jatropha curcas casbene synthase (jccbs, ncbi reference sequence: np_001292945.1)  (GenScript corporation)

Journal: ACS Synthetic Biology

Article Title: Development of Novel Riboswitches for Synthetic Biology in the Green Alga Chlamydomonas

doi: 10.1021/acssynbio.0c00082

Figure Lengend Snippet: Riboswitch regulation of casbene production. Casbene production was achieved by introducing the casbene synthase expression cassette into the nuclear genome of the Chlamydomonas UVM4 strain. (a) Schematic of the expression cassette shows the contributing parts assembled in the following order, HSP70/RBCS2 promoter, 22 nt RBCS2 5′UTR, CrTