达托霉素生物合成研究进展

达托霉素是由玫瑰孢链霉菌(Streptomyces roseosporus)生产的一种环脂肽类抗生素, 具有强大的抗革兰氏阳性致病细菌的作用, 是继“抗生素最后一道防线”万古霉素后的新型抗生素。本文主要对达托霉素的结构、作用机制、合成基因簇及合成机制等当前的研究成果进行综述, 并且总结了利用组合生物学对达托霉素进行结构改造的策略, 以此来研究结构与活性之间的关系, 并寻找更广谱高效的抗生素。最后, 本文总结了提高达托霉素产量的策略, 为工业上降低达托霉素生产成本提供理论参考。     

Daptomycin,a Bacterial Lipopeptide Synthesized by a Nonribosomal Machinery

Daptomycin (Cubicin®) is a branched cyclic lipopeptide antibiotic of nonribosomal origin and the prototype of the acidic lipopeptide family. It was approved in 2003 for the nontopical treatment of skin structure infections caused by Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), and in 2006 for the treatment of bacteremia. Understanding the ribosome-independent biosynthesis of daptomycin assembly will provide opportunities for the generation of daptomycin derivatives with an altered pharmaceutical spectrum to address upcoming daptomycin-resistant pathogens. Herein, the structural properties of daptomycin, its biosynthesis, recent efforts for the generation of structural diversity, and its proposed mode of action are discussed.     

The action mechanism of daptomycin

Daptomycin is a lipopeptide antibiotic produced by the soil bacterium Streptomyces roseosporus that is clinically used to treat severe infections with Gram-positive bacteria. In this review, we discuss the mode of action of this important antibiotic. Although daptomycin is structurally related to amphomycin and similar lipopeptides that inhibit peptidoglycan biosynthesis, experimental studies have not produced clear evidence that daptomycin shares their action mechanism. Instead, the best characterized effect of daptomycin is the permeabilization and depolarization of the bacterial cell membrane. This activity, which can account for daptomycin’s bactericidal effect, correlates with the level of phosphatidylglycerol (PG) in the membrane. Accordingly, reduced synthesis of PG or its increased conversion to lysyl-PG promotes bacterial resistance to daptomycin. While other resistance mechanisms suggest that daptomycin may indeed directly interfere with cell wall synthesis or cell division, such effects still await direct experimental confirmation. Daptomycin’s complex structure and biosynthesis have hampered the analysis of its structure activity relationships. Novel methods of total synthesis, including a recent one that is carried out entirely on a solid phase, will enable a more thorough and systematic exploration of the sequence space.     

In silico aided metabolic engineering of <Emphasis Type="Italic">Streptomyces roseosporus</Emphasis> for daptomycin yield improvement

In silico metabolic network models are valuable tools for strain improvement with desired properties. In this work, based on the comparisons of each pathway flux under two different objective functions for the reconstructed metabolic network of Streptomyces roseosporus, three potential targets of zwf2 (code for glucose-6-phosphate hydrogenase), dptI (code for α-ketoglutarate methyltransferase), and dptJ (code for tryptophan oxygenase) were identified and selected for the genetic modifications. Overexpression of zwf2, dptI, and dptJ genes increased the daptomycin concentration up to 473.2, 452.5, and 489.1 mg/L, respectively. Furthermore, co-overexpression of three genes in series resulted in a 34.4% higher daptomycin concentration compared with the parental strain, which ascribed to the synergistic effect of the enzymes responsible for daptomycin biosynthesis. Finally, the engineered strain enhanced the yield of daptomycin up to 581.5 mg/L in the fed-batch culture, which was approximately 43.2% higher than that of the parental strain. These results demonstrated that the metabolic network based on in silico prediction would be accurate, reasonable, and practical for target gene identification and strain improvement.     

