Polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas stutzeri 1317 had different substrate specificities

The whole polyhydroxyalkanoate (PHA) synthesis gene locus of Pseudomonas stutzeri strain 1317 containing PHA synthase genes phaC1Ps, phaC2Ps and PHA depolymerase gene phaZPs was cloned using a PCR cloning strategy. The sequence analysis results of the phaC1Ps, phaC2Ps and phaZPs showed high homology to the corresponding pha loci of the known Pseudomonas strains, respectively. PhaC1Ps and PhaC2Ps were functionally expressed in recombinant Escherichia coli strains and their substrate specificity was compared. The results demonstrated that PhaC1Ps and PhaC2Ps from P. stutzeri 1317 had different substrate specificities when expressed in E. coli. In details, PhaC2Ps could incorporate both short-chain-length 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates (mcl 3HA) into PHA, while PhaC1Ps only favored mcl 3HA for polymerization.     

聚羟基脂肪酸酯纳米微球：结构特征、生物合成及其在生物技术和生物医药领域的应用

聚羟基脂肪酸酯（polyhydroxyalkanoate）PHA 纳米微球是很多微生物在营养失衡的情况下,在体内合成的一种可生物降解的细胞内聚酯,主要作为微生物的碳源及能量储备。天然 PHA 微球的内部是由疏水的聚酯链构成的疏水核心,其外层是由磷脂界膜及膜上嵌入或附着的包括 PHA合酶 PhaC 和 PHA 颗粒相关蛋白 PhaP 等蛋白构成的边界层。PhaC 通过共价键连接在PHA微球表面,而 PhaP 通过疏水相互作用吸附在 PHA 微球表面。通过将外源性功能蛋白与 PhaC 或 PhaP 进行融合表达,在重组微生物体内就能直接合成表面带有功能蛋白的纳米微球复合体。由于该纳米微球在微生物细胞内是以独立的包涵体形式存在,因此通过细胞破碎及离心等方法就能简便、有效地使其从细胞中分离并得以纯化。鉴于 PHA 微球这种表面易被修饰改造的特性,越来越多的功能蛋白通过与 PHA 微球表面蛋白（PhaC 或 PhaP）的融合表达,呈递在了 PHA 微球表面,使其成为一种廉价、高效的蛋白固定化及呈递的新技术。本文在介绍了 PHA 微球的结构特性及生物合成的基础上,着重综述了目前关于功能化 PHA 微球在蛋白纯化、固定化酶、生物分离、靶向递药、疾病诊断、成像技术及新型疫苗开发方面的研究现状及其未来在生物医药等领域的广泛应用前景。     

Molecular cloning and functional analysis of two polyhydroxyalkanoate synthases from two strains of Aeromonas hydrophila spp

Polyhydroxyalkanoate (PHA) synthase genes (phaC) were cloned from two Aeromonas hydrophila strains named WQ and 4AK5, respectively. Both strains are able to produce PHBHHx copolyesters consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx). Sequence analysis showed that there was only 2 bp difference between these two PHA synthase genes, corresponding to two-amino acid difference at positions of 437 and 458 of the two synthases. PHA productivity and its monomer content produced by A. hydrophila WQ and A. hydrophila 4AK5 were quite different. A. hydrophila WQ accumulated 33% PHBHHx of its cell dry weight (CDW) with 5 mol% 3HHx in the copolyester when cultured in lauric acid for 48 h. Yet A. hydrophila 4AK5 was able to produce 43% PHBHHx of the CDW with 14 mol% 3HHx under the same condition. Hetero-expression of PHA synthase genes of A. hydrophila WQ and A. hydrophila 4AK5, respectively, in Escherichia coli XL1-Blue led to PHBHHx accumulation of 24% and 39% of the CDW and the 3HHx content in PHBHHx were 6 and 15 mol%, respectively. This indicated that the function of these two PHA synthases were different due to these two different residues at positions of 437 and 458. Site specific mutation was carried out to change these two amino acid residues. Results showed that the changes on either of the two amino acids negatively affected the PHA productivity.     

