Michael Christopher Jewett

All Publications

• Carbon-negative production of acetone and isopropanol by gas fermentation at industrial pilot scale NATURE BIOTECHNOLOGY Liew, F., Nogle, R., Abdalla, T., Rasor, B. J., Canter, C., Jensen, R. O., Wang, L., Strutz, J., Chirania, P., De Tissera, S., Mueller, A. P., Ruan, Z., Gao, A., Tran, L., Engle, N. L., Bromley, J. C., Daniell, J., Conrado, R., Tschaplinski, T. J., Giannone, R. J., Hettich, R. L., Karim, A. S., Simpson, S. D., Brown, S. D., Leang, C., Jewett, M. C., Kopke, M. 2022; 40 (3): 335-+

  Abstract

  Many industrial chemicals that are produced from fossil resources could be manufactured more sustainably through fermentation. Here we describe the development of a carbon-negative fermentation route to producing the industrially important chemicals acetone and isopropanol from abundant, low-cost waste gas feedstocks, such as industrial emissions and syngas. Using a combinatorial pathway library approach, we first mined a historical industrial strain collection for superior enzymes that we used to engineer the autotrophic acetogen Clostridium autoethanogenum. Next, we used omics analysis, kinetic modeling and cell-free prototyping to optimize flux. Finally, we scaled-up our optimized strains for continuous production at rates of up to ~3 g/L/h and ~90% selectivity. Life cycle analysis confirmed a negative carbon footprint for the products. Unlike traditional production processes, which result in release of greenhouse gases, our process fixes carbon. These results show that engineered acetogens enable sustainable, high-efficiency, high-selectivity chemicals production. We expect that our approach can be readily adapted to a wide range of commodity chemicals.

  View details for DOI 10.1038/s41587-021-01195-w

  View details for Web of Science ID 000758993700004

  View details for PubMedID 35190685

  View details for PubMedCentralID 7356534

• On-demand biomanufacturing of protective conjugate vaccines SCIENCE ADVANCES Stark, J. C., Jaroentomeechai, T., Moeller, T. D., Hershewe, J. M., Warfel, K. F., Moricz, B. S., Martini, A. M., Dubner, R. S., Hsu, K. J., Stevenson, T. C., Jones, B. D., DeLisa, M. P., Jewett, M. C. 2021; 7 (6)

  Abstract

  Conjugate vaccines are among the most effective methods for preventing bacterial infections. However, existing manufacturing approaches limit access to conjugate vaccines due to centralized production and cold chain distribution requirements. To address these limitations, we developed a modular technology for in vitro conjugate vaccine expression (iVAX) in portable, freeze-dried lysates from detoxified, nonpathogenic Escherichia coli. Upon rehydration, iVAX reactions synthesize clinically relevant doses of conjugate vaccines against diverse bacterial pathogens in 1 hour. We show that iVAX-synthesized vaccines against Francisella tularensis subsp. tularensis (type A) strain Schu S4 protected mice from lethal intranasal F. tularensis challenge. The iVAX platform promises to accelerate development of new conjugate vaccines with increased access through refrigeration-independent distribution and portable production.

  View details for DOI 10.1126/sciadv.abe9444

  View details for Web of Science ID 000615369000039

  View details for PubMedID 33536221

  View details for PubMedCentralID PMC7857678

• In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design. Nature chemical biology Karim, A. S., Dudley, Q. M., Juminaga, A., Yuan, Y., Crowe, S. A., Heggestad, J. T., Garg, S., Abdalla, T., Grubbe, W. S., Rasor, B. J., Coar, D. N., Torculas, M., Krein, M., Liew, F. E., Quattlebaum, A., Jensen, R. O., Stuart, J. A., Simpson, S. D., Köpke, M., Jewett, M. C. 2020; 16 (8): 912-919

  Abstract

  The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate production and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, we then scaled up our highest-performing pathway, which improved in vivo 3-HB production in Clostridium by 20-fold to 14.63 ± 0.48 g l-1. We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.

  View details for DOI 10.1038/s41589-020-0559-0

  View details for PubMedID 32541965

  View details for PubMedCentralID 373084

• BioBits (TM) Bright: A fluorescent synthetic biology education kit SCIENCE ADVANCES Stark, J. C., Huang, A., Nguyen, P. Q., Dubner, R. S., Hsu, K. J., Ferrante, T. C., Anderson, M., Kanapskyte, A., Mucha, Q., Packett, J. S., Patel, P., Patel, R., Qaq, D., Zondor, T., Burke, J., Martinez, T., Miller-Berry, A., Puppala, A., Reichert, K., Schmid, M., Brand, L., Hill, L. R., Chellaswamy, J. F., Faheem, N., Fetherling, S., Gong, E., Gonzalzles, E., Granito, T., Koritsaris, J., Binh Nguyen, Ottman, S., Palffy, C., Patel, A., Skweres, S., Slaton, A., Woods, T., Donghia, N., Pardee, K., Collins, J. J., Jewett, M. C. 2018; 4 (8): eaat5107

