(54e) High Production of Fatty Alcohols By Escherichia coli Modified with Tandem Repeats Assisted Genome Editing (TRAGE) Method

Genome editing can introduce predetermined sequence changes to the target genome. In this work, a new genome editing method of tandem repeat assisted genome editing (TRAGE) was developed. This method can realize seamlessly deleting, substituting or inserting targeted genes in genome by one round of genetic manipulation with high fidelity.

We knocked out tesC, tesB and tesA sequentially for inducing fatty acid starvation to enhance fatty alcohol production. The deletion of tesC increased the fatty alcohol titer from 480 to 710 mg/L. Meanwhile, the fatty acid production dropped from 30 mg/L to 15 mg/L. The deletions of all three acyl-ACP thioesterases improved fatty alcohol tilter to 760 mg/L. At the same point, the fatty acid concentration was 8 mg/L.

We performed whole-genome transcriptional analysis. The expression levels of most genes from glycolysis module and fatty acid synthesis module were upregulated. Most genes from TCA cycle and fatty acid degradation module were downregulated.

Deletions of ldhA, pta and ackA from KLCB coupled with over expression of FAR were performed. The fatty alcohol tilter was increased from 756 mg/L to 2024 mg/L. Fed-batch fermentations were performed in a 3-L Bioflo 110 fermentor. Total fatty alcohol accumulation reached a maximum of 6.33 g/L after 50 h. Two saturated fatty alcohols (C14:0 and C16:0) and two unsaturated ones (C16:1 and C18:1) are the major components. C14:0 (2.42 g/L) and C16:1 (1.81 g/L) are the two most abundant fatty alcohols, constituting 38.2% and 28.6% of total fatty alcohols, respectively.