Because the sugar moieties of natural products are primarily O-linked, the hydrolytic sensitivity... more Because the sugar moieties of natural products are primarily O-linked, the hydrolytic sensitivity of the glycosidic linkage limits their therapeutic application. One potential solution to this problem is to replace the labile O-glycosidic bond with an enzymatically and chemically stable C-glycosidic bond. In this study, computational analysis of the O-glycosyltransferase LanGT2 and the C-glycosyltransferase UrdGT2 was used to predict the changes necessary to switch the O-glycosylating enzyme to a C-glycosyltransferase. By screening rationally designed LanGT2 mutants a number of LanGT2 variants with C-glycosyltransferase activity were identified. One variant, having 10 amino acid substitutions, revealed the primary region that determines O-versus C-glycosylation. By modeling the active site of this mutant and probing the role of active site residues with alanine substitutions, this work also illuminates the mechanistic features of O-and C-glycosylation.
Angewandte Chemie (International ed. in English), Jan 7, 2015
The structures of the O-glycosyltransferase LanGT2 and the engineered, CC bond-forming variant L... more The structures of the O-glycosyltransferase LanGT2 and the engineered, CC bond-forming variant LanGT2S8Ac show how the replacement of a single loop can change the functionality of the enzyme. Crystal structures of the enzymes in complex with a nonhydrolyzable nucleotide-sugar analogue revealed that there is a conformational transition to create the binding sites for the aglycon substrate. This induced-fit transition was explored by molecular docking experiments with various aglycon substrates.
Inducible expression is a versatile genetic tool for controlling gene transcription, determining ... more Inducible expression is a versatile genetic tool for controlling gene transcription, determining gene functions and other uses. Herein, we describe our attempts to create several inducible systems based on a cumate or a resorcinol switch, a hammerhead ribozyme, the LacI repressor, and isopropyl β-dthiogalactopyranoside (IPTG). We successfully developed a new cumate (p-isopropylbenzoic acid)-inducible gene switch in actinobacteria that is based on the CymR regulator, the operator sequence (cmt) from the Pseudomonas putida cumate degradation operon and P21 synthetic promoter. Resorcinol-inducible expression system is also functional and is composed of the RolR regulator and the PA3 promoter fused with the operator (rolO) from the Corynebacterium glutamicum resorcinol catabolic operon. Using the gusA (βglucuronidase) gene as a reporter, we showed that the newly generated expression systems are tightly regulated and hyperinducible. The activity of the uninduced promoters is negligible in both cases. Whereas the induction factor reaches 45 for Streptomyces albus in the case of cumate switch and 33 in the case of resorcinol toggle. The systems are also dosedependent, which allows the modulation of gene expression even from a single promoter. In addition, the cumate system is versatile, given that it is functional in different actinomycetes. Finally, these systems are nontoxic and inexpensive, as these are characteristics of cumate and resorcinol, and they are easy to use because inducers are water-soluble and easily penetrate cells. Therefore, the P21-cmt-CymR and PA3-rolO-RolR systems are powerful tools for engineering actinobacteria.
Pathogenic antibiotic-resistant bacteria are an unprecedented threat to health care worldwide. Th... more Pathogenic antibiotic-resistant bacteria are an unprecedented threat to health care worldwide. The range of antibiotics active against these bacteria is narrow; it includes teicoplanin, a "last resort" drug, which is produced by the filamentous actinomycete Actinoplanes teichomyceticus. In this report, we determine the functions of tei15* and tei16*, pathway-specific regulatory genes that code for StrR-and LuxR-type transcriptional factors, respectively. The products of these genes are master switches of teicoplanin biosynthesis, since their inactivation completely abolished antibiotic production. We show that Tei15* positively regulates the transcription of at least 17 genes in the cluster, whereas the targets of Tei16* still remain unknown. Integration of tei15* or tei16* under the control of the aminoglycoside resistance gene aac(3)IV promoter into attBϕC31 site of the A. teichomyceticus chromosome increased teicoplanin productivity to nearly 1 g/L in TM1 industrial medium. The expression of these genes from the moderate copy number episomal vector pKC1139 led to 3-4 g/L teicoplanin, while under the same conditions, wild type produced approximately 100 mg/L. This shows that a significant increase in teicoplanin production can be achieved by a single step of genetic manipulation of the wild-type strain by increasing the expression of the tei regulatory genes. This confirms that natural product yields can be increased using rational engineering once suitable genetic tools have been developed. We propose that this new technology for teicoplanin overproduction might now be transferred to industrial mutants of A. teichomyceticus.
