Kiyotaka Y. Hara

Catalysis and rotation of F1 motor: Cleavage of ATP at the catalytic site occurs in 1 ms before 40° substep rotation

F1, a water-soluble portion of FoF1-ATP synthase, is an ATP hydrolysis-driven rotary motor. The central γ-subunit rotates in the α3β3 cylinder by repeating the following four stages of rotation: ATP-binding 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 F1 subcomplex (α3β3γ) from thermophilic Bacillus PS3 under conditions where cleavage of ATP at the catalytic site is decelerated: hydrolysis of ATP by the catalytic-site mutant F1 and hydrolysis of a slowly hydrolyzable substrate ATPγS (adenosine 5′-[γ-thio]triphosphate) by wild-type F1. 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 ATPγS hydrolysis by the mutant F1 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 F1 occurs in 1 ms during the interim dwell, and we call this interim dwell catalytic dwell.

A review of enzymes and microbes for lignocellulosic biorefinery and the possibility of their application to consolidated bioprocessing technology

The biorefinery manufacturing process for producing chemicals and liquid fuels from biomass is a promising approach for securing energy and resources. To establish cost-effective fermentation of lignocellulosic biomass, the consolidation of sacccharification and fermentation processes is a desirable strategy, but requires the development of microorganisms capable of cellulose/hemicellulose hydrolysis and target chemical production. Such an endeavor requires a large number of prerequisites to be realized, including engineering microbial strains with high cellulolytic activity, high product yield, productivities, and titers, ability to use many carbon sources, and resistance to toxic compounds released during the pretreatment of lignocellulosic biomass. Researchers have focused on either engineering naturally cellulolytic microorganisms to improve product-related properties or modifying non-cellulolytic organisms with high product yields to become cellulolytic. This article reviews recent advances in the development of microorganisms for the production of renewable chemicals and advanced biofuels, as well as ethanol, from lignocellulosic materials through consolidated bioprocessing.

Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems

INTRODUCTION To combat invading pathogens, cells develop an adaptive immune response by changing their own genetic information. In vertebrates, the generation of genetic variation (somatic hypermutation) is an essential process for diversification and affinity maturation of antibodies that function to detect and sequester various foreign biomolecules. The activation-induced cytidine deaminase (AID) carries out hypermutation by modifying deoxycytidine bases in the variable region of the immunoglobulin locus that produces antibody. AID-generated deoxyuridine in DNA is mutagenic as it can be miss-recognized as deoxythymine, resulting in C to T mutations. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) is a prokaryotic adaptive immune system that records and degrades invasive foreign DNA or RNA. The CRISPR/Cas system cleaves and incorporates foreign DNA/RNA segments into the genomic region called the CRISPR array. The CRISPR array is transcribed to produce crispr-RNA that serves as guide RNA (gRNA) for recognition of the complementary foreign DNA/RNA in a ribonucleoprotein complex with Cas proteins, which degrade the target. The CRISPR/Cas system has been repurposed as a powerful genome editing tool, because it can be programmed to cleave specific DNA sequence by providing custom gRNAs. RATIONALE Although the precise mechanism by which AID specifically mutates the immunoglobulin locus remains elusive, targeting of AID activity is facilitated by the formation of a single-stranded DNA region, such as a transcriptional RNA/DNA hybrid (R-loop). The CRISPR/Cas system can be engineered to be nuclease-inactive. The nuclease-inactive form is capable of unfolding the DNA double strand in a protospacer adjacent motif (PAM) sequence-dependent manner so that the gRNA binds to complementary target DNA strand and forms an R-loop. The nuclease-deficient CRISPR/Cas system may serve as a suitable DNA-targeting module for AID to catalyze site-specific mutagenesis. RESULTS To determine whether AID activity can be specifically targeted by the CRISPR/Cas system, we combined dCas9 (a nuclease-deficient mutant of Cas9) from Streptococcus pyogenes and an AID ortholog, PmCDA1 from sea lamprey, to form a synthetic complex (Target-AID) by either engineering a fusion between the two proteins or attaching a SH3 (Src 3 homology) domain to the C terminus of dCas9 and a SHL (SH3 interaction ligand) to the C terminus of PmCDA1. Both of these complexes performed highly efficient site-directed mutagenesis. The mutational spectrum was analyzed in yeast and demonstrated that point mutations were dominantly induced at cytosines within the range of three to five bases surrounding the –18 position upstream of the PAM sequence on the noncomplementary strand to gRNA. The toxicity associated with the nuclease-based CRISPR/Cas9 system was greatly reduced in the Target-AID complexes. Combination of PmCDA1 with the nickase Cas9(D10A) mutant, which retains cleavage activity for noncomplementary single-stranded DNA, was more efficient in yeast but also induced deletions as well as point mutations in mammalian cells. Addition of the uracil DNA glycosylase inhibitor protein, which blocks the initial step of the uracil base excision repair pathway, suppressed collateral deletions and further improved targeting efficiency. Potential off-target effects were assessed by whole-genome sequencing of yeast as well as deep sequencing of mammalian cells for regions that contain mismatched target sequences. These results showed that off-target effects were comparable to those of conventional CRISPR/Cas systems, with a reduced risk of indel formation. CONCLUSION By expanding the genome editing potential of the CRISPR/Cas9 system by deaminase-mediated hypermutation, Target-AID demonstrated a very narrow range of targeted nucleotide substitution without the use of template DNA. Nickase Cas9 and uracil DNA glycosylase inhibitor protein can be used to boost the targeting efficiency. The reduced cytotoxicity will be beneficial for use in cells that are sensitive to artificial nucleases. Use of other types of nucleotide-modifying enzymes and/or other CRISPR-related systems with different PAM requirements will expand our genome-editing repertoire and capacity. A crippled CRISPR/Cas targets AID. In vertebrate adaptive immunity, cytosine deaminase (AID or PmCDA1) induces somatic hypermutation at single-stranded DNA regions formed during transcription. The bacterial CRISPR/Cas9 immunity system recognizes and cleaves invasive DNA in a gRNA-dependent manner. AID and nuclease-deficient CRISPR/Cas9 are engineered to form a hybrid complex (Target-AID) that performs programmable cytosine mutations in a range of a few bases surrounding the –18 position upstream of PAM sequence of the noncomplementary DNA strand. 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.

