Journal
ACS SYNTHETIC BIOLOGY
Volume 10, Issue 12, Pages 3561-3575Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.1c00492
Keywords
Synechococcus elongatus UTEX 2973; lysine; cadaverine; glutarate
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Funding
- National Science Foundation [CBET-1705409]
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This study focused on engineering UTEX 2973 to overproduce L-lysine, cadaverine, and glutarate, utilizing both chromosomal and episomal expression methods. By introducing a feedback-resistant aspartate kinase and a lysine exporter, along with extending the lysine pathway to produce glutarate and cadaverine, significant improvements in production yields were achieved. Further efforts to overcome pathway and transport bottlenecks will be crucial for optimizing these bioproduction processes.
Amino acids and related targets are typically produced by well-characterized heterotrophs including Corynebacterium glutamicum and Escherichia coli. Cyanobacteria offer an opportunity to supplant these sugar-intensive processes by instead directly utilizing atmospheric CO2 and sunlight. Synechococcus elongatus UTEX 2973 (hereafter UTEX 2973) is a particularly promising photoautotrophic platform due to its fast growth rate. Here, we first engineered UTEX 2973 to overproduce L-lysine (hereafter lysine), after which both cadaverine and glutarate production were achieved through further pathway engineering. To facilitate metabolic engineering, the relative activities of a subset of previously uncharacterized promoters were investigated, in each case, while also comparing the effects of both chromosomal (from neutral site NS3) and episomal (from pAM4788) expressions. Using these parts, lysine overproduction in UTEX 2973 was engineered by introducing a feedback-resistant copy of aspartate kinase (encoded by lysC(fbr)) and a lysine exporter (encoded by ybjE), both from E. con. While chromosomal expression resulted in lysine production up to just 325.3 +/- 14.8 mg/L after 120 h, this was then increased to 556.3 +/- 62.3 mg/L via plasmid-based expression, also surpassing prior reports of photoautotrophic lysine bioproduction. Lastly, additional products of interest were then targeted by modularly extending the lysine pathway to glutarate and cadaverine, two 5-carbon, bioplastic monomers. By this approach, glutarate has so far been produced at final titers reaching 67.5 +/- 2.2 mg/L by 96 h, whereas cadaverine has been produced at up to 55.3 +/- 6.7 mg/L. Overcoming pathway and/or transport bottlenecks, meanwhile, will be important to improving upon these initial outputs.
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