Sortase A-assisted metabolic enzyme ligation in Escherichia coli

We demonstrated the metabolic enzyme ligation by sortase A-mediated ligation (sortagging) for the redirection of metabolic flux thorough metabolic channeling. Staphylococcal sortase A (SrtA) is utilized for the ligation of metabolic enzymes. SrtA is transpeptidase, which recognizes Leu-Pro-Xaa-Thr-Gly sequences (LP tag) and cleaves between Thr and Gly, and subsequently links amino group of oligoglycine (G tag) thorough a native peptide bond. Sortagging enables to conjugate protein with other molecules in a site-specific manner. Minimal modifications of protein with short peptide tags; LP tag and G tag are only required for site-specific ligation. Hence, sortagging has been utilized for preparing a variety of bioconjugation not only in vitro but also in vivo.1 In current study, we hypothesize that SrtA-mediated metabolic enzyme ligation in cytoplasm of Escherichia coli facilitates processing metabolic intermediate, and redirects metabolic fluxes to desired pathway. As proof of concept, we constructed acetate producing E. coli with engineered endogenous metabolic pathway, which redirect central metabolic fluxes to acetate producing flux by the induction of chemical additives (Figure 1). The expression of SrtA was controlled by Lac operating promoter, metabolic channeling was videlicet occurred by the addition of IPTG. Acetyl-CoA was chosen as the intermediate model because acetyl-CoA is one of the most important central metabolic intermediates, which is converted to alcohols, fatty acids, and mevalonate derivatives. In this study, we tested covalent linking of pyruvate-formate lyase and phosphate acetyltransferase by sortase A-mediated ligation and evaluated the production of acetate. The time point of addition of IPTG was not critical for facilitating metabolic enzyme ligation, and acetate production increased upon expression of sortase A. These results show that sortase A-mediated enzyme ligation enhances an acetate-producing flux in E. coli. We have validated that sortase A-mediated enzyme ligation offers a metabolic channeling approach to redirect a central flux to a desired flux.2 Please click Additional Files below to see the full abstract

Metabolic engineering of S. pombe via CRISPR-Cas9 genome editing for lactic acid production from glucose and cellobiose

We constructed D-lactic acid (D-LA) producing Schizosaccharomyces pombe using CRISPR-Cas9 system. Two PDC genes, intact L-LDH, a minor gene of alcohol dehydrogenase (SPBC337.11) were disrupted to attenuate ethanol production pathway. To increase the cellular supply of acetyl-CoA, an important metabolite for growth, we introduced bacterial acetylating acetaldehyde dehydrogenase enzyme genes. Two kinds of acetaldehyde dehydrogenase genes from Escherichia coli, mhpF and eutE, were expressed. D-LA production was achieved by expressing D-lactate dehydrogenase gene from Lactobacillus plantarum. The engineered strains efficiently consumed glucose and produced 25.2 g/liter of D-LA from 35.5 g/liter of consumed glucose with the yield of 0.71 g-D-LA / g-glucose. Finally, we expressed beta-glucosidase by cell surface display techniques, and the resultant strain produced 24.4 g/L of D-LA from 30 g/L of cellobiose in minimal medium with the yield of 0.68 g-D-LA / g-glucose. This is the first report to generate metabolically engineered S. pombe strain using CRISPR-Cas9 system and we showed the possibility of S. pombe for the production host cell of value-added chemicals

Metabolic design of Escherichia coli for muconic acid production

Adipic acid(AA) is a versatile bulk chemical to be used for raw materials such as nylon 6,6. Currently, AA biosynthesis from bio-resources have received a lot of attention in recent years as environment-friendly and renewable AA production process. Muconic acid(MA), also known as 2,4-hexadienedioic acid, is expected as a biosynthesis precursor of AA. There are Several studies on MA biosynthesis using Escherichia coli introduced foreign genes. In those studies, MA is synthesized from intermediate products of shikimate pathway. However, the production volume is not sufficient and it is a hindrance to industrialization. In this study, we aimed to the high efficiency biosynthesis of MA using metabolic designed Escherichia coli. First, we designed the metabolism to increase the accumulation of phosphoenolpyruvic acid (PEP), which is one of the starting materials of the shikimate pathway. Next, we determined the optimal MA synthetic pathway branched from the shikimate pathway. Specifically, we examined three types of MA production pathway with PEP accumulation strain as parent and selected the pathway with the highest MA production. Finally, we examined efficient production of MA using fusion proteins. Shikimate pathway protein and MA production pathway protein were combined to direct carbon flux into MA production

Direct ethanol production from cellulosic materials using a diploid strain of Saccharomyces cerevisiae with optimized cellulase expression

