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1.
Biplav Shrestha Dipesh Dhakal Sumangala Darsandhari Ramesh Prasad Pandey Anaya Raj Pokhrel Hum Nath Jnawali Jae Kyung Sohng 《Biotechnology and Bioprocess Engineering》2017,22(4):359-365
Heterologous expression can enhance production of diverse secondary metabolites by redirecting precursor pools towards compound of interest. In this study, Streptomyces venezuelae YJ028 was utilized as the heterologous host for the expression of four structural clavulanic acid biosynthesis genes, which encode carboxyethylarginine synthase (ceas2), β-lactam synthetase (bls2), clavaminate synthase (cas2), and proclavaminate amidinohydrolase (pah2). These genes were cloned into pIBR25 expression vector containing ermE* promoter to generate pBS4. The cas2 gene was also cloned into pSET152 to generate pCas2. It was then integrated into the genome of S. venezuelae YJ028. Upon metabolite profiling of recombinant strains by ultra-pressure liquid chromatography-photodiode array (UPLC-PDA) and high resolution liquid chromatography quadruple time-offlight electrospray ionization mass spectrometry (HR-LC-QTOF-ESI/MS), the production of following clavulanic acid intermediates in S. venezuelae recombinant were confirmed: deoxygaunidinoproclavaminic acid, guanidinoproclavaminic acid, and dihydroclavaminic acid. This work demonstrates the production of β-lactam intermediates of the clavulanic acid pathway by heterologous expression in S. venezuelae YJ028. 相似文献
2.
Won Seok Jung Eunji Kim Young Ji Yoo Yeon Hee Ban Eun Ji Kim Yeo Joon Yoon 《Applied microbiology and biotechnology》2014,98(8):3701-3713
Streptomyces venezuelae has an inherent advantage as a heterologous host for polyketide production due to its fast rate of growth that cannot be endowed easily through metabolic engineering. However, the utility of S. venezuelae as a host has been limited thus far due to its inadequate intracellular reserves of the (2S)-ethylmalonyl-CoA building block needed to support the biosynthesis of polyketides preventing the efficient production of the desired metabolite, such as tylactone. Here, via precursor supply engineering, we demonstrated that S. venezuelae can be developed into a more efficient general heterologous host for the quick production of polyketides. We first identified and functionally characterized the ethylmalonyl-CoA pathway which plays a major role in supplying the (2S)-ethylmalonyl-CoA extender unit in S. venezuelae. Next, S. venezuelae was successfully engineered to increase the intracellular ethylmalonyl-CoA concentration by the deletion of the meaA gene encoding coenzyme B12-dependent ethylmalonyl-CoA mutase in combination with ethylmalonate supplementation and was engineered to upregulate the expression of the heterologous tylosin PKS by overexpression of the pathway specific regulatory gene pikD. Thus, a dramatic increase (~10-fold) in tylactone production was achieved. In addition, the detailed insights into the role of the ethylmalonyl-CoA pathway, which is present in most streptomycetes, provides a general strategy to increase the ethylmalonyl-CoA supply for polyketide biosynthesis in the most prolific family of polyketide-producing bacteria. 相似文献
3.
Jung WS Lee SK Hong JS Park SR Jeong SJ Han AR Sohng JK Kim BG Choi CY Sherman DH Yoon YJ 《Applied microbiology and biotechnology》2006,72(4):763-769
Tylosin polyketide synthase (Tyl PKS) was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin PKS gene cluster using two compatible low-copy plasmids, each under the control of a pikAI promoter. The mutant strain produced 0.5 mg/l of the 16-membered ring macrolactone, tylactone, after a 4-day culture, which is a considerably reduced culture period to reach the maximum production level compared to other Streptomyces hosts. To improve the production level of tylactone, several precursors for ethylmalonyl-CoA were fed to the growing medium, leading to a 2.8-fold improvement (1.4 mg/ml); however, switching the pikAI promoter to an actI promoter had no observable effect. In addition, a small amount of desosamine-glycosylated tylactone was detected from the extract of the mutant strain, revealing that the native glycosyltransferase DesVII displayed relaxed substrate specificity in accepting the 16-membered ring macrolactone to produce the glycosylated tylactone. These results demonstrate a successful attempt for a heterologous expression of Tyl PKS in S. venezuelae and introduce S. venezuelae as a rapid heterologous expression system for the production of secondary metabolites. 相似文献
4.