Manipulation of kynurenine pathway for enhanced daptomycin production in Streptomyces roseosporus

Daptomycin is a cyclic lipopeptide natural product produced by Stretptomyces roseosporus, displaying good bactericidal activity against a wide range of gram‐positive pathogens. Daptomycin contains a 13 amino acid and kynurenine (Kyn) is essential for optimal activity of daptomycin. In this study, we characterized the Kyn pathway in S. roseosporus and investigated its role in supplying precursor for daptomycin biosynthesis. Two genes (dptJ and tdo) coding for tryptophan‐2,3‐dioxgenase existed in the chromosome. dptJ is located in the daptomycin biosynthetic gene cluster, while tdo is in other locus. Disruption of dptJ or tdo resulted in reduced yield by ～50%. The introduction of an additional copy of dptJ but not tdo led to enhanced production of daptomycin by 110%. Furthermore, disruption of kyn encoding kynureninase showed improved daptomycin productivity by 30%. Our results demonstrated that the enhancement of Kyn supply through metabolic engineering approach is an efficient way to increase daptomycin production. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:847–852, 2013     

Whole genome characterization of the mechanisms of daptomycin resistance in clinical and laboratory derived isolates of Staphylococcus aureus

Background

Daptomycin remains one of our last-line anti-staphylococcal agents. This study aims to characterize the genetic evolution to daptomycin resistance in S. aureus.

Methods

Whole genome sequencing was performed on a unique collection of isogenic, clinical (21 strains) and laboratory (12 strains) derived strains that had been exposed to daptomycin and developed daptomycin-nonsusceptibility. Electron microscopy (EM) and lipid membrane studies were performed on selected isolates.

Results

On average, six coding region mutations were observed across the genome in the clinical daptomycin exposed strains, whereas only two mutations on average were seen in the laboratory exposed pairs. All daptomycin-nonsusceptible strains had a mutation in a phospholipid biosynthesis gene. This included mutations in the previously described mprF gene, but also in other phospholipid biosynthesis genes, including cardiolipin synthase (cls2) and CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase (pgsA). EM and lipid membrane composition analyses on two clinical pairs showed that the daptomycin-nonsusceptible strains had a thicker cell wall and an increase in membrane lysyl-phosphatidylglycerol.

Conclusion

Point mutations in genes coding for membrane phospholipids are associated with the development of reduced susceptibility to daptomycin in S. aureus. Mutations in cls2 and pgsA appear to be new genetic mechanisms affecting daptomycin susceptibility in S. aureus.     

园艺作物花青素合成调控研究进展

花青素是一类保护植物免受生物和非生物胁迫的重要次生代谢产物,因其赋予植物丰富的色彩和对人体的保健功能而受到广泛关注。花青素合成调控机理的相关研究是目前园艺作物分子生物学研究的前沿课题,对于园艺作物花青素含量的提高、种质品质的提升等具有重要的意义。结合国内外园艺作物中花青素生物合成调控方面的最新研究进展,介绍了环境因素、酶与激素、DNA甲基化与泛素化和调控基因等对花青素生物合成的作用,以及花青素抵御外界胁迫的功能机制,综述了近年来园艺作物中花青素生物合成调控的研究成果,以期利用基因工程为提升园艺作物的色彩丰富度提供理论参考。     

Molecular Cloning and Physical Mapping of the Daptomycin Gene Cluster from Streptomyces roseosporus

The daptomycin biosynthetic gene cluster of Streptomyces roseosporus was analyzed by Tn5099 mutagenesis, molecular cloning, partial DNA sequencing, and insertional mutagenesis with cloned segments of DNA. The daptomycin biosynthetic gene cluster spans at least 50 kb and is located about 400 to 500 kb from one end of the ~7,100-kb linear chromosome. We identified two peptide synthetase coding regions interrupted by a 10- to 20-kb region that may encode other functions in lipopeptide biosynthesis.     

