Achieving high-level production in a heterologous host requires the introduction of a long pathway and stable expression of the involved genes. For the latter, genome integration is a better choice than plasmid expression, especially considering that many non-model microbial hosts do not have readily usable plasmids. The question is which locus in the genome could favor gene integration and expression. Genome is organized into the accessible euchromatin regions and tightly packed heterochromatin regions. Genes located in the heterochromatins are normally silent because they cannot be accessed by transcriptional machinery. Unfortunately, for the majority of the microbial hosts, how their genomes are packed is not known. To address this issue, we leverage the cut-and-paste feature of the transposon system to insert the heterologous pathway flanked by the two terminal transposon recognition sequences randomly to the highly accessible genomic regions. Using the 14-kb shikimate pathway as an example, we identified genomic hotspots favoring pathway integration and increased the production of shikimate from 1.8 g/L (plasmid-based production) to 6.2 g/L in one-step. More importantly, this strategy also efficiently reduced false positives (caused by the integration of only the selection marker instead of the pathway) and improved the efficiency of identifying top-performing variants from 15% to 97%.<br/>The two biggest challenges of engineering these non-model microbes are (1) the lack of genetic manipulation tools and (2) the missing knowledge of genotype-phenotype correlation, which constrain the efficiency of further strain development to improve the productivity. The transposon may hop into the genes that negatively impact the production of the target compound. Therefore, disruption of their functions will lead to increased product yields. Currently, we are expanding this success to achieve rapid strain engineering in three species to produce nutraceuticals and biopolymer precursors. Demonstrating the general applicability of our strain engineering solution will push the frontier of the biochemical engineering field with a radical technology to accelerate strain engineering for other interesting products.<br/>

Leveraging transposon technology to accelerate the development process of microbial cell factories for high-level production of bio-based chemicals

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.