Park SR Park JW Jung WS Han AR Ban YH Kim EJ Sohng JK Sim SJ Yoon YJ 《Applied microbiology and biotechnology》2008,81(1):109-117
Epothilones, produced from the myxobacterium Sorangium cellulosum, are potential anticancer agents that stabilize microtubules in a similar manner to paclitaxel. The entire epothilone biosynthetic
gene cluster was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin polyketide synthase gene cluster. The resulting strains produced approximately 0.1 μg/l
of epothilone B as a sole product after 4 days cultivation. Deletion of an epoF encoding the cytochrome P450 epoxidase gave rise to a mutant that selectively produces 0.4 μg/l of epothilone D. To increase
the production level of epothilones B and D, an additional copy of the positive regulatory gene pikD was introduced into the chromosome of both S. venezuleae mutant strains. The resulting strains showed enhanced production of corresponding compounds (approximately 2-fold). However,
deletion of putative transport genes, orf3 and orf14 in the epothilone D producing S. venezuelae mutant strain, led to an approximately 3-fold reduction in epothilone D production. These results introduce S. venezuelae as an alternative heterologous host for the production of these valuable anticancer agents and demonstrate the possibility
of engineering this strain as a generic heterologous host for the production of polyketides and hybrid polyketide-nonribosomal
peptides. 相似文献
5.
Thapa LP Oh TJ Lee HC Liou K Park JW Yoon YJ Sohng JK 《Applied microbiology and biotechnology》2007,76(6):1357-1364
The pSKC2 cosmid, which has 32 kb and 28 open-reading frames, was isolated from Streptomyces kanamyceticus ATCC12853 as the gene cluster of kanamycin. This gene cluster includes the minimal biosynthetic genes of kanamycin with the
resistance and regulatory genes. It was heterologously expressed in Streptomyces venezuelae YJ003, which has the advantage of fast growth, good efficiency of the transformation host, and rapid production of the aminoglycosides
antibiotic. The isolated compound was analyzed by electrospray ionization–mass spectrometry, liquid chromatography–mass spectrometry,
high-performance liquid chromatography, and tandem mass spectrometry and shows a molecular weight of 485 as kanamycin A. 相似文献
6.
7.
Salvador Peirú Eduardo Rodríguez Hugo G. Menzella John R. Carney Hugo Gramajo 《Microbial biotechnology》2008,1(6):476-486
Significant achievements in polyketide gene expression have made Escherichia coli one of the most promising hosts for the heterologous production of pharmacologically important polyketides. However, attempts to produce glycosylated polyketides, by the expression of heterologous sugar pathways, have been hampered until now by the low levels of glycosylated compounds produced by the recombinant hosts. By carrying out metabolic engineering of three endogenous pathways that lead to the synthesis of TDP sugars in E. coli, we have greatly improved the intracellular levels of the common deoxysugar intermediate TDP‐4‐keto‐6‐deoxyglucose resulting in increased production of the heterologous sugars TDP‐L‐mycarose and TDP‐d ‐desosamine, both components of medically important polyketides. Bioconversion experiments carried out by feeding 6‐deoxyerythronolide B (6‐dEB) or 3‐α‐mycarosylerythronolide B (MEB) demonstrated that the genetically modified E. coli B strain was able to produce 60‐ and 25‐fold more erythromycin D (EryD) than the original strain K207‐3, respectively. Moreover, the additional knockout of the multidrug efflux pump AcrAB further improved the ability of the engineered strain to produce these glycosylated compounds. These results open the possibility of using E. coli as a generic host for the industrial scale production of glycosylated polyketides, and to combine the polyketide and deoxysugar combinatorial approaches with suitable glycosyltransferases to yield massive libraries of novel compounds with variations in both the aglycone and the tailoring sugars. 相似文献
8.