Actinoplanes teichomyceticus is the only known producer of the valuable glycopeptide antibiotic t... more Actinoplanes teichomyceticus is the only known producer of the valuable glycopeptide antibiotic teicoplanin. Random mutagenesis and selection were extensively applied to teicoplanin producers, while the gene engineering methods were not used, because of the paucity of genetic tools for A. teichomyceticus. Particularly, availability of promoters of different strength that are functional in Actinoplanes would be very useful for overexpression of beneficial genes. Here we report the use of a glucuronidase reporter system (gusA) for studying transcriptional activity in A. teichomyceticus and describe the behavior of a set of heterologous promoters in this strain. We reveal several elements that exceed in their strength the wellestablished Streptomyces promoter ermEp, underscoring the utility of the gusA reporter for Actinoplanes sp. Remarkable overproduction of teicoplanin was achieved by constructing strains carrying additional copies of the regulatory gene tcp28 under the control of one of the two most active promoters, moeE5p and actp, discovered in this study.
Streptomycetes are typical soil-dwelling bacteria with intricate morphological and biochemical di... more Streptomycetes are typical soil-dwelling bacteria with intricate morphological and biochemical differentiation of colonies, resulting in onset of secondary metabolite production. Compounds produced during these stages of differentiation comprise nearly two-thirds of bioactive molecules synthesized by microorganisms, including antibiotics, antitumor agents and immunosuppressants. 1,2 The biosynthesis of antibiotics and other secondary metabolites is controlled by interactions of both global and pathway-specific regulators. However, in all cases, the influence of the environment is reflected by the activity of pathway-specific regulatory genes, which are located within a biosynthetic gene cluster and which control the expression of biosynthetic genes. 3 On account of high structural and functional similarity, such regulators were grouped into two families, namely Streptomyces antibiotics regulatory proteins (SARP), mostly found within the aromatic polyketides clusters, and large ATP-binding regulators of LuxR family (LAL), controlling the production of macrolides and glycopeptides. 4-6 Recent efforts in cloning and characterization of biosynthetic gene clusters for new groups of secondary metabolites revealed novel classes of transcriptional factors that differ from typical SARPs or LAL representatives. Avilamycins are secondary metabolites produced by Streptomyces viridochromogenes Tu57. They are active against Gram-positive bacteria. It is known that avilamycins bind to the 23S rRNA in the region proximal to the channel, where tRNA enters the A-site and blocks the protein synthesis. 9 Avilamycin resistance is mediated by 23S rRNA methylation and active avilamycin transport. The avilamycin resistance genes together with the structural genes were identified and cloned as an entire biosynthetic gene cluster containing 54 open reading frames. 11 The biosynthetic steps leading to the formation of the avilamycin molecule were established by the analysis of secondary metabolites produced by mutants. 11,12 Within the cluster also, two putative regulatory genes named aviC1 and aviC2 were identified. Both genes are located in close proximity to each other and are transcribed in the same direction ( ). Genes are separated by a 271-bp non-coding region. We succeeded in identifying the presence of putative bacterial promoter sequences upstream of aviC1 initiation codon by the use of BPROM bacterial promoter prediction server (Softberry Inc., Mount Kisco, NY, USA). It consists of a putative À10 box GGTTTTCAT (Score 34) and a À35 box ATGCGA (Score 12). Another putative promoter sequence is located upstream of the aviC2 translation start site consisting of a À10 box GCCCATGAT (Score 31) and a À35 box at TTTCTA (Score 34) similar to consensus Streptomycetes promoters. 13
Analysis of the α-lipomycin biosynthesis gene cluster of Streptomyces aureofaciens Tü117 led to t... more Analysis of the α-lipomycin biosynthesis gene cluster of Streptomyces aureofaciens Tü117 led to the identification of five putative regulatory genes, which are congregated into a subcluster. Analysis of the lipReg1-4 and lipX1 showed that they encode components of twocomponent signal transduction systems (LipReg1 and LipReg2), multiple antibiotics resistance-type regulator (LipReg3), large ATP-binding regulators of the LuxR family-type regulator (LipReg4), and small ribonuclease (LipRegX1), respectively. A combination of targeted gene disruptions, complementation experiments, lipomycin production studies, and gene expression analysis via RT-PCR suggests that all regulatory lip genes are involved in αlipomycin production. On the basis of the obtained data, we propose that LipReg2 controls the activity of LipReg1, which in its turn govern the expression of the α-lipomycin pathway-specific regulatory gene lipReg4. The ribonuclease gene lipX1 and the transporter regulator lipReg3 appear to work independently of genes lipReg1, lipReg2, and lipReg4.
