Peter Meiser

Myxovirescin A biosynthesis is directed by hybrid polyketide synthases/nonribosomal peptide synthetase, 3-hydroxy-3-methylglutaryl-CoA synthases, and trans-acting acyltransferases.

Myxococcus xanthus DK1622 is shown to be a producer of myxovirescin (antibiotic TA) antibiotics. The myxovirescin biosynthetic gene cluster spans at least 21 open reading frames (ORFs) and covers a chromosomal region of approximately 83 kb. In silico analysis of myxovirescin ORFs in conjunction with genetic studies suggests the involvement of four type I polyketide synthases (PKSs; TaI, TaL, TaO, and TaP), one major hybrid PKS/NRPS (Ta‐1), and a number of monofunctional enzymes similar to the ones involved in type II fatty‐acid biosyntesis (FAB). Whereas deletion of either taI or taL causes a dramatic drop in myxovirescin production, deletion of both genes (ΔtaIL) leads to the complete loss of myxovirescin production. These results suggest that both TaI and TaL PKSs might act in conjunction with a methyltransferase, reductases, and a monooxygenase to produce the 2‐hydroxyvaleryl–S–ACP starter that is proposed to act as the biosynthetic primer in the initial condensation reaction with glycine. Polymerization of the remaining 11 acetates required for lactone formation is directed by 12 modules of Ta‐1, TaO, and TaP megasynthetases. All modules, except for the first module of TaL, lack cognate acyltransferase (AT) domains. Furthermore, deletion of a discrete tandem AT—encoded by taV—blocks myxovirescin production; this suggests an “in trans” mode of action. To embellish the macrocycle with methyl and ethyl moieties, assembly of the myxovirescin scaffold is proposed to switch twice from PKS to 3‐hydroxy‐3‐methylglutaryl–CoA (HMG–CoA)‐like biochemistry during biosynthesis. Disruption of the S‐adenosylmethionine (SAM)‐dependent methyltransferase, TaQ, shifts production toward two novel myxovirescin analogues, designated myxovirescin Qa and myxovirescin Qc. NMR analysis of purified myxovirescin Qa revealed the loss of the methoxy carbon atom. This novel analogue lacks bioactivity against E. coli.

The unique DKxanthene secondary metabolite family from the myxobacterium Myxococcus xanthus is required for developmental sporulation

Under starvation conditions myxobacteria form multicellular fruiting bodies in which vegetative cells differentiate into heat- and desiccation-resistant myxospores. Myxobacteria in general are a rich source of secondary metabolites that often exhibit biological activities rarely found in nature. Although the involvement of a yellow compound in sporulation and fruiting body formation of Myxococcus xanthus was described almost 30 years ago, the chemical principle of the pigment remained elusive. This work presents the isolation and structure elucidation of a unique class of pigments that were named DKxanthenes (DKX). The corresponding biosynthetic gene cluster was identified, and DKX-negative mutants were constructed to investigate the physiological role of DKX during development. In these mutants, fruiting body formation was delayed. Moreover, severely reduced amounts of viable spores were observed after 120 h of starvation, whereas no viable spores were formed at all after 72 h. The addition of purified DKX to the mutants resulted in the formation of viable spores after 72 h. Even though an antioxidative activity could be assigned to DKX, the true biochemical mechanism underlying the complementation remains to be elucidated.

Mutasynthesis-derived myxalamids and origin of the isobutyryl-CoA starter unit of myxalamid B.

Myxalamids are potent inhibitors of the eukaryotic electron transport chain produced by different myxobacteria. Here, we describe the identification of the myxalamid biosynthesis gene cluster from Myxococcus xanthus. Additionally, new myxalamids (5–13) have been obtained by mutasynthesis from bkd mutants of M. xanthus and Stigmatella aurantiaca. Moreover, as these bkd mutants are still able to produce myxalamid B (2), the origin of the isobutyryl‐CoA (IB‐CoA) starter unit required for its biosynthesis has been determined. In a M. xanthus bkd mutant, IB‐CoA originates from valine, but in S. aurantiaca this starter unit is derived from α‐oxidation of iso‐odd fatty acids, thereby connecting primary and secondary metabolism.

