Biomedical subjects

Juha Hakala

Publications and source records attributed to Juha Hakala.

5 recordsLinked to original sources

PubMed2003

Studies on a second and third ring cyclization in anthracycline biosynthesis.

This paper focuses on study of second and third ring cyclization in anthracycline biosynthesis by a heterologous gene expression. Firstly, anthracycline non-producing Streptomyces peucetius mutant, D2 was heterologously complemented to produce daunomycins with plasmids pSgs44 and pSYE66, which contain putative cyclase genes of S. galilaeus and S. nogalater, respectively. A point mutation in the cyclase gene dpsY of D2 has changed glycine to serine resulting inactivation of the enzyme. Secondly, the putative cyclase gene snoaM from S. nogalater, was expressed in a gene cassette in S. lividans TK24 and S. coelicolor CH999 to study the influence of the cyclase gene on auramycinone production and the impact of endogenous genes on production profiles. The results obtained confirms that a cyclase closing the second and third ring of a polyketide is essential in anthracycline biosynthesis.

Anthracyclines↗

PubMed2002

Cloning and characterization of Streptomyces galilaeus aclacinomycins polyketide synthase (PKS) cluster.

We have cloned and sequenced polyketide synthase (PKS) genes from the aclacinomycin producer Streptomyces galilaeus ATCC 31,615. The sequenced 13.5-kb region contained 13 complete genes. Their organization as well as their protein sequences showed high similarity to those of other type II PKS genes. The continuous region included the genes for the minimal PKS, consisting of ketosynthase I (aknB), ketosynthase II (aknC), and acyl carrier protein (aknD). These were followed by the daunomycin dpsC and dpsD homologues (aknE2 and F, respectively), which are rare in type II PKS clusters. They are associated with the unusual starter unit, propionate, used in the biosynthesis of aklavinone, a common precursor of aclacinomycin and daunomycin. Accordingly, when aclacinomycins minimal PKS genes were substituted for those of nogalamycin in the plasmid carrying genes for auramycinone biosynthesis, aklavinone was produced in the heterologous hosts. In addition to the minimal PKS, the cloned region included the PKS genes for polyketide ketoreductase (aknA), aromatase (aknE1) and oxygenase (aknX), as well as genes putatively encoding an aklanonic acid methyl transferase (aknG) and an aklanonic acid methyl ester cyclase (aknH) for post-polyketide steps were found. Moreover, the region carried genes for an activator (aknI), a glycosyl transferase (aknK) and an epimerase (aknL) taking part in deoxysugar biosynthesis.

Aclarubicin↗

PubMed2002

Characterization of mutations in aclacinomycin A-non-producing Streptomyces galilaeus strains with altered glycosylation patterns.

In this study a set of Streptomyces galilaeus ATCC 31615 mutants was characterized, which are incapable of synthesizing some or all of the deoxyhexose sugars of aclacinomycin A. Complementation experiments with the the mutant strains H026, H038, H039, H054, H063, H065 and H075 were carried out with glycosylation genes previously derived from the wild-type S. galilaeus. Mutations in strains H038, H063 and H075 were complemented with single PCR-amplified genes. Furthermore, amplification and sequencing of the corresponding genes from the mutant strains revealed single point mutations in the sequences. First, in H038 a transition mutation in aknQ, encoding a putative dTDP-hexose 3-ketoreductase, causes an amino acid substitution from glycine to aspartate, suppressing the biosynthesis of both 2-deoxyfucose and rhodinose and thus leading to the accumulation of aclacinomycin T with rhodosamine as its only sugar. Second, in H063, which accumulates aklavinone without a sugar moiety, amino acid substitution occurs, with threonine being substituted by isoleucine in dTDP-glucose synthase, the first enzyme participating in deoxyhexose biosynthesis, encoded by aknY. Third, a nonsense mutation in aknP leads to truncated dTDP-hexose 3-dehydratase in H075, which is incapable of synthesizing rhodinose. In addition, mutants H054 and H065, which accumulate aclacinomycins without aminosugars, were complemented by a gene for an aminotransferase, aknZ. Characterization of the nature of the mutations adds to the usefulness and value of the mutants in the analysis of gene function and in the creation of novel compounds by combinatorial biosynthesis. Furthermore, these results strengthen the assignments of akn gene products and enlighten the biosynthetic pathway for deoxyhexoses.

Aclarubicin↗

PubMed2002

Molecular evolution of aromatic polyketides and comparative sequence analysis of polyketide ketosynthase and 16S ribosomal DNA genes from various streptomyces species.

A 613-bp fragment of an essential ketosynthase gene from the biosynthetic pathway of aromatic polyketide antibiotics was sequenced from 99 actinomycetes isolated from soil. Phylogenetic analysis showed that the isolates clustered into clades that correspond to the various classes of aromatic polyketides. Additionally, sequencing of a 120-bp fragment from the gamma-variable region of 16S ribosomal DNA (rDNA) and subsequent comparative sequence analysis revealed incongruity between the ketosynthase and 16S rDNA phylogenetic trees, which strongly suggests that there has been horizontal transfer of aromatic polyketide biosynthesis genes. The results show that the ketosynthase tree could be used for DNA fingerprinting of secondary metabolites and for screening interesting aromatic polyketide biosynthesis genes. Furthermore, the movement of the ketosynthase genes suggests that traditional marker molecules like 16S rDNA give misleading information about the biosynthesis potential of aromatic polyketides, and thus only molecules that are directly involved in the biosynthesis of secondary metabolites can be used to gain information about the biodiversity of antibiotic production in different actinomycetes.

Anti-Bacterial Agents↗

PubMed2000

Elucidation of anthracyclinone biosynthesis by stepwise cloning of genes for anthracyclines from three different Streptomyces spp.

The anthracycline skeleton is biosynthesized by aromatic (type II) polyketide synthases. Furthermore, three post-polyketide steps are needed to form the basic aglycone of anthracyclines. Auramycinone was produced in Streptomyces lividans by introducing nine structural genes from three different anthracycline-producing Streptomyces species. The genes used to construct the auramycinone biosynthesis cluster were derived from nogalamycin-, daunomycin- and aclacinomycin-producing Streptomyces strains. The biosynthetic stages were divided into polyketide and post-polyketide steps on the assumption that the first stable intermediate would be nogalonic acid, named analogously to aklanonic acid, the precursor of several anthracyclines. Single genes were cloned in the expression construct in the order determined by the proposed biosynthetic pathway. This facilitated investigation of the products formed in the heterologous host after addition of each separate gene to the construct. The results thus elucidate the biosynthesis steps, products and the genes responsible for the reactions needed to build up an anthracyclinone.

Anthracyclines↗