Developing Genetic Editing Tools for Lysobacter

Authors

Wei Su, University of Nebraska-LinclonFollow

First Advisor

Liangcheng Du

Date of this Version

Spring 4-14-2016

Document Type

Article

Comments

A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Chemistry, Under the Supervision of Professor Liangcheng Du. Lincoln, Nebraska: April, 2016

Copyright (c) 2016 Wei Su

Abstract

Lysobacter species are a group of environmental bacteria that are prolific in producing bioactive natural products. However, very little genetic engineering has been conducted in Lysobacter. The main reason is the limited genetic tools available for Lysobacter. CRISPR/Cas9 is a novel and powerful genome editing tool that has not been tested in Lysobacter. Herein, we first tested CRISPR/Cas9 for specific inactivation of a key biosynthetic gene for phenazine biosynthesis in L. antibioticus OH13. The distinct colors of phenazines provide a convenient visual selection for gene disruption mutants. The results showed that CRISPR/Cas9 was able to function in Lysobacter. Next, we applied the CRISPR/Cas9 system to L. enzymogenes OH11 to enhance the expression of the WAP operon, which is responsible for the biosynthesis of WAP-8294A, a group of cyclic lipodepsipeptides with potent anti-MRSA activity. The yield of these compounds is extremely low in L. enzymogenes, which has hampered the study of their structure-and-activity relation and application. We designed six different CRISPR containing vectors and successfully increased the expression of the WAP biosynthetic genes. The system we used was CRISPR/dCas9-ω, which contained the sgRNA and an inactivated dCas9 that was linked to an omega subunit (ω). The sgRNA was designed to direct this dCas9-ω fusion to a proper position of the WAP promoter, which would lead to a proper binding of ω subunit with RNA polymerase. The consequence is an enhanced transcription of the target genes. This is the first time that CRISPR/Cas9 has been applied in Lysobacter species.

Advisor: Dr. Liangcheng Du