Two functionally redundant Sfp-type 4'-phosphopantetheinyl transferases differentially activate biosynthetic pathways in Myxococcus xanthus.

Phosphopantetheinyl transferases (PPTases) represent a superfamily of enzymes that are essential for the post-translational modification of carrier proteins involved in the biosynthesis of primary and secondary metabolites, such as fatty acids, polyketides and nonribosomal peptides. These carrier proteins are activated by the transfer of a 4’-phosphopantetheine (Ppant) cofactor from coenzyme A to a conserved serine residue of the apo form of the protein, in a Mg-dependent reaction. Chain-extension intermediates are bound to the protein by a thioester linkage to the Ppant prosthetic group; this enables them to be shuttled around the individual active sites of the multienzyme complexes. Several organisms are known to possess multiple types of PPTase enzymes, which exhibit specificity for distinct biosynthetic pathways. Acyl carrier protein synthase (AcpS)-type PPTases usually activate fatty acid synthases (FASs) and type II polyketide synthases (PKSs). These enzymes consist of approximately 120 amino acids, and exhibit a homotrimeric structure. 3] In contrast, Sfp-type PPTases (named after the prototype PPTase Sfp from Bacillus subtilis) typically modify carrier proteins that are responsible for the biosynthesis of secondary metabolites, such as type I PKS, nonribosomal peptide synthetase (NRPS) systems and their hybrids. Furthermore, Sfp-type PPTases have also been shown to participate in fungal lysine biosynthesis, b-alanine conjugation and cyanobacterial heterocyst differentiation. Sfp-type PPTases are approximately twice the size of AcpStype PPTases, which suggests that they evolved by gene duplication from an AcpS ancestor. Sfp exhibits broad substrate tolerance towards different types of carrier proteins, a feature that has enabled its exploitation in various biotechnological applications. Indeed, some genomes contain only a Sfp-type PPTase; this strongly supports its function in both primary and secondary metabolism. For example, inactivation of the single PPTase-encoding gene pcpS in Pseudomonas aeruginosa could only be accomplished when a copy of the E. coli acpS gene was simultaneously introduced in trans on the chromosome. In contrast, disruption of the Sfp-type PPTase gene mtaA in Stigmatella aurantiaca DW4/3-1 was not lethal, due to the presence of at least one additional (AcpS-type) PPTase (Figure 1 C), but production of all known secondary metabolites was abolished. In E. coli, the defect caused by a mutated, dysfunctional AcpS could be restored by over-expression of a second E. coli PPTase, YhhU, the exact function of which has yet to be determined.

Heterologous expression of the plant cysteine protease bromelain and its inhibitor in Pichia pastoris

Expression of proteases in heterologous hosts remains an ambitious challenge due to severe problems associated with digestion of host proteins. On the other hand, proteases are broadly used in industrial applications and resemble promising drug candidates. Bromelain is an herbal drug that is medicinally used for treatment of oedematous swellings and inflammatory conditions and consists in large part of proteolytic enzymes. Even though various experiments underline the requirement of active cysteine proteases for biological activity, so far no investigation succeeded to clearly clarify the pharmacological mode of action of bromelain. The potential role of proteases themselves and other molecules of this multi‐component extract currently remain largely unknown or ill defined. Here, we set out to express several bromelain cysteine proteases as well as a bromelain inhibitor molecule in order to gain defined molecular entities for subsequent studies. After cloning the genes from its natural source Ananas comosus (pineapple plant) into Pichia pastoris and subsequent fermentation and purification, we obtained active protease and inhibitor molecules which were subsequently biochemically characterized. Employing purified bromelain fractions paves the way for further elucidation of pharmacological activities of this natural product.