Enhancement of natamycin production on Streptomyces gilvosporeus by chromosomal integration of the Vitreoscilla hemoglobin gene (vgb)

Oxygen deficiency is a critical factor during the fermentation production of natamycin. In order to alleviate oxygen limitation and enhance the yield of natamycin, the vgb gene, encoding Vitreoscilla hemoglobin (VHb) was inserted into pSET152 with its native promoter and integrated into the chromosome of Streptomyces gilvosporeus (S. gilvosporeus). The expression of VHb was determined by Western blotting. The activity of expressed VHb was confirmed by the observation of VHb-specific CO-difference spectrum with a maximal absorption at 419 nm for the recombinant. Integration of the empty plasmid pSET152 did not affect natamycin production of S. gilvosporeus. While the vgb-harboring strain exhibited high natamycin productivity, reaching 3.31 g/L in shake flasks and 8.24 g/L in 1-L fermenters. Compared to the wild strain, expression of VHb, increased the natamycin yield of the strain bearing vgb by 131.3 % (jar fermenter scale) and 175 % (shake flask scale), respectively, under certain oxygen-limiting condition. Addition of an extra copy of the vgb gene in S. gilvosporeus-vgb2 did not enhance the natamycin production obviously. These results provided a superior natamycin-producing strain which can be directly used in industry and a useful strategy for increasing yields of other metabolites in industrial strains.     

Intergeneric Conjugation Escherichia coli–Streptomyces globisporus1912 Using Integrative Plasmid pSET152 and Its Derivatives

Streptomyces globisporus1912 produces a novel angucycline antitumor antibiotic landomycin E (LE). To study the LE biosynthetic gene cluster in detail, a system for the conjugal transfer of the integrative plasmid pSET152 fromEscherichia coliinto S. globisporus1912 has been developed. It was shown that this plasmid integrates into two sites of the S. globisporuschromosome and is stably inherited under nonselective conditions. pSET152⁺exconjugants of the strain 1912 are characterized by a significant decrease in LE synthesis (by 50–90%). A negative effect of pSET152 integration on antibiotic production was observed even upon the use of the recipient strain with increased LE synthesis, although in this case, the level of LE production in exconjugants was 120–150% of that in the original strain 1912. Based on pSET152, a vector system for gene knockouts in S. globisporuswas developed. The effectivity of this system was shown in the example of disruption of the lndAgene encoding the key enzyme of LE synthesis (-ketoacylsynthase). Inactivation of this gene was shown to lead to the cessation of LE biosynthesis.     

Metabolic Engineering of Rational Screened Saccharopolyspora spinosa for the Enhancement of Spinosyns A and D Production

Spinosyns A and D are potent ingredient for insect control with exceptional safety to non-target organisms. It consists of a 21-carbon tetracyclic lactone with forosamine and tri-O-methylated rhamnose which are derived from S-adenosylmethionine. Although previous studies have revealed the involvement of metK1 (S-adenosylmethionine synthetase), rmbA (glucose-1-phosphate thymidylyltransferase), and rmbB (TDP-D-glucose-4, 6-dehydratase) in the biosynthesis of spinosad, expression of these genes into rational screened Saccharopolyspora spinosa (S. spinosa MUV) has not been elucidated till date. In the present study, S. spinosa MUV was developed to utilize for metabolic engineering. The yield of spinosyns A and D in S. spinosa MUV was 244 mg L⁻¹ and 129 mg L⁻¹, which was 4.88-fold and 4.77-fold higher than that in the wild-type (50 mg L⁻¹ and 27 mg L⁻¹), respectively. To achieve the better production; positive regulator metK1-sp, rmbA and rmbB genes from Streptomyces peucetius, were expressed and co-expressed in S. spinosa MUV under the control of strong ermE* promoter, using an integration vector pSET152 and expression vector pIBR25, respectively. Herewith, the genetically engineered strain of S. spinosa MUV, produce spinosyns A and D up to 372/217 mg L⁻¹ that is 7.44/8.03-fold greater than that of wild type. This result demonstrates the use of metabolic engineering on rationally developed high producing natural variants for the production.     

