Coding

Part:BBa_K5218025

Designed by: Amy Fu, Lichuan Chen, Xiaojuan Wang   Group: iGEM24_BGI-MammothEdu-South   (2024-09-03)


NoRtTAL

Tyrosine ammonia lyase (TAL) CDS from Rhodotorula toruloides without codon optimization.

Base Pairs：2148 bp

Function：Tyrosine ammonia-lyase (TAL) that catalyzes the formation of p-coumaric acid from tyrosine.

Figure.1: TAL plays a role in the pathway of caffeic acid biosynthesis.
Figure adopted from L. Liu et al, 2019 [1]

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2090
    Illegal BamHI site found at 352
    Illegal XhoI site found at 403
    Illegal XhoI site found at 466
    Illegal XhoI site found at 484
    Illegal XhoI site found at 562
    Illegal XhoI site found at 763
    Illegal XhoI site found at 1006
    Illegal XhoI site found at 1753
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 922
    Illegal NgoMIV site found at 1168
    Illegal NgoMIV site found at 1336
    Illegal NgoMIV site found at 1479
    Illegal NgoMIV site found at 1813
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 463
    Illegal BsaI site found at 793
    Illegal BsaI site found at 799
    Illegal BsaI.rc site found at 1924
    Illegal SapI site found at 753

Introduction

Ammonia-lyases catalyze the deamination of amino acids. Members of this family include histidine ammonia lyase (HAL) that converts histidine to urocanic acid, phenylalanine ammonia lyase (PAL) that converts phenylalanine to cinnamate (CA), and tyrosine ammonia lyase (TAL) that converts tyrosine to p-coumaric acid. p-coumaric acid is an important precursor in many metabolic pathways, one of which is the production of caffeic acid with the catalysis of coumarate 3-hydroxylase (C3H).

PALs, as key rate-limiting enzymes, are commonly found in plants, algae, fungi and bacteria[2]. The presence of TAL enzymes have been reported in a few microorganisms. TAL from Rhodosporidium toruloides was demonstrated to synthesize p-coumaric acid from L-tyrosine and obtained a yield of 117.5 mg/L [1]. TAL from Rhodobacter capsulatus has 32% identity with plant PAL sequence of Pinus taeda[3]. iGEM13_Uppsala team registered a TAL gene from rhodobacter sphaeroides (Part:BBa_K1033000) and characterized the enzyme with spectrophotometry and chromatography.

Rhodotorula toruloides is a species of oleaginous yeast. It is a red basidiomycete isolated from the wood pulp of conifers and naturally accumulates carotenoids, neutral lipids, and enzymes relevant to the chemical and pharmaceutical industries (Wikipedia).

The engineering objective of this project is to generate brighter autoluminescent plants using synthetic biology approaches. Team BGI-MammothEdu-South 2024 selected a variety of candidate genes from different species and tested their functions in Fungal Bioluminescence Pathway (FBP) via eGFP reporter system (pS1300-GFP plasmid). The RtTAL protein ID is CDR39392.1 (NCBI). Team BGI-MammothEdu-South retrieved the RtTAL CDS from published article[1] and obtained the sequence through De novo DNA synthesis.

Reference

1.Liu, Langqing, Liu, Hong, Zhang Wei, Yao, Mingdong, Li, Bingzhi, Liu, Duo, Yuan Yingjin. (2019) Engineering the biosynthesis of caffeic acid in saccharomyces cerevisiae with heterologous enzyme combinations. Engineering, 5(2), 9.

2.Zhang Fulin, Wang Juan, Li Xianguo, Zhang Jun, Liu Yuxiang , Chen Yijia, Yu Qinghui, Li Ning (2023) Genome-wide identification and expression analyses of phenylalanine ammonia-lyase gene family members from tomato (Solanum lycopersicum) reveal their role in root-knot nematode infection. Frontiers in Plant Science, 14:1204990

3.Kyndt J.A., Meyer T.E., Cusanovich M.A. and Van Beeumen J.J. (2002) Characterization of a bacterial tyrosine ammonia lyase, a biosynthetic enzyme for the photoactive yellow protein, FEBS Letters, 512

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