Because the sugar moieties of natural products are primarily O-linked, the hydrolytic sensitivity... more Because the sugar moieties of natural products are primarily O-linked, the hydrolytic sensitivity of the glycosidic linkage limits their therapeutic application. One potential solution to this problem is to replace the labile O-glycosidic bond with an enzymatically and chemically stable C-glycosidic bond. In this study, computational analysis of the O-glycosyltransferase LanGT2 and the C-glycosyltransferase UrdGT2 was used to predict the changes necessary to switch the O-glycosylating enzyme to a C-glycosyltransferase. By screening rationally designed LanGT2 mutants a number of LanGT2 variants with C-glycosyltransferase activity were identified. One variant, having 10 amino acid substitutions, revealed the primary region that determines O-versus C-glycosylation. By modeling the active site of this mutant and probing the role of active site residues with alanine substitutions, this work also illuminates the mechanistic features of O-and C-glycosylation.
Angewandte Chemie (International ed. in English), Jan 7, 2015
The structures of the O-glycosyltransferase LanGT2 and the engineered, CC bond-forming variant L... more The structures of the O-glycosyltransferase LanGT2 and the engineered, CC bond-forming variant LanGT2S8Ac show how the replacement of a single loop can change the functionality of the enzyme. Crystal structures of the enzymes in complex with a nonhydrolyzable nucleotide-sugar analogue revealed that there is a conformational transition to create the binding sites for the aglycon substrate. This induced-fit transition was explored by molecular docking experiments with various aglycon substrates.
Inducible expression is a versatile genetic tool for controlling gene transcription, determining ... more Inducible expression is a versatile genetic tool for controlling gene transcription, determining gene functions and other uses. Herein, we describe our attempts to create several inducible systems based on a cumate or a resorcinol switch, a hammerhead ribozyme, the LacI repressor, and isopropyl β-dthiogalactopyranoside (IPTG). We successfully developed a new cumate (p-isopropylbenzoic acid)-inducible gene switch in actinobacteria that is based on the CymR regulator, the operator sequence (cmt) from the Pseudomonas putida cumate degradation operon and P21 synthetic promoter. Resorcinol-inducible expression system is also functional and is composed of the RolR regulator and the PA3 promoter fused with the operator (rolO) from the Corynebacterium glutamicum resorcinol catabolic operon. Using the gusA (βglucuronidase) gene as a reporter, we showed that the newly generated expression systems are tightly regulated and hyperinducible. The activity of the uninduced promoters is negligible in both cases. Whereas the induction factor reaches 45 for Streptomyces albus in the case of cumate switch and 33 in the case of resorcinol toggle. The systems are also dosedependent, which allows the modulation of gene expression even from a single promoter. In addition, the cumate system is versatile, given that it is functional in different actinomycetes. Finally, these systems are nontoxic and inexpensive, as these are characteristics of cumate and resorcinol, and they are easy to use because inducers are water-soluble and easily penetrate cells. Therefore, the P21-cmt-CymR and PA3-rolO-RolR systems are powerful tools for engineering actinobacteria.
Pathogenic antibiotic-resistant bacteria are an unprecedented threat to health care worldwide. Th... more Pathogenic antibiotic-resistant bacteria are an unprecedented threat to health care worldwide. The range of antibiotics active against these bacteria is narrow; it includes teicoplanin, a "last resort" drug, which is produced by the filamentous actinomycete Actinoplanes teichomyceticus. In this report, we determine the functions of tei15* and tei16*, pathway-specific regulatory genes that code for StrR-and LuxR-type transcriptional factors, respectively. The products of these genes are master switches of teicoplanin biosynthesis, since their inactivation completely abolished antibiotic production. We show that Tei15* positively regulates the transcription of at least 17 genes in the cluster, whereas the targets of Tei16* still remain unknown. Integration of tei15* or tei16* under the control of the aminoglycoside resistance gene aac(3)IV promoter into attBϕC31 site of the A. teichomyceticus chromosome increased teicoplanin productivity to nearly 1 g/L in TM1 industrial medium. The expression of these genes from the moderate copy number episomal vector pKC1139 led to 3-4 g/L teicoplanin, while under the same conditions, wild type produced approximately 100 mg/L. This shows that a significant increase in teicoplanin production can be achieved by a single step of genetic manipulation of the wild-type strain by increasing the expression of the tei regulatory genes. This confirms that natural product yields can be increased using rational engineering once suitable genetic tools have been developed. We propose that this new technology for teicoplanin overproduction might now be transferred to industrial mutants of A. teichomyceticus.