Elsa Pimienta Julio C Ayala Caridad Rodríguez Astrid Ramos Lieve Van Mellaert Carlos Vallín Jozef Anné 《Microbial cell factories》2007,6(1):20
Background
Streptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. It is naturally secreted by several strains of beta-haemolytic streptococci. The low yields obtained in SK production, lack of developed gene transfer methodology and the pathogenesis of its natural host have been the principal reasons to search for a recombinant source for this important therapeutic protein. We report here the expression and secretion of SK by the Gram-positive bacterium Streptomyces lividans. The structural gene encoding SK was fused to the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi) signal sequence or to the Streptomyces lividans xylanase C (xlnC) signal sequence. The native Vsi protein is translocated via the Sec pathway while the native XlnC protein uses the twin-arginine translocation (Tat) pathway. 相似文献9.
Díaz M Adham SA Ramón D Gil JA Santamaría RI 《Applied microbiology and biotechnology》2004,65(4):401-406
The Aspergillus nidulans gene xlnA coding for the fungal xylanase X22 has been cloned and expressed in two heterologous bacterial hosts: Streptomyces lividans and Brevibacterium lactofermentum. Streptomyces strains yielded 10 units/ml of xylanase when the protein was produced with its own signal peptide, and 19 units/ml when its signal peptide was replaced by the one for xylanase Xys1 from Streptomyces halstedii. B. lactofermentum was also able to produce xylanase X22, affording 6 units/ml upon using either the Aspergillus xlnA signal peptide or Streptomyces xysA. These production values are higher than those previously reported for the heterologous expression of the A. nidulans xlnA gene in Saccharomyces cerevisiae (1 unit/ml). Moreover, the X22 enzyme produced by Streptomyces lividans showed oenological properties, indicating that this Streptomyces recombinant strain is a good candidate for the production of this enzyme at the industrial scale. 相似文献
10.
Yang D Zhu X Wu X Feng Z Huang L Shen B Xu Z 《Applied microbiology and biotechnology》2011,89(6):1709-1719
iso-Migrastatin (iso-MGS) has been actively pursued recently as an outstanding candidate of antimetastasis agents. Having
characterized the iso-MGS biosynthetic gene cluster from its native producer Streptomyces platensis NRRL 18993, we have recently succeeded in producing iso-MGS in five selected heterologous Streptomyces hosts, albeit the low titers failed to meet expectations and cast doubt on the utility of this novel technique for large-scale
production. To further explore and capitalize on the production capacity of these hosts, a thorough investigation of these
five engineered strains with three fermentation media for iso-MGS production was undertaken. Streptomyces albus J1074 and Streptomyces lividans K4-114 were found to be preferred heterologous hosts, and subsequent analysis of carbon and nitrogen sources revealed that
sucrose and yeast extract were ideal for iso-MGS production. After the initial optimization, the titers of iso-MGS in all
five hosts were considerably improved by 3–18-fold in the optimized R2YE medium. Furthermore, the iso-MGS titer of S. albus J1074 (pBS11001) was significantly improved to 186.7 mg/L by a hybrid medium strategy. Addition of NaHCO3 to the latter finally afforded an optimized iso-MGS titer of 213.8 mg/L, about 5-fold higher than the originally reported
system. With S. albus J1074 (pBS11001) as a model host, the expression of iso-MGS gene cluster in four different media was systematically studied
via the quantitative RT–PCR technology. The resultant comparison revealed the correlation of gene expression and iso-MGS production
for the first time; synchronous expression of the whole gene cluster was crucial for optimal iso-MGS production. These results
reveal new insights into the iso-MGS biosynthetic machinery in heterologous hosts and provide the primary data to realize
large-scale production of iso-MGS for further preclinical studies. 相似文献
11.
Rodriguez E Ward S Fu H Revill WP McDaniel R Katz L 《Applied microbiology and biotechnology》2004,66(1):85-91
Development of host microorganisms for heterologous expression of polyketide synthases (PKS) that possess the intrinsic capacity to overproduce polyketides with a broad spectrum of precursors supports the current demand for new tools to create novel chemical structures by combinatorial engineering of modular and other classes of PKS. Streptomyces fradiae is an ideal host for development of generic polyketide-overproducing strains because it contains three of the most common precursors—malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA—used by modular PKS, and is a host that is amenable to genetic manipulation. We have expanded the utility of an overproducing S. fradiae strain for engineered biosynthesis of polyketides by engineering a biosynthetic pathway for methoxymalonyl-ACP, a fourth precursor used by many 16-membered macrolide PKS. This was achieved by introducing a set of five genes, fkbG–K from Streptomyces hygroscopicus, putatively encoding the methoxymalonyl-ACP biosynthetic pathway, into the S. fradiae chromosome. Heterologous expression of the midecamycin PKS genes in this strain resulted in 1 g/l production of a midecamycin analog. These results confirm the ability to engineer unusual precursor pathways to support high levels of polyketide production, and validate the use of S. fradiae for overproduction of 16-membered macrolides derived from heterologous PKS that require a broad range of precursors. 相似文献
12.
