Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance.

Ribosome engineering, originally applied to Streptomyces lividans, has been widely utilized for strain improvement, especially for the activation of bacterial secondary metabolism. This study assessed ribosome engineering technology to modulate primary metabolism, taking butanol production as a representative example. The introduction into Clostridium saccharoperbutylacetonicum of mutations conferring resistance to butanol (ButR) and of the str mutation (SmR; a mutation in the rpsL gene encoding ribosomal protein S12), conferring high-level resistance to streptomycin, increased butanol production 1.6-fold, to 16.5 g butanol/L. Real-time qPCR analysis demonstrated that the genes involved in butanol metabolism by C. saccharoperbutylacetonicum were activated at the transcriptional level in the drug-resistant mutants, providing a mechanism for the higher yields of butanol by the mutants. Moreover, the activity of enzymes butyraldehyde dehydrogenase (AdhE) and butanol dehydrogenases (BdhAB), the key enzymes involved in butanol synthesis, was both markedly increased in the ButR SmR mutant, reflecting the significant up-regulation of adhE and bdhA at transcriptional level in this mutant strain. These results demonstrate the efficacy of ribosome engineering for the production of not only secondary metabolites but of industrially important primary metabolites. The possible ways to overcome the reduced growth rate and/or fitness cost caused by the mutation were also discussed.

Applicability of ribosome engineering to vitamin B12 production by Propionibacterium shermanii.

Ribosome engineering has been widely utilized for strain improvement, especially for the activation of bacterial secondary metabolism. This study assessed ribosome engineering technology to modulate primary metabolism, taking vitamin B12 production as a representative example. The introduction into Propionibacterium shermanii of mutations conferring resistance to rifampicin, gentamicin, and erythromycin, respectively, increased per cell production (µg/L/OD600) of vitamin B12 5.2-fold, although net production (µg/L) was unchanged, as the cell mass of the mutants was reduced. Real-time qPCR analysis demonstrated that the genes involved in vitamin B12 fermentation by P. shermanii were activated at the transcriptional level in the drug-resistant mutants, providing a mechanism for the higher yields of vitamin B12 by the mutants. These results demonstrate the efficacy of ribosome engineering for the production of not only secondary metabolites but of industrially important primary metabolites.

Metabolic perturbation to enhance polyketide and nonribosomal peptide antibiotic production using triclosan and ribosome-targeting drugs.

Although transcriptional activation of pathwayspecific positive regulatory genes and/or biosynthetic genes is primarily important for enhancing secondary metabolite production, reinforcement of substrate supply, as represented by primary metabolites, is also effective. For example, partial inhibition of fatty acid synthesis with ARC2 (an analog of triclosan) was found to enhance polyketide antibiotic production. Here, we demonstrate that this approach is effective even for industrial high-producing strains, for example enhancing salinomycin production by 40%, reaching 30.4 g/l of salinomycin in an industrial Streptomyces albus strain. We also hypothesized that a similar approach would be applicable to another important antibiotic group, nonribosomal peptide (NRP) antibiotics. We therefore attempted to partially inhibit protein synthesis by using ribosome-targeting drugs at subinhibitory concentrations (1/50∼1/2 of MICs), which may result in the preferential recruitment of intracellular amino acids to the biosynthesis of NRP antibiotics rather than to protein synthesis. Among the ribosome-targeting drugs examined, chloramphenicol at subinhibitory concentrations was most effective at enhancing the production by Streptomyces of NRP antibiotics such as actinomycin, calcium-dependent antibiotic (CDA), and piperidamycin, often resulting in an almost 2-fold increase in antibiotic production. Chloramphenicol activated biosynthetic genes at the transcriptional level and increased amino acid pool sizes 1.5- to 6-fold, enhancing the production of actinomycin and CDA. This "metabolic perturbation" approach using subinhibitory concentrations of ribosome-targeting drugs is a rational method of enhancing NRP antibiotic production, being especially effective in transcriptionally activated (e.g., rpoB mutant) strains. Because this approach does not require prior genetic information, it may be widely applicable for enhancing bacterial production of NRP antibiotics and bioactive peptides.

