Regulation Of Biosynthetic Pathway Research Articles

Bacillaene, sharp objects consist in the arsenal of antibiotics produced by Bacillus.

Bacillus species act as plant growth-promoting rhizobacteria (PGPR) that can produce a large number of bioactive metabolites. Bacillaene, a linear polyketide/nonribosomal peptide produced by Bacillus strains, is synthesized by the trans-acyltransferase polyketide synthetase. The complexity of the chemical structure, particularity of biosynthesis, potent bioactivity, and the important role of competition make Bacillus an ideal antibiotic weapon to resist other microbes and maintain the optimal rhizosphere environment. This review provides an updated view of the structural features, biological activity, biosynthetic regulators of biosynthetic pathways, and the important competitive role of bacillaene during Bacillus survival.

An operator-based expression toolkit for Bacillus subtilis enables fine-tuning of gene expression and biosynthetic pathway regulation

SignificanceA gene regulatory system is an important tool for the engineering of biosynthetic pathways of organisms. Here, we report the development of an inducible-ON/OFF regulatory system using a malO operator as a key element. We identified and modulated sequence, position, numbers, and spacing distance of malO operators, generating a series of activating or repressive promoters with tunable strength. The stringency and robustness are both guaranteed in this system, a maximal induction factor of 790-fold was achieved, and nine proteins from different organisms were expressed with high yields. This system can be utilized as a gene switch, promoter enhancer, or metabolic valve in synthetic biology applications. This operator-based engineering strategy can be employed for developing similar regulatory systems in different microorganisms.

Candidate genes involved in cuticular hydrocarbon differentiation between cryptic, parabiotic ant species

Insect cuticular hydrocarbons (CHCs) are highly diverse and have multiple functions, including communication and waterproofing. CHC profiles form species-specific, complex blends of up to 150 compounds. Especially in ants, even closely related species can have largely different profiles, raising the question how CHC differences are mirrored in the regulation of biosynthetic pathways. The neotropical ants Crematogaster levior and Camponotus femoratus both consist of two cryptic species each that are morphologically similar, but express strongly different CHC profiles. This is ideal to study the molecular basis of CHC differences. We thus investigated gene expression differences in fat-body transcriptomes of these ants. Despite common garden conditions, we found several thousand differentially expressed transcripts within each cryptic species pair. Many of these were related to metabolic processes, probably accounting for physiological differences. Moreover, we identified candidate genes from five gene families involved in CHC biosynthesis. By assigning candidate transcripts to orthologs in Drosophila, we inferred which CHCs might be influenced by differential gene expression. Expression of these candidate genes was often mirrored in the CHC profiles. For example, Cr. levior A, which has longer CHCs than its cryptic sister species, had a higher expression of elongases and a lower expression of fatty acyl- CoA reductases. This study is one of the first to identify CHC candidate genes in ants and will provide a basis for further research on the genetic basis of CHC biosynthesis.

Development and optimization of N-acetylneuraminic acid biosensors in Bacillus subtilis.

Transcriptional factor (TF)-based metabolite-responsive biosensors are important tools for screening engineered enzymes with desired properties and for the dynamic regulation of biosynthetic pathways. However, TF-based biosensor construction is often constrained by undesired effects of TF-binding site sequence insertion on gene expression and unpredictable optimal TF expression levels. In the present study, a stepwise TF-based biosensor construction approach was developed using an N-acetylneuraminic acid (NeuAc) biosensor for Bacillus subtilis, as a case study. Specifically, 12 promoters with various strengths were selected as the first promoter library. Next, binding site sequences for the NanR were inserted into various positions of the selected promoter sequences to develop the second promoter library, resulting in 6 engineered promoters containing TF-binding site sequences (NanO), without major effects on promoter strength. NanR expression cassettes with different expression levels were further integrated to construct the biosensor library, yielding 9 NeuAc biosensors with efficient repression in the absence of NeuAc. Finally, biosensor activation was characterized by testing fold changes in expression levels of the green fluorescent protein reporter in the presence of NeuAc in vivo, which revealed 61-fold activation when NeuAc was present. The NeuAc biosensor developed in this study can be used for screening engineered enzymes for enhanced NeuAc biosynthesis in B. subtilis.

