阿卡波糖生物合成调控蛋白的钓取与功能解析

[目的] 发现游动放线菌Actinoplanes sp.SE50/110中阿卡波糖生物合成的调控因子,并提高其产量。[方法] 首先,利用DNA亲和层析技术,钓取与阿卡波糖生物合成基因簇2个双向启动子区域结合的调控蛋白。然后,在阿卡波糖产生菌QQ-2中强化表达或敲除这些调控蛋白编码基因,进行体内功能验证。同时,利用大肠杆菌BL21（DE3）异源表达获得可溶性蛋白,通过凝胶阻滞实验验证蛋白与启动子区域的结合能力。[结果] 经DNA亲和层析及蛋白质质谱分析,钓取出9个与双向启动子P_WV和P_AB结合的调控蛋白。在QQ-2中分别强化表达和缺失这9个调控基因后发现,基因ACPL_1889的强化表达使阿卡波糖产量提高25%,而该基因的缺失使产量降低22%;基因ACPL_5445、ACPL_3989的强化表达使阿卡波糖产量分别降低12%和39%,而这两个基因的缺失使产量分别提高15%和8%。对阿卡波糖生物合成基因转录水平的检测发现,强化表达基因ACPL_1889使acbA、acbB、acbW、acbV的转录水平升高,而缺失该基因使这4个基因的转录水平降低;敲除基因ACPL_5445使这4个基因转录水平均有提高;强化表达基因ACPL_3989使这4个基因的转录水平均下降,而其敲除使acbW和acbA的转录水平分别提高了约100倍和40倍。在凝胶阻滞实验中,ACPL_1889与ACPL_3989均能与acb基因簇的启动子区域结合。最后将正调控基因的强化表达和负调控基因的敲除进行组合,使阿卡波糖产量提升32%。[结论] 本研究发现了9个与阿卡波糖生物合成基因簇的启动子区域结合的调控蛋白,通过体内、体外实验证明ACPL_1889为阿卡波糖生物合成的正调控因子、ACPL_5445和ACPL_3989为负调控因子,不但为揭示阿卡波糖生物合成的转录调控机制奠定了基础,而且这些调控基因的改造显著提升了阿卡波糖的产量。     

游动放线菌8-22中treY基因敲除对于降低阿卡波糖C组分的作用

【目的】在阿卡波糖发酵过程中,C组分的存在严重影响阿卡波糖产品的质量,研究拟通过基因改造降低阿卡波糖C组分。【方法】通过构建treY同框敲除质粒pUAmT-YUD,以接合转移方法将其转入阿卡波糖工业菌株8-22,经同源重组将treY基因内部编码182个氨基酸的序列敲除,从而得到treY基因失活的突变株Y810。【结果】发酵结果显示突变菌株中C组分较出发菌株下降了约10倍,而阿卡波糖本身的效价末受影响。【结论】敲除treY基因可大幅降低阿卡波糖C组分的含量。研究的实施将大大简化阿卡波糖的纯化步骤,提升产品品质,降低生产成本,从而提高工业化生产的市场竞争力。研究同时还对游动放线菌的接合转移条件进行了优化,大大提高了转化效率。     

游动放线菌属的一个新种

游动放线菌Y80—610菌株在所采用的合成及有机培养基内产生紫色基内菌丝体,它与游动放线菌属的所有已知种有显著区别,认为是个新种,定名为紫色游动放线菌(Actinoplanesviolaceus n．Sp．)     

创新霉素产生菌——济南游动放线菌的分类鉴定

放线菌3945是从山东济南土壤中分离出来的,产生一种新的抗菌素——创新霉素,形成孢囊,孢囊孢子微游动,极生鞭毛。在合成与有机培养基上,基内菌丝为紫色到深褐紫,培养基染成相应颜色。根据形态、培养特征、生理特性及抗菌性质的研究,放线菌3945有别于游动放线菌属(Actinoplancs)中的已知种,经鉴定认为是个新种,名之为济南游动放线菌(Actino-planes tainanensis n. sp.)。     

阿卡波糖的生物合成途径研究进展

阿卡波糖(Acarbose)作为第一个用于临床治疗Ⅱ型糖尿病的α-葡萄糖苷酶抑制剂类药物,自上市以来,由于其高效、安全而得到广泛应用.目前工业化生产所用的生产茵株都来自Actinoplanes sp.SE50/110,其合成途径随着acb基因簇的发现已经基本研究清楚.在另外一种阿卡波糖产生菌Streptomyces glaucescens GLA.O内,同样发现了与acb基因簇具有高度相似性的gac基因簇,其合成途径与acb基因簇遵循相同的路径,而又有显著的差别.就这两种合成途径以及阿卡波糖的产生、转运和代谢进行综述.     