Identification of a Novel Lincomycin Resistance Mutation Associated with Activation of Antibiotic Production in Streptomyces coelicolor A3(2).

Comparative genome sequencing analysis of a lincomycin-resistant strain of Streptomyces coelicolor A3(2) and the wild-type strain identified a novel mutation conferring a high level of lincomycin resistance. Surprisingly, the new mutation was an in-frame DNA deletion in the genes SCO4597 and SCO4598, resulting in formation of the hybrid gene linR. SCO4597 and SCO4598 encode two histidine kinases, which together with SCO4596, encoding a response regulator, constitute a unique two-component system. Sequence analysis indicated that these three genes and their arrangement patterns are ubiquitous among all Streptomyces genomes sequenced to date, suggesting these genes play important regulatory roles. Gene replacement showed that this mutation was responsible for the high level of lincomycin resistance, the overproduction of the antibiotic actinorhodin, and the enhanced morphological differentiation of this strain. Moreover, heterologous expression of the hybrid gene linR in Escherichia coli conferred resistance to lincomycin in this organism. Introduction of the hybrid gene linR in various Streptomyces strains by gene engineering technology may widely activate and/or enhance antibiotic production.

Actinopyrone D, a new downregulator of the molecular chaperone GRP78 from Streptomyces sp.

A new downregulator of the molecular chaperone GRP78, actinopyrone D, was isolated together with a known related compound, PM050463, from Streptomyces sp. RAG92. The molecular formula of actinopyrone D was established as C25H36O4 by high-resolution FAB-MS. NMR spectroscopic analysis revealed the structure of actinopyrone D, which consists of an α-methoxy-γ-pyrone ring and a C17 side chain containing a cis olefin moiety. Actinopyrone D and PM050463 dose-dependently inhibited 2-deoxyglucose-induced luciferase expression in HT1080 human fibrosarcoma cells transfected with a luciferase reporter plasmid containing the GRP78 promoter. Actinopyrone D inhibited GRP78 protein expression and induced cell death under endoplasmic reticulum stress.

Functional analysis of hatomarubigin biosynthesis genes and production of a new hatomarubigin using a heterologous expression system.

The function of hatomarubigin biosynthesis genes was analyzed by heterologous expression of the hrb gene cluster. Streptomyces lividans carrying a gene cluster consisting of 25 genes (hrbR1-hrbX) with hrbY was found to produce all the known hatomarubigins including hatomarubigin D, which has a unique dimeric angucycline with a methylene linkage. Gene disruption was used in this heterologous expression system to analyze the function of hrbF, a gene with no homology to any known angucycline biosynthesis genes. A new metabolite was detected in the fermented broth of S. lividans expressing the hrb genes lacking hrbF and was designated hatomarubigin F. This compound was identified as 5-hydroxyhatomarubigin E by NMR spectroscopic analysis, suggesting that HrbF regulates the regiospecificity of oxygenation enzymes.

Cloning and heterologous expression of the thioviridamide biosynthesis gene cluster from Streptomyces olivoviridis.

Thioviridamide is a unique peptide antibiotic containing five thioamide bonds from Streptomyces olivoviridis. Draft genome sequencing revealed a gene (the tvaA gene) encoding the thioviridamide precursor peptide. The thioviridamide biosynthesis gene cluster was identified by heterologous production of thioviridamide in Streptomyces lividans.

Shining new light on the Hawthorne illumination experiments.

OBJECTIVE: This study provides an historical and statistical analysis of archival data from the Hawthorne illumination experiments. BACKGROUND: Previous accounts of the illumination experiments are fraught with inconsistencies because they have been based on secondary sources. The general consensus has been that variations in light levels had no effect on worker productivity at Hawthorne. All reports and data were thought to have been destroyed, but an archive at Cornell University was found to contain copies of the original documentation and much of the data from all three illumination experiments. Conclusions were originally drawn from visual comparisons of productivity graphs, and the data have never been properly statistically analyzed. METHOD: Archival reports, notes, photographs, and letters on the experiments were consulted. Productivity data were extracted from the tables and graphs in the reports and statistically analyzed for each experiment. RESULTS: Previously unpublished details of the illumination experiments emerged. An effect of lighting on productivity was found in the first treatment sequence for the first experiment, but this finding was not confirmed in the second sequence or in the second and third experiments. CONCLUSION: Experimental results provided inconsistent evidence of an association between light levels and productivity. All three experiments were found to be seriously flawed. APPLICATION: This study challenges popular accounts of the "Hawthorne effect," and the shortcomings of these experiments also have implications for the design of field studies.