Chromosomal integration of the Vitreoscilla hemoglobin gene and its physiological actions in Tremella fuciformis

The Vitreoscilla hemoglobin (VHb) gene was expressed in yeast-like conidia (YLCs) of Tremella fuciformis (T. fuciformis) to increase cell density in submerged fermentation by enhancing oxygen uptake. With the intention of doing this, an integrated expression vector containing the VHb gene and the hygromycin B phosphotransferase (hph) gene derived from Escherichia coli (E. coli) as the selectable marker was constructed, and then transformed into protoplasts of YLCs from T. fuciformis with restriction enzyme-mediated DNA integration (REMI). Hygromycin-resistant transformants had been generated during the transformation. Molecular evidences including PCR assay, Southern blotting, and Western blot analysis indicated the VHb gene had been integrated into the genome of transgenic T. fuciformis strains and was expressed successfully. Shake-flask fermentation and bioreactor cultivation results showed that the expression of VHb in this fungus could enhance growth of YLCs. The final cell density was higher in the culture of VHb-expressing strain than that of the wild-type strain. Moreover, these results also suggested that CaMV35S promoter was capable of driving the expression of heterologous genes in T. fuciformis.     

斑贝链霉菌接合转移系统的构建及透明颤菌血红蛋白基因的表达对其次级代谢的影响

以pSET152和pHZ1358为出发质粒,通过供体大肠杆菌ET12567(pUZ8002)和S17-1接合转移斑贝链霉菌(Streptomycesbambergiensis),构建和优化了接合转移体系。采用OOE-PCR技术构建含ermEp*与vhb结构基因融合片段的整合型表达载体pBIB2005,转化ET12567(pUZ8002)后,属间接合转移至斑贝链霉菌。通过PCR、CO结合差光谱验证vhb基因在斑贝链霉菌中整合表达。摇瓶发酵显示VHb蛋白能改善菌体对氧的需求,一定程度上促进细胞生长,提高抗生素产量。     

藤黄生孢链霉菌NRRL 2401遗传操作系统和基因文库的构建北大核心CSCD

【目的】建立藤黄生孢链霉菌NRRL 2401的遗传操作系统和基因文库,以便筛选次级代谢产物生物合成基因。【方法】利用大肠杆菌和链霉菌的属间接合转移的方法,以整合型载体pPM927、pSET152和复制型载体pJTU1278构建链霉菌遗传操作系统。以pCClFOS^(TM)载体,大肠杆菌EP1300^(TM)-T1~R为宿主菌构建Fosmid文库。随后,设计引物,利用"板池-行池-单克隆"的三级PCR方法对文库进行快速筛选。【结果】pPM927、pSET152和pJTU1278均成功转入藤黄生孢链霉菌NRRL 2401,其中pSET152载体的转化效率最高。构建了稳定高效的藤黄生孢链霉菌NRRL 2401的基因文库,含2880个克隆,平均插入片段大小约为35 kb,空载率小于1%,文库覆盖率为99.99%,覆盖基因组16.5倍。同时,初步筛选出可能含有吲哚霉素生物合成基因簇的9个阳性克隆。【结论】成功构建了稳定高效的藤黄生孢链霉菌NRRL 2401遗传操作系统和高质量的基因文库,为克隆该菌中次级代谢产物生物合成基因簇以及进一步遗传改造奠定了基础。     

Interspecies conjugation of Escherichia coli-Streptomyces globisporus 1912 using integrative plasmid pSET152 and its derivatives]

Streptomyces globisporus 1912 produces a novel angucycline antitumor antibiotic landomycin E (LE). To study the LE biosynthetic gene cluster in detail, a system for the conjugal transfer of the integrative plasmid pSET152 from Escherichia coli into S. globisporus 1912 has been developed. It was shown that this plasmid integrates into two sites of the S. globisporus chromosome and is stably inherited under nonselective conditions. pSET152+ exconjugants of the strain 1912 are characterized by a significant decrease in LE synthesis (by 50-90%). A negative effect of pSET152 integration on antibiotic production was observed even upon the use of the recipient strain with increased LE synthesis, although in this case, the level of LE production in ex-conjugants was 120-150% of that in the original strain 1912. Based on pSET152, a vector system for gene knockouts in S. globisporus was developed. The effectivity of this system was shown in the example of disruption of the lndA gene encoding the key enzyme of LE synthesis (beta-ketoacylsynthase). Inactivation of this gene was shown to lead to the cessation of LE biosynthesis.     

A cluster of genes for the biosynthesis of spinosyns, novel macrolide insect control agents produced by Saccharopolyspora spinosa

Spinosyns A and D are the active ingredients in a family of insect control agents produced by fermentation of Saccharopolyspora spinosa. Spinosyns are 21–carbon tetracyclic lactones to which are attached two deoxysugars. Most of the genes involved in spinosyn biosynthesis are clustered in an 74 kb region of the S. spinosa genome. This region has been characterized by DNA sequence analysis and by targeted gene disruptions. The spinosyn biosynthetic gene cluster contains five large genes encoding a type I polyketide synthase, and 14 genes involved in modification of the macrolactone, or in the synthesis, modification and attachment of the deoxysugars. Four genes required for rhamnose biosynthesis (two of which are also required for forosamine biosynthesis) are not present in the cluster. A pathway for the biosynthesis of spinosyns is proposed.     