达托霉素生产工艺及其衍生物组合生物合成

作为首个进入临床应用的环脂肽类抗生素,达托霉素自2003年上市以来,适应症不断增加,市场前景良好。达托霉素的制备工艺与结构修饰,已经成为了近年抗感染用药研发的一个热点。追溯了达托霉素的发现历程,生产工艺沿革。重点介绍了达托霉素的发酵工艺,合成基因簇研究进展,以及组合生物技术在达托霉素结构修饰中的应用。     

A glutamic acid 3-methyltransferase encoded by an accessory gene locus important for daptomycin biosynthesis in Streptomyces roseosporus

In many peptide antibiotics, modified amino acids are important for biological activity. The amino acid 3-methyl-glutamic acid (3mGlu) has been found only in three cyclic lipopeptide antibiotics: daptomycin and the A21978C family produced by Streptomyces roseosporus, calcium-dependent antibiotic produced by Streptomyces coelicolor and A54145 produced by Streptomyces fradiae. We studied the non-ribosomal peptide synthetase genes involved in A21978C biosynthesis and the downstream genes, dptG, dptH, dptI and dptJ predicted to encode a conserved protein of unknown function, a thioesterase, a methyltransferase (MTase) and a tryptophan 2,3-dioxygenase respectively. Deletion of dptGHIJ reduced overall lipopeptide yield and led to production of a series of novel A21978C analogues containing Glu12 instead of 3mGlu12. Complementation by only dptI, or its S. coelicolor homologue, glmT, restored the biosynthesis of the 3mGlu-containing compounds in the mutant. Compared with A21978C, the Glu12-containing derivatives were less active against Staphylococcus aureus. Further genetic analyses showed that members of the dptGHIJ locus cooperatively contributed to optimal A21978C production; deletion of dptH, dptI or dptJ genes reduced the yield significantly, while expression of dptIJ or dptGHIJ from the strong ermEp* promoter substantially increased lipopeptide production. The results indicate that these genes play important roles in the biosynthesis of daptomycin, and that dptI encodes a Glu MTase.     

植物花色素苷生物合成的转录调控

转录调节是植物花色素苷生物合成途径中调节其结构基因表达的重要环节之一。近十多年来,通过调节转录因子的表达激活或抑制有效地调控植物中花色素苷的合成成了众多植物学家研究的重点。现简要介绍了花色素苷的合成运输过程与液泡的沉积扣押、转录因子与调控及转录调节在遗传改良中的应用等方面的研究进展。     

Purification of daptomycin binding proteins (DBPs) from the membrane of Enterococcus hirae

Daptomycin binding proteins (DBPs) are membrane proteins which act as daptomycin targets. Daptomycin is a cyclic lipopeptide antibiotic which is active against Gram-positive bacteria and was shown to be the first inhibitor of lipoteichoic acid (LTA) synthesis. It was found that the antibiotic did not penetrate the bacterial cytoplasm but bound membranes with a non-covalent bond and in particular some proteins which were called DBPs. DBPs were indicated as enzymes involved in LTA synthesis whose binding and inhibition by daptomycin is responsible for the observed effect on bacterial LTA synthesis. The purification of DBPs will make it possible not only to shed light on the biosynthesis of the cell wall polymer but will also provide innovative targets for selection of new antibacterial compounds. In this study, the purification of DBPs is described. Affinity chromatography was used with daptomycin as the ligand. Final elution of DBPs from daptomycin-coupled resin was performed using either 0.1% SDS or 3 M NaCl. Polyacrylamide gel electrophoresis of the eluted protein fractions consistently showed four protein bands (ranging from 55 to 66 kDa) in denaturating conditions and two protein bands (60 and 66 kDa) in non-denaturating conditions. Isoelectrofocusing analysis of the same sample consistently revealed two bands with pIs around 5. That these purified proteins were really the desired DBPs is demonstrated by the retention of daptomycin-binding capability they displayed.     

Synthetic biology,genome mining,and combinatorial biosynthesis of NRPS-derived antibiotics: a perspective

Combinatorial biosynthesis of novel secondary metabolites derived from nonribosomal peptide synthetases (NRPSs) has been in slow development for about a quarter of a century. Progress has been hampered by the complexity of the giant multimodular multienzymes. More recently, advances have been made on understanding the chemical and structural biology of these complex megaenzymes, and on learning the design rules for engineering functional hybrid enzymes. In this perspective, I address what has been learned about successful engineering of complex lipopeptides related to daptomycin, and discuss how synthetic biology and microbial genome mining can converge to broaden the scope and enhance the speed and robustness of combinatorial biosynthesis of NRPS-derived natural products for drug discovery.