纳他霉素发酵培养基及发酵条件的优化

采用Plackett-Burman法、最陡爬坡实验和响应面实验(Box-Behnken设计法)相结合的方法对褐黄孢链霉菌合成纳他霉素的发酵培养基及发酵条件进行优化。结果表明,培养基中的蛋白胨、pH和摇瓶装液量是影响纳他霉素产量的主要因素。优化后的培养基组成为葡萄糖50 g/L、蛋白胨19.5 g/L、酵母粉7 g/L、pH 7.4~7.5;发酵条件为装液量60 mL/500 mL、接种量15%、发酵温度29℃、摇床转速200 r/min、发酵周期96 h。此条件下,纳他霉素的产量较优化前提高了94%,达到2.19 g/L。     

基于前体供给途径遗传改造提高褐黄孢链霉菌纳他霉素的产量

纳他霉素(natamycin)是一种高效、广谱、安全的抗真菌剂,广泛应用于食品防腐与医药领域。纳他霉素可由多种链霉菌发酵产生。它是以乙酰辅酶A、丙二酰辅酶A及甲基丙二酰辅酶A为前体经Ⅰ型聚酮合酶(polyketide synthase,PKS)催化合成的多烯大环内酯类化合物。本研究以纳他霉素产生菌——褐黄孢链霉菌为研究材料,分别对不同前体分子供给途径中的关键酶进行过表达,并确定影响纳他霉素产量的关键前体供给途径。研究结果发现：通过过表达乙酰辅酶A合成酶(acetyl-CoA synthase,ACS)加强乙酰辅酶A合成途径,以及通过过表达甲基丙二酰辅酶A变位酶(methylmalonyl-CoA mutase,MCM)加强甲基丙二酰辅酶A合成途径,重组菌株纳他霉素产量分别比野生型菌株提高了44.19%和20.51%。共过表达ACS和MCM,重组菌株纳他霉素产量获得进一步提升(达1123.34mg/L),比野生型菌株提高了66.29%。上述发现为通过前体代谢工程的策略构建纳他霉素工业高产菌株提供了参考,也为其他聚酮类天然产物高产工程菌株的构建提供了借鉴。     

ABC转运蛋白SgnA/B促进纳他霉素胞外转运与高产

纳他霉素是一种天然、广谱、高效的多烯大环内酯类还原性抗真菌剂,广泛应用于食品真菌污染的防治和临床真菌感染的治疗。纳他霉素胞外转运效率可能是限制褐黄孢链霉菌(Streptomyces gilvosporeus)发酵高产纳他霉素的重要因素。通过生物信息学及分子对接技术分析纳他霉素胞外转运蛋白SgnA/B,发现SgnA和SgnB两个异源二聚体组成的ABC转运蛋白是内向开口构象的转运蛋白,且2个结合位点与纳他霉素结合能力有强弱差异,更有利于纳他霉素的胞外转运。本研究以纳他霉素生产菌株——褐黄孢链霉菌F607为出发菌株,构建了sgnA/B基因超表达菌株F-EX,以分析sgn A/B基因超表达对纳他霉素合成及胞外转运的影响。研究发现,纳他霉素对数合成期的F-EX菌株不仅提高了纳他霉素胞外/胞内比,其120 h发酵总产量也提高了12.5%,达到7.38 g/L。最后,通过转录组测序发现,sgnA/B基因超表达除提高纳他霉素胞外转运效率外,还影响了与多种氨基酸、丙酸盐、糖、五碳化合物代谢和TCA循环相关基因的表达。研究表明,强化纳他霉素胞外转运有利于纳他霉素的合成,是提高褐黄孢链霉菌纳他霉素产量的有效...     

