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1.
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
2.
To develop a system for combinatorial biosynthesis of glycosylated macrolides, Streptomyces venezuelae was genetically manipulated to be deficient in the production of its macrolide antibiotics by deletion of the entire biosynthetic
gene cluster encoding the pikromycin polyketide synthases and desosamine biosynthetic enzymes. Two engineered deoxysugar biosynthetic
pathways for the biosynthesis of thymidine diphosphate (TDP)- d-quinovose or TDP- d-olivose in conjunction with the glycosyltransferase–auxiliary protein pair DesVII/DesVIII derived from S. venezuelae were expressed in the mutant strain. Feeding the representative 12-, 14-, and 16-membered ring macrolactones including 10-deoxymethynolide,
narbonolide, and tylactone, respectively, to each mutant strain capable of producing TDP- d-quinovose or TDP- d-olivose resulted in the successful production of the corresponding quinovose- and olivose-glycosylated macrolides. In mutant
strains where the DesVII/DesVIII glycosyltransferase–auxiliary protein pair was replaced by TylMII/TylMIII derived from Streptomyces fradiae, quinovosyl and olivosyl tylactone were produced; however, neither glycosylated 10-deoxymethynolide nor narbonolide were
generated, suggesting that the glycosyltransferase TylMII has more stringent substrate specificity toward its aglycones than
DesVII. These results demonstrate successful generation of structurally diverse hybrid macrolides using a S. venezuelae in vivo system and provide further insight into the substrate flexibility of glycosyltransferases.
Won Seok Jung and Ah Reum Han contributed equally to this work. 相似文献
3.
A series of large chromosomal deletions in Streptomyces hygroscopicus 10-22 were aligned on the physical map of the wild-type strain and the mutants were assessed for their ability to produce the aminocyclitol antibiotic 5102-I (jinggangmycin). Twenty-eight mutants were blocked for jinggangmycin production and all of them were found to lack a 300 kb AseI-F fragment of the wild-type chromosome. An ordered cosmid library of the 300 kb AseI-F fragment was made and one of the cosmids conferred jinggangmycin productivity to Streptomyces lividans ZX1. Three of the overlapping cosmids (18G7, 5H3 and 9A2) also hybridized to the valA gene of the validamycin pathway from S. hygroscopicus 5008 as a probe. This gene resembles acbC from Actinoplanes sp. 50/110, which encodes a C7-cyclitol synthase that catalyses the transformation of sedoheptulose 7-phosphate into 2-5-epi-valiolone for acarbose biosynthesis. The valA/acbC-homolog ( orf1) of S. hygroscopicus 10-22 was shown to be essential for jinggangmycin biosynthesis as an engineered mutant with a specific in-frame deletion removing a 609 bp sequence internal to orf1 completely abolished jinggangmycin production and the corresponding knock-out mutant (JXH4) could be complemented for jinggangmycin production by the introduction of an orf1-containing construct. Concurrently, the identities of the genes common to S. hygroscopicus strains 10-22 and 5008 prompted a comparison of the chemical structures of jinggangmycin and validamycin, which led to a clear demonstration that they are identical.The first two authors contributed equally to this study. 相似文献
4.
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 (2 S)-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 (2 S)-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 B 12-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
5.
The Saccharomyces cerevisiae PMR1 gene encodes a Ca 2+-ATPase localized in the Golgi. We have investigated the effects of PMR1 disruption in S. cerevisiae on the glycosylation and secretion of three heterologous glycoproteins, human α 1-antitrypsin (α 1-AT), human antithrombin III (ATHIII), and Aspergillus niger glucose oxidase (GOD). The pmr1 null mutant strain secreted larger amounts of ATHIII and GOD proteins per a unit cell mass than the wild type strain. Despite
a lower growth rate of the pmr1 mutant, two-fold higher level of human ATHIII was detected in the culture supernatant from the pmr1 mutant compared to that of the wild-type strain. The pmr1 mutant strain secreted α 1-AT and the GOD proteins mostly as core-glycosylated forms, in contrast to the hyperglycosylated proteins secreted in the
wild-type strain. Furthermore, the core-glycosylated forms secreted in the pmr1 mutant migrated slightly faster on SDS-PAGE than those secreted in the mnn9 deletion mutant and the wild type strains. Analysis of the recombinant GOD with anti-α1,3-mannose antibody revealed that
GOD secreted in the pmr1 mutant did not have terminal α1,3-linked mannoses unlike those secreted in the mnn9 mutant and the wild type strains. The present results indicate that the pmr1 mutant, with the super-secretion phenotype, is useful as a host system to produce recombinant glycoproteins lacking high-mannose
outer chains. 相似文献
6.