中长链聚羟基脂肪酸酯（mcl PHA）在嗜水气单胞菌I型PHA合酶缺失突变株中的合成

拥有Ⅰ型聚羟基脂肪酸酯(PHA)合酶基因的嗜水气单胞菌CGMCC0911株可利用月桂酸而不能利用葡萄糖作为碳源积累PHBHHx。将氯霉素抗性基因(Cm)插入到该基因中,获得带有I型PHA合酶断裂基因(phaC::Cm)的自杀质粒pFH10。自杀质粒pFH10通过接合作用转入嗜水气单胞菌CGMCC0911株中并发生体内同源重组,Cm被整合到基因组上,获得Ⅰ型PHA合酶缺失突变株。DNA序列测定证明了这一结果。GC分析表明,突变株不再产生PHBHHx,但却可利用月桂酸或葡萄糖积累中长链PHA,明显表明野生型嗜水气单胞菌基因组中存在另一个编码Ⅱ型PHA合酶的基因,且只有Ⅰ型PHA合酶被钝化后,这个功能被隐藏的Ⅱ型PHA合酶才可在细胞中发挥作用。     

Cloning and analysis of the polyhydroxyalkanoic acid synthase gene from an Acinetobacter sp.: Evidence that the gene is both plasmid and chromosomally located

Abstract The polyhydroxyalkanoic acid (PHA) synthase gene ( phaC_Ac ) of a species of Acinetobacter isolated from an activated sludge treatment plant was cloned by heterologous complementation in a poly-β-hydroxybutyrate (PHB) negative mutant of Alcaligenes eutrophus . Nucleotide sequence analysis of phaC_Ac revelaed an open reading frame of 1770 bp with potential to encode a 67.7 kDa protein. The deduced amino acid sequence displays high similarity to other PHA synthase proteins. Probing with an internal region of phaC_Ac revealed that the PHA sythase gene may be present in more than one copy and may occur at both plasmid and chromosomal locations in Acinetobacter spp. This is the first organisms for which evidence has been presented to suggest that a gene involved in PHA metabolism is plasmid-encoded. Purification of PHB granules from sucrose gradients identified proteins of 38 kDa, 41 kDa and 64 kDa which may have a role in PHB metabolism.     

Detection of polyhydroxyalkanoate synthase activity on a polyacrylamide gel

This study presents a method to detect active polyhydroxyalkanoate (PHA) synthase on a polyacrylamide gel that combines the polyhydroxybutyrate (PHB) polymerization reaction with Sudan Black B staining. After separation of the protein samples on a modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the slab gel was submerged in a buffer containing β-hydroxybutyryl-coenzyme A (3-HBCoA) as substrate and incubated at room temperature for in vitro PHB polymerization. The active PHA synthase catalyzed 3-HBCoA into the PHB polymer and was stained with Sudan Black B. The active PHA synthase appeared as a dark blue band. The activity staining was of high sensitivity, capable of detecting 3.9 ng (0.273 mU) of Cupriavidus necator H16 PHA synthase purified from recombinant Escherichia coli. The detection sensitivity of activity staining was comparable to that of Western blotting analysis. Furthermore, the high sensitivity of activity staining enabled specific detection of the active PHA synthase in the crude extract of wild-type strain C. necator H16. This study provides a rapid, sensitive, and highly specific method for detecting active PHA synthase in gel. The method could be applied to detecting PHA synthase from wild-type bacteria and to the process of enzyme purification.     

Development of Halomonas TD01 as a host for open production of chemicals

Genetic engineering of Halomonas spp. was seldom reported due to the difficulty of genetic manipulation and lack of molecular biology tools. Halomonas TD01 can grow in a continuous and unsterile process without other microbial contaminations. It can be therefore exploited for economic production of chemicals. Here, Halomonas TD01 was metabolically engineered using the gene knockout procedure based on markerless gene replacement stimulated by double-strand breaks in the chromosome. When gene encoding 2-methylcitrate synthase in Halomonas TD01 was deleted, the conversion efficiency of propionic acid to 3-hydroxyvalerate (3HV) monomer fraction in random PHBV copolymers of 3-hydroxybutyrate (3HB) and 3HV was increased from around 10% to almost 100%, as a result, cells were grown to accumulate 70% PHBV in dry weight (CDW) consisting of 12 mol% 3HV from 0.5 g/L propionic acid in glucose mineral medium. Furthermore, successful deletions on three PHA depolymerases eliminate the possible influence of PHA depolymerases on PHA degradation in the complicated industrial fermentation process even though significant enhanced PHA content was not observed. In two 500 L pilot-scale fermentor studies lasting 70 h, the above engineered Halomonas TD01 grew to 112 g/L CDW containing 70 wt% P3HB, and to 80 g/L CDW with 70 wt% P(3HB-co-8 mol% 3HV) in the presence of propionic acid. The cells grown in shake flasks even accumulated close to 92% PHB in CDW with a significant increase of glucose to PHB conversion efficiency from around 30% to 42% after 48 h cultivation when pyridine nucleotide transhydrogenase was overexpressed. Halomonas TD01 was also engineered for producing a PHA regulatory protein PhaR which is a robust biosurfactant.     