庆丰霉素的研究 Ⅵ.庆丰霉素的深层发酵

本研究的目的是探索庆丰霉素深层发酵的适宜条件。结果指出:庆丰霉素深层发酵的最适培养基成份为:玉米淀粉4％,糊精0.3％,黄豆饼粉2.0％,花生饼粉2.5％,硝酸钾0.2％,氯化钠0.3％,碳酸钙0.4％。适量的花生饼粉和硝酸钾对庆丰霉素的产生均有明显的促进作用,最适发酵温度为33℃。用庆丰链霉菌M15菌株进行摇瓶及发酵罐发酵,庆丰霉素的效价均达4500～5000微克/毫升。 铁对庆丰霉素产生有抑制作用,培养基中加入葡萄糖表现出葡萄糖效应。 对花生饼粉及硝酸钾促进庆丰霉素合成和铁及葡萄糖抑制庆丰霉素合成的可能作用机理进行了讨论。     

链霉菌A048产几丁质酶最佳发酵工艺研究*

将链霉菌A048在完全培养基中培养至对数生长末期,离心洗涤收集菌丝体,然后接种入发酵产酶培养基中,进行二步发酵工艺生产几丁质酶,几丁质酶活力比一步发酵工艺提高1.1倍,发酵周期共54h,比一步发酵工艺缩短66h;把菌丝体与几丁质粉共固定化,接入发酵产酶培养基中培养36h,几丁质酶活力比一步发酵工艺提高1.8倍,发酵周期缩短54h;在二步发酵工艺中另添加0.4%纤维素,几丁质酶活力可提高4倍,比一步发酵工艺提高10倍,酶活力达18.52U/mL。采用几丁质和纤维素双因子诱导二步发酵工艺可能是链霉菌A048生     

异源表达afsRScla全局性调控基因提高工业菌株纳他霉素产量

前期研究表明棒状链霉菌的全局性调控基因afsRScla异源表达可以激活变铅青链霉菌中两种抗生素的合成。本研究将包含afsRScla基因的质粒pHL851整合到纳他霉素工业生产菌株褐黄孢链霉菌TZ1401的基因组中,使纳他霉素产量提高38%,达到3.56g/L。qRT-PCR检测6个纳他霉素生物合成基因的转录情况,发现其转录水平提高1.9-2.7倍,表明afsRScla通过正调控纳他霉素生物合成基因的转录,从而提高纳他霉素的产量。本研究结果对afsRScla在抗生素工业生产菌株的高产育种应用具有借鉴意义。     

链霉菌A048产几丁质酶最佳发酵工艺研究

将链霉菌A048在完全培养基中培养至对数生长末期,离心洗涤收集菌丝体,然后接种入发酵产酶培养基中,进行二步发酵工艺牛产几丁质酶,几丁质酶活力比一步发酵工艺提高1．1倍,发酵周期共54h,比一步发酵工艺缩短66h;把菌丝体与几丁质粉共固定化,接入发酵产酶培养基中培养36h,几丁质酶活力比一步发酵工艺提高1．8倍,发酵周期缩短54h;在二步发酵工岂中另添加0．4％纤维素,几丁质酶活力可提高4倍,比一步发酵工艺提高10倍,酶活力达18．52U／mL。采用几丁质和纤维索双因子诱导二步发酵工艺可能是链霉菌A048生产几丁质酶的最佳工艺。     

响应面法优化链霉菌S10A09发酵产纤维素酶条件

在筛选纤维素酶活菌株时,发现一株放线菌链霉菌属S10A09具有较高的纤维素酶活力。为了获得高酶活纤维素酶,将Plackett-Burman(PB)筛选和中心组合设计(CCD)以及响应面分析法相结合,考察影响链霉菌属S10A09发酵生产滤纸酶的发酵条件。Plackett-Burman结果表明,羧甲基纤维素钠(CMC-Na)和(NH_4)_2SO_4是影响S10A09发酵产纤维素酶活高低的主要因素。CCD实验优化后产酶最优发酵培养基(g/L)为CMC-Na 2.57、(NH_4)_2SO_411.31、KH_2PO_4 0.2、MgSO_41、FeSO_40.01。优化后,滤纸酶活(FPA)达到125.96 U/mL,接近优化前的3倍。     

