In this study, we present a minimal template design and accompanying methods to produce assayable quantities of custom sequence proteins within 24 hr from receipt of inexpensive gene fragments from a DNA synthesis vendor. This is done without the conventional steps of plasmid cloning or cell‐based amplification and expression. Instead the linear template is PCR amplified, circularized, and isothermally amplified using a rolling circle polymerase. The resulting template can be used directly with cost‐optimized, scalably‐manufactured Escherichia coli extract and minimal supplement reagents to perform cell‐free protein synthesis (CFPS) of the template protein. We demonstrate the utility of this template design and 24 hr process with seven fluorescent proteins (sfGFP, mVenus, mCherry, and four GFP variants), three enzymes (chloramphenicol acetyltransferase, a chitinase catalytic domain, and native subtilisin), a capture protein (anti‐GFP nanobody), and 2 antimicrobial peptides (BP100 and CA(1–7)M(2–9)). We detected each of these directly from the CFPS reaction using colorimetric, fluorogenic, and growth assays. Of especial note, the GFP variant sequences were found from genomic screening data and had not been expressed or characterized before, thus demonstrating the utility of this approach for phenotype characterization of sequenced libraries. We also demonstrate that the rolling circle amplified version of the linear template exhibits expression similar to that of a complete plasmid when expressing sfGFP in the CFPS reaction. We evaluate the cost of this approach to be $61/mg sfGFP for a 4 hr reaction. We also detail limitations of this approach and strategies to overcome these, namely proteins with posttranslational modifications.

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Lactic acid bacteria play an important role in a variety of biochemical and biomedical applications. This thesis focuses on methods to improve the use of lactic acid bacteria for use in a variety of different applications. The thesis starts by discussing techniques in improving gut health and treating gut dysbiosis related disorders. The primary focus of these techniques is the use of prebiotic substrates and the use of livebiotherapeutics and probiotics. Lactic acid bacteria are reasonable candidates for creating genetically modified live-biotherapeutics or probiotics. Focusing further, the thesis indicates the numerous live-biotherapeutic applications of Lactococcus lactis (L. lactis), the primary organism of interest throughout the rest of the thesis.After describing previous applications of modified L. lactis, the thesis shifts to improving the organism's ease of use and opens up further applications of modified L. lactis. The first research task was to improve the capability of L. lactis to metabolize biorenewable sugars and produce valuable biorenewable chemicals. This research endeavor focused on enabling levoglucosan metabolism in L. lactis. The sugar, levoglucosan, is a primary component of the carbohydrate profile in pyrolysized biomass. Introducing levoglucosan metabolism in L. lactis enabled the organism to consume more of the available carbohydrates in bio-oil, a component of pyrolysized biomass. Furthermore, this levoglucosan consuming L. lactis was modified to convert it's primary fermentation product, lactic acid, to a more valuable biorenewable chemical, 1,2-propanediol. This research endeavor enabled the metabolism of bio-oil carbohydrates and subsequent conversion to lactic acid and 1,2-propanediol in L. lactis.