Previously, we achieved approximately 30-fold enhanced secretion of the protease-sensitive model protein human growth hormone
(hGH) by multiple gene deletion of seven obstructive proteases in the fission yeast Schizosaccharomyces pombe. However, intracellular retention of secretory hGH was found in the resultant multiprotease-deficient strains. As a solution,
genetic modification of the intracellular trafficking pathway that is related to intracellular retention of hGH was attempted
on a protease octuple deletant strain. Vacuolar accumulation of the intracellularly retained hGH was identified by secretory
expression of hGH fused with EGFP, and three vacuolar protein sorting ( vps)-deficient strains, vps10Δ, vps22Δ, and vps34Δ, were determined on account of their hGH secretion efficiency. The mutant vps10Δ was found to be effective for hGH secretion, which suggested a role for vps10 in the vacuolar accumulation of the intracellularly retained hGH. Finally, vps10 deletion was performed on the protease octuple deletant strain, which led to an approximately 2-fold increase in hGH secretion.
This indicated the possible application of secretory-pathway modification and multiple protease deletion for improving heterologous
protein secretion from the fission yeast S. pombe. 相似文献
7.
A new actinomycete strain, isolated from soil in China, strongly inhibited in vitro proliferation of human hepatoma, chronic
myelogenous leukemia, and colonic carcinoma cell lines. The strain, designated L033, was identified as a strain of Streptomyces avermitilis based on cultural property, morphology, carbon source utilization, 16s rRNA gene analysis, and DNA–DNA relatedness studies.
The anticancer component from L033 was purified to homogeneity by preparative positive-phase high-performance liquid chromatography
and crystallization. Nuclear magnetic resonance and mass spectrometric analysis showed that this compound had the same structure
as oligomycin A. Different with other reported naturally occurring strains of S. avermitilis, L033 produced high quantity of oligomycin A (maximal 1,461 μg/ml). Therefore, L033 was considered of great potential as
an industrial oligomycin-A-producing strain.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
8.
Two genes, orf6 and orf9 located in the L-oleandrose sugar biosynthetic gene cluster of Streptomyces antibioticus Tü99. NovU has been characterized as C-5 methyltrnaferase involved in noviose biosynthetic pathway. We have cloned and heterologously
expressed the orf6, orf9, and novU genes in S. venezuelae YJ003-OTBP1. This established the function of orf6 and orf9 as 4-ketoreductase and 3-epimerase, respectively. All of analytical data of the noviosylated 10-deoxymethynolide also is
in support of proving their functions. Furthermore biosynthetic pathway 5,5- gem-dimethyl-6-deoxyglucose (TDP-Lnoviose) has been proposed. 相似文献
10.
Using metabolic engineering, we developed Streptomyces venezuelae YJ028 as an efficient heterologous host to increase the malonyl-CoA pool to be directed towards enhanced production of various
polyketides. To probe the applicability of newly developed hosts in the heterologous production of polyketides, we expressed
type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase, in these hosts. Flaviolin production was doubled by
expression of acetyl-CoA carboxylase (ACCase) and 4-fold by combined expression of ACCase, metK1-sp and afsR-sp. Thus, the newly developed Streptomyces venezuelae YJ028 hosts produce heterologous polyketides more efficiently than the parent strain. 相似文献
11.
Along with traditional random mutagenesis-driven strain improvement, cloning and heterologous expression of Streptomyces secondary metabolite gene clusters have become an attractive complementary approach to increase its production titer, of
which regulation is typically under tight control via complex multiple regulatory networks present in a metabolite low-producing
wild-type strain. In this study, we generated a polyketide non-producing strain by deleting the entire actinorhodin cluster
from the chromosome of a previously generated S. coelicolor mutant strain, which was shown to stimulate actinorhodin biosynthesis through deletion of two antibiotic downregulators as
well as a polyketide precursor flux downregulator (Kim et al. in Appl Environ Microbiol 77:1872–1877, 2011). Using this engineered S. coelicolor mutant strain as a surrogate host, a model minimal polyketide pathway for aloesaponarin II, an actinorhodin shunt product,
was cloned in a high-copy conjugative plasmid, followed by functional pathway expression and quantitative metabolite analysis.
Aloesaponarin II production was detected only in the presence of a pathway-specific regulatory gene, actII- ORF4, and its production level was the highest in the actinorhodin cluster-deleted and downregulator-deleted mutant strain, implying
that this engineered polyketide pathway-free and regulation-optimized S. coelicolor mutant strain could be used as a general surrogate host for efficient expression of indigenous or foreign polyketide pathways
derived from diverse actinomycetes in nature. 相似文献
12.