Pause and rotation of F 1 -ATPase during catalysis

F1-ATPase is a rotary motor enzyme in which a single ATP molecule drives a 120° rotation of the central γ subunit relative to the surrounding α3β3 ring. Here, we show that the rotation of F1-ATPase spontaneously lapses into long (≈30 s) pauses during steady-state catalysis. The effects of ADP-Mg and mutation on the pauses, as well as kinetic comparison with bulk-phase catalysis, strongly indicate that the paused enzyme corresponds to the inactive state of F1-ATPase previously known as the ADP-Mg inhibited form in which F1-ATPase fails to release ADP-Mg from catalytic sites. The pausing position of the γ subunit deviates from the ATP-waiting position and is most likely the recently found intermediate 90° position.

The role of the betaDELSEED motif of F1-ATPase: propagation of the inhibitory effect of the epsilon subunit.

In F1-ATPase, a rotary motor enzyme, the region of the conserved DELSEED motif in the β subunit moves and contacts the rotor γ subunit when the nucleotide fills the catalytic site, and the acidic nature of the motif was previously assumed to play a critical role in rotation. Our previous work, however, disproved the assumption (Hara, K. Y., Noji, H., Bald, D., Yasuda, R., Kinosita, K., Jr., and Yoshida, M. (2000) J. Biol. Chem. 275, 14260–14263), and the role of this motif remained unknown. Here, we found that the ε subunit, an intrinsic inhibitor, was unable to inhibit the ATPase activity of a mutant thermophilic F1-ATPase in which all of the five acidic residues in the DELSEED motif were replaced with alanines, although the ε subunit in the mutant F1-ATPase assumed the inhibitory form. In addition, the replacement of basic residues in the C-terminal region of the ε subunit by alanines caused a decrease of the inhibitory effect. Partial replacement of the acidic residues in the DELSEED motif of the β subunit or of the basic residues in the C-terminal α-helix of the ε subunit induced a partial effect. We here conclude that the ε subunit exerts its inhibitory effect through the electrostatic interaction with the DELSEED motif of the β subunit.

Optimizing biodiesel production in marine Chlamydomonas sp. JSC4 through metabolic profiling and an innovative salinity-gradient strategy.

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.

Development of microbial cell factories for bio-refinery through synthetic bioengineering.