Inhibition of histone deacetylase causes reduction of appressorium formation in the rice blast fungus Magnaporthe oryzae.

Post-translational modifications (PTMs) are important for cellular functions. The regulation of histone acetyltransferases (HATs) and histone deacetylases (HDACs) is one of important PTMs for epigenetic control, protein activity and protein stability. The regulation of acetylation of the N-terminal histone tails of core histone affects gene expression. Two class I HDAC genes and two class II HDAC genes have been identified in the Magnaporthe oryzae genome. Treatment with Rpd3/Hda1 family (classical) HDAC inhibitor inhibited the appressorium differentiation of M. oryzae. Treatment with trichostatin A, a classical HDAC inhibitor, also decreased pathogenesis. Furthermore, analyses of HDAC mutants indicated that MoHda1 and MoHos2 were required for vegetative growth and conidiation, and MoHos2 was required for appressorium formation. Disruption MoRPD3 was unsuccessful, as in the case with Aspergillus nidulans RpdA. These data indicated that HDACs have important roles in the asexual differentiation of M. oryzae.

MgLig4, a homolog of Neurospora crassa Mus-53 (DNA ligase IV), is involved in, but not essential for, non-homologous end-joining events in Magnaporthe grisea.

In many eukaryotic organisms, the non-homologous end-joining (NHEJ) system is a major pathway for the repair of DNA double-strand breaks (DSBs). DNA ligase IV is a component of the NHEJ system and is strictly required for the NHEJ system in Saccharomyces cerevisiae and in Neurospora crassa. To investigate the functions of DNA Ligase IV in Magnaporthe grisea, we generated deletion mutants of MGLIG4, which encodes a homolog of N. crassa DNA Ligase IV. Mutants (mglig4) showed no defects in asexual or sexual growth, and were fully pathogenic. Compared to the wild-type, mglig4 exhibited weak sensitivity to a DNA-damaging agent, camptothecin. In addition, the frequency of targeted-gene replacement was relatively elevated in mglig4, although this varied in a gene-dependent manner. Surprisingly, non-homologous integration of DNA was frequently observed in mglig4 transformants. Our results demonstrate that MgLig4 is involved in, but not essential for, the NHEJ system in M. grisea.

4-O-acetylation and 3-O-acetylation of trichothecenes by trichothecene 15-O-acetyltransferase encoded by Fusarium Tri3.

In the biosynthesis of Fusarium trichothecenes, the C-3 hydroxyl group of isotrichodermol must be acetylated by TRI101 for subsequent pathway genes to function. Despite the importance of this 3-O-acetylation step in biosynthesis, Tri101 is both physically and evolutionarily unrelated to other Tri genes in the trichothecene gene cluster. To gain insight into the evolutionary history of the cluster, we purified recombinant TRI3 (rTRI3), one of the two cluster gene-encoded trichothecene O-acetyltransferases, and examined to determine whether this 15-O-acetyltransferase can add an acetyl to the C-3 hydroxyl group of isotrichodermol. When a high concentration of rTRI3 was used in the assay (final concentration, 50 microM), we observed 3-O-acetylation activity against isotrichodermol that was more than 10(5) times less efficient than the known 15-O-acetylation activity against 15-deacetylcalonectrin. The rTRI3 protein also exhibited 4-O-acetylation activity when nivalenol was used as a substrate; in addition to 15-acetylnivalenol, di-acetylated derivatives, 4,15-diacetylnivalenol, and, to a lesser extent, 3,15-diacetylnivalenol, were also detected at high enzyme concentrations. The significance of the trace trichothecene 3-O-acetyltransferase activity detected in rTRI3 is discussed in relation to the evolution of the trichothecene gene cluster.