenic streptococci.     

Increased riboflavin production from activated bleaching earth by a mutant strain of Ashbya gossypii

The production of riboflavin from vegetable oil was increased using a mutant strain of Ashbya gossypii. This mutant was generated by treating the wild-type strain with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Riboflavin production was 10-fold higher in the mutant compared to the wild-type strain. The specific intracellular catalase activity after 3 d of culture was 6-fold higher in the mutant than in the wild-type strain. For the mutant, riboflavin production in the presence of 40 mM hydrogen peroxide was 16% less than that in the absence of hydrogen peroxide, whereas it was 56% less for the wild-type strain. The isocitrate lyase (ICL) activity of the mutant was 0.26 mU/mg of protein during the active riboflavin production phase, which was 2.6-fold higher than the wild-type strain. These data indicate that the mutant utilizes the carbon flux from the TCA cycle to the glyoxylate cycle more efficiently than the wild-type strain, resulting in enhanced riboflavin production. This novel mutant has the potential to be of use for industrial-scale riboflavin production from waste-activated bleaching earth (ABE), thereby transforming a useless material into a valuable bioproduct.