培养条件对绿木霉菌几丁质酶分泌水平的调节

木霉菌（Trichoderma spp．）是一类重要的植病生防因子,该菌产生的包括几丁质酶在内的细胞壁降解酶,在木霉重寄生中起重要作用。该文采用正交试验方法研究了葡萄糖浓度、铵盐浓度、胶状几丁质三个因素及其交互作用对绿木霉菌（Trichoderma,Virens）几丁质酶分泌水平的影响,结果表明,低浓度葡萄糖（0．1％）和低浓度铵盐（10mmol／L）有利于几丁质酶的分泌,几丁质酶活性最高可达到6．83U;高浓度葡萄糖对几丁质酶的分泌有明显抑制作用,在3％葡萄精浓度条件下几丁质酶的分泌水平均小于0．45U,在高铵盐浓度（100mmo/L）时,几丁质酶分泌水平在0．59～1．29U;胶状几丁质的添加可在术霉菌丝体生长早期（培养前72h）诱导几丁质酶的分泌;葡萄糖与铵盐的交互作用能够显著提高几丁质酶的分泌水平。     

产几丁质酶菌株S68-CM5产酶条件优化研究

为提高黏质沙雷氏菌株S68-CM5产几丁质酶能力,对产酶发酵条件进行优化研究。利用Plackett-Burman设计和响应面法对培养基和发酵条件进行摸索。结果显示,获得最佳发酵产酶培养基:胶体几丁质1.5%,牛肉膏7 g/L,酵母膏2 g/L,葡萄糖8 g/L,氯化钠3.5 g/L,蛋白胨2 g/L,磷酸氢二钾3.5 g/L;最佳产酶培养条件为:p H6.88,温度27.32℃,摇床转数155.82r/min,培养时间60 h,接种量1%,装液量50 m L/250 m L。优化后产酶量达到7.131 U/m L,比优化前产酶量提高了1.43倍。     

Construction of a Streptomyces lydicus A01 transformant with a chit42 gene from Trichoderma harzianum P1 and evaluation of its biocontrol activity against Botrytis cinerea

Streptomyces lydicus A01 and Trichoderma harzianum P1 are potential biocontrol agents of fungal diseases in plants. S. lydicus A01 produces natamycin to bind the ergosterol of the fungal cell membrane and inhibits the growth of Botrytis cinerea. T. harzianum P1, on the other hand, features high chitinase activity and decomposes the chitin in the cell wall of B. cinerea. To obtain the synergistic biocontrol effects of chitinase and natamycin on Botrytis cinerea, this study transformed the chit42 gene from T. harzianum P1 to S. lydicus A01. The conjugal transformant (CT) of S. lydicus A01 with the chit42 gene was detected using polymerase chain reaction (PCR). Associated chitinase activity and natamycin production were examined using the 3, 5-dinitrosalicylic acid (DNS) method and ultraviolet spectrophotometry, respectively. The S. lydicus A01-chit42 CT showed substantially higher chitinase activity and natamycin production than its wild type strain (WT). Consequently, the biocontrol effects of S. lydicus A01-chit42 CT on B. cinerea, including inhibition to spore germination and mycelial growth, were highly improved compared with those of the WT. Our research indicates that the biocontrol effect of Streptomyces can be highly improved by transforming the exogenous resistance gene, i.e. chit42 from Trichoderma, which not only enhances the production of antibiotics, but also provides a supplementary function by degrading the cell walls of the pathogens.     