Engineering a growth-phase-dependent biosynthetic pathway for carotenoid production in Saccharomyces cerevisiae.

Metabolic engineering is usually focused on static control of microbial cell factories to efficient production of interested chemicals, though heterologous pathways compete with endogenous metabolism. However, products like carotenoids may cause metabolic burden on engineering strains, thus limiting product yields and influencing strain growth. Herein, a growth-phase-dependent regulation was developed to settle this matter, and its efficiency was verified using the heterogenous biosynthesis of lycopene in Saccharomyces cerevisiae as an example. Through growth-phase-dependent control of the lycopene biosynthetic pathway, limited step in MVA pathway, and competitive squalene pathway, production yield was increased by approximately 973-fold (from 0.034- to 33.1-mg/g CDW) and 1.48g/L of production was obtained by one-stage fermentation in a 5-L bioreactor. Our study not only introduces an economically approach to the production of carotenoids, but also provides an example of dynamic regulation of biosynthetic pathways for metabolic engineering.

Co-expression of anti-miR319g and miRStv_11 lead to enhanced steviol glycosides content in Stevia rebaudiana

BackgroundmiRNAs are major regulators of gene expression and have proven their role in understanding the genetic regulation of biosynthetic pathways. Stevioside and rebaudioside-A, the two most abundant and sweetest compounds found in leaf extract of Stevia rebaudiana, have been used for many years in treatment of diabetes. It has been found that the crude extract is more potent than the purified extract. Stevioside, being accumulated in higher concentration, imparts licorice like aftertaste. Thus, in order to make the sweetener more potent and palatable, there is a need to increase the intrinsic concentration of steviol glycosides and to alter the ratio of rebaudioside-A to stevioside. Doing so would significantly increase the quality of the sweeteners, and the potential to be used on a wider scale. To do so, in previous report, miRNAs associated with genes of steviol glycosides biosynthetic pathway were identified in S. rebaudiana. In continuation to that in this study, the two miRNAs (miR319g and miRStv_11) targeting key genes of steviol glycosides biosynthetic pathway were modulated and their impact was evaluated on steviol glycosides contents.ResultsThe over-expression results showed that miRStv_11 induced, while miR319g had repressive action on its target genes. The knock-down constructs for miR319g and miRStv_11 were then prepared and it was demonstrated that the expression of anti-miR319g produced inhibitory effect on its target miRNA, resulting in enhanced expression of its target genes. On the other hand, anti-miRStv_11 resulted in down-regulation of miRStv_11 and its target gene. Further miRStv_11 and anti-miR319gwere co-expressed which resulted in significant increase in stevioside (24.5%) and rebaudioside-A (51%) contents.ConclusionIn conclusion, the role of miR319g and miRStv_11 was successfully validated in steviol gycosides biosynthetic pathway gene regulation and their effect on steviol gycosides contents. In this study, we found the positively correlated miRNA-mRNA interaction network in plants, where miRStv_11 enhanced the expression of KAH gene. miRNAs knock-down was also successfully achieved using antisense precursors. Overall, this study thus reveals more complex nature and fundamental importance of miRNAs in biosynthetic pathway related gene networks and hence, these miRNAs can be successfully employed to enhance the ratio of rebaudioside-A to stevioside, thus enhancing the sweetening indices of this plant and making it more palatable.

A CRISPR-Cas9 Strategy for Activating the Saccharopolyspora erythraea Erythromycin Biosynthetic Gene Cluster with Knock-in Bidirectional Promoters.

