An improved ternary vector system for Agrobacterium-mediated rapid maize transformation.

KEY MESSAGE: A simple and versatile ternary vector system that utilizes improved accessory plasmids for rapid maize transformation is described. This system facilitates high-throughput vector construction and plant transformation. The super binary plasmid pSB1 is a mainstay of maize transformation. However, the large size of the base vector makes it challenging to clone, the process of co-integration is cumbersome and inefficient, and some Agrobacterium strains are known to give rise to spontaneous mutants resistant to tetracycline. These limitations present substantial barriers to high throughput vector construction. Here we describe a smaller, simpler and versatile ternary vector system for maize transformation that utilizes improved accessory plasmids requiring no co-integration step. In addition, the newly described accessory plasmids have restored virulence genes found to be defective in pSB1, as well as added virulence genes. Testing of different configurations of the accessory plasmids in combination with T-DNA binary vector as ternary vectors nearly doubles both the raw transformation frequency and the number of transformation events of usable quality in difficult-to-transform maize inbreds. The newly described ternary vectors enabled the development of a rapid maize transformation method for elite inbreds. This vector system facilitated screening different origins of replication on the accessory plasmid and T-DNA vector, and four combinations were identified that have high (86-103%) raw transformation frequency in an elite maize inbred.

Synthetic chromosome arms function in yeast and generate phenotypic diversity by design.

Recent advances in DNA synthesis technology have enabled the construction of novel genetic pathways and genomic elements, furthering our understanding of system-level phenomena. The ability to synthesize large segments of DNA allows the engineering of pathways and genomes according to arbitrary sets of design principles. Here we describe a synthetic yeast genome project, Sc2.0, and the first partially synthetic eukaryotic chromosomes, Saccharomyces cerevisiae chromosome synIXR, and semi-synVIL. We defined three design principles for a synthetic genome as follows: first, it should result in a (near) wild-type phenotype and fitness; second, it should lack destabilizing elements such as tRNA genes or transposons; and third, it should have genetic flexibility to facilitate future studies. The synthetic genome features several systemic modifications complying with the design principles, including an inducible evolution system, SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution). We show the utility of SCRaMbLE as a novel method of combinatorial mutagenesis, capable of generating complex genotypes and a broad variety of phenotypes. When complete, the fully synthetic genome will allow massive restructuring of the yeast genome, and may open the door to a new type of combinatorial genetics based entirely on variations in gene content and copy number.

Consequences of a telomerase-related fitness defect and chromosome substitution technology in yeast synIX strains.

We describe the complete synthesis, assembly, debugging, and characterization of a synthetic 404,963 bp chromosome, synIX (synthetic chromosome IX). Combined chromosome construction methods were used to synthesize and integrate its left arm (synIXL) into a strain containing previously described synIXR. We identified and resolved a bug affecting expression of EST3, a crucial gene for telomerase function, producing a synIX strain with near wild-type fitness. To facilitate future synthetic chromosome consolidation and increase flexibility of chromosome transfer between distinct strains, we combined chromoduction, a method to transfer a whole chromosome between two strains, with conditional centromere destabilization to substitute a chromosome of interest for its native counterpart. Both steps of this chromosome substitution method were efficient. We observed that wild-type II tended to co-transfer with synIX and was co-destabilized with wild-type IX, suggesting a potential gene dosage compensation relationship between these chromosomes.

Thermostabilization of Pichia stipitis xylitol dehydrogenase by mutation of structural zinc-binding loop.

Xylitol dehydrogenase from Pichia stipitis (PsXDH) is one of the key enzymes for the bio-ethanol fermentation system from xylose. Previously, we constructed the C4 mutant (S96C/S99C/Y102C) with enhanced thermostability by introduction of structural zinc. In this study, for further improvement of PsXDH thermostability, we constructed the appropriate structural zinc-binding loop by comparison with other polyol dehydrogenase family members. A high thermostability of PsXDH was obtained by subsequent site-directed mutagenesis of the structural zinc-binding loop. The best mutant in this study (C4/F98R/E101F) showed a 10.8 degrees C higher thermal transition temperature (T(CD)) and 20.8 degrees C higher half denaturation temperature (T(1/2)) compared with wild-type.

Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein engineered NADP+-dependent xylitol dehydrogenase.

Effects of reversal coenzyme specificity toward NADP+ and thermostabilization of xylitol dehydrogenase (XDH) from Pichia stipitis on fermentation of xylose to ethanol were estimated using a recombinant Saccharomyces cerevisiae expressing together with a native xylose reductase from P. stipitis. The mutated XDHs performed the similar enzyme properties in S. cerevisiae cells, compared with those in vitro. The significant enhancement(s) was found in Y-ARSdR strain, in which NADP+-dependent XDH was expressed; 86% decrease of unfavorable xylitol excretion with 41% increased ethanol production, when compared with the reference strain expressing the wild-type XDH.

