Composite
davBA-AK

Part:BBa_K3776006

Designed by: Helene Heling Huang   Group: iGEM21_HK_SSC   (2021-10-01)


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 800
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1889
    Illegal BglII site found at 4844
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE 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
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
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