The regulation of biosynthetic pathways is a universal strategy for industrial strains that overproduce metabolites. Erythromycin produced by Saccharopolyspora erythraea has extensive clinical applications. In this study, promoters of the erythromycin biosynthesis gene cluster were tested by reporter mCherry. The SACE_0720 ( eryBIV)-SACE_0721 ( eryAI) spacer was selected as a target regulatory region, and bidirectional promoters with dual single guide RNAs (sgRNAs) were knocked-in using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 method. qPCR results indicated that knock-in of Pj23119-PkasO, which replaced the native promoter, enabled biosynthetic gene cluster activation, with eryBIV and eryAI expression increased 32 and 79 times, respectively. High performance liquid chromatography results showed that, compared with the wild-type strain, the yield of erythromycin was increased (58.3%) in bidirectional promoter knock-in recombinant strains. On the basis of the activated strain Ab::Pj23119-PkasO, further investigation showed that CRISPR-based interference of sdhA gene affected erythromycin biosynthesis and cell growth. Finally, regulating the culture temperature to optimize the inhibition intensity of sdhA further increased the yield by 15.1%. In summary, this study showed that bidirectional promoter knock-in and CRISPR interference could regulate gene expression in S.erythraea. This strategy has potential application for biosynthetic gene cluster activation and gene regulation in Actinobacteria.

Features of Protein-Protein with Protein-RNA Interactions in Bacteria and Fungi

Various protein-protein and protein-RNA interactions are extant which are fundamental for the regulation of biosynthetic pathways, metabolic and cellular processes in bacteria, fungi and other microorganisms, but their role and significance must be clearly elucidated. To effectively and efficiently compete in their natural habitat or niche, bacteria, fungi, and other microorganisms utilize regulatory mechanisms in metabolic production and other cellular processes; thus controlling overproduction and excretion of the secondary metabolites of excess concentrations into the environment. The knowledge and information regarding fungal natural product biosynthesis have been retarded in comparison to the corresponding extant bacterial biosynthetic pathways due to salient externalities, constraints and challenges.

Development of Artificial Riboswitches for Monitoring of Naringenin In Vivo.

Microbial strains are considered promising hosts for production of flavonoids because of their rapid growth rate and suitability for large-scale manufacturing. However, productivity and titer of current recombinant strains still do not meet the requirements of industrial processes. Genetically encoded biosensors have been applied for high-throughput screening or dynamic regulation of biosynthetic pathways for enhancing the performance of microbial strains that produce valuable chemicals. Currently, few protein sensor-regulators for flavonoids exist. Unlike the protein-based trans-regulating controllers, riboswitches can respond to their ligands faster and eliminate off-target effects. Here, we developed artificial riboswitches that activate gene expression in response to naringenin, an important flavonoid. RNA aptamers for naringenin were developed using SELEX and cloned upstream of a dual selectable marker gene to construct a riboswitch library. Two in vivo selection routes were applied separately to the library by supplementing naringenin at two different concentrations during enrichments to modulate the operational ranges of the riboswitches. The selected riboswitches were responsive to naringenin and activated gene expression up to 2.91-fold. Operational ranges of the riboswitches were distinguished on the basis of their selection route. Additionally, the specificity of the riboswitches was assessed, and their applicability as dynamic regulators was confirmed. Collectively, the naringenin riboswitches reported in this work will be valuable tools in metabolic engineering of microorganisms for the production of flavonoids.