Rewriting the blueprint of life by synthetic genomics and genome engineering.

Advances in DNA synthesis and assembly methods over the past decade have made it possible to construct genome-size fragments from oligonucleotides. Early work focused on synthesis of small viral genomes, followed by hierarchical synthesis of wild-type bacterial genomes and subsequently on transplantation of synthesized bacterial genomes into closely related recipient strains. More recently, a synthetic designer version of yeast Saccharomyces cerevisiae chromosome III has been generated, with numerous changes from the wild-type sequence without having an impact on cell fitness and phenotype, suggesting plasticity of the yeast genome. A project to generate the first synthetic yeast genome--the Sc2.0 Project--is currently underway.

TALEN-mediated generation and genetic correction of disease-specific human induced pluripotent stem cells.

Generation and precise genetic correction of patient-derived hiPSCs have great potential in regenerative medicine. Such targeted genetic manipulations can now be achieved using gene-editing nucleases. Here, we report generation of cystic fibrosis (CF) and Gaucher's disease (GD) hiPSCs respectively from CF (homozygous for CFTRΔF508 mutation) and Type II GD [homozygous for ß-glucocerebrosidase (GBA) 1448T>C mutation] patient fibroblasts, using CCR5- specific TALENs. Site-specific addition of loxP-flanked Oct4/Sox2/Klf4/Lin28/Nanog/eGFP gene cassette at the endogenous CCR5 site of patient-derived disease-specific primary fibroblasts induced reprogramming, giving rise to both monoallele (heterozygous) and biallele CCR5-modified hiPSCs. Subsequent excision of the donor cassette was done by treating CCR5-modified CF and GD hiPSCs with Cre. We also demonstrate site-specific correction of sickle cell disease (SCD) mutations at the endogenous HBB locus of patient-specific hiPSCs [TNC1 line that is homozygous for mutated ß- globin alleles (ßS/ßS)], using HBB-specific TALENs. SCD-corrected hiPSC lines showed gene conversion of the mutated ßS to the wild-type ßA in one of the HBB alleles, while the other allele remained a mutant phenotype. After excision of the loxP-flanked DNA cassette from the SCD-corrected hiPSC lines using Cre, we obtained secondary heterozygous ßS/ßA hiPSCs, which express the wild-type (ßA) transcript to 30-40% level as compared to uncorrected (ßS/ßS) SCD hiPSCs when differentiated into erythroid cells. Furthermore, we also show that TALEN-mediated generation and genetic correction of disease-specific hiPSCs did not induce any off-target mutations at closely related sites.

Total synthesis of a functional designer eukaryotic chromosome.

Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871-base pair designer eukaryotic chromosome, synIII, which is based on the 316,617-base pair native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, transfer RNAs, transposons, and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in a-mater derivatives resulting from loss of the MATα allele on synIII. The complete design and synthesis of synIII establishes S. cerevisiae as the basis for designer eukaryotic genome biology.

A CRISPR way to engineer the human genome.

RNA-guided genome engineering based on the type II prokaryotic CRISPR/Cas system provides an efficient and versatile method for targeted manipulation of mammalian genomes.

Assembling DNA fragments by USER fusion.

Recent advances in DNA synthesis technology make it possible to design and synthesize DNA fragments of several kb in size. However, the process of assembling the smaller DNA fragments into a larger DNA segment is still a cumbersome process. In this chapter, we describe the use of the uracil specific excision reaction (USER)-mediated approach for rapid and efficient assembly of multiple DNA fragments both in vitro and in vivo (using Escherichia coli). For USER fusion in vitro assembly, each of the individual building blocks (BBs), 0.75 kb in size (that are to be assembled), was amplified using the appropriate forward and reverse primers containing a single uracil (U) and DNA polymerase. The overlaps between adjoining BBs were 8-13 base pairs. An equimolar of the amplified BBs were mixed together and treated by USER enzymes to generate complementary 3' single-strand overhangs between adjoining BBs, which were then ligated and amplified simultaneously to generate the larger 3-kb segments. The assembled fragments were then cloned into plasmid vectors and sequenced to confirm their identity. For USER fusion in vivo assembly in E. coli, USER treatment of the BBs was performed in the presence of a synthetic plasmid, which had 8-13 base pair overlaps at the 5'-end of the 5' BB and at the 3'-end of the 3' BB in the mixture. The USER treated product was then transformed directly into E. coli to efficiently and correctly reconstitute the recombinant plasmid containing the desired target insert. The latter approach was also used to rapidly assemble three different target genes into a vector to form a new synthetic plasmid construct.