The Role of Ceramide Synthases in the Pathogenicity of Cryptococcus neoformans

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Peculiarities of PHA granules preparation and PHA depolymerase activity determination

An extensive amount of knowledge on biochemistry of poly(3-hydroxyalkanoic acid) (PHA) synthesis and on its biodegradation has accumulated during the last two decades. Numerous genes encoding enzymes involved in the formation of PHA and in PHA degradation (PHA depolymerases) were cloned and characterized from many microorganisms. A large variety of methods exists for determination of PHA depolymerase activity and for preparation of the polymeric substrate (PHA). Unfortunately, results obtained with these different methods cannot be compared directly because they highly depend on the assay method applied and on the history of PHA granules preparation. In this contribution, the peculiarities, advantages, disadvantages and limitations of existing PHA depolymerase assay methods are described.     

小麦淀粉粒束缚淀粉合成酶基因多态性的分子鉴定

运用6％的SDS-PAGE对14个小麦品种成熟籽粒Wx蛋白的多态性进行了鉴定,结果表明,14个小麦品种根据其Wx蛋白的缺失情况可分为6种组合类型。另外,根据Wx-A1、Wx-D1和Wx-D1这3个位点基因序列和变异情况分别设计了PCR引物,扩增结果表明：Wx-A1位点突变材料扩增产物为327bp,正常材料中扩增不到该特异带;在Wx-B1位点扩增出187bp目标带,突变材料没有该扩增产物;在Wx-D1位点扩增出约700bp目标带,突变材料没有该特异带。与前人的研究结果相比,Wx-B1引物在3个位点的扩增产物长度更短,差异更大,在2％琼脂糖胶上即可清楚分开,缩短了鉴定时间,提高了效率,为大规模筛选优质面条小麦品种提供了可能。     

Identification, cloning and sequence analysis of the poly(3-hydroxyalkanoic acid) synthase gene of the Gram-positive bacterium Rhodococcus ruber

The first polyhydroxyalkanoic acid (PHA) synthase gene (phbCRr) of a Gram-positive bacterium was cloned from a genomic library of Rhodococcus ruber in the broad-host-range plasmid vector pRK404. The hybrid plasmid harboring phbCRr allowed the expression of polyhydroxybutyric acid (PHB) synthase activity and restored the ability of PHB synthesis in a PHB-negative mutant of Alcaligenes eutrophus. Nucleotide sequence analysis of phbCRr revealed an open reading frame of 1686 bp starting with the rare codon TTG and encoding a protein of relative molecular mass 61,371. The deduced amino acid sequence of phbCRr exhibited homologies to the primary structures of the PHA synthases of A. eutrophus and Pseudomonas oleovorans. Preparation of PHA granules by discontinuous density gradient centrifugation of crude cellular extracts revealed four major bands in an SDS polyacrylamide gel. A Mr 61,000 protein was identified as the PHA synthase of R. ruber by N-terminal amino acid sequence determination.     

Polyhydroxyalkanoate biosynthesis in Bacillus cereus SPV under varied limiting conditions and an insight into the biosynthetic genes involved

Aims:  A new strain of Bacillus, Bacillus cereus SPV, was found to be capable of using a wide range of carbon sources for the production of polyhydroxyalkanoates (PHAs) ( Valappil et al. 2007b ). Limiting nutrient in the culture conditions is crucial for PHA production. In this study, B.   cereus SPV was grown in different culture conditions with limitation of potassium, nitrogen, sulphur and phosphorous to establish the impact of nutritional limitation on PHA production.
Methods and Results:  The PHA yields obtained were found to be 13·4, 38, 13·15 and 33·33% dcw for potassium, nitrogen, sulphur and phosphorus limitations, respectively. Gas chromatography–mass spectrometry analysis of the isolated polymers showed the presence of P(3HB) under nitrogen, sulphur and phosphate-limiting conditions and P(3HB-3HV) copolymer under potassium limiting conditions. This ability of B. cereus SPV to accumulate different PHA monomers from structurally unrelated carbon sources led to an interest in the molecular analysis of PHA biosynthesis in this organism. To achieve this, PCR was used to identify the polyhydroxyalkanoate biosynthetic genes in B. cereus SPV.
Conclusion:  Sequence analysis of the PCR products from B. cereus SPV revealed the sequence of the putative biosynthetic genes, and possible regions involved in substrate binding.
The nucleotide sequence reported in this paper is in the GenBank nucleotide sequence database under accession number DQ486135 .
Significance and Impact of the Study:  This is the first report comparing the capability of B. cereus SPV to produce PHAs under different culture conditions of potassium, nitrogen, sulfur and phosphate limitations. The results in this study suggest the unique ability of B. cereus SPV to supply both 3HB and 3HV monomers from a structurally unrelated carbon source, glucose.     