游动放线菌科的研究IlI．游动放线菌属的三个新种

从北京土壤中分离出5株放线菌,经过形态,培养特征、生理生化、细胞壁组份及DNA中GC％含量的研究,这5株菌都形成孢囊,孢囊孢子有鞭毛,能游动,细胞壁组份11型,属于游动放线菌科中游动放线菌属。经与国内外已知种比较,定为三个新种：丛鞭毛游动放线菌新种(Actinoplanes tuftoflagellus n. sp．);梨形游动放线菌新种(Actinoplanes pyriforinis n. sp.)绛红褐游动放线菌新种(Actinoplanes purpeobrunneus n. sp.)。     

游动放线菌科分类的研究II.游动放线菌属的四个新种

从我国土壤中分离出35株游动放线菌,细胞壁组份为II型,即含有内消旋二氨基庚二酸和甘氨酸。根据形态、培养特征和生理生化特性的系统研究,定为4个新种：桔橙游动放线菌新种(Actlnoplanem ourantlacus n. sp. 71—C38);粉红孢囊游动放线凶新种(Actinoplanesroseosporangius n. sp. 71-C29);淡桔橙游动放线菌新种(ActlnoplaneJ pallido-aurantiacusn. Sp. C);赭红孢囊游动放线菌新种(Actinoplanes rutilosporangius n. sp．71-C6)。     

游动放线菌属和小瓶菌属的新种

从昆明地区采集的土壤样品中分离到Y79 21及Y79-15两株放线菌。Y79-21菌株无气生菌丝体,在基内菌丝体顶端形成球形或椭圆形孢囊,孢囊孢于能游动,细胞壁组份II型,属于游动放线菌属(Actinoplanes Couch,1950)[1],但与该属的已知种不同,定为新种,命名为云南游动放线菌(Acunoplanes yunnanensts n. sp.)。     

性状加权编码在放线菌数值分类上的应用

本文提出了一种新的微生物形态性状的加权编码方式。使用这种加权编码方式及平均链锁法对放线菌游动放线菌属(Actinoplanes)和小瓶菌属(Ampullariella)共116株进行了聚类分析。结果表明:通过性状加权处理可以使游动放线菌属和小瓶菌属得以区分。     

环氧聚胺——一种细胞固定化的新型交联剂

近年来随着人们对酶和微生物细胞固定化研究的逐渐深入,对固定化的各种载体材料和固定化方法也进行广泛研究。本文报道一种固定化酶的新型交联剂——环氧聚胺(Epoxy-polyamine简称E.P.A.)用于游动放线菌葡萄糖异构酶和大肠杆菌青霉素酰化酶交联固定化的研究结果。一、材料和方法 1.菌体及试剂 (1)菌体含青霉素酰化酶之大肠杆菌,由华北制药厂提供。含葡萄糖异构酶之游动放线菌,由上海新型发酵厂提供。 (2)环氧聚胺上海市化学试剂商店。     

游动放线菌中orf20基因敲除对雷莫拉宁合成的影响

雷莫拉宁生物合成基因簇中orf20与已知的卤化酶基因高度同源.本研究在大肠杆菌中构建同框敲除质粒pSM-3,将其转入游动放线菌Actinoplanes sp.ATCC 33076,通过同源重组双交换的方法将orf20基因内部编码64个氨基酸的序列敲除,筛选得到双交换阻断突变株SIPI-A.2001 dorf20(Δor...     

Identification, cloning, expression, and characterization of the extracellular acarbose-modifying glycosyltransferase, AcbD, from Actinoplanes sp. strain SE50.

An extracellular enzyme activity in the culture supernatant of the acarbose producer Actinoplanes sp. strain SE50 catalyzes the transfer of the acarviosyl moiety of acarbose to malto-oligosaccharides. This acarviosyl transferase (ATase) is encoded by a gene, acbD, in the putative biosynthetic gene cluster for the alpha-glucosidase inhibitor acarbose. The acbD gene was cloned and heterologously produced in Streptomyces lividans TK23. The recombinant protein was analyzed by enzyme assays. The AcbD protein (724 amino acids) displays all of the features of extracellular alpha-glucosidases and/or transglycosylases of the alpha-amylase family and exhibits the highest similarities to several cyclodextrin glucanotransferases (CGTases). However, AcbD had neither alpha-amylase nor CGTase activity. The AcbD protein was purified to homogeneity, and it was identified by partial protein sequencing of tryptic peptides. AcbD had an apparent molecular mass of 76 kDa and an isoelectric point of 5.0 and required Ca(2+) ions for activity. The enzyme displayed maximal activity at 30 degrees C and between pH 6.2 and 6.9. The K(m) values of the ATase for acarbose (donor substrate) and maltose (acceptor substrate) are 0.65 and 0.96 mM, respectively. A wide range of additional donor and acceptor substrates were determined for the enzyme. Acceptors revealed a structural requirement for glucose-analogous structures conserving only the overall stereochemistry, except for the anomeric C atom, and the hydroxyl groups at positions 2, 3, and 4 of D-glucose. We discuss here the function of the enzyme in the extracellular formation of the series of acarbose-homologous compounds produced by Actinoplanes sp. strain SE50.     