Assembling large DNA segments in yeast.

As described in a different chapter in this volume, the uracil-specific excision reaction (USER) fusion method can be used to assemble multiple small DNA fragments (∼0.75-kb size) into larger 3-kb DNA segments both in vitro and in vivo (in Escherichia coli). However, in order to assemble an entire synthetic yeast genome (Sc2.0 project), we need to be able to assemble these 3-kb pieces into larger DNA segments or chromosome-sized fragments. This assembly into larger DNA segments is carried out in vivo, using homologous recombination in yeast. We have successfully used this approach to assemble a 40-kb chromosome piece in the yeast Saccharomyces cerevisiae. A lithium acetate (LiOAc) protocol using equimolar amount of overlapping smaller fragments was employed to transform yeast. In this chapter, we describe the assembly of 3-kb fragments with an overlap of one building block (∼750 base pairs) into a 40-kb DNA piece.

The positive effect of the decreased NADPH-preferring activity of xylose reductase from Pichia stipitis on ethanol production using xylose-fermenting recombinant Saccharomyces cerevisiae.

We focused on the effects of a mutation of xylose reductase from Pichia stipitis (PsXR) on xylose-to-ethanol fermentation using recombinant Saccharomyces cerevisiae transformed with PsXR and PsXDH (xylitol dehydrogenase from P. stipitis) genes. Based on inherent NADH-preferring XR and several site-directed mutagenetic studies using other aldo-keto reductase enzymes, we designed several single PsXR mutants. K270R showing decreased NADPH-preferring activity without a change in NADH-preferring activity was found to be a potent mutant. Strain Y-K270R transformed with K270R PsXR and wild-type PsXDH showed a 31% decrease in unfavorable xylitol excretion with 5.1% increased ethanol production as compared to the control in the fermentation of 15 g l(-1) xylose and 5 g l(-1) glucose.

Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis.

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis (PsXR and PsXDH, respectively) has the ability to convert xylose to ethanol together with the unfavourable excretion of xylitol, which may be due to intercellular redox imbalance caused by the different coenzyme specificity between NADPH-preferring XR and NAD(+)-dependent XDH. In this study, we focused on the effect(s) of mutated NADH-preferring PsXR in fermentation. The R276H and K270R/N272D mutants were improved 52- and 146-fold, respectively, in the ratio of NADH/NADPH in catalytic efficiency [(k(cat)/K(m) with NADH)/(k(cat)/K(m) with NADPH)] compared with the wild-type (WT), which was due to decrease of k(cat) with NADPH in the R276H mutant and increase of K(m) with NADPH in the K270R/N272D mutant. Furthermore, R276H mutation led to significant thermostabilization in PsXR. The most positive effect on xylose fermentation to ethanol was found by using the Y-R276H strain, expressing PsXR R276H mutant and PsXDH WT: 20 % increase of ethanol production and 52 % decrease of xylitol excretion, compared with the Y-WT strain expressing PsXR WT and PsXDH WT. Measurement of intracellular coenzyme concentrations suggested that maintenance of the of NADPH/NADP(+) and NADH/NAD(+) ratios is important for efficient ethanol fermentation from xylose by recombinant S. cerevisiae.

Construction of various mutants of xylose metabolizing enzymes for efficient conversion of biomass to ethanol.

We applied protein engineering to construct an efficient biomass-ethanol conversion system using Saccharomyces cerevisiae. Intercellular redox imbalance caused by the different coenzyme specificity of xylose reductase (XR) and xylitol dehydrogenase (XDH) has been thought to be one of the main factors of xylitol excretion. Introduction of NADH-dependant XR generated in this study reduced the xylitol excretion probably because of maintaining the intercellular redox balance. Ethanol fermentation was measured in batch culture under anaerobic conditions. The best strain R276H produced a maximum of 5.94 g/l ethanol with yield of 0.43 g/g from 5 g glucose/l plus 15 g xylose/l.

Site-directed mutagenesis of a yeast gene for improvement of enzyme thermostability.

Enzyme stability is one of the critical factors to construct an efficient biological conversion system. Xylitol dehydrogenase (XDH) from Pichia stipitis is one of the key enzymes for bio-ethanol fermentation system from xylose. Previously, we tried to improve thermostability of XDH by introduction of structural zinc into the enzyme and successfully obtained a mutant, named C4 mutant, with an increased unfolding temperature (J. Biol. Chem., 280:10340-10349, 2005). We focused on further improvement of the thermostability of XDH in this study and employed subsequent site directed mutagenesis in structural zinc binding region for stabilizing the structural zinc binding loop. Two variants (C4/F98R and C4/E101F) showed higher thermostability than C4 mutant judged by thermal inactivation of enzyme activity and thermal transition temperature.