Identification and characterization of Streptomyces ghanaensis ATCC14672 integration sites for three actinophage-based plasmids

Streptomyces ghanaensis produces the antibiotic moenomycin A, which is the only known direct inhibitor of bacterial peptidoglycan glycosyltransferases (transglycosylases). Recent progress in understanding moenomycin biosynthesis opens the door to the generation of novel moenomycins via biocombinatorial approaches. To realize the promise of such an approach, one needs better knowledge of the S. ghanaensis genome and diverse genetic tools for stable expression of recombinant constructs in this strain. In this respect, we report the intergeneric Escherichia coli–S. ghanaensis conjugal transfer of plasmids pRT801 and pSOK804 based on the actinophage BT1 and VWB integrase systems, respectively. The attB sites for these two plasmids and for pSET152 were characterized. In particular, sequencing revealed that a putative Arg-tRNA gene serves as an integration site for both phage VWB and pSAM2-like actinomycete integrative and conjugative element recently suggested to be widespread and functional in actinomycetes. The stability of the studied plasmids and their neutrality with respect to antibiotic production warrant their use for manipulations of S. ghanaensis genome.     

Reconstitution of a mini-gene cluster combined with ribosome engineering led to effective enhancement of salinomycin production in Streptomyces albus

Salinomycin, an FDA-approved polyketide drug, was recently identified as a promising anti-tumour and anti-viral lead compound. It is produced by Streptomyces albus, and the biosynthetic gene cluster (sal) spans over 100 kb. The genetic manipulation of large polyketide gene clusters is challenging, and approaches delivering reliable efficiency and accuracy are desired. Herein, a delicate strategy to enhance salinomycin production was devised and evaluated. We reconstructed a minimized sal gene cluster (mini-cluster) on pSET152 including key genes responsible for tailoring modification, antibiotic resistance, positive regulation and precursor supply. These genes were overexpressed under the control of constitutive promoter P_kasO* or P_neo. The pks operon was not included in the mini-cluster, but it was upregulated by SalJ activation. After the plasmid pSET152::mini-cluster was introduced into the wild-type strain and a chassis host strain obtained by ribosome engineering, salinomycin production was increased to 2.3-fold and 5.1-fold compared with that of the wild-type strain respectively. Intriguingly, mini-cluster introduction resulted in much higher production than overexpression of the whole sal gene cluster. The findings demonstrated that reconstitution of sal mini-cluster combined with ribosome engineering is an efficient novel approach and may be extended to other large polyketide biosynthesis.     

A gene cloning system for the aranciamycin producer strain Streptomyces echinatus Lv 22

Streptomyces echinatus Lv 22 (=DSM 40730) produces an anthracycline antibiotic aranciamycin. Development of DNA transfer using conjugation from Escherichia coli into this strain is described. Various replicative plasmids (pKC1139, pKC1218E, pSOK1O1, pCHZ101) as well as actinophage (φC31- and VWB-based vectors pSET152 and pVWB, respectively, were transferred from E. coli ET12567 (pUB307) into the S. echinatus at a frequency ranging from 2.4 × 10^?3 to 1.6 × 10^?4. The transconjugants did not differ from wild type in their ability to produce aranciamycin and morphological features. There is one attB site for pSET152 and pVWB integrative plasmids in the S. echinatus chromosome. Developed DNA transfer system was used for expression of heterologous regulatory genes in S. echinatus cells. Expression of relA gene of ppGpp synthetase increased antibiotic production in S. echinatus. The absA2 gene of S. ghanaensis appears to play a negative role in the control of aranciamycin biosynthesis. Additional copies of absA2 leads to inhibition of aranciamycin’s production. absB and afsS had no effect on aranciamycin biosynthesis as well as on the morphological features of S. echinatus. Obtained results indicate efficiency of the developed system for gene cloning in S. echinatus.     

