Phenotypic diversification by enhanced genome restructuring after induction of multiple DNA double-strand breaks.

DNA double-strand break (DSB)-mediated genome rearrangements are assumed to provide diverse raw genetic materials enabling accelerated adaptive evolution; however, it remains unclear about the consequences of massive simultaneous DSB formation in cells and their resulting phenotypic impact. Here, we establish an artificial genome-restructuring technology by conditionally introducing multiple genomic DSBs in vivo using a temperature-dependent endonuclease TaqI. Application in yeast and Arabidopsis thaliana generates strains with phenotypes, including improved ethanol production from xylose at higher temperature and increased plant biomass, that are stably inherited to offspring after multiple passages. High-throughput genome resequencing revealed that these strains harbor diverse rearrangements, including copy number variations, translocations in retrotransposons, and direct end-joinings at TaqI-cleavage sites. Furthermore, large-scale rearrangements occur frequently in diploid yeasts (28.1%) and tetraploid plants (46.3%), whereas haploid yeasts and diploid plants undergo minimal rearrangement. This genome-restructuring system (TAQing system) will enable rapid genome breeding and aid genome-evolution studies.

Selection of Transcripts Affecting Initial Growth Rate of Rice Backcrossed Inbred Lines Using RNA Sequencing Data.

Seedling growth is an important factor for direct seeding of rice. However, the genetic and transcriptomic factors involved in this process are largely unknown. In this study, transcripts affecting shoot weight were identified in rice (Oryza sativa L.) using RNA sequencing (RNA-Seq) data from 20 backcrossed inbred lines (BILs) and their parental cultivars. The selection frequency of the genes for the regression model was determined using repeated analysis of random subsets of the transcriptome. The qLTG3-1gene, controlling low-temperature germinability, and short grain 1 gene (SG1), known to decrease organ elongation, showed high frequency. The quantitative trait loci (QTLs) analysis performed for BILs revealed that qLTG3-1 was included in the QTLs for shoot weight but SG1 was not. No nucleotide polymorphisms were found in the coding region of SG1 in either of the parental cultivars. Quantitative real-time PCR showed that SG1 expression was negatively correlated with shoot weight for all 104 BILs analyzed in this study. Expression QTL (eQTLs) analysis showed an eQTL for SG1 expression located in the same region as the QTL for shoot weight. However, no eQTLs were detected on the same chromosome as SG1, suggesting that nucleotide polymorphisms around the gene do not affect its expression in analyzed growth stage. Overall, these results indicate that RNA-Seq is a useful tool for identifying transcripts that can be related to seedling growth rate.

Overexpression of a novel Arabidopsis PP2C isoform, AtPP2CF1, enhances plant biomass production by increasing inflorescence stem growth.

In contrast to mammals, higher plants have evolved to express diverse protein phosphatase 2Cs (PP2Cs). Of all Arabidopsis thaliana PP2Cs, members of PP2C subfamily A, including ABI1, have been shown to be key negative regulators of abscisic acid (ABA) signalling pathways, which regulate plant growth and development as well as tolerance to adverse environmental conditions. However, little is known about the enzymatic and signalling roles of other PP2C subfamilies. Here, we report a novel Arabidopsis subfamily E PP2C gene, At3g05640, designated AtPP2CF1. AtPP2CF1 was dramatically expressed in response to exogenous ABA and was expressed in vascular tissues and guard cells, similar to most subfamily A PP2C genes. In vitro enzymatic activity assays showed that AtPP2CF1 possessed functional PP2C activity. However, yeast two-hybrid analysis revealed that AtPP2CF1 did not interact with PYR/PYL/RCAR receptors or three SnRK2 kinases, which are ABI1-interacting proteins. This was supported by homology-based structural modelling demonstrating that the putative active- and substrate-binding site of AtPP2CF1 differed from that of ABI1. Furthermore, while overexpression of ABI1 in plants induced an ABA-insensitive phenotype, Arabidopsis plants overexpressing AtPP2CF1 (AtPP2CF1oe) were weakly hypersensitive to ABA during seed germination and drought stress. Unexpectedly, AtPP2CF1oe plants also exhibited increased biomass yield, mainly due to accelerated growth of inflorescence stems through the activation of cell proliferation and expansion. Our results provide new insights into the physiological significance of AtPP2CF1 as a candidate gene for plant growth production and for potential application in the sustainable supply of plant biomass.

Analysis of gene-disruption mutants of a sucrose phosphate synthase gene in rice, OsSPS1, shows the importance of sucrose synthesis in pollen germination.