Synthetic bioengineering is a strategy for developing useful microbial strains with innovative biological functions. Novel functions are designed and synthesized in host microbes with the aid of advanced technologies for computer simulations of cellular processes and the system-wide manipulation of host genomes. Here, we review the current status and future prospects of synthetic bioengineering in the yeast Saccharomyces cerevisiae for bio-refinery processes to produce various commodity chemicals from lignocellulosic biomass. Previous studies to improve assimilation of xylose and production of glutathione and butanol suggest a fixed pattern of problems that need to be solved, and as a crucial step, we now need to identify promising targets for further engineering of yeast metabolism. Metabolic simulation, transcriptomics, and metabolomics are useful emerging technologies for achieving this goal, making it possible to optimize metabolic pathways. Furthermore, novel genes responsible for target production can be found by analyzing large-scale data. Fine-tuning of enzyme activities is essential in the latter stage of strain development, but it requires detailed modeling of yeast metabolic functions. Recombinant technologies and genetic engineering are crucial for implementing metabolic designs into microbes. In addition to conventional gene manipulation techniques, advanced methods, such as multicistronic expression systems, marker-recycle gene deletion, protein engineering, cell surface display, genome editing, and synthesis of very long DNA fragments, will facilitate advances in synthetic bioengineering.

Improvement of glutathione production by metabolic engineering the sulfate assimilation pathway of Saccharomyces cerevisiae

Glutathione (GSH) is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae. In this study, we demonstrated that engineering in sulfate assimilation metabolism can significantly improve GSH production. The intracellular GSH content of MET14 and MET16 over-expressing strains increased up to 1.2 and 1.4-fold higher than that of the parental strain, respectively, whereas those of APA1 and MET3 over-expressing strains decreased. Especially, in the MET16 over-expressing strain, the volumetric GSH concentration was up to 1.7-fold higher than that of the parental strain as a result of the synergetic effect of the increases in the cell concentration and the intracellular GSH content. Additionally, combinatorial mutant strains that had been engineered to contain both the sulfur and the GSH synthetic metabolism synergistically increased the GSH production. External addition of cysteine to S. cerevisiae is well known as a way to increase the intracellular GSH content; however, it results a decrease in cell growth. This study showed that the engineering of sulfur metabolism in S. cerevisiae proves more valuable than addition of cysteine as a way to boost GSH production due to the increases in both the intracellular GSH content and the cell growth.

Glutathione production by efficient ATP‐regenerating Escherichia coli mutants

There is an ongoing demand to improve the ATP-regenerating system for industrial ATP-driven bioprocesses because of the low efficiency of ATP regeneration. To address this issue, we investigated the efficiency of ATP regeneration in Escherichia coli using the Permeable Cell Assay. This assay identified 40 single-gene deletion strains that had over 150% higher total cellular ATP synthetic activity relative to the parental strain. Most of them also showed higher ATP-driven glutathione synthesis. The deleted genes of the identified strains that showed increased efficiency of ATP regeneration for glutathione production could be divided into the following four groups: (1) glycolytic pathway-related genes, (2) genes related to degradation of ATP or adenosine, (3) global regulatory genes, and (4) genes whose contribution to the ATP regeneration is unknown. Furthermore, the high glutathione productivity of DeltanlpD, the highest glutathione-producing mutant strain, was due to its reduced sensitivity to the externally added ATP for ATP regeneration. This study showed that the Permeable Cell Assay was useful for improving the ATP-regenerating activity of E. coli for practical applications in various ATP-driven bioprocesses, much as that of glutathione production.

Development of bio-based fine chemical production through synthetic bioengineering

AbstractFine chemicals that are physiologically active, such as pharmaceuticals, cosmetics, nutritional supplements, flavoring agents as well as additives for foods, feed, and fertilizer are produced by enzymatically or through microbial fermentation. The identification of enzymes that catalyze the target reaction makes possible the enzymatic synthesis of the desired fine chemical. The genes encoding these enzymes are then introduced into suitable microbial hosts that are cultured with inexpensive, naturally abundant carbon sources, and other nutrients. Metabolic engineering create efficient microbial cell factories for producing chemicals at higher yields. Molecular genetic techniques are then used to optimize metabolic pathways of genetically and metabolically well-characterized hosts. Synthetic bioengineering represents a novel approach to employ a combination of computer simulation and metabolic analysis to design artificial metabolic pathways suitable for mass production of target chemicals in host strains. In the present review, we summarize recent studies on bio-based fine chemical production and assess the potential of synthetic bioengineering for further improving their productivity.