The generation of genetic variation (somatic hypermutation) is an essential process for the adaptive immune system in vertebrates. We demonstrate the targeted single-nucleotide substitution of DNA using hybrid vertebrate and bacterial immune systems components. Nuclease-deficient type II CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) and the activation-induced cytidine deaminase (AID) ortholog PmCDA1 were engineered to form a synthetic complex (Target-AID) that performs highly efficient target-specific mutagenesis. Specific point mutation was induced primarily at cytidines within the target range of five bases. The toxicity associated with the nuclease-based CRISPR/Cas9 system was greatly reduced. Although combination of nickase Cas9(D10A) and the deaminase was highly effective in yeasts, it also induced insertion and deletion (indel) in mammalian cells. Use of uracil DNA glycosylase inhibitor suppressed the indel formation and improved the efficiency.

F1, a water-soluble portion of FoF1-ATP synthase, is an ATP hydrolysis-driven rotary motor. The central ␥-subunit rotates in the ␣3␤3 cylinder by repeating the following four stages of rotation: ATPbinding dwell, rapid 80°substep rotation, interim dwell, and rapid 40°substep rotation. At least two 1-ms catalytic events occur in the interim dwell, but it is still unclear which steps in the ATPase cycle, except for ATP binding, correspond to these events. To discover which steps, we analyzed rotations of F 1 subcomplex (␣3␤3␥) from thermophilic Bacillus PS3 under conditions where cleavage of ATP at the catalytic site is decelerated: hydrolysis of ATP by the catalytic-site mutant F 1 and hydrolysis of a slowly hydrolyzable substrate ATP␥S (adenosine 5-[␥-thio]triphosphate) by wild-type F 1. In both cases, interim dwells were extended as expected from bulk phase kinetics, confirming that cleavage of ATP takes place during the interim dwell. Furthermore, the results of ATP␥S hydrolysis by the mutant F 1 ensure that cleavage of ATP most likely corresponds to one of the two 1-ms events and not some other faster undetected event. Thus, cleavage of ATP on F 1 occurs in 1 ms during the interim dwell, and we call this interim dwell catalytic dwell. Fo F 1 -ATP synthase is an enzyme ubiquitous from bacteria to animals and plants. It synthesizes ATP from ADP and inorganic phosphate by using ⌬ H ϩ -driven proton flow through a membrane (1, 2). F 0 F 1 -ATP synthase can easily be separated into two major portions: water-soluble F 1 and membraneembedded F o . The isolated F 1 (␣ 3 ␤ 3 ␥␦) has an ATP hydrolysis activity and is often called F 1 -ATPase (3, 4). The crystal structure of F 1 shows that the rod-shaped ␥-subunit is surrounded by a cylinder made of three ␣-and three ␤-subunits arranged alternatively (5). The catalytic sites are located in ␤-subunits but residues from adjacent ␣-subunits also contribute. It has been thought that F o F 1 -ATP synthase is a complex of F o motor and F 1 motor that share a common rotor: a downhill proton flow through F o drives rotation of the rotor, causing conformational changes in F 1 that result in ATP synthesis. Conversely, ATP hydrolysis in F 1 causes a reverse rotation of the rotor that enforces F o to pump protons in the reverse direction (6). The rotor is made of a c-subunit ring of F o (7-11) and ␥ subunits of F 1 (12-15).We have visualized and analyzed the ATP-driven rotation of the ␥-subunit in the minimum assembly of F 1 motor, ␣ 3 ␤ 3 ␥ subcomplex (hereafter in this article, this subcomplex is called F 1 ) (13,16). To date, the following features have been established. The ␥-subunit makes a 120°step per one ATP consumption (17), which is further divided into 90°and 30°substeps (18). The dwelling time before the 90°substep rotation depends on ATP concentration and disappears beyond the limit of time resolution of the observation methods as ATP concentration ([ATP]) increases. Therefore, the dwell before the 90°substep rotation is a dwell for ATP binding, and the 90°substep rota...

