Synthetic biology and metabolic engineering of actinomycetes for natural product discovery

Actinomycetes are one of the most valuable sources of natural products with industrial and medicinal importance. After more than half a century of exploitation, it has become increasingly challenging to find novel natural products with useful properties as the same known compounds are often repeatedly re-discovered when using traditional approaches. Modern genome mining approaches have led to the discovery of new biosynthetic gene clusters, thus indicating that actinomycetes still harbor a huge unexploited potential to produce novel natural products. In recent years, innovative synthetic biology and metabolic engineering tools have greatly accelerated the discovery of new natural products and the engineering of actinomycetes. In the first part of this review, we outline the successful application of metabolic engineering to optimize natural product production, focusing on the use of multi-omics data, genome-scale metabolic models, rational approaches to balance precursor pools, and the engineering of regulatory genes and regulatory elements. In the second part, we summarize the recent advances of synthetic biology for actinomycetal metabolic engineering including cluster assembly, cloning and expression, CRISPR/Cas9 technologies, and chassis strain development for natural product overproduction and discovery. Finally, we describe new advances in reprogramming biosynthetic pathways through polyketide synthase and non-ribosomal peptide synthetase engineering. These new developments are expected to revitalize discovery and development of new natural products with medicinal and other industrial applications.     

Poly(ADP-ribose) polymerase-1: association with nuclear lamins in rodent liver cells

The distribution of poly(ADP-ribose) polymerase-1 (PARP-1) over different nuclear compartments was studied by nuclear fractionation procedures and Western analysis revealing a prominent role of the nuclear matrix. This structure is operationally defined by the solubility properties of the A- and B-type lamins under defined experimental conditions. We consistently observed that most of the nuclear matrix-associated PARP-1 partitioned, in an active form, with the insoluble, lamin-enriched protein fractions that were prepared by a variety of established biochemical procedures. These PARP-1-protein interactions resisted salt extraction, disulfide reduction, RNase and DNase digestion. An inherent ability of PARP-1 to reassemble with the lamins became evident after a cycle of solubilization/dialysis using either urea or Triton X-100 and disulfide reduction, indicating that these interactions were dominated by hydrophobic forces. Together with in vivo crosslinking and co-immunoprecipitation experiments our results show that the lamins are prominent PARP-1-binding partners which could contribute to the functional sequestration of the enzyme on the nuclear matrix.     

Brownian dynamics simulation of helix-capping motifs

Helix-capping motifs are believed to play an important role in stabilizing alpha-helices and defining helix start and stop signals. We performed microsecond scale Brownian dynamics simulations to study ten XAAD sequences, with X = (A,E,I,L,N,Q,S,T,V,Y), to examine their propensity to form helix capping motifs and correlate these results with those obtained from analyzing a structural database of proteins. For the widely studied capping box motif S**D, where the asterisk can be any amino acid residue, the simulations suggested that one of the two hydrogen bonds proposed earlier as a stabilizing factor might not be as important. On the other hand, side-chain interactions between the capping residue and the third residue downstream on the polypeptide chain might also play a role in stabilizing this motif. These results are consistent with explicit-solvent molecular dynamics simulations of two capping box motifs found in the proteins BPTI and alpha-dendrotoxin. Principal component analysis of the SAAD trajectory showed that the first three principal components, after those corresponding to translational-rotational motion were removed, accounted for more than half of the conformational fluctuations. The first component separated the conformational space into two parts with the all-helical conformation and the capping box motif lying largely in one part. The second component, on the other hand, could be used to describe conformational transitions between the all-helical form and the capping box motif.     

Retinoid signalling and gene expression in neuroblastoma cells: RXR agonist and antagonist effects on CRABP-II and RARbeta expression

9-cis Retinoic acid (RA) induces gene expression in neuroblastoma cells more effectively and with different kinetics than other RA isomers, and could be acting in part through Retinoid X Receptors (RXRs). The aim of this study was to characterise the effects of an RXR agonist and RXR homodimer antagonist on the induction of cellular RA binding protein II (CRABP-II) and RA receptor-beta (RARbeta) in neuroblastoma cells in response to different retinoids. The RXR agonist, LDG1069, was as effective as all-trans RA in inducing gene expression, but less effective than 9-cis RA. The RXR-homodimer antagonist, LG100754, inhibited the induction of CRABP-II mRNA in SH-SY5Y neuroblastoma cells by 9-cis RA or the RXR-specific agonist LGD1069, but had no effect when used with all-trans RA. Conversely, LG100754 did not inhibit induction of RARbeta mRNA by 9-cis or all-trans RA, or by LGD1069. RAR- and RXR-specific ligands used together induced CRABP-II and RARbeta as effectively as 9-cis RA. These results demonstrate the value of combining RXR- and RAR-specific ligands to regulate RA-inducible gene expression. The possibility that RXR-homodimers mediate, in part, the induction of CRABP-II by 9-cis RA and RXR-specific ligands is discussed.     

Interhelical hydrogen bonds and spatial motifs in membrane proteins: polar clamps and serine zippers

Polar and ionizable amino acid residues are frequently found in the transmembrane (TM) regions of membrane proteins. In this study, we show that they help to form extensive hydrogen bond connections between TM helices. We find that almost all TM helices have interhelical hydrogen bonding. In addition, we find that a pair of contacting TM helices is packed tighter when there are interhelical hydrogen bonds between them. We further describe several spatial motifs in the TM regions, including "Polar Clamp" and "Serine Zipper," where conserved Ser residues coincide with tightly packed locations in the TM region. With the examples of halorhodopsin, calcium-transporting ATPase, and bovine cytochrome c oxidase, we discuss the roles of hydrogen bonds in stabilizing helical bundles in polytopic membrane proteins and in protein functions.     

Pigmentation pattern formation in butterflies: experiments and models

Butterfly pigmentation patterns are one of the most spectacular and vivid examples of pattern formation in biology. They have attracted much attention from experimentalists and theoreticians, who have tried to understand the underlying genetic, chemical and physical processes that lead to patterning. In this paper, we present a brief review of this field by first considering the generation of the localised, eyespot, patterns and then the formation of more globally controlled patterns. We present some new results applied to pattern formation on the wing of the mimetic butterfly Papilio dardanus.     

NMR analysis of cleaved Escherichia coli thioredoxin (1-73/74-108) and its P76A variant: cis/trans peptide isomerization

Inspection of high resolution three-dimensional (3D) structures from the protein database reveals an increasing number of cis-Xaa-Pro and cis-Xaa-Yaa peptide bonds. However, we are still far from being able to predict whether these bonds will remain cis upon single-site substitution of Pro or Yaa and/or cleavage of a peptide bond close to it in the sequence. We have chosen oxidized Escherichia coli thioredoxin (Trx), a member of the Trx superfamily with a single alpha/beta domain and cis P76 to determine the effect of single-site substitution and/or cleavage on this isomer. Standard two-dimensional (2D) NMR analysis were performed on cleaved Trx (1-73/74-108) and its P76A variant. Analysis of the NOE connectivities indicates remarkable similarity between the secondary and supersecondary structure of the noncovalent complexes and Trx. Analysis of the 2D version of the HCCH-TOCSY and HMQC-NOESY-HMQC and 13C-filtered HMQC-NOESY spectra of cleaved Trx with uniformly 13C-labeled 175 and P76 shows surprising conservation of both cis P76 and packing of 175 against W31. A similar NMR analysis of its P76A variant provides no evidence for cis A76 and shows only subtle local changes in both the packing of 175 and the interstrand connectivities between its most protected hydrophobic strands (beta2 and beta4). Indeed, a molecular simulation model for the trans P76A variant of Trx shows only subtle local changes around the substitution site. In conclusion, cleavage of R73 is insufficient to provoke cis/trans isomerization of P76, but cleavage and single-site substitution (P76A) favors the trans isomer.     

Enhancement ofcis,cis-muconate productivity by overexpression of catechol 1,2-dioxygenase inPseudomonas putida BCM114

For enhancement ofcis,cis-muconate productivity from benzoate, catechol 1,2-dioxygenase (C12O) which catalyzes the rate-limiting step (catechol conversion tocis,cis-muconate) was cloned and expressed in recombinantPseudomonas putida BCM114. At higher benzoate concentrations (more than 15 mM),cis,cis-muconate productivity gradually decreased and unconverted catechol was accumulated up to 10 mM in the case of wildtypeP. putida BM014, whereascis,cis-muconate productivity continuously increased and catechol was completely transformed tocis,cis-muconate forP. putida BCM114. Specific C12O activity ofP. putida BCM114 was about three times higher than that ofP. putida BM014, and productivity was enhanced more than two times.