Expanding the bio-capabilities of Lactococcus lactis through efficient genome modification

Rothstein, Samuel
Major Professor
Thomas J Mansell
Committee Member
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Chemical and Biological Engineering

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 live-biotherapeutics 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. The next research effort shifts the focus from carbohydrate metabolism to introducing inducible promoter systems in several strains of L. lactis. The use of inducible promoters and inducible gene expression systems is a key component to biomedical applications of L. lactis. This research demonstrated an in-trans antibiotic resistance selection method to integrate the requisite genes into the L. lactis genome that are required for inducible gene expression. The inducible gene expression systems integrated into the genome in this research were nisin (NICE), T7 RNA polymerase, and xylose induced gene expression. This study not only developed a system for integrating these inducible gene expression cassettes, but demonstrated that this method is useful for modifying/improving these gene cassettes. Two important examples of this feature was the development of a nisin and theophylline AND gated gene expression and the evaluation of a reduced xylose operon which severed the link between xylose metabolism and xylose induced gene expression. The development of this method for integrating gene induction systems into the L. lactis genome relied upon the use of homologous recombination. The use of homologous recombination, though common, is not the only tool used to integrate DNA in the genome of lactic acid bacteria (LAB). The next part of the thesis shifts focus specifically to techniques used to modify the genome of LAB. This review thoroughly investigates the use of homologous recombination and various forms of lambda red recombination in several LAB. It was found that though more powerful than homologous recombination, lambda red recombination in LAB is generally specific and is best used with a strong selection technique, such as CRISPR-Cas9 targeting or antibiotic resistance. Lastly, the thesis shifts focus to identifying recombinant L. lactis through the use of an inducible CRISPR-Cas9 selection system. This research focus hinges on two key aspects of identifying recombinant L. lactis: improving the selection efficiency of CRISPR-Cas9 and boosting recombination efficiency. The selection efficiency of CRISPR-Cas9 in L. lactis was improved through evaluating a few published mutants of the Cas9 protein. Additionally, the selection efficiency was improved through the use of the nisin-theophylline AND gate that was previously established in the thesis. Though mutants were successfully identified through the improvements in the selection efficiency, the research effort turned to improving the recombination efficiency. Though CRISPR-Cas9 proved to be a powerful selection tool, a single modification to the Cas9 protein made the DNA blunt cutting enzyme into a nickase. This change in Cas9 function still enabled effective selection, but substantially improved the recombination efficiency. The change in Cas9 function into a nickase gave a substantial increase in the probability of identifying a recombinant L. lactis. Taken all the research thrusts together, this thesis focused on molding the L. lactis genome and expanding the carbohydrate profile of L. lactis. Through this thesis, it has been shown that these research thrusts open the use of L. lactis in different biochemical and biomedical applications. These research thrusts show that the substrates used by L. lactis can be expanded, new inducible gene expression systems can be used, and further genome modifications can be conducted with CRISPR-Cas9 tools.