聚酮是一大类具有重要生物活性的天然产物,其生物合成途径复杂多样。利用异源宿主合成聚酮化合物要比使用天然生产菌有很多优点。异源宿主的选择是异源生物合成聚酮的关键。这种宿主必须能够大量表达大分子聚酮合成酶(300 kDa或更大)且能够大规模的转译后修饰这些蛋白;还要能够形成大量的像丙二酰CoA、甲基丙二酰CoA等细胞内起始单元。随着各种技术的不断进步,异源宿主很可能成为大规模生产聚酮化合物的一个强有力平台。本文对聚酮合成酶,异源生产聚酮的优点、条件和应用都有所阐述。 相似文献
13.
M. Lopatniuk B. Ostash A. Luzhetskyy S. Walker V. Fedorenko 《Russian Journal of Genetics》2014,50(4):360-365
Heterologous expression of the moenomycin biosynthesis gene cluster (moe) would be one of the ways to reach this goal. Here, we report the generation of a number of novel heterologous streptomycete hosts producing nosokomycin A2 (one of the members of Mm family) and determine their potential for the antibiotic production. The rpoB point mutation in the model strain of Streptomyces coelicolor (strain M1152) significantly improved nosokomycin A2 production compared to parental strains (M145 and M512), while double rpoBrpsL mutation in the same species (strain M1154) decreased it. Our results point to the previously unanticipated epistatic interactions between mutations that individually are known to be highly beneficial for antibiotic production. We also showed here for the first time that facultative chemolitotrophic streptomycete S. thermospinosisporus and chloramphenicol producer S. venezuelae can be used as the hosts for moe genes. 相似文献
14.
15.
Rapid engineering of polyketide overproduction by gene transfer to industrially optimized strains 总被引:3,自引:0,他引:3
Rodriguez E Hu Z Ou S Volchegursky Y Hutchinson CR McDaniel R 《Journal of industrial microbiology & biotechnology》2003,30(8):480-488
Development of natural products for therapeutic use is often hindered by limited availability of material from producing organisms.
The speed at which current technologies enable the cloning, sequencing, and manipulation of secondary metabolite genes for
production of novel compounds has made it impractical to optimize each new organism by conventional strain improvement procedures.
We have exploited the overproduction properties of two industrial organisms—Saccharopolyspora erythraea and Streptomyces fradiae, previously improved for erythromycin and tylosin production, respectively—to enhance titers of polyketides produced by genetically
modified polyketide synthases (PKSs). An efficient method for delivering large PKS expression vectors into S. erythraea was achieved by insertion of a chromosomal attachment site (attB) for φC31-based integrating vectors. For both strains, it was discovered that only the native PKS-associated promoter was
capable of sustaining high polyketide titers in that strain. Expression of PKS genes cloned from wild-type organisms in the
overproduction strains resulted in high polyketide titers whereas expression of the PKS gene from the S. erythraea overproducer in heterologous hosts resulted in only normal titers. This demonstrated that the overproduction characteristics
are primarily due to mutations in non-PKS genes and should therefore operate on other PKSs. Expression of genetically engineered
erythromycin PKS genes resulted in production of erythromycin analogs in greatly superior quantity than obtained from previously
used hosts. Further development of these hosts could bypass tedious mutagenesis and screening approaches to strain improvement
and expedite development of compounds from this valuable class of natural products. 相似文献
16.
