Part:BBa_K3776006
davB-davA-AK
This part allows overproduction of L-lysine by a feedback insensitive AK and conversion of L-lysine to 5-aminovaleramide (5-AVA) by L-Lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) in Synechococcus elongatus UTEX 2973. Promoters Pcpc560, 6803PsbA2 and terminator TrbcL are from the genome Synechocystis sp. PCC6803.
In the L-lysine biosynthetic pathway in cyanobacteria, aspartate kinase (AK) catalyzes the conversion of L-aspartate to L- aspartyl-phosphate and is subject to negative feedback by L-lysine, having decreased activity when there are high levels of L-lysine. Introducing a feedback insensitive AK circumvents L-lysine regulation and enhances the synthesis of L-lysine(Klompe et al., 2019) for synthesis of 5-AVA by DavB and DavA. DavB catalyzes L-lysine to 5-aminovaleramide and DavA catalyzes 5-aminovaleramide to 5-AVA (Li et al., 2018).
Being a strong promoter, Pcpc560(Li et al., 2018) was chosen to initiate transcription of DavB and DavA to maximize 5-AVA synthesis. A relatively weaker, but still moderately strong, promoter 6803PsbA2(Li et al., 2018) was chosen to initiate transcription of the feedback insensitive AK to avoid putting too much metabolic burden on S.elongatus UTEX 2973 while still enhancing L-lysine synthesis.
References
Klompe, S. E., Vo, P. L. H., Halpin-Healy, T. S., & Sternberg, S. H. (2019). Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration. Nature, 571(7764), 219–225. https://doi.org/10.1038/s41586-019-1323-z
Liu, P., Zhang, H., Lv, M., Hu, M., Li, Z., Gao, C., Xu, P., & Ma, C. (2014). Enzymatic production of 5-aminovalerate from l-lysine using l-lysine monooxygenase and 5-aminovaleramide amidohydrolase. Scientific Reports, 4(1). https://doi.org/10.1038/srep05657
Li, S., Sun, T., Xu, C., Chen, L., & Zhang, W. (2018). Development and optimization of genetic toolboxes for a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973. Metabolic Engineering, 48, 163–174. https://doi.org/10.1016/j.ymben.2018.06.002
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 800
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1889
Illegal BglII site found at 4844 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2049
Illegal NgoMIV site found at 3816
Illegal AgeI site found at 1456
Illegal AgeI site found at 1705
Illegal AgeI site found at 3040
Illegal AgeI site found at 5043 - 1000COMPATIBLE WITH RFC[1000]
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