Maltodextrin-binding proteins from diverse bacteria and archaea are potent solubility enhancers

In the biosynthesis of the C7-cyclitol moiety, valienol, of the -glucosidase inhibitor acarbose in Actinoplanes sp. SE50/110 various cyclitol phosphates, such as 1-epi-valienol-7-phosphate, are postulated precursors. In the cell extracts of Actinoplanes SE50/110 we found a new kinase activity which specifically phosphorylates 1-epi-valienol; other C7-cyclitol analogs were only weakly or not phosphorylated. The purified product of the kinase reaction turned out to be 1-epi-valienol-7-phosphate in analyses by nuclear magnetic resonance spectroscopy. The enzyme seems not to be encoded by an acb gene and, therefore, plays a role in a salvage pathway rather than directly in the de novo biosynthesis of acarbose.     

Gene cluster responsible for validamycin biosynthesis in Streptomyces hygroscopicus subsp. jinggangensis 5008

A gene cluster responsible for the biosynthesis of validamycin, an aminocyclitol antibiotic widely used as a control agent for sheath blight disease of rice plants, was identified from Streptomyces hygroscopicus subsp. jinggangensis 5008 using heterologous probe acbC, a gene involved in the cyclization of D-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone of the acarbose biosynthetic gene cluster originated from Actinoplanes sp. strain SE50/110. Deletion of a 30-kb DNA fragment from this cluster in the chromosome resulted in loss of validamycin production, confirming a direct involvement of the gene cluster in the biosynthesis of this important plant protectant. A sequenced 6-kb fragment contained valA (an acbC homologue encoding a putative cyclase) as well as two additional complete open reading frames (valB and valC, encoding a putative adenyltransferase and a kinase, respectively), which are organized as an operon. The function of ValA was genetically demonstrated to be essential for validamycin production and biochemically shown to be responsible specifically for the cyclization of D-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone in vitro using the ValA protein heterologously overexpressed in E. coli. The information obtained should pave the way for further detailed analysis of the complete biosynthetic pathway, which would lead to a complete understanding of validamycin biosynthesis.     

Reduced formation of byproduct component C in acarbose fermentation by Actinoplanes sp. CKD485-16

Acarbose fermentation was conducted by cultivation of Actinoplanes sp. CKD485-16. Approximately 2,300 mg/L of acarbose was produced at the end of cultivation along with 600 mg/L of the acarbose byproduct component C. Maltose, a known moiety of acarbose, should be maintained at high concentration levels in culture broths for efficient acarbose production. The acarbose yield increased with an increasing osmolality of the culture medium, with a maximum value of 3,200 mg/L obtained at 500 mOsm/kg. Component C was also produced in proportion to the osmolality. Conversion of acarbose to component C was accomplished with resting whole cells. Inhibitors of the conversion of acarbose to component C were sought since component C is probably derived from acarbose. Valienamine was found to be a potent inhibitor, resulting in a more than 90% reduction in component C formation at a 10 microM concentration. Effects were similar in a 1,500-L pilot fermentor with acarbose and component C yields of 3,490 and 43 mg/L at 500 mOsm/kg, respectively.     