Abstract 3371: Aldh expressing stem cells mediate tumor initiation and metastasis in triple negative breast cancers across different ethnicities

Abstract TNBC is the only subtype of breast cancer for which there are no approved targeted therapies. In the US, its incidence is highest in women with African ancestry (AA); in western sub-Saharan Africa, single-institution studies show that TNBC constitutes 40- 80% of all breast cancers. Given the Caucasian/AA survival disparity in breast cancer, there is an urgent need to find actionable targets in TNBC of all ethnicities, but especially in TNBC in AA, which are suspected to be more aggressive. Breast cancer stem cells, the small population of cells that have been shown to mediate breast tumor initiation, metastasis, and resistance to conventional therapy have also been reported to mediate the heterogeneity of TNBC and are especially abundant in TNBC in AA women. Here, we sought to better understand the biology of TNBC by finding genes and pathways that are differentially expressed in the stem cell population of patient derived xenografts (PDX) from TNBC from Ghanaian (G), AA and Caucasian (C) women and the effect of these differentially expressed genes on the stem cell phenotype in these primary tumors. We isolated the ALDH+ and the CD44+/CD24- stem cell populations from the bulk cells from 15 PDXs using flow cytometry. We performed RNA sequencing (Illumina HiSeq platform) on the isolated populations and bulk cells (45). Comprehensive bioinformatics analyses led to the identificationof highly significantly differentially expressed genes and pathways between the cell populations. By principal component analysis, the tumors were very heterogeneous. However, the ALDH+ cells separated out from the CD44+/CD24- and the bulk cells. We identified 14 genes that were simultaneously differentially expressed between the ALDH+ vs the CD44+/CD24- as well as ALDH+ VS bulk (p-value <0.001, FDR < 0.05). The 3 most significant genes were MMP2 and PCDH7, both known to be involved in breast cancer metastasis and CPXM1, a carboxylase. Inhibiting MMP2 expression in the PDX cells grown in suspension resulted in significant reduction in the ALDH+ cell population. Also, the ALDH+ and not the CD44+/CD24- cells formed spheres in serum free media. The WNT, MAPK and TGF-beta pathways known to mediate metastasis were all significantly up-regulated in the ALDH+ population with down regulation of biosynthetic pathways which were up-regulated in the CD44+/CD24- population. Further studies are ongoing on pathway modulation in ALDH1+ cells based on these findings. Our data demonstrate that the ALDH+ stem cell population is enriched for tumor initiating cells in aggressive TNBCs across different ethnicities. These cells may play a role in mediating the aggressive behavior of TNBC’s found in African women. Citation Format: Evelyn M. Jiagge, Shukmei Wong, Rabia Gilani, Sean Mcdermott, Lisa Newman, Jessica Bensenhaver, Max Wicha, John Carpten, Sofia Merajver. Aldh expressing stem cells mediate tumor initiation and metastasis in triple negative breast cancers across different ethnicities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3371. doi:10.1158/1538-7445.AM2017-3371

Rewiring carotenoid biosynthesis in plants using a viral vector

Plants can be engineered to sustainably produce compounds of nutritional, industrial or pharmaceutical relevance. This is, however, a challenging task as extensive regulation of biosynthetic pathways often hampers major metabolic changes. Here we describe the use of a viral vector derived from Tobacco etch virus to express a whole heterologous metabolic pathway that produces the health-promoting carotenoid lycopene in tobacco tissues. The pathway consisted in three enzymes from the soil bacteria Pantoea ananatis. Lycopene is present at undetectable levels in chloroplasts of non-infected leaves. In tissues infected with the viral vector, however, lycopene comprised approximately 10% of the total carotenoid content. Our research further showed that plant viruses that express P. ananatis phytoene synthase (crtB), one of the three enzymes of the heterologous pathway, trigger an accumulation of endogenous carotenoids, which together with a reduction in chlorophylls eventually result in a bright yellow pigmentation of infected tissues in various host-virus combinations. So, besides illustrating the potential of viral vectors for engineering complex metabolic pathways, we also show a yellow carotenoid-based reporter that can be used to visually track infection dynamics of plant viruses either alone or in combination with other visual markers.

Biology of Heme in Mammalian Erythroid Cells and Related Disorders.