<p>Abstract</p> <p>Background</p> <p>Hydrolysis of cellulose requires the action of the cellulolytic enzymes endoglucanase, cellobiohydrolase and β-glucosidase. The expression ratios and synergetic effects of these enzymes significantly influence the extent and specific rate of cellulose degradation. In this study, using our previously developed method to optimize cellulase-expression levels in yeast, we constructed a diploid <it>Saccharomyces cerevisiae </it>strain optimized for expression of cellulolytic enzymes, and attempted to improve the cellulose-degradation activity and enable direct ethanol production from rice straw, one of the most abundant sources of lignocellulosic biomass.</p> <p>Results</p> <p>The engineered diploid strain, which contained multiple copies of three cellulase genes integrated into its genome, was precultured in molasses medium (381.4 mU/g wet cell), and displayed approximately six-fold higher phosphoric acid swollen cellulose (PASC) degradation activity than the parent haploid strain (63.5 mU/g wet cell). When used to ferment PASC, the diploid strain produced 7.6 g/l ethanol in 72 hours, with an ethanol yield that achieved 75% of the theoretical value, and also produced 7.5 g/l ethanol from pretreated rice straw in 72 hours.</p> <p>Conclusions</p> <p>We have developed diploid yeast strain optimized for expression of cellulolytic enzymes, which is capable of directly fermenting from cellulosic materials. Although this is a proof-of-concept study, it is to our knowledge, the first report of ethanol production from agricultural waste biomass using cellulolytic enzyme-expressing yeast without the addition of exogenous enzymes. Our results suggest that combining multigene expression optimization and diploidization in yeast is a promising approach for enhancing ethanol production from various types of lignocellulosic biomass.</p

Cocktail δ-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains

<p>Abstract</p> <p>Background</p> <p>The filamentous fungus <it>T. reesei </it>effectively degrades cellulose and is known to produce various cellulolytic enzymes such as β-glucosidase, endoglucanase, and cellobiohydrolase. The expression levels of each cellulase are controlled simultaneously, and their ratios and synergetic effects are important for effective cellulose degradation. However, in recombinant <it>Saccharomyces cerevisiae</it>, it is difficult to simultaneously control many different enzymes. To construct engineered yeast with efficient cellulose degradation, we developed a simple method to optimize cellulase expression levels, named cocktail δ-integration.</p> <p>Results</p> <p>In cocktail δ-integration, several kinds of cellulase expression cassettes are integrated into yeast chromosomes simultaneously in one step, and strains with high cellulolytic activity (i.e., expressing an optimum ratio of cellulases) are easily obtained. Although the total integrated gene copy numbers of cocktail δ-integrant strain was about half that of a conventional δ-integrant strain, the phosphoric acid swollen cellulose (PASC) degradation activity (64.9 mU/g-wet cell) was higher than that of a conventional strain (57.6 mU/g-wet cell). This suggests that optimization of the cellulase expression ratio improves PASC degradation activity more so than overexpression.</p> <p>Conclusions</p> <p>To our knowledge, this is the first report on the expression of cellulase genes by δ-integration and optimization of various foreign genes by δ-integration in yeast. This method should be very effective and easily applied for other multi-enzymatic systems using recombinant yeast.</p

Predominant Magnetic States in Hubbard Model on Anisotropic Triangular Lattices

Using an optimization variational Monte Carlo method, we study the half-filled-band Hubbard model on anisotropic triangular lattices, as a continuation of the preceding study [J. Phys. Soc. Jpn 75, 074707 (2006)]. We introduce two new trial states: (i) A coexisting state of (\pi,\pi)-antiferromagnetic (AF) and a d-wave singlet gaps, in which we allow for a band renormalization effect, and (ii) a state with an AF order of 120^\circ spin structure. In both states, a first-order metal-to-insulator transition occurs at smaller U/t than that of the pure d-wave state. In insulating regimes, magnetic orders always exist; an ordinary (\pi,\pi)-AF order survives up to t'/t\sim 0.9 (U/t=12), and a 120^\circ-AF order becomes dominant for t'/t \gsim 0.9. The regimes of the robust superconductor and of the nonmagnetic insulator the preceding study proposed give way to these magnetic domains.Comment: 11 pages, 14 figure

Neurosecretory protein GL stimulates food intake, de novo lipogenesis, and onset of obesity.

Mechanisms underlying the central regulation of food intake and fat accumulation are not fully understood. We found that neurosecretory protein GL (NPGL), a newly-identified neuropeptide, increased food intake and white adipose tissue (WAT) in rats. NPGL-precursor gene overexpression in the hypothalamus caused increases in food intake, WAT, body mass, and circulating insulin when fed a high calorie diet. Intracerebroventricular administration of NPGL induced de novo lipogenesis in WAT, increased insulin, and it selectively induced carbohydrate intake. Neutralizing antibody administration decreased the size of lipid droplets in WAT. Npgl mRNA expression was upregulated by fasting and low insulin levels. Additionally, NPGL-producing cells were responsive to insulin. These results point to NPGL as a novel neuronal regulator that drives food intake and fat deposition through de novo lipogenesis and acts to maintain steady-state fat level in concert with insulin. Dysregulation of NPGL may be a root cause of obesity