Recently, the epothilone polyketide synthase (PKS) was successfully introduced into a heterologous production host for the
large-scale production of epothilone D. We have found that at least three other epothilones can also be produced as the major
fermentation product of this recombinant strain by supplementation of specific substrates to the production media. Addition
of acetate or propionate to the media results in modulation of the epothilone D:C ratio, whereas addition of l-serine with either acetate or propionate yields epothilone H 1 or H 2 as the major product. This strategy permits production of at least four novel epothilones by culturing a single host with
a genetically modified epothilone PKS in various media. Journal of Industrial Microbiology & Biotechnology (2002) 28, 17–20 DOI: 10.1038/sj/jim/7000209
Received 20 June 2001/ Accepted in revised form 03 September 2001 相似文献
13.
Aspergillus strains are being considered as potential hosts for recombinant heterologous protein production because of their excellent
extracellular enzyme production characteristics. However, Aspergillus proteases are problematic in that they modify and degrade the heterologous proteins in the extracellular medium. In previous
studies we observed that media adjustments and maintenance of a filamentous morphology greatly reduced protease activity and
that a low concentration of the aspartic protease inhibitor pepstatin inhibited the latter protease activity to the extent
of approximately 90%. In this paper we report that when the serine protease inhibitor chymostatin is used in combination with
pepstatin 99–100% of total protease activity in Aspergillus cultures is inhibited. In protease assays a concentration of 30 μM chymostatin combined with 0.075 μM pepstatin was required
for maximum inhibition. Inhibitor concentrations of chymostatin and pepstatin of 120 and 0.3 μM, respectively, when added
to Aspergillus cultures, has no significant effect on biomass production, glucose utilization or culture pH pattern. The potential of using
these protease inhibitors in cultures of recombinant Aspergillus strains producing heterologous proteins will now be investigated to determine if the previously observed recombinant protein
denaturing effects of Aspergillus proteases can be negated. 相似文献
14.
Mithramycin A is an antitumor compound used for treatment of several types of cancer including chronic and acute myeloid leukemia, testicular carcinoma, hypercalcemia and Paget’s disease. Selective modifications of this molecule by combinatorial biosynthesis and biocatalysis opened the possibility to produce mithramycin analogues with improved properties that are currently under preclinical development. The mithramycin A biosynthetic gene cluster from Streptomyces argillaceus ATCC12956 was cloned by transformation assisted recombination in Saccharomyces cerevisiae and heterologous expression in Streptomyces lividans TK24 was evaluated. Mithramycin A was efficiently produced by S. lividans TK24 under standard fermentation conditions. To improve the yield of heterologously produced mithramycin A, a collection of derivative strains of S. lividans TK24 were constructed by sequential deletion of known potentially interfering secondary metabolite gene clusters using a protocol based on the positive selection of double crossover events with blue pigment indigoidine-producing gene. Mithramycin A production was evaluated in these S. lividans strains and substantially improved mithramycin A production was observed depending on the deleted gene clusters. A collection of S. lividans strains suitable for heterologous expression of actinomycetes secondary metabolites were generated and efficient production of mithramycin A with yields close to 3 g/L, under the tested fermentation conditions was achieved using these optimized collection of strains. 相似文献
15.
The GPD2 gene, encoding NAD +-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol-producing strain of Saccharomyces cerevisiae, was deleted. And then, either the non-phosphorylating NADP +-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Bacillus cereus, or the NADP +-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Kluyveromyces lactis, was expressed in the obtained mutant AG2 deletion of GPD2, respectively. The resultant recombinant strain AG2A ( gpd2Δ P
PGK
- gapN) exhibited a 48.70 ± 0.34% (relative to the amount of substrate consumed) decrease in glycerol production and a 7.60 ± 0.12%
(relative to the amount of substrate consumed) increase in ethanol yield, while recombinant AG2B ( gpd2Δ P
PGK
- GAPDH) exhibited a 52.90 ± 0.45% (relative to the amount of substrate consumed) decrease in glycerol production and a 7.34 ± 0.15%
(relative to the amount of substrate consumed) increase in ethanol yield compared with the wild-type strain. More importantly,
the maximum specific growth rates ( μ
max) of the recombinant AG2A and AG2B were higher than that of the mutant gpd2Δ and were indistinguishable compared with the wild-type strain in anaerobic batch fermentations. The results indicated that
the redox imbalance of the mutant could be partially solved by expressing the heterologous genes. 相似文献
16.
This paper describes the effect of the heterologous expression of tomatinase from Fusarium oxysporum f. sp lycopersici in Saccharomyces cerevisiae. The gene FoTom1 under the control of the S. cerevisiae phosphoglycerate kinase (PGK1) promoter was cloned into pYES2. S. cerevisiae strain Y45 was transformed with this vector and URA3 transformant strains were selected for resistance to α-tomatine. Two
transformants were randomly selected for further study (designated Y45-1 and Y45-2). Control strain Y45 was inhibited at 50 μM
α-tomatine, in contrast, transformants Y45-1 and Y45-2 did not show inhibition at 200 μM. Tomatinase activity was detected
by HPLC monitoring tomatine disappearance and tomatidine appearance in the supernatants of culture medium. Maximum tomatinase
activity was observed in the transformants after 6 h, remaining constant during the following 24 h. No tomatinase activity
was detected in the parental strain. Moreover, the transformants were able to grow and produce ethanol in a mix of Agave
tequilana Weber var. azul and Agave salmiana must, contrary to the Y45 strain which was unable to grow and ferment under these conditions. 相似文献
17.