Heme is a prosthetic group comprising ferrous iron (Fe2+) and protoporphyrin IX and is an essential cofactor in various biological processes such as oxygen transport (hemoglobin) and storage (myoglobin) and electron transfer (respiratory cytochromes) in addition to its role as a structural component of hemoproteins. Heme biosynthesis is induced during erythroid differentiation and is coordinated with the expression of genes involved in globin formation and iron acquisition/transport. However, erythroid and nonerythroid cells exhibit distinct differences in the heme biosynthetic pathway regulation. Defects of heme biosynthesis in developing erythroblasts can have profound medical implications, as represented by sideroblastic anemia. This review will focus on the biology of heme in mammalian erythroid cells, including the heme biosynthetic pathway as well as the regulatory role of heme and human disorders that arise from defective heme synthesis.

Phylum-specific regulation of resistomycin production in a Streptomyces sp. via microbial coculture.

Actinomycete genomes are encoded with immense potential to produce secondary metabolites, however standard laboratory culture experiments rarely provide the conditions under which associated biosynthetic pathways are expressed. Despite years of research attempting to access these pathways and aside from a few well-studied bacterial quorum sensing systems, little is known about the specificity of secondary metabolite regulation in bacteria, such as the conditions under which a bacterium produces an antibiotic and the extent to which it does so in recognition of a particular species in the immediate environment. In the current study, we observed that the cocultivation of a Streptomyces sp. (strain B033) with four pathogenic strains of the phylum Proteobacteria resulted in the production of the antibiotic resistomycin. After further coculture experiments, we determined that Proteobacteria induced the production of resistomycin in B033 at significantly higher rates (65%) than strains from the phyla Firmicutes (5.9%) and Actinobacteria (9.1%), supporting that the regulation of secondary metabolism in bacteria can be dependent on the species present in the immediate environment. These results suggest a lack of promiscuity of antibiotic biosynthetic pathway regulation and indicate that it is feasible to mine existing microbial strain libraries for antibiotics in a phylum-specific manner.

Transcriptional control of vitamin C defective 2 and tocopherol cyclase genes by light and plastid-derived signals: The partial involvement of GENOMES UNCOUPLED 1

Previous findings have suggested that light and plastid-derived signals are involved in the regulation of biosynthetic pathways for l-ascorbic acid (AsA) and tocopherols (Toc). Photosynthetic electron transport (PET) activity, plastid gene expression (PGE), and the tetrapyrrole metabolism have been identified as signals that regulate nuclear gene expression through the GENOMES UNCOUPLED 1 (GUN1) protein. Here, we examined the effects of disrupting GUN1 on these pathways. The expression of vitamin C defective 2 (VTC2) and tocopherol cyclase (TC) genes, which encode key enzymes in the AsA and Toc biosynthetic pathways, respectively, was affected by illumination and darkness in parallel with the levels of both these antioxidants. However, the GUN1 disruption had no effect on these biosynthetic pathways under light-dark conditions. All treatments that inhibited PET, PGE, and the tetrapyrrole metabolism interrupted both biosynthetic pathways; however, this was partially mitigated by the GUN1 disruption. The expression patterns of VTC2 and TC reflected the levels of both antioxidants under most of the conditions examined. Our results suggest that the transcriptional control of VTC2 and TC by light and plastid-derived signals is important for the regulation of the biosynthetic pathways, and that GUN1 is at least partially involved in the plastid-derived signals-dependent regulation.

Molecular cloning, characterization, and expression analysis of LeMYB1 from Lithospermum erythrorhizon