A TP synthase of mitochondria, chloroplasts, and bacteria catalyzes ATP synthesis coupled with a transmembrane proton flow (1-4). The enzyme consists of a membraneembedded, proton-conducting portion (F 0 ) and a protruding portion (F 1 ) in which catalytic sites for ATP synthesis͞hydrolysis exist. The isolated F 1 portion has ATPase activity; hence, it is often called F 1 -ATPase. It is composed of five different subunits with a stoichiometry of ␣ 3 ␤ 3 ␥␦. The ␣ 3 ␤ 3 ␥ subcomplex is the minimum ATPase-active complex, which has catalytic features similar to F 1 -ATPase. In the crystal structure (5), the central ␥ subunit is surrounded by an ␣ 3 ␤ 3 cylinder where three ␣ and three ␤ subunits are arranged alternately, and the six nucleotide binding sites are located at the ␣͞␤ subunit interfaces. Three of the binding sites are catalytic, and the ␤ subunits provide most of the catalytic residues. The other three are noncatalytic, and the ␣ subunits provide most residues contributing nucleotide binding.It has been postulated that the energy of the proton flow liberated at F 0 is transformed into the energy of ATP synthesis at F 1 through rotation of the central ␥ subunit and vice versathe energy of ATP hydrolysis can be converted into the energy of proton pumping through reverse rotation of the ␥ subunit (6). By using an ␣ 3 ␤ 3 ␥ subcomplex of thermophilic F 1 -ATPase (F 1 -ATPase) immobilized on a glass surface, we have observed ATP hydrolysis-driven rotation of the fluorescent actin filament attached to the ␥ subunit (7).At nanomolar ATP concentration, F 1 -ATPase binds and hydrolyzes a single ATP molecule, makes a 120°rotation, and waits for the next ATP molecule. As the ATP concentration increases, the ATP-waiting period becomes shorter until it is finally undetectable, and rotation of the actin filament becomes apparently continuous over hundreds of revolutions (8). However, when the rotation was observed for long periods, occasional pauses of rotation were recognized, even at high ATP concentrations (7, 9). Here, we show that these pauses occur at an intermediate step of rotation and mostly correspond to the ADP-Mg inhibition, which has been observed in bulk-phase kinetics as a general feature of the F 1 -ATPases (and ATP synthases). Slow interconversion between rotating and pausing states thus contributes to the attenuation of ATPase during steady-state catalysis. Materials and MethodsProtein Preparation. Escherichia coli strains used were JM109 (10) for preparation of plasmids, CJ236 (11) for generating uracilcontaining single-stranded plasmids for site-directed mutagenesis, and JM103⌬ (uncB-uncD) for expression of the mutant complexes of F 1 from the thermophilic Bacillus PS3. Plasmids M13mp18 and pKAGB1 (12), which carried genes for the ␣, ␤, and ␥ subunits of F 1 from the thermophilic Bacillus PS3, were used for mutagenesis and for gene expression, respectively. Site-directed mutagenesis was accomplished as described by Kunkel et al. (11). The plasmid pKAGB1͞␣C193S͞␥S107C͞ ␤His10tag has been described ...

No abstract

BackgroundBiodiesel production from marine microalgae has received much attention as microalgae can be cultivated on non-arable land without the use of potable water, and with the additional benefits of mitigating CO2 emissions and yielding biomass. However, there is still a lack of effective operational strategies to promote lipid accumulation in marine microalgae, which are suitable for making biodiesel since they are mainly composed of saturated and monounsaturated fatty acids. Moreover, the regulatory mechanisms involved in lipid biosynthesis in microalgae under environmental stress are not well understood.ResultsIn this work, the combined effects of salinity and nitrogen depletion stresses on lipid accumulation of a newly isolated marine microalga, Chlamydomonas sp. JSC4, were explored. Metabolic intermediates were profiled over time to observe transient changes during the lipid accumulation triggered by the combination of the two stresses. An innovative cultivation strategy (denoted salinity-gradient operation) was also employed to markedly improve the lipid accumulation and lipid quality of the microalga, which attained an optimal lipid productivity of 223.2 mg L-1 d-1 and a lipid content of 59.4% per dry cell weight. This performance is significantly higher than reported in most related studies.ConclusionsThis work demonstrated the synergistic integration of biological and engineering technologies to develop a simple and effective strategy for the enhancement of oil production in marine microalgae.