Actinoplanes teichomyceticus is the only known producer of the valuable glycopeptide antibiotic t... more Actinoplanes teichomyceticus is the only known producer of the valuable glycopeptide antibiotic teicoplanin. Random mutagenesis and selection were extensively applied to teicoplanin producers, while the gene engineering methods were not used, because of the paucity of genetic tools for A. teichomyceticus. Particularly, availability of promoters of different strength that are functional in Actinoplanes would be very useful for overexpression of beneficial genes. Here we report the use of a glucuronidase reporter system (gusA) for studying transcriptional activity in A. teichomyceticus and describe the behavior of a set of heterologous promoters in this strain. We reveal several elements that exceed in their strength the wellestablished Streptomyces promoter ermEp, underscoring the utility of the gusA reporter for Actinoplanes sp. Remarkable overproduction of teicoplanin was achieved by constructing strains carrying additional copies of the regulatory gene tcp28 under the control of one of the two most active promoters, moeE5p and actp, discovered in this study.
Streptomycetes are typical soil-dwelling bacteria with intricate morphological and biochemical di... more Streptomycetes are typical soil-dwelling bacteria with intricate morphological and biochemical differentiation of colonies, resulting in onset of secondary metabolite production. Compounds produced during these stages of differentiation comprise nearly two-thirds of bioactive molecules synthesized by microorganisms, including antibiotics, antitumor agents and immunosuppressants. 1,2 The biosynthesis of antibiotics and other secondary metabolites is controlled by interactions of both global and pathway-specific regulators. However, in all cases, the influence of the environment is reflected by the activity of pathway-specific regulatory genes, which are located within a biosynthetic gene cluster and which control the expression of biosynthetic genes. 3 On account of high structural and functional similarity, such regulators were grouped into two families, namely Streptomyces antibiotics regulatory proteins (SARP), mostly found within the aromatic polyketides clusters, and large ATP-binding regulators of LuxR family (LAL), controlling the production of macrolides and glycopeptides. 4-6 Recent efforts in cloning and characterization of biosynthetic gene clusters for new groups of secondary metabolites revealed novel classes of transcriptional factors that differ from typical SARPs or LAL representatives. Avilamycins are secondary metabolites produced by Streptomyces viridochromogenes Tu57. They are active against Gram-positive bacteria. It is known that avilamycins bind to the 23S rRNA in the region proximal to the channel, where tRNA enters the A-site and blocks the protein synthesis. 9 Avilamycin resistance is mediated by 23S rRNA methylation and active avilamycin transport. The avilamycin resistance genes together with the structural genes were identified and cloned as an entire biosynthetic gene cluster containing 54 open reading frames. 11 The biosynthetic steps leading to the formation of the avilamycin molecule were established by the analysis of secondary metabolites produced by mutants. 11,12 Within the cluster also, two putative regulatory genes named aviC1 and aviC2 were identified. Both genes are located in close proximity to each other and are transcribed in the same direction ( ). Genes are separated by a 271-bp non-coding region. We succeeded in identifying the presence of putative bacterial promoter sequences upstream of aviC1 initiation codon by the use of BPROM bacterial promoter prediction server (Softberry Inc., Mount Kisco, NY, USA). It consists of a putative À10 box GGTTTTCAT (Score 34) and a À35 box ATGCGA (Score 12). Another putative promoter sequence is located upstream of the aviC2 translation start site consisting of a À10 box GCCCATGAT (Score 31) and a À35 box at TTTCTA (Score 34) similar to consensus Streptomycetes promoters. 13
Analysis of the α-lipomycin biosynthesis gene cluster of Streptomyces aureofaciens Tü117 led to t... more Analysis of the α-lipomycin biosynthesis gene cluster of Streptomyces aureofaciens Tü117 led to the identification of five putative regulatory genes, which are congregated into a subcluster. Analysis of the lipReg1-4 and lipX1 showed that they encode components of twocomponent signal transduction systems (LipReg1 and LipReg2), multiple antibiotics resistance-type regulator (LipReg3), large ATP-binding regulators of the LuxR family-type regulator (LipReg4), and small ribonuclease (LipRegX1), respectively. A combination of targeted gene disruptions, complementation experiments, lipomycin production studies, and gene expression analysis via RT-PCR suggests that all regulatory lip genes are involved in αlipomycin production. On the basis of the obtained data, we propose that LipReg2 controls the activity of LipReg1, which in its turn govern the expression of the α-lipomycin pathway-specific regulatory gene lipReg4. The ribonuclease gene lipX1 and the transporter regulator lipReg3 appear to work independently of genes lipReg1, lipReg2, and lipReg4.
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Papers by Liliya Horbal