游动放线菌Actinoplanes sp.SE50/110中阿卡波糖脱氧氨基糖单元的生物合成

【目的】解析Actinoplanes sp.SE50/110(简称SE50/110)中阿卡波糖脱氧氨基糖单元的生物合成机制。【方法】经过BLASTp分析,推测了Acb A、Acb B和Acb V负责阿卡波糖脱氧氨基糖单元的生物合成。首先,本研究在SE50/110中分别构建了acb A、acb B和acb V的同框缺失和回补突变株。然后,利用大肠杆菌BL21(DE3)/p Gro7分别对Acb A、Acb B和Acb V成功实现了可溶性表达。最后,以D-葡萄糖-1-磷酸为起始底物,通过体外催化反应,研究脱氧氨基糖单元的生物合成过程和相关蛋白的酶学性质。【结果】在SE50/110中分别缺失acb A、acb B和acb V基因后,相应突变株均丧失了阿卡波糖的合成能力,将acb A、acb B和acb V基因分别回补后,各菌株又恢复了阿卡波糖的合成能力,证明了它们均为阿卡波糖生物合成的必需基因。在体外酶促反应中,D-葡萄糖-1-磷酸-胸腺嘧啶转移酶Acb A催化D-葡萄糖-1-磷酸和d TTP合成d TDP-D-葡萄糖,对D-葡萄糖-1-磷酸的Km值为(0.185±0.053)mmol/L,Vmax为(2.366±0.217)μmol/(min·mg);对d TTP的Km值为(4.964±1.089)mmol/L,Vmax为(60.310±5.419)μmol/(min·mg)。d TDP-D-葡萄糖-4,6-脱水酶Acb B催化d TDP-D-葡萄糖转化为d TDP-4-酮基-6-脱氧-D-葡萄糖,Km值和Vmax分别为(0.353±0.089)mmol/L和(306.401±28.740)μmol/(min·mg)。氨基转移酶Acb V催化d TDP-4-酮基-6-脱氧-D-葡萄糖生成d TDP-4-氨基-4,6-双脱氧-D-葡萄糖,Km值和Vmax分别为(1.411±0.293)mmol/L和(3.447±0.279)μmol/(min·mg)。【结论】本研究阐明了阿卡波糖脱氧氨基糖单元的生物合成过程,为全面解析阿卡波糖生物合成途径奠定了基础。同时,测定了相关酶的动力学参数,为代谢工程改造SE50/110,提高阿卡波糖产量提供了重要的理论依据。     

Crystal structures of receptors involved in small molecule transport across membranes

This paper briefly reviews contemporary protein crystallography and focuses on six receptor proteins of membrane-intrinsic ATP binding cassette (ABC) transporters. Three of these receptors are specific for carbohydrates and three for amino acids. The receptor GacH of the transporter GacFGH from Streptomyces glaucescens is specific for acarbose and its homologs, and MalE of Salmonella typhimurium is specific for maltose but also forms a complex with acarbose, and the third receptor is the highly specific d-galactose receptor AcbH of the transporter AcbFGH from Actinoplanes sp. Concerning the receptors for amino acids, ArtJ belongs to the ArtJ-(MP)(2) transporter of Geobacillus stearotermophilus and recognizes and binds to positively charged arginine, lysine, and histidine with different sizes of side chains, contrasting the receptors Ngo0372 and Ngo2014 from Neisseria gonorrhaeae that are highly specific for cystine and cysteine, respectively. The differences in the rather unspecific receptors GacH, MalE and ArtJ are compared with the highly specific receptors AcbH, Ngo0372 and Ngo2014.     

A new RNASeq-based reference transcriptome for sugar beet and its application in transcriptome-scale analysis of vernalization and gibberellin responses

Background

Actinoplanes sp. SE50/110 is known as the wild type producer of the alpha-glucosidase inhibitor acarbose, a potent drug used worldwide in the treatment of type-2 diabetes mellitus. As the incidence of diabetes is rapidly rising worldwide, an ever increasing demand for diabetes drugs, such as acarbose, needs to be anticipated. Consequently, derived Actinoplanes strains with increased acarbose yields are being used in large scale industrial batch fermentation since 1990 and were continuously optimized by conventional mutagenesis and screening experiments. This strategy reached its limits and is generally superseded by modern genetic engineering approaches. As a prerequisite for targeted genetic modifications, the complete genome sequence of the organism has to be known.

Results

Here, we present the complete genome sequence of Actinoplanes sp. SE50/110 [GenBank:CP003170], the first publicly available genome of the genus Actinoplanes, comprising various producers of pharmaceutically and economically important secondary metabolites. The genome features a high mean G + C content of 71.32% and consists of one circular chromosome with a size of 9,239,851 bp hosting 8,270 predicted protein coding sequences. Phylogenetic analysis of the core genome revealed a rather distant relation to other sequenced species of the family Micromonosporaceae whereas Actinoplanes utahensis was found to be the closest species based on 16S rRNA gene sequence comparison. Besides the already published acarbose biosynthetic gene cluster sequence, several new non-ribosomal peptide synthetase-, polyketide synthase- and hybrid-clusters were identified on the Actinoplanes genome. Another key feature of the genome represents the discovery of a functional actinomycete integrative and conjugative element.

Conclusions

The complete genome sequence of Actinoplanes sp. SE50/110 marks an important step towards the rational genetic optimization of the acarbose production. In this regard, the identified actinomycete integrative and conjugative element could play a central role by providing the basis for the development of a genetic transformation system for Actinoplanes sp. SE50/110 and other Actinoplanes spp. Furthermore, the identified non-ribosomal peptide synthetase- and polyketide synthase-clusters potentially encode new antibiotics and/or other bioactive compounds, which might be of pharmacologic interest.