Heterologous expression of tylosin polyketide synthase and production of a hybrid bioactive macrolide in Streptomyces venezuelae

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.     

Metabolic engineering of <Emphasis Type="Italic">Streptomyces venezuelae</Emphasis> for malonyl-CoA biosynthesis to enhance heterologous production of polyketides

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.     

Heterologous production of epothilones B and D in Streptomyces venezuelae

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.     

Bioconversion of 12-, 14-, and 16-membered ring aglycones to glycosylated macrolides in an engineered strain of <Emphasis Type="Italic">Streptomyces venezuelae</Emphasis>

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.     

Engineered biosynthesis of 16-membered macrolides that require methoxymalonyl-ACP precursors in Streptomyces fradiae

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.     

Engineering 4-coumaroyl-CoA derived polyketide production in Yarrowia lipolytica through a β-oxidation mediated strategy

Polyketides are a diverse class of molecules sought after for their valuable properties, including as potential pharmaceuticals. Previously, we demonstrated that the oleaginous yeast Yarrowia lipolytica is an optimal host for production of the simple polyketide, triacetic acid lactone (TAL). We here expand the capacities of this host by overcoming previous media challenges and enabling production of more complex polyketides. Specifically, we employ a β-oxidation related strategy to improve polyketide production directly from defined media. Beyond TAL production, we establish biosynthesis of the 4-coumaroyl-CoA derived polyketides: naringenin, resveratrol, and bisdemethoxycurcumin, as well as the diketide intermediate, (E)-5-(4-hydroxyphenyl)-3-oxopent-4-enoic acid. In this background, we enable high-level de novo production of naringenin through import of both a heterologous pathway and a mutant Y. lipolytica allele. In doing so, we generated an averaged maximum titer of 898 mg/L naringenin, the highest titer reported to date in any host. These results demonstrate that Y. lipolytica is an ideal polyketide production host for more complex 4-coumaroyl-CoA derived products.     

异源生物合成聚酮化合物的研究进展

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。     

Metabolic pathway engineering for complex polyketide biosynthesis in Saccharomyces cerevisiae

Polyketides are a diverse group of natural products with significance in human and veterinary medicine. Because polyketides are structurally complex molecules and fermentation is the most commercially viable route of production, a generic heterologous host system for high-level polyketide production is desirable. Saccharomyces cerevisiae has been shown to be an excellent production host for a simple polyketide, yielding 1.7 g of 6-methylsalicylic acid per liter of culture in un-optimized shake-flask fermentations. However, a barrier to the heterologous production of more complex 'modular' polyketides in S. cerevisiae is the lack of required polyketide precursor pathways. In this work, we describe the introduction into S. cerevisiae of pathways for the production of methylmalonyl-coenzyme A (CoA), a precursor for complex polyketides, by both propionyl-CoA-dependent and propionyl-CoA-independent routes. Furthermore, we demonstrate that the methylmalonyl-CoA produced in the engineered yeast strains is used in vivo for the production of a polyketide product, a triketide lactone.     

Engineered polyketide biosynthesis and biocatalysis in Escherichia coli

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.     

Minimal polyketide pathway expression in an actinorhodin cluster-deleted and regulation-stimulated <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

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.     

Quorum sensing-based metabolic engineering of the precursor supply in Streptomyces coelicolor to improve heterologous production of neoaureothin

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.     

Metabolically engineered Escherichia coli for efficient production of glycosylated natural products

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.     

Biotechnological potential of the ethylmalonyl-CoA pathway

The ethylmalonyl-CoA pathway is central to the carbon metabolism of many α-proteobacteria, like Rhodobacter sphaeroides and Methylobacterium extorquens as well as actinomycetes, like Streptomyces spp. Its function is to convert acetyl-CoA, a central carbon intermediate, to other precursor metabolites for cell carbon biosynthesis. In contrast to the glyoxylate cycle—another widely distributed acetyl-CoA assimilation strategy—the ethylmalonyl-CoA pathway contains many unique CoA-ester intermediates, such as (2R)- and (2S)-ethylmalonyl-CoA, (2S)-methylsuccinyl-CoA, mesaconyl-(C1)-CoA, and (2R, 3S)-methylmalyl-CoA. With this come novel catalysts that interconvert these compounds. Among these unique enzymes is a novel carboxylase that reductively carboxylates crotonyl-CoA, crotonyl-CoA carboxylase/reductase, and (3S)-malyl-CoA thioesterase. The latter represents the first example of a non-Claisen condensation enzyme of the malate synthase superfamily and defines a new class of thioesterases apart from the hotdog-fold and α/β-fold thioesterases. The biotechnological implications of the ethylmalonyl-CoA pathway are tremendous as one looks to tap into the potential of using these new intermediates and catalysts to produce value-added products.     

