Yusuke Sasaki

High titer, rate, yield (TRY), and scalability are challenging metrics to achieve due to trade-offs between carbon use for growth and production. To achieve these metrics, we take the minimal cut set (MCS) approach that predicts metabolic reactions for elimination to couple metabolite production strongly with growth. We compute MCS solution-sets for a non-native product indigoidine, a sustainable pigment, in Pseudomonas putida KT2440, an emerging industrial microbe. From the 63 solution-sets, our omics guided process identifies one experimentally feasible solution requiring 14 simultaneous reaction interventions. We implement a total of 14 genes knockdowns using multiplex-CRISPRi. MCS-based solution shifts production from stationary to exponential phase. We achieve 25.6 g/L, 0.22 g/l/h, and ~50% maximum theoretical yield (0.33 g indigoidine/g glucose). These phenotypes are maintained from batch to fed-batch mode, and across scales (100-ml shake flasks, 250-ml ambr®, and 2-L bioreactors).

Journal Article Phase I Study and Pharmacokinetics of CPT-11 With 5-Day Continuous Infusion Get access Yuichiro Ohe, Yuichiro Ohe * Department of Internal Medicine, National Cancer Center HospitalTokyo, Japan *Correspondence to : Yuichiro Ohe, M.D., Department of Internal Medicine, National Cancer Center Hospital, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104,Japan Search for other works by this author on: Oxford Academic PubMed Google Scholar Yasutsuna Sasaki, Yasutsuna Sasaki Department of Internal Medicine, National Cancer Center HospitalTokyo, Japan Search for other works by this author on: Oxford Academic PubMed Google Scholar Tetsu Shinkai, Tetsu Shinkai Department of Internal Medicine, National Cancer Center HospitalTokyo, Japan Search for other works by this author on: Oxford Academic PubMed Google Scholar Kenji Eguchi, Kenji Eguchi Department of Internal Medicine, National Cancer Center HospitalTokyo, Japan Search for other works by this author on: Oxford Academic PubMed Google Scholar Tomohide Tamura, Tomohide Tamura Department of Internal Medicine, National Cancer Center HospitalTokyo, Japan Search for other works by this author on: Oxford Academic PubMed Google Scholar Akira Kojima, Akira Kojima Department of Internal Medicine, National Cancer Center HospitalTokyo, Japan Search for other works by this author on: Oxford Academic PubMed Google Scholar Hiroshi Kunikane, Hiroshi Kunikane Department of Internal Medicine, National Cancer Center HospitalTokyo, Japan Search for other works by this author on: Oxford Academic PubMed Google Scholar Hiroaki Okamoto, Hiroaki Okamoto Department of Internal Medicine, National Cancer Center HospitalTokyo, Japan Search for other works by this author on: Oxford Academic PubMed Google Scholar Atsuya Karato, Atsuya Karato Department of Internal Medicine, National Cancer Center HospitalTokyo, Japan Search for other works by this author on: Oxford Academic PubMed Google Scholar Hironobu Ohmatsu, Hironobu Ohmatsu Department of Internal Medicine, National Cancer Center HospitalTokyo, Japan Search for other works by this author on: Oxford Academic PubMed Google Scholar ... Show more Fumihiko Kanzawa, Fumihiko Kanzawa Pharmacology Division, National Cancer Center Research InstituteTokyo Search for other works by this author on: Oxford Academic PubMed Google Scholar Nagahiro Saijo Nagahiro Saijo Pharmacology Division, National Cancer Center Research InstituteTokyo Search for other works by this author on: Oxford Academic PubMed Google Scholar JNCI: Journal of the National Cancer Institute, Volume 84, Issue 12, 17 June 1992, Pages 972–974, https://doi.org/10.1093/jnci/84.12.972 Published: 17 June 1992 Article history Revision received: 03 April 1992 Accepted: 13 April 1992 Published: 17 June 1992 Received: 30 December 1992

Abstract Background Many microbes used for the rapid discovery and development of metabolic pathways have sensitivities to final products and process reagents. Isopentenol (3-methyl-3-buten-1-ol), a biogasoline candidate, has an established heterologous gene pathway but is toxic to several microbial hosts. Reagents used in the pretreatment of plant biomass, such as ionic liquids, also inhibit growth of many host strains. We explored the use of Corynebacterium glutamicum as an alternative host to address these constraints. Results We found C. glutamicum ATCC 13032 to be tolerant to both the final product, isopentenol, as well to three classes of ionic liquids. A heterologous mevalonate-based isopentenol pathway was engineered in C. glutamicum . Targeted proteomics for the heterologous pathway proteins indicated that the 3-hydroxy-3-methylglutaryl-coenzyme A reductase protein, HmgR, is a potential rate-limiting enzyme in this synthetic pathway. Isopentenol titers were improved from undetectable to 1.25 g/L by combining three approaches: media optimization; substitution of an NADH-dependent HmgR homolog from Silicibacter pomeroyi ; and development of a C. glutamicum ∆poxB ∆ldhA host chassis. Conclusions We describe the successful expression of a heterologous mevalonate-based pathway in the Gram-positive industrial microorganism, C. glutamicum, for the production of the biogasoline candidate, isopentenol. We identified critical genetic factors to harness the isopentenol pathway in C. glutamicum . Further media and cultivation optimization enabled isopentenol production from sorghum biomass hydrolysates.