  Abstract

  Synthetic biology offers opportunities for experiential educational activities at the intersection of the life sciences, engineering, and design. However, implementation of hands-on biology activities in classrooms is challenging because of the need for specialized equipment and expertise to grow living cells. We present BioBits™ Bright, a shelf-stable, just-add-water synthetic biology education kit with easy visual outputs enabled by expression of fluorescent proteins in freeze-dried, cell-free reactions. We introduce activities and supporting curricula for teaching the central dogma, tunable protein expression, and design-build-test cycles and report data generated by K-12 teachers and students. We also develop inexpensive incubators and imagers, resulting in a comprehensive kit costing

  View details for DOI 10.1126/sciadv.aat5107

  View details for Web of Science ID 000443498100062

  View details for PubMedID 30083609

  View details for PubMedCentralID PMC6070313

• Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids NATURE COMMUNICATIONS Martin, R. W., Des Soye, B. J., Kwon, Y., Kay, J., Davis, R. G., Thomas, P. M., Majewska, N. I., Chen, C. X., Marcum, R. D., Weiss, M., Stoddart, A. E., Amiram, M., Charna, A., Patel, J. R., Isaacs, F. J., Kelleher, N. L., Hong, S., Jewett, M. C. 2018; 9: 1203

  Abstract

  Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-L-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation ( ≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.

  View details for DOI 10.1038/s41467-018-03469-5

  View details for Web of Science ID 000428165400002

  View details for PubMedID 29572528

  View details for PubMedCentralID PMC5865108

• Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery. Nature communications Jaroentomeechai, T. n., Stark, J. C., Natarajan, A. n., Glasscock, C. J., Yates, L. E., Hsu, K. J., Mrksich, M. n., Jewett, M. C., DeLisa, M. P. 2018; 9 (1): 2686

  Abstract

  The emerging discipline of bacterial glycoengineering has made it possible to produce designer glycans and glycoconjugates for use as vaccines and therapeutics. Unfortunately, cell-based production of homogeneous glycoproteins remains a significant challenge due to cell viability constraints and the inability to control glycosylation components at precise ratios in vivo. To address these challenges, we describe a novel cell-free glycoprotein synthesis (CFGpS) technology that seamlessly integrates protein biosynthesis with asparagine-linked protein glycosylation. This technology leverages a glyco-optimized Escherichia coli strain to source cell extracts that are selectively enriched with glycosylation components, including oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs). The resulting extracts enable a one-pot reaction scheme for efficient and site-specific glycosylation of target proteins. The CFGpS platform is highly modular, allowing the use of multiple distinct OSTs and structurally diverse LLOs. As such, we anticipate CFGpS will facilitate fundamental understanding in glycoscience and make possible applications in on demand biomanufacturing of glycoproteins.

  View details for DOI 10.1038/s41467-018-05110-x

  View details for PubMedID 30002445

  View details for PubMedCentralID PMC6043479

• Protein synthesis by ribosomes with tethered subunits NATURE Orelle, C., Carlson, E. D., Szal, T., Florin, T., Jewett, M. C., Mankin, A. S. 2015; 524 (7563): 119-U289

  Abstract

  The ribosome is a ribonucleoprotein machine responsible for protein synthesis. In all kingdoms of life it is composed of two subunits, each built on its own ribosomal RNA (rRNA) scaffold. The independent but coordinated functions of the subunits, including their ability to associate at initiation, rotate during elongation, and dissociate after protein release, are an established model of protein synthesis. Furthermore, the bipartite nature of the ribosome is presumed to be essential for biogenesis, since dedicated assembly factors keep immature ribosomal subunits apart and prevent them from translation initiation. Free exchange of the subunits limits the development of specialized orthogonal genetic systems that could be evolved for novel functions without interfering with native translation. Here we show that ribosomes with tethered and thus inseparable subunits (termed Ribo-T) are capable of successfully carrying out protein synthesis. By engineering a hybrid rRNA composed of both small and large subunit rRNA sequences, we produced a functional ribosome in which the subunits are covalently linked into a single entity by short RNA linkers. Notably, Ribo-T was not only functional in vitro, but was also able to support the growth of Escherichia coli cells even in the absence of wild-type ribosomes. We used Ribo-T to create the first fully orthogonal ribosome-messenger RNA system, and demonstrate its evolvability by selecting otherwise dominantly lethal rRNA mutations in the peptidyl transferase centre that facilitate the translation of a problematic protein sequence. Ribo-T can be used for exploring poorly understood functions of the ribosome, enabling orthogonal genetic systems, and engineering ribosomes with new functions.

  View details for DOI 10.1038/nature14862

  View details for Web of Science ID 000359002300044

  View details for PubMedID 26222032