Engineered EryF hydroxylase improving heterologous polyketide erythronolide B production in Escherichia coli

In the last two decades, the production of complex polyketides such as erythromycin and its precursor 6-deoxyerythronolide B (6-dEB) was demonstrated feasible in Escherichia coli. Although the heterologous production of polyketide skeleton 6-dEB has reached 210 mg l⁻¹ in E. coli, the yield of its post-modification products erythromycins remains to be improved. Cytochrome P450EryF catalyses the C6 hydroxylation of 6-dEB to form erythronolide B (EB), which is the initial rate-limiting modification in a multi-step pathway to convert 6-dEB into erythromycin. Here, we engineered hydroxylase EryF to improve the production of heterologous polyketide EB in E. coli. By comparative analysis of various versions of P450EryFs, we confirmed the optimal SaEryF for the biosynthesis of EB. Further mutation of SaEryF based on the crystal structure of SaEryF and homology modelling of AcEryF and AeEryF afforded the enhancement of EB production. The designed mutant of SaEryF, I379V, achieved the yield of 131 mg l⁻¹ EB, which was fourfold to that produced by wild-type SaEryF. Moreover, the combined mutagenesis of multiple residues led to further boost the EB concentration by another 41%, which laid the foundation for efficient heterologous biosynthesis of erythromycin or other complex polyketides.     

Investigation of early tailoring reactions in the oxytetracycline biosynthetic pathway

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases. The amidated tetracycline backbone is biosynthesized by the minimal polyketide synthases and an amidotransferase homologue OxyD. Biosynthesis of the key intermediate 6-methylpretetramid requires two early tailoring steps, which are cyclization of the linearly fused tetracyclic scaffold and regioselective C-methylation of the aglycon. Using a heterologous host (CH999)/vector pair, we identified the minimum set of enzymes from the oxytetracycline biosynthetic pathway that is required to afford 6-methylpretetramid in vivo. Only two cyclases (OxyK and OxyN) are necessary to completely cyclize and aromatize the amidated tetracyclic aglycon. Formation of the last ring via C-1/C-18 aldol condensation does not require a dedicated fourth-ring cyclase, in contrast to the biosynthetic mechanism of other tetracyclic aromatic polyketides, such as daunorubicin and tetracenomycin. Acetyl-derived polyketides do not undergo spontaneous fourth-ring cyclization and form only anthracene carboxylic acids as demonstrated both in vivo and in vitro. OxyF was identified to be the C-6 C-methyltransferase that regioselectively methylates pretetramid to yield 6-methylpretetramid. Reconstitution of 6-methylpretetramid in a heterologous host sets the stage for a more systematic investigation of additional tetracycline downstream tailoring enzymes and is a key step toward the engineered biosynthesis of tetracycline analogs.     

Engineering the precursor pool to modulate the production of pamamycins in the heterologous host S. albus J1074

Pamamycins, a group of polyketides originally discovered in Streptomyces alboniger, induce sporulation in Streptomyces and inhibit the growth of Gram-positive bacteria, Mycobacterium tuberculosis and fungi. The pamamycin biosynthetic gene cluster encodes 6 ketosynthases that utilize a variety of three-carbon to five-carbon CoA thioesters as starter and extender units. This promiscuity in production results in an up to 18 different derivatives during fermentation. For more-selective production and simplified purification, we aimed to modify the precursor supply to narrow the spectrum of the produced derivatives. Eight genes potentially responsible for the supply of two major precursors, 2-S-methylmalonyl-CoA and 2-S-ethylmalonyl-CoA, were identified using the NCBI Basic Local Alignment Search Tool (BLAST) against the genome of the heterologous host S. albus J1074. Knockout mutants of the identified genes were constructed and their impact on intracellular CoA ester concentrations and on the production of pamamycins was determined. The created mutants enabled us to conclusively identify the ethylmalonyl-CoA supplying routes and their impact on the production of pamamycin. Furthermore, we gained significant information on the origin of the methylmalonyl-CoA supply in Streptomyces albus.     

Biosynthesis of glycosylated derivatives of tylosin in Streptomyces venezuelae

Streptomyces venezuelae YJ028, bearing a deletion of the entire biosynthetic gene cluster encoding the pikromycin polyketide synthases and desosamine biosynthetic enzymes, was used as a bioconversion system for combinatorial biosynthesis of glycosylated derivatives of tylosin. Two engineered deoxysugar biosynthetic pathways for the biosynthesis of TDP-3-O-demethyl-D-chalcose or TDP-Lrhamnose in conjunction with the glycosyltransferaseauxiliary protein pair DesVII/DesVIII were expressed in a S. venezuelae YJ028 mutant strain. Supplementation of each mutant strain capable of producing TDP-3-O-demethyl- D-chalcose or TDP-L-rhamnose with tylosin aglycone tylactone resulted in the production of the 3-O-demethyl- D-chalcose, D-quinovose, or L-rhamnose-glycosylated tylactone.     

Heterologous production of clavulanic acid intermediates in <Emphasis Type="Italic">Streptomyces venezuelae</Emphasis>

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.     

Biosynthesis of Yersiniabactin, a Complex Polyketide-Nonribosomal Peptide, Using Escherichia coli as a Heterologous Host

The medicinal value associated with complex polyketide and nonribosomal peptide natural products has prompted biosynthetic schemes dependent upon heterologous microbial hosts. Here we report the successful biosynthesis of yersiniabactin (Ybt), a model polyketide-nonribosomal peptide hybrid natural product, using Escherichia coli as a heterologous host. After introducing the biochemical pathway for Ybt into E. coli, biosynthesis was initially monitored qualitatively by mass spectrometry. Next, production of Ybt was quantified in a high-cell-density fermentation environment with titers reaching 67 ± 21 (mean ± standard deviation) mg/liter and a volumetric productivity of 1.1 ± 0.3 mg/liter-h. This success has implications for basic and applied studies on Ybt biosynthesis and also, more generally, for future production of polyketide, nonribosomal peptide, and mixed polyketide-nonribosomal peptide natural products using E. coli.