Protein Engineering of Chit42 Towards Improvement of Chitinase and Antifungal Activities

The antagonism of Trichoderma strains usually correlates with the secretion of fungal cell wall degrading enzymes such as chitinases. Chitinase Chit42 is believed to play an important role in the biocontrol activity of Trichoderma strains as a biocontrol agent against phytopathogenic fungi. Chit42 lacks a chitin-binding domain (ChBD) which is involved in its binding activity to insoluble chitin. In this study, a chimeric chitinase with improved enzyme activity was produced by fusing a ChBD from T. atroviride chitinase 18–10 to Chit42. The improved chitinase containing a ChBD displayed a 1.7-fold higher specific activity than chit42. This increase suggests that the ChBD provides a strong binding capacity to insoluble chitin. Moreover, Chit42-ChBD transformants showed higher antifungal activity towards seven phytopathogenic fungal species.     

Expression of Paenibacillus polymyxa β-1,3-1,4-glucanase in Streptomyces lydicus A01 improves its biocontrol effect against Botrytis cinerea

Streptomyces lydicus strain A01, which can produce natamycin and chitinase, has a significant inhibition effect on gray mold disease caused by Botrytis cinerea. However, it has no detectable glucanase activity. Strain A21 isolated from the snow covered high altitude area in Tibet, China, also has a high antagonistic activity against B. cinerea. It displayed an obvious halo on lichen polysaccharides plates by congo red staining, indicating a strong glucanase activity. A21 was identified as Paenibacillus polymyxa using 16S rDNA gene analysis and biochemical and physiological analysis. To obtain the synergistic antifungal effects of natamycin, chitinase, and glucanases on B. cinerea, this study transformed the β-1,3-1,4-glucanase gene from P. polymyxa A21 to S. lydicus A01. The engineered S. lydicus AG01 showed substantially high glucanase activity, and had similar natamycin production and chitinase activity as the wild-type strain A01. Compared to the wild-type strain A01, the antifungal effects of S. lydicus AG01 on B. cinerea, including inhibition of spore germination and mycelial growth, were highly improved. The improved biocontrol effect of S. lydicus AG01 is likely attributed to the heterologous expression of glucanase from P. polymyxa, which acted synergistically with natamycin and chitinase to increase the antifungal activity of the strain.     

Effects of cultivation conditions on the production of natamycin with Streptomyces gilvosporeus LK-196

Natamycin is a very attractive antifungal agent with wide applications in medical and food industries. In order to improve the productivity of natamycin, the effects of cultivation conditions were investigated with Streptomyces gilvosporeus LK-196 in the shake flasks and 30-L fermentors. The results showed that dissolved oxygen and shear force would affluence the biosynthesis of natamycin significantly. The high concentration of natamycin (2.03g/L) was achieved under the suitable culture conditions in the shake flask scale. Further investigations in 30-L fermentors showed that the optimal pH was controlled at 6.0 during the whole bioprocess, and the dissolved oxygen level should be more than 30% by adjusting the aeration and agitation rates for high production of natamycin. Under these optimal conditions the high concentration of natamycin (3.94g/L) was achieved with Str. gilvosporeus LK-196 in the 30-L fermentor. Finally, the high-level fermentation process was successfully scaled up to 1000-L fermentors and 18,000-L fermentors in the pilot plant.     

Heterologous coexpression of Vitreoscilla hemoglobin and Bacillus megaterium glucanase in Streptomyces lydicus A02 enhanced its production of antifungal metabolites

Streptomyces lydicus A02 is a novel producer of commercially important polyene macrocyclic antibiotic natamycin and a potential biocontrol agent to several plant fungal diseases, including wilt caused by Fusarium oxysporum f. spp. To improve the natamycin production and the antifungal activity of S. lydicus A02, we coexpressed gene vgb encoding Vitreoscilla hemoglobin (VHb) and bglC encoding Bacillus megaterium L103 glucanase, both under the control of the strong constitutive ermE* promoter, in S. lydicus A02. Our results showed that coexpressing VHb and glucanase improved cell growth, and the engineered strain produced 26.90% more biomass than the wild-type strain after 72 h fermentation in YSG medium. In addition, coexpressing genes encoding VHb and glucanase led to increased natamycin production, higher endogenous chitinase activity and exogenous glucanase activity, as well as enhanced antifungal activity in the engineered S. lydicus AVG02 and AGV02, regardless of the position of the two genes on the plasmids. Compared with model strains, few reports have successfully coexpressed VHb and other foreign proteins in industrial strains. Our results illustrated an effective approach for improving antifungal activity in an industrial strain by the rational engineering of combined favorable factors.     