MYB transcription factors (TFs) are known to have important functions in regulating the biosynthesis of secondary metabolites in plants. In this study, LeMYB1, a member of the MYB gene family of Lithospermum erythrorhizon, was cloned via the rapid amplification of cDNA ends. The alignment of the predicted translations of LeMYB1 with other MYB proteins revealed that LeMYB1 contained an N-terminal R2R3 repeat and a high degree of amino acid identity to NtMYBJS1 which is involved in jasmonic acid signalling and phenylpropanoid biosynthetic pathway regulation. To determine the expression pattern of LeMYB1, its promoter was cloned and the sequence analysis was performed. The results revealed a number of potential regulatory motifs related to tissue-specific gene expression and abiotic and biotic stress responses. Real-time PCR results suggest that LeMYB1 was induced transiently during the early stage when L. erythrorhizon cells were transferred from a B5 growth medium to a M9 production medium for shikonin formation. Exogenous methyl jasmonate (MeJA), an effective inducer of shikonin biosynthesis, induced the rapid LeMYB1 expression. In contrast, a treatment with ibuprofen (IBU), an inhibitor of jasmonate biosynthesis, significantly inhibited the LeMYB1 expression. Another inhibitor of shikonin formation, 2,4-dichlorophenoxyacetic acid (2,4-D), also markedly repressed the expression of LeMYB1. Tissue-specific expression analysis showed that LeMYB1 mRNA was predominantly accumulated in roots where shikonin was synthesized. Thus, the LeMYB1 gene may be a valuable member of the R2R3-MYB family in L. erythrorhizon and is possibly involved in the regulation of shikonin biosynthesis.

Taxoids profiling of suspension Taxus chinensis var. mairei cells in response to shear stress

To develop an optimal bioprocess for paclitaxel (Taxols) supply, taxoid biosynthetic pathway regulation must be better understood. The main taxoid metabolites (paclitaxel, baccatin III, taxol C, etc.) in Taxus cell culture showed great difference under shear stress. However, the regulating mechanism of taxoids metabolism under shear stress remained elusive. Here an efficient metabolic profiling approach combined with multivariate analysis was employed to profile taxoids changes of Taxus cells under laminar shear stress. A total of 21 taxoids were identified and quantified by ultra-performance liquid chromatography coupled with Q-TOF mass spectrometry. The result showed the contents of paclitaxel and baccatin III were reduced by shear stress, indicating the inhibitory effect of shear stress on paclitaxel biosynthesis. The levels of other taxoids uninvolved in paclitaxel biosynthesis were decreased except several metabolites. Further analysis of mapping measured taxoids concentrations onto paclitaxel biosynthesis pathway illustrating proposed intermediates and “off-pathway” metabolites revealed shear stress might disrupt the appropriate cyclization process of geranylgeranyl-pyrophosphate, aggravate the inappropriate order of hydroxylations and acylations, and not be good for functional group oxetane formation. These findings revealed the possible mechanism for shear stress limiting paclitaxel production and might have important biotechnological applications to increase the yields of paclitaxel and relevant precursors.

Molecular mechanism of elicitor-induced tanshinone accumulation in Salvia miltiorrhiza hairy root cultures

To develop an optimal bioprocess for the production of tanshinone which is mainly used for the treatment of cardiocerebral vascular disease, the tanshinone biosynthetic pathway regulation must be better understood. In this paper, expression of tanshinone biosynthetic pathway related genes as well as tanshinone accumulation in Salvia miltiorrhiza hairy root cultures were investigated, in response to biotic and abiotic elicitors, respectively. Our results showed tanshinone accumulation in S. miltiorrhiza hairy roots was highly regulated by the coordination of the expression of several genes involved in tanshinone biosynthesis pathway. Our results showed a positive correlation between gene expression and tanshinone accumulation, suggesting that tanshinone accumulation may be the result of the coexpression up-regulation of several genes involved in tanshinone biosynthesis under the treatment of various elicitors. Meantime, SmHMGR, SmDXS2, SmFPPS, SmGGPPS and SmCPS were identified as the potential key enzymes in the pathway for targeted metabolic engineering to increase accumulation of tanshinone in S. miltiorrhiza hairy roots. This is the first report integrating comprehensively the transcript and metabolite biosynthesis of tanshinone in S. miltiorrhiza hairy roots.

Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway

Flavonoids are secondary metabolites involved in several aspects of plant development and defence. They colour fruits and flowers, favouring seed and pollen dispersal, and contribute to plant adaptation to environmental conditions such as cold or UV stresses, and pathogen attacks. Because they affect the quality of flowers (for horticulture), fruits and vegetables, and their derivatives (colour, aroma, stringency, etc.), flavonoids have a high economic value. Furthermore, these compounds possess pharmaceutical properties extremely attractive for human health. Thanks to easily detectable mutant phenotypes, such as modification of petal pigmentation and seeds exhibiting transparent testa, the enzymes involved in the flavonoid biosynthetic pathway have been characterized in several plant species. Conserved features as well as specific differences have been described. Regulation of structural gene expression appears tightly organized in a spatial and temporal way during plant development, and is orchestrated by a ternary complex involving transcription factors from the R2R3-MYB, basic helix-loop-helix (bHLH), and WD40 classes. This MYB-bHLH-WD40 (MBW) complex regulates the genes that encode enzymes specifically involved in the late steps of the pathway leading to the biosynthesis of anthocyanins and condensed tannins. Although several genes encoding transcription factors from these three families have been identified, many gaps remain in our understanding of the regulation of this biosynthetic pathway, especially about the respective roles of bHLH and WD40 proteins. A better knowledge of the regulatory mechanisms of the flavonoid pathway is likely to favour the development of new biotechnological tools for the generation of value-added plants with optimized flavonoid content.

Mapping of an anthocyanin-regulating MYB transcription factor and its expression in red and green pear, Pyrus communis

'Max Red Bartlett' is a red bud mutation of the yellow pear (Pyrus communis L.) cultivar 'Williams' (known as 'Bartlett' in North America). Anthocyanins are the most important pigments for red colour in fruits. Synthesis of anthocyanins is mediated by a number of well-characterized enzymes that include chalcone synthase (CHS), flavanone-3-hydroxylase (F3H), dihydroflavonol-4-reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose:flavonoid-3-O-glucosyltransferase (UFGT). Expression of the genes encoding these five enzymes was examined in pear fruit skin in order to elucidate the molecular mechanism for red coloration. In addition, the gene PcMYB10, encoding an R2R3 MYB transcription factor involved in anthocyanin biosynthetic pathway regulation, was isolated from both 'Williams' and 'Max Red Bartlett'. Analysis of the deduced amino acid sequence suggests that this gene is an ortholog of anthocyanin regulators known in other plant species. Its expression level was significantly higher in 'Max Red Bartlett' (red pear) compared with the original yellow variety 'Williams'. Although the map position of PcMYB10 corresponds to that of MdMYBa and MdMYB10, which control pigmentation of apple fruit skin, PcMYB10 is not directly responsible for red versus yellow colour in the two pear varieties, as the mutation underlying this difference maps to a different region of the pear genome.

Manipulation of pathway regulation in Streptomyces globisporus for overproduction of the enediyne antitumor antibiotic C-1027

Manipulation of pathway regulation is an efficient strategy to increase specific secondary metabolite production. Here we successfully improved production of both the enediyne antitumor antibiotic C-1027 and a heptaebe, an early metabolite of the C-1027 pathway, by manipulating the three regulatory genes, sgcR1, sgcR2, and sgcR3, within the C-1027 biosynthetic gene cluster. SgcR3 has previously been established as an activator, and we now propose that SgcR1 and SgcR2 are also positive regulators based on their up-regulation effects on titer and/or timing of heptaene and C-1027 production in Streptomyces globisporus. Specifically, overexpression of sgcR1 significantly improved production of the heptaene (about 5-fold) and C-1027 (2- to 3-fold) compared to the wild-type strain. However, the titers of heptaene and C-1027 were not increased by overexpressing all three activators together, underscoring the complexity of C-1027 biosynthetic pathway regulation. The possibility of exploiting the heptaene as a readily identifiable and unique indicator for rapidly detecting enediyne production was also assessed.