Genes for the biosynthesis of spinosyns: applications for yield improvement in Saccharopolyspora spinosa

Spinosyns A and D are the active ingredients in an insect control agent produced by fermentation of Saccharopolyspora spinosa. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. The spinosyn biosynthesis genes, except for the rhamnose genes, are located in a cluster that spans 74 kb of the S. spinosa genome. DNA sequence analysis, targeted gene disruptions and bioconversion studies identified five large genes encoding type I polyketide synthase subunits, and 14 genes involved in sugar biosynthesis, sugar attachment to the polyketide or cross-bridging of the polyketide. Four rhamnose biosynthetic genes, two of which are also necessary for forosamine biosynthesis, are located outside the spinosyn gene cluster. Duplication of the spinosyn genes linked to the polyketide synthase genes stimulated the final step in the biosynthesis — the conversion of the forosamine-less pseudoaglycones to endproducts. Duplication of genes involved in the early steps of deoxysugar biosynthesis increased spinosyn yield significantly. Journal of Industrial Microbiology & Biotechnology (2001) 27, 399–402. Received 31 May 2001/ Accepted in revised form 09 July 2001     

Studies on the bacterial hemoglobin fromVitreoscilla

Summary Vitreoscilla contained a homodimeric bacterial hemoglobin (VtHb). The purification of this protein yielded VtmetHb which exhibited electronic and electron paramagnetic resonance (EPR) spectra, showing that it existed predominantly in a high-spin ferric form, both axial and rhombic components being present. The preparations also contained variable amounts of low-spin components. There was no evidence that these high-spin and low-spin forms were in equilibrium. The former were reducible by NADH catalyzed by the NADH-metVtHb reductase, and the latter were not. High ionic strength and high pH led to the formation of low-spin metVtHb; both treatments were reversible. Cyanide and imidazole liganded to VtHb resulted in the conversion of high-spin to low-spin ferric heme centers, each with characteristic electronic and EPR spectra. Some preparations of VtHb exhibited EPR signals consistent with a sulfur ligand bound to the ferric site. When VtHb was treated with NADH plus the reductase in the presence of oxygen, the intensity of the high-spin EPR signals decreased significantly. No reduction occurred in the absence of oxygen, suggesting a possible role for the superoxide anion. Dithionite treatment of VtHb resulted in a slow reduction, but the main product of the reaction of dithionite-reduced VtHb with oxygen was VtmetHb, not VtHbO₂. EPR spectra of whole cells ofVitreoscilla exhibited a variety of intense signals at low and high magnetic field, theg-values being consistent with the presence of high-spin ferric heme proteins, in addition to an iron-containing superoxide dismutase (FeSOD) and iron-sulfur proteins. EPR spectra of the cytosol fraction ofVitreoscilla showed the expected resonances for VtmetHb and FeSOD.Abbreviations A absorbance - DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetate - EPR electron paramagnetic resonance - HiPIP high-potential iron protein - SDS sodium dodecyl sulfate - SOD superoxide dismutase - VtHb Vitreoscilla hemoglobin - VtmetHb oxidizedVitreoscilla hemoglobin - VtHbO₂ oxygenatedVitreoscilla hemoglobin     

Engineering of ethanolic E. coli with the Vitreoscilla hemoglobin gene enhances ethanol production from both glucose and xylose

Escherichia coli strain FBR5, which has been engineered to direct fermentation of sugars to ethanol, was further engineered, using three different constructs, to contain and express the Vitreoscilla hemoglobin gene (vgb). The three resulting strains expressed Vitreoscilla hemoglobin (VHb) at various levels, and the production of ethanol was inversely proportional to the VHb level. High levels of VHb were correlated with an inhibition of ethanol production; however, the strain (TS3) with the lowest VHb expression (approximately the normal induced level in Vitreoscilla) produced, under microaerobic conditions in shake flasks, more ethanol than the parental strain (FBR5) with glucose, xylose, or corn stover hydrolysate as the predominant carbon source. Ethanol production was dependent on growth conditions, but increases were as high as 30%, 119%, and 59% for glucose, xylose, and corn stover hydrolysate, respectively. Only in the case of glucose, however, was the theoretical yield of ethanol by TS3 greater than that achieved by others with FBR5 grown under more closely controlled conditions. TS3 had no advantage over FBR5 regarding ethanol production from arabinose. In 2 L fermentors, TS3 produced about 10% and 15% more ethanol than FBR5 for growth on glucose and xylose, respectively. The results suggest that engineering of microorganisms with vgb/VHb could be of significant use in enhancing biological production of ethanol.     