2-Deoxystreptamine is a core aglycon that is vital to backbone formation in various aminoglycosides. This core structure can be modified to develop hybrid types of aminoglycoside antibiotics. We obtained three genes responsible for 2-deoxystreptamine production, neo7, neo6, and neo5, which encode 2-deoxy-scyllo-inosose synthase, L-glutamine: 2-deoxy-scyllo-inosose aminotransferase, and dehydrogenase, respectively, from the neomycin gene cluster. These genes were cloned into pIBR25, a Streptomyces expression vector, resulting in pNDOS. The recombinant pNDOS was transformed into a non-aminoglycoside-producing host, Streptomyces venezuelae YJ003, for heterologous expression. Based on comparisons of the retention time on LC-ESI/MS and ESIMS data with those of the 2-deoxystreptamine standard, a compound produced by S. venezuelae YJ003/pNDOS was found to be 2-deoxystreptamine. 相似文献
17.
Do-Kyung Kim Boncheol Gu Duck Gyun Kim Min-Kyu Oh 《Biotechnology and bioengineering》2023,120(7):2039-2044
Streptomyces are important industrial bacteria that produce pharmaceutically valuable polyketides. However, mass production on an industrial scale is limited by low productivity, which can be overcome through metabolic engineering and the synthetic biology of the host strain. Recently, the introduction of an auto-inducible expression system depending on microbial physiological state has been suggested as an important tool for the industrial-scale production of polyketides. In this study, titer improvement by enhancing the pool of CoA-derived precursors required for polyketide production was driven in a quorum sensing (QS)-dependent manner. A self-sustaining and inducer-independent regulatory system, named the QS-based metabolic engineering of precursor pool (QMP) system, was constructed, wherein the expression of genes involved in precursor biosynthesis was regulated by the QS-responsive promoter, scbAp. The QMP system was applied for neoaureothin production in a heterologous host, Streptomyces coelicolor M1152, and productivity increased by up to 4-fold. In particular, the engineered hyperproducers produced high levels of neoaureothin without adversely affecting cell growth. Overall, this study showed that self-regulated metabolic engineering mediated by QS has the potential to engineer strains for polyketide titer improvement. 相似文献
18.
Polyketide natural products show great promise as medicinal agents. Typically the products of microbial secondary biosynthesis, polyketides are synthesized by an evolutionarily related but architecturally diverse family of multifunctional enzymes called polyketide synthases. A principal limitation for fundamental biochemical studies of these modular megasynthases, as well as for their applications in biotechnology, is the challenge associated with manipulating the natural microorganism that produces a polyketide of interest. To ameliorate this limitation, over the past decade several genetically amenable microbes have been developed as heterologous hosts for polyketide biosynthesis. Here we review the state of the art as well as the difficulties associated with heterologous polyketide production. In particular, we focus on two model hosts, Streptomyces coelicolor and Escherichia coli. Future directions for this relatively new but growing technological opportunity are also discussed. 相似文献
19.
Biosynthesis of polyketides in heterologous hosts. 总被引:3,自引:0,他引:3
Polyketide natural products show great promise as medicinal agents. Typically the products of microbial secondary biosynthesis, polyketides are synthesized by an evolutionarily related but architecturally diverse family of multifunctional enzymes called polyketide synthases. A principal limitation for fundamental biochemical studies of these modular megasynthases, as well as for their applications in biotechnology, is the challenge associated with manipulating the natural microorganism that produces a polyketide of interest. To ameliorate this limitation, over the past decade several genetically amenable microbes have been developed as heterologous hosts for polyketide biosynthesis. Here we review the state of the art as well as the difficulties associated with heterologous polyketide production. In particular, we focus on two model hosts, Streptomyces coelicolor and Escherichia coli. Future directions for this relatively new but growing technological opportunity are also discussed. 相似文献
20.
Polyketides are important bioactive natural products biosynthesized by bacteria, fungi, and plants. The enzymes that synthesize
polyketides are collectively referred to as polyketide synthases (PKSs). Because many of the natural hosts that produce polyketides
are difficult to culture or manipulate, establishing a universal heterologous host that is genetically tractable has become
an important goal toward the engineered biosynthesis of polyketides and analogues. Here, we summarize the recent progresses
in engineering Escherichia coli as a heterologous host for reconstituting PKSs of different types. Our increased understanding of PKS enzymology and structural
biology, combined with new tools in protein engineering, metabolic engineering, and synthetic biology, has firmly established
E. coli as a powerful host for producing polyketides. 相似文献