Genome editing and synthesis platforms which facilitate the Construction of cell factories

We have developed the platform technologies such as genome editing and a large gene cluster synthesis systems and are going to integrate to set up the automated systems for efficient construction of microbial cell factories. By tethering the DNA deaminase activity to nuclease-deficient CRISPR/Cas9 system, we have developed a genome editing tool that enables targeted point mutagenesis. An AID orthologue PmCDA1 was attached to nuclease-deficient mutant of Cas9 (D10A and H840A) to perform highly efficient and target-specific nucleotide editing. This hybrid system, termed Target-AID, induced cytosine point mutation in 3-5 bases range at the distal site within target sequence. Use of nickase Cas9 (D10A), which retains single-strand cleaving activity, greatly increase the efficiency, although it also occasionally induces insertion/deletion (indel) in higher eukaryotes. Uracil-DNA glycosylase inhibitor further increase the efficiency and reduced the indel formation. The toxicity associated with Cas9 has been greatly diminished, enabling application of this technique to wider range of organisms including yeast, bacteria, animals and plants. In addition, by tethering Glycosilase activity to nuclease-deficient CRISPR/Cas9 system, we have developed a genome editing tool that enables targeted randam mutagenesis. We have also developed an efficient DNA assembly method, namely, Ordered Gene Assembly in B. subtilis (OGAB) method. OGAB method can assemble more than 50 DNA fragments in one-step using B. subtilis. Thanks to this high processability, even in construction of long DNA (~100 kb), material DNA fragments can be kept in chemical DNA synthesis-friendly and sequencing-friendly small size (\u3c 2 kb). Since there is no in vitro DNA synthesis step that may cause unexpected mutation(s), long DNA by OGAB method using sequence-confirmed material DNA thus contains essentially no mutation. We are now constructing user friendly DNA synthesis system by integrating new automation system, such like a liquid handling robot that is specifically developed for OGAB method These technologies might lead to new pipelines through which functional genomes are cleated with much faster speed to construct microbial cell factories to produce variety of biofuels and chemicals

Biosensing Techniques in Yeast: G-Protein Signaling and Protein-Protein Interaction Assays for Monitoring Ligand Stimulation and Oligomer Formation of Heterologous GPCRs

Guanine nucleotide-binding proteins (G-proteins) act as transducers of external stimuli for intracellular signaling, and control various cellular processes in cooperation with seven transmembrane G-protein-coupled receptors (GPCRs). Because GPCRs constitute the largest family of eukaryotic membrane proteins and enable the selective recognition of a diverse range of molecules (ligands), they are the major molecular targets in pharmaceutical and medicinal fields. In addition, GPCRs have been known to form heteromers as well as homomers, which may result in vast physiological diversity and provide opportunities for drug discovery. G-proteins and their signal transduction machinery are universally conserved in eukaryotes; thereby, the yeast Saccharomyces cerevisiae has been used to construct artificial in vivo GPCR biosensors. In this chapter, we focus on the yeast-based GPCR biosensors that can detect ligand stimulation and oligomer formation, and summarize their techniques using the G-protein signaling and protein-protein interaction assays

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

Microbial platform to synthesize chorismate derivatives via metabolic engineering approach

A synthetic metabolic pathway suitable for the production of chorismate derivatives was designed in Escherichia coli. An L-phenylalanine-overproducing E. coli strain was engineered to enhance the availability of phosphoenolpyruvate (PEP), which is a key precursor in the biosynthesis of aromatic compounds in microbes. Two major reactions converting PEP to pyruvate were inactivated. Using this modified E.coli as a base strain, we tested our system by carrying out the production of salicylate, a high-demand aromatic chemical. The titer of salicylate reached 11.5 g/L in batch culture after 48 h cultivation in a 1-liter jar fermentor, and the yield from glucose as the sole carbon source exceeded 40% (mol/mol). In this test case, we found that pyruvate was synthesized primarily via salicylate formation and the reaction converting oxaloacetate to pyruvate. In order to demonstrate the generality of our designed strain, we employed this platform for the production of each of 7 different chorismate derivatives. Each of these industrially important chemicals was successfully produced to levels of 1-3 g/L in test tube-scale culture. In addition, by extending chorismate pathway, we successfully achieved maleate production, which is one of significant dicarboxylic acid as well as succinate and malate. A novel synthetic pathway of maleate was constructed in our base strain, and the productivity reached 7.1 g/L. This is the first report about maleate production using genetically engineered micro-organisms

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