Improvement of the ability to produce spinosad in <Emphasis Type="Italic">Saccharopolyspora spinosa</Emphasis> through the acquisition of drug resistance and genome shuffling

Spinosyns, a secondary metabolite from the fermentation of Saccharopolyspora spinosa, exhibits evident insecticidal activity. The most active components of the spinosyns family are spinosyns A and D, which are macrocyclic lactone antibiotics. Spinosad is a defined combination of the two principal fermentation factors, spinosyns A and D. Spinosad is used on grain storage, vegetable and fruit crops, ornamentals, and turf for pest control because it is toxic to many insects, but relatively nontoxic to mammals. In this study, we combined drug resistance screening and genome shuffling to achieve rapid improvement of spinosad yield of S. spinosa. The starting mutant population was generated by UV irradiation of S. spinosa ATCC 49460 protoplasts, which were then screened for erythromycin or neomycin resistance. Two mutant strains, Ery-13 (erythromycin resistant) and Neo-127 (neomycin resistant), were selected according to their spinosad yield. The highest titers of Ery-13 and Neo-127 strain reached 188 μg/ml and 165 μg/ml, respectively, which are 3.7-fold and 3.3-fold higher than that of the parental strain ATCC 49460. After four rounds of genome shuffling, an improved recombinant EN4-33 with both erythromycin and neomycin resistance was obtained. The highest spinosad yield of the recombinant EN4-33 reached 332 μg/ml, which is 6.6-fold higher than that of ATCC 49460. Results demonstrated that combining genome shuffling with antibiotics resistance screening is an effective approach for the molecular breeding of high-producing strains.     

Enhanced production of acetoin and butanediol in recombinant Enterobacter aerogenes carrying Vitreoscilla hemoglobin gene

Microbial production of butanediol and acetoin has received increasing interest because of their diverse potential practical uses. Although both products are fermentative in nature, their optimal production requires a low level of oxygen. In this study, the use of a recombinant oxygen uptake system on production of these metabolites was investigated. Enterobacter aerogenes was transformed with a pUC8-based plasmid carrying the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). The presence of vgb and production of VHb by this strain resulted in an increase in viability from 72 to 96 h in culture, but no overall increase in cell mass. Accumulation of the fermentation products acetoin and butanediol were enhanced (up to 83%) by the presence of vgb/VHb. This vgb/VHb related effect appears to be due to an increase of flux through the acetoin/butanediol pathway, but not at the expense of acid production.     

Four‐stage dissolved oxygen strategy based on multi‐scale analysis for improving spinosad yield by Saccharopolyspora spinosa ATCC49460

Dissolved oxygen (DO) is an important influencing factor in the process of aerobic microbial fermentation. Spinosad is an aerobic microbial‐derived secondary metabolite. In our study, spinosad was used as an example to establish a DO strategy by multi‐scale analysis, which included a reactor, cell and gene scales. We changed DO conditions that are related to the characteristics of cell metabolism (glucose consumption rate, biomass accumulation and spinosad production). Consequently, cell growth was promoted by maintaining DO at 40% in the first 24 h and subsequently increasing DO to 50% in 24 h to 96 h. In an in‐depth analysis of the key enzyme genes (gtt, spn A, spn K and spn O), expression of spinosad and specific Adenosine Triphosphate (ATP), the spinosad yield was increased by regulating DO to 30% within 96 h to 192 h and then changing it to 25% in 192 h to 240 h. Under the four‐phase DO strategy, spinosad yield increased by 652.1%, 326.1%, 546.8%, and 781.4% compared with the yield obtained under constant DO control at 50%, 40%, 30%, and 20% respectively. The proposed method provides a novel way to develop a precise DO strategy for fermentation.     

Butenyl-spinosyns, a natural example of genetic engineering of antibiotic biosynthetic genes

Spinosyns, a novel class of insect active macrolides produced by Saccharopolyspora spinosa, are used for insect control in a number of commercial crops. Recently, a new class of spinosyns was discovered from S. pogona NRRL 30141. The butenyl-spinosyns, also called pogonins, are very similar to spinosyns, differing in the length of the side chain at C-21 and in the variety of novel minor factors. The butenyl-spinosyn biosynthetic genes (bus) were cloned on four cosmids covering a contiguous 110-kb region of the NRRL 30141 chromosome. Their function in butenyl-spinosyn biosynthesis was confirmed by a loss-of-function deletion, and subsequent complementation by cloned genes. The coding sequences of the butenyl-spinosyn biosynthetic genes and the spinosyn biosynthetic genes from S. spinosa were highly conserved. In particular, the PKS-coding genes from S. spinosa and S. pogona have 91–94% nucleic acid identity, with one notable exception. The butenyl-spinosyn gene sequence codes for one additional PKS module, which is responsible for the additional two carbons in the C-21 tail. The DNA sequence of spinosyn genes in this region suggested that the S. spinosa spnA gene could have been the result of an in-frame deletion of the S. pogona busA gene. Therefore, the butenyl-spinosyn genes represent the putative parental gene structure that was naturally engineered by deletion to create the spinosyn genes.