The molecular function of an isoform of sucrose phosphate synthase (SPS) in rice, OsSPS1, was investigated using gene-disruption mutant lines generated by retrotransposon insertion. The progeny of the heterozygote of disrupted OsSPS1 (SPS1(+/-)) segregated into SPS1(+/+), SPS1(+/-), and SPS1(-/-) at a ratio of 1:1:0. This distorted segregation ratio, together with the expression of OsSPS1 in the developing pollen revealed by quantitative RT-PCR analysis and promoter-beta-glucuronidase (GUS) fusion assay, suggested that the disruption of OsSPS1 results in sterile pollen. This hypothesis was reinforced by reciprocal crosses of SPS1(+/-) plants with wild-type plants in which the disrupted OsSPS1 was not paternally transmitted to the progeny. While the pollen grains of SPS(+/-) plants normally accumulated starch during their development, pollen germination on the artificial media was reduced to half of that observed in the wild-type control. Overall, our data suggests that sucrose synthesis via OsSPS1 is essential in pollen germination in rice.

The promoter activities of sucrose phosphate synthase genes in rice, OsSPS1 and OsSPS11, are controlled by light and circadian clock, but not by sucrose.

Although sucrose plays a role in sugar sensing and its signaling pathway, little is known about the regulatory mechanisms of the expressions of plant sucrose-related genes. Our previous study on the expression of the sucrose phosphate synthase gene family in rice (OsSPSs) suggested the involvement of sucrose sensing and/or circadian rhythm in the transcriptional regulation of OsSPS. To examine whether the promoters of OsSPSs can be controlled by sugars and circadian clock, we produced transgenic rice plants harboring a promoter-luciferase construct for OsSPS1 or OsSPS11 and analyzed the changes in the promoter activities by monitoring bioluminescence from intact transgenic plants in real-time. Transgenic plants fed sucrose, glucose, or mannitol under continuous light conditions showed no changes in bioluminescence intensity; meanwhile, the addition of sucrose increased the concentration of sucrose in the plants, and the mRNA levels of OsSPS remained constant. These results suggest that these OsSPS promoters may not be regulated by sucrose levels in the tissues. Next, we investigated the changes in the promoter activities under 12-h light/12-h dark cycles and continuous light conditions. Under the light-dark cycle, both OsSPS1 and OsSPS11 promoter activities were low in the dark and increased rapidly after the beginning of the light period. When the transgenic rice plants were moved to the continuous light condition, both P OsSPS1 ::LUC and P OsSPS11 ::LUC reporter plants exhibited circadian bioluminescence rhythms; bioluminescence peaked during the subjective day with a 27-h period: in the early morning as for OsSPS1 promoter and midday for OsSPS11 promoter. These results indicate that these OsSPS promoters are controlled by both light illumination and circadian clock and that the regulatory mechanism of promoter activity differs between the two OsSPS genes.

Tissue specificity and diurnal change in gene expression of the sucrose phosphate synthase gene family in rice.

The rice genome contains 5 isogenes for sucrose phosphate synthase (SPS), the key enzyme in sucrose synthesis; however, little is known about their transcriptional regulation. In order to determine the expression patterns of the SPS gene family in rice plants, we conducted an expression analysis in various tissues and developmental stages by real-time quantitative RT-PCR. At the transcript level, the rice SPS genes, particularly SPS1, were preferentially expressed in source tissues, whereas SPS2, SPS6, and SPS8 were expressed equally in source and sink tissues. We also investigated diurnal changes in SPS gene expression, SPS activity, and soluble sugar content in leaf blades. Interestingly, the expression of all the SPS genes, particularly that of SPS1 and SPS11, tended to be higher at night when the activation state of the SPS proteins was low, and the mRNA levels of SPS1 and SPS6 were negatively correlated with sucrose content. Furthermore, the temporal patterns of SPS gene expression and sugar content under continuous light conditions suggested the involvement of endogenous rhythm and/or sucrose sensing in the transcriptional regulation of SPS genes. Our data revealed differential expression patterns in the rice SPS gene family and part of the complex mechanisms of their transcriptional control.

Production of geranylgeraniol on overexpression of a prenyl diphosphate synthase fusion gene in Saccharomyces cerevisiae.