Isolation and identification of granule-associated proteins relevant for poly(3-hydroxyalkanoic acid) biosynthesis in Chromatium vinosum D

Abstract Poly(3-hydroxybutyric acid) granules, which harbored only four major granule-associated proteins as revealed by SDS polyacrylamide gel electrophoresis, were isolated from crude cellular extracts of Chromatium vinosum D by centrifugation in a linear sucrose gradient. N-Terminal amino acid sequence determination identified two proteins of M _r 41 000 and M _{r 40 000} as the phaE _Cv and phaC _Cv translational products, respectively, of C. vinosum D. In a previous study it was shown that both proteins are required for the expression opf poly(3-hydroxyalkanoic acid) synthase activity. The N-terminus of the third protein ( M _r 17 000) exhibited no homology to other proteins. Lysozyme, which was during purification of the granules, exhibited a strong affinity to PHB granules and was identified as the fourth protein enriched with the granules.     

Advanced bacterial polyhydroxyalkanoates: Towards a versatile and sustainable platform for unnatural tailor-made polyesters

Polyhydroxyalkanoates (PHAs) are biopolyesters that generally consist of 3-, 4-, 5-, and 6-hydroxycarboxylic acids, which are accumulated as carbon and energy storage materials in many bacteria in limited growth conditions with excess carbon sources. Due to the diverse substrate specificities of PHA synthases, the key enzymes for PHA biosynthesis, PHAs with different material properties have been synthesized by incorporating different monomer components with differing compositions. Also, engineering PHA synthases using in vitro-directed evolution and site-directed mutagenesis facilitates the synthesis of PHA copolymers with novel material properties by broadening the spectrum of monomers available for PHA biosynthesis. Based on the understanding of metabolism of PHA biosynthesis, recombinant bacteria have been engineered to produce different types of PHAs by expressing heterologous PHA biosynthesis genes, and by creating and enhancing the metabolic pathways to efficiently generate precursors for PHA monomers. Recently, the PHA biosynthesis system has been expanded to produce unnatural biopolyesters containing 2-hydroxyacid monomers such as glycolate, lactate, and 2-hydroxybutyrate by employing natural and engineered PHA synthases. Using this system, polylactic acid (PLA), one of the major commercially-available bioplastics, can be synthesized from renewable resources by direct fermentation of recombinant bacteria. In this review, we discuss recent advances in the development of the PHA biosynthesis system as a platform for tailor-made polyesters with novel material properties.     

植物萜类合酶研究进展

随着植物中许多有价值的萜类化合物被发现和应用于人类生活 ,萜类生物合成途径的研究倍受重视。萜类合酶催化单萜、倍半萜和二萜生物合成 ,即分别催化GPP、FPP和GGPP形成单萜、倍半萜和二萜。本文叙述了近年来在植物萜类合酶催化机理、克隆策略和萜类生物工程的研究进展     

Cloning of poly(3-hydroxybutyric acid) synthase genes of Rhodobacter sphaeroides and Rhodospirillum rubrum and heterologous expression in Alcaligenes eutrophus

From genomic libraries of the purple non-sulfur bacteria Rhodospirillum rubrum Ha and Rhodobacter sphaeroides ATCC 17023 in the broad-host range cosmid pVK100, we cloned a 15- and a 14-kbp HindIII restriction fragment, respectively. Each of these fragments restored the ability to accumulate poly(3-hydroxybutyrate) (PHB), in the PHB-negative mutant Alcaligenes eutrophus PHB-4. These hybrid cosmids also complemented PHB-negative mutants derived from wild-type R. rubrum or R. sphaeroides. Both fragments hybridized with the PHB synthase structural gene of A. eutrophus H16 and conferred the ability to express PHB synthase activity. Only the 15-kbp HindIII fragment from R. rubrum conferred on the mutant PHB-4 the ability to form large PHB granules (length up to 3.5 microns).     

红霉素生物合成的生物化学和基因学

从基因重组技术问世以来,对红霉素(大环内酯类抗生素)生物合成的生物化学和基因学的研究到目前已经累积了大量的研究结果。就这2个领域中对红霉素生物合成的研究历史、研究现状和研究前景(组合生物合成)作以简要的介绍。