Designing a new chitinase with more chitin binding and antifungal activity

Chitinases have the ability of chitin digestion that constitutes a main compound of the cell wall in many of the phytopathogens such as fungi. Chitinase Chit42 from Trichoderma atroviride PTCC5220 is considered to play an important role in the biocontrol activity of this fungus against plant pathogens. Chit42 lacks a chitin binding domain (ChBD). We have produced a chimeric chitinase with stronger chitin-binding capacity by fusing to Chit42 a ChBD from Serratia marcescens Chitinase B. The fusion of ChBD improved the affinity to crystalline and colloidal chitin and also the enzyme activity of the chimeric chitinase when compared with the native Chit42. The chimeric chitinase showed higher antifungal activity toward phytopathogenic fungi.     

A comparative study of transgenic canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) harboring either chimeric or native <Emphasis Type="Italic">Chit</Emphasis>42 genes against phytopathogenic fungi

Canola (Brassica napus L.), an agro-economically important crop in the world, is sensitive to many fungal pathogens. One strategy to combat fungal diseases is genetic engineering through transferring genes encoding the pathogenesis-related (PR) proteins such as chitinase which cause the chitin degradation of fungal cell wall. Chitinase Chit42 from Trichoderma atroviride (PTCC5220) plays an important role in biocontrol and has high antifungal activity against a wide range of phytopathogenic fungi. This enzyme lacks a chitin binding domain (ChBD) which is involved in binding activity to insoluble chitin. In the present study, we investigated the effect of chitin binding domain fused to Chit42 when compared with native Chit42. These genes were over-expressed under the CaMV35S promoter in B. napus, R line Hyola 308. Transformation of cotyledonary petioles was achieved by pBISM2 and pBIKE1 constructs containing chimeric and native Chit42 genes respectively, via Agrobacterium method. The insertion of transgenes in T₀ generation was verified through polymerase chain reaction (PCR) and Southern blot analysis. Antifungal activity of expressed chitinase in transgenic plants was also investigated by bioassays. The transgenic canola expressing chimeric chitinase showed stronger inhibition against phytopathogenic fungi that indicates the role of chitin binding domain.     

Antifungal thiopeptide cyclothiazomycin B1 exhibits growth inhibition accompanying morphological changes via binding to fungal cell wall chitin

Cyclothiazomycin B1 (CTB1) is an antifungal cyclic thiopeptide isolated from the culture broth of Streptomyces sp. HA 125-40. CTB1 inhibited the growth of several filamentous fungi including plant pathogens along with swelling of hyphae and spores. The antifungal activity of CTB1 was weakened by hyperosmotic conditions, and hyphae treated with CTB1 burst under hypoosmotic conditions, indicating increased cell wall fragility. CTB1-sensitive fungal species contain high levels of cell wall chitin and/or chitosan. Unlike nikkomycin Z, a competitive inhibitor of chitin synthase (CHS), CTB1 did not inhibit CHS activity. Although CTB1 inhibited CHS biosynthesis, the same result was also obtained with a non-specific proteins inhibitor, cycloheximide, which did not reduce cell wall rigidity. These results indicate that the primary target of CTB1 is not CHS, and we concluded that CTB1 antifungal activity was independent of this sole inhibition. We found that CTB1 bound to chitin but did not bind to β-glucan and chitosan. The results of the present study suggest that CTB1 induces cell wall fragility by binding to chitin, which forms the fungal cell wall. The antifungal activity of CTB1 could be explained by this chitin-binding ability.