An acyclic diterpene alcohol, (E,E,E)-geranylgeraniol (GGOH), is one of the important compounds used as perfume and pharmacological agents. A deficiency of squalene (SQ) synthase activity allows yeasts to accumulate an acyclic sesquiterpene alcohol, (E,E)-farnesol, in their cells. Since sterols are essential for the growth of yeasts, a deficiency of SQ synthase activity makes the addition of supplemental sterols to the culture media necessary. To develop a GGOH production method not requiring any supplemental sterols, we overexpressed HMG1 encoding hydroxymethylglutaryl-CoA reductase and the genes of two prenyl diphosphate synthases, ERG20 and BTS1, in Saccharomyces cerevisiae. A prototrophic diploid coexpressing HMG1 and the ERG20-BTS1 fusion accumulated GGOH with neither disruption of the SQ synthase gene nor the addition of any supplemental sterols. The GGOH content on the diploid cultivation in a 5-l jar fermenter reached 138.8 mg/l under optimal conditions.

Overproduction of geranylgeraniol by metabolically engineered Saccharomyces cerevisiae.

(E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter(-1)) rather than GGOH (0.2 mg liter(-1)) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter(-1) GGOH and 6.5 mg liter(-1) squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter(-1) GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering.

Alkaline pH enhances farnesol production by Saccharomyces cerevisiae.

External environments affect prenyl alcohol production by squalene synthetase-deficient mutant Saccharomyces cerevisiae ATCC 64031. Cultivation of the yeast in medium with an initial pH ranging from 7.0 to 8.0 increased the amount of secreted farnesol (FOH). In contrast, acidic medium with a pH below 4.0 increased the intracellular FOH and its isomer nerolidol. These effects of alkaline pH were also observed on constant pH cultivation in a jar fermenter. On cultivation for 133 h, the FOH production reached 102.8 mg/l.

Prenyl alcohol production by expression of exogenous isopentenyl diphosphate isomerase and farnesyl diphosphate synthase genes in Escherichia coli.

Isopentenyl diphosphate isomerase (idi) and farnesyl diphosphate synthase (ispA) genes were overexpressed in Escherichia coli. The resulting transformant showed 6.8-fold higher production of farnesol (389 microg/l). In a similar manner, overexpression of idi and mutated ispA led to high production of geranylgeraniol (128 microg/l).

Overexpression of the gene encoding HMG-CoA reductase in Saccharomyces cerevisiae for production of prenyl alcohols.

To develop microbial production method for prenyl alcohols (e.g., (E,E)-farnesol (FOH), (E)-nerolidol (NOH), and (E,E,E)-geranylgeraniol (GGOH)), the genes encoding enzymes in the mevalonate and prenyl diphosphate pathways were overexpressed in Saccharomyces cerevisiae, and the resultant transformants were evaluated as to the production of these alcohols. Overexpression of the gene encoding hydroxymethylglutaryl (HMG)-CoA reductase was most effective among the genes tested. A derivative of S. cerevisiae ATCC 200589, which was selected through screening, was found to be the most suitable host for the production. On cultivation of the resultant transformant, in which the HMG-CoA reductase gene was overexpressed, in a 5-liter bench-scale jar fermenter for 7 d, the production of FOH, NOH, and GGOH reached 145.7, 98.8, and 2.46 mg/l, respectively.

Various oils and detergents enhance the microbial production of farnesol and related prenyl alcohols.

The object of this research was improvement of prenyl alcohol production with squalene synthase-deficient mutant Saccharomyces cerevisiae ATCC 64031. On screening of many kinds of additives, we found that oils and detergents significantly enhanced the extracellular production of prenyl alcohols. Soybean oil showed the most prominent effect among the additives tested. Its effect was accelerated by a high concentration of glucose in the medium. The combination of these cultivation conditions led to the production of more than 28 mg/l of farnesol in the soluble fraction of the broth. The addition of these compounds to the medium was an effective method for large-scale production of prenyl alcohols with microorganisms.

Accumulation of prenyl alcohols by terpenoid biosynthesis inhibitors in various microorganisms.

Squalene synthase inhibitors significantly accelerate the production of farnesol by various microorganisms. However, farnesol production by Saccharomyces cerevisiae ATCC 64031, in which the squalene synthase gene is deleted, was not affected by the inhibitors, indicating that farnesol accumulation is enhanced in the absence of squalene synthase activity. The combination of diphenylamine as an inhibitor of carotenoid biosynthesis and a squalene synthase inhibitor increases geranylgeraniol production by a yeast, Rhodotorula rubra NBRC 0870. An ent-kauren synthase inhibitor also enhances the production of farnesol and geranylgeraniol by a filamentous fungus, Gibberella fujikuroi NBRC 30336. These results indicate that the inhibition of downstream enzymes from prenyl diphosphate synthase leads to the production of farnesol and geranylgeraniol.