Nonomuraea strain ATCC 39727 produces the glycopeptide A40926, used for manufacturing dalbavancin, currently in advanced clinical trials.
From the gene cluster involved in A40926 biosynthesis, a strain deleted in dbv23 was constructed. This mutant can produce only the glycopeptides lacking the O-linked acetyl residue at position 6 of the mannose moiety, while, under identical fermentation conditions, the wild-type
strain produces mostly glycopeptides carrying an acetylated mannose. Furthermore, the total amount of glycopeptides produced
by the mutant strain was found to be approximately twice that of the wild type. The reduced level of glycopeptides observed
in the wild-type strain may be due to an inhibitory effect exerted by the acetylated compound on the biosynthesis of A40926.
Indeed, spiking production cultures with ≥1 μg/ml of the acetylated glycopeptide inhibited A40926 production in the mutant
strain. 相似文献
18.
In this study, the glucose 6-phosphate dehydrogenase gene (XOO2314) was inactivated in order to modulate the intracellular
glucose 6-phosphate, and its effects on xanthan production in a wild-type strain of Xanthomonas oryzae were evaluated. The intracellular glucose 6-phosphate was increased from 17.6 to 99.4 μmol g −1 (dry cell weight) in the gene-disrupted mutant strain. The concomitant increase in the glucose 6-phosphate was accompanied
by an increase in xanthan production of up to 2.23 g l −1 (culture medium). However, in defined medium supplemented with 0.4% glucose, the growth rate of the mutant strain was reduced
to 52.9% of the wild-type level. Subsequently, when a family B ATP-dependent phosphofructokinase from Escherichia coli was overexpressed in the mutant strain, the growth rate was increased to 142.9%, whereas the yields of xanthan per mole of
glucose remained approximately the same. 相似文献
19.
Virginiae butanolide (VB) is a member of the γ-butyrolactone autoregulators and triggers the production of streptogramin antibiotics
virginiamycin M 1 and S in Streptomyces virginiae. A VB biosynthetic gene ( barS2) was localized in a 10-kb regulatory island which controls the virginiamycin biosynthesis/resistance of S. virginiae, and analyzed by gene disruption/complementation. The barS2 gene is flanked by barS1, another VB biosynthetic gene catalyzing stereospecific reduction of an A-factor-type precursor into a VB-type compound,
and barX encoding a pleiotropic regulator for virginiamycin biosynthesis. The deduced product of barS2 possessed moderate similarity to a putative dehydrogenase of Streptomyces venezuelae, encoded by jadW
2
located in similar gene arrangement to that in the regulatory island of S. virginiae. A barS2-disruptant (strain IC152), created by means of homologous recombination, showed no differences in growth in liquid medium
or morphology on solid medium compared to a wild-type strain, suggesting that BarS2 does not play any role in primary metabolism
or morphological differentiation of S. virginiae. In contrast, no initiation of virginiamycin production or VB production was detected with the strain IC152 until 18 h of
cultivation, at which time full production of virginiamycin occurs in the wild-type strain. The delayed virginiamycin production
of the strain IC152 was fully restored to the level of the wild-type strain either by the exogenous addition of VB or by complementation
of the intact barS2 gene, indicating that the lack of VB production at the initiation phase of virginiamycin production is the sole reason for
the defect of virginiamycin production, and the barS2 gene is of primary importance for VB biosynthesis in S. virginiae.
An erratum to this article can be found at 相似文献
20.
Pseudomonas putida KT2440 is an emerging biomanufacturing host amenable for use with renewable carbon streams including aromatics such as para-coumarate. We used a pooled transposon library disrupting nearly all (4,778) non-essential genes to characterize this microbe under common stirred-tank bioreactor parameters with quantitative fitness assays. Assessing differential fitness values by monitoring changes in mutant strain abundance identified 33 gene mutants with improved fitness across multiple stirred-tank bioreactor formats. Twenty-one deletion strains from this subset were reconstructed, including GacA, a regulator, TtgB, an ABC transporter, and PP_0063, a lipid A acyltransferase. Thirteen deletion strains with roles in varying cellular functions were evaluated for conversion of para-coumarate, to a heterologous bioproduct, indigoidine. Several mutants, such as the Δ gacA strain improved fitness in a bioreactor by 35 fold and showed an 8-fold improvement in indigoidine production (4.5 g/L, 0.29 g/g, 23% of maximum theoretical yield) from para-coumarate as the carbon source. 相似文献
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