Adenosine-5′-triphosphate (ATP) is consumed as a biological energy source by many intracellular reactions. Thus, the intracellular ATP supply is required to maintain cellular homeostasis. The dependence on the intracellular ATP supply is a critical factor in bioproduction by cell factories. Recent studies have shown that changing the ATP supply is critical for improving product yields. In this review, we summarize the recent challenges faced by researchers engaged in the development of engineered cell factories, including the maintenance of a large ATP supply and the production of cell factories. The strategies used to enhance ATP supply are categorized as follows: addition of energy substrates, controlling pH, metabolic engineering of ATP-generating or ATP-consuming pathways, and controlling reactions of the respiratory chain. An enhanced ATP supply generated using these strategies improves target production through increases in resource uptake, cell growth, biosynthesis, export of products, and tolerance to toxic compounds.

F 1 -ATPase is a rotary motor protein, and ATP hydrolysis generates torque at the interface between the ␥ subunit, a rotor shaft, and the ␣ 3 ␤ 3 substructure, a stator ring. The region of conserved acidic "DELSEED" motif of the ␤ subunit has a contact with ␥ subunit and has been assumed to be involved in torque generation. Using the thermophilic ␣ 3 ␤ 3 ␥ complex in which the corresponding sequence is DELSDED, we replaced each residue and all five acidic residues in this sequence with alanine. In addition, each of two conserved residues at the counterpart contact position of ␥ subunit was also replaced. Surprisingly, all of these mutants rotated with as much torque as the wild-type. We conclude that side chains of the DELSEED motif of the ␤ subunit do not have a direct role in torque generation.F 1 , together with the membrane-embedded proton-conducting unit F 0 , forms the F 0 F 1 -ATP synthase that reversibly couples transmembrane proton flow to ATP synthesis/hydrolysis (1-6). Isolated F 1 has ATP-hydrolyzing activity, F 1 -ATPase, and has a subunit structure ␣ 3 ␤ 3 ␥␦⑀ in which the central ␥ subunit with coiled-coil structure is surrounded by the ␣ 3 ␤ 3 hexagonal ring structure (7). The ␣ and ␤ subunits have amino acid sequences homologous with each other, a similar folding topology, and noncatalytic and catalytic nucleotide binding sites, respectively. F 1 is by itself a rotary motor molecule. Using the ␣ 3 ␤ 3 ␥ complex, a minimum stable ATPase-active complex of F 1 from thermophilic Bacillus PS3 (TF 1 ) 1 (8 -10), rotation of the ␥ subunit relative to the ␣ 3 ␤ 3 ring was visualized under an optical microscope as rotation of a fluorescent actin filament attached to the ␥ subunit of the immobilized ␣ 3 ␤ 3 ␥ complex (11). The torque of the rotation is invariably ϳ40 pN⅐nm for actin filaments with various lengths, and at low ATP concentrations, rotation driven by a single ATP hydrolysis was observed as a discrete 120°step (12).Since the rotation of the ␥ subunit was established (11-18), the mechanism of how ATP hydrolysis on the ␤ subunits drives rotation of the ␥ subunit has attracted keen interest. It is obvious that torque should be generated at the interface between the ␥ subunit and the ␣ 3 ␤ 3 ring. In the crystal structure of F 1 from bovine mitochondria (MF 1 ), three ␤ subunits are in different states; one ␤ (␤ TP ) has an ATP analog, Mg-AMP-PNP, at its catalytic site, another ␤ (␤ DP ) has Mg-ADP, the third ␤ E has none. The structures of ␤ TP and ␤ DP are very similar to each other and they are in the "closed" conformation, in which the carboxyl-terminal helical domain is lifted close to the nucleotide binding domain and in contact with the ␥ subunit. In contrast, ␤ E adopts the "open" conformation, in which the crevice for substrate binding is open and the carboxyl-terminal domain is apart from the ␥ subunit. It was shown that this structure of The carboxyl-terminal domain of the ␤ subunit contains the acidic cluster sequence, known as the DELSEED motif. This sequence has been well conserved i...

We determined the genome sequence of the thermotolerant yeast Kluyveromyces marxianus strain NBRC1777. The genome of strain NBRC1777 is composed of 4,912 open reading frames (ORFs) on 8 chromosomes, with a total size of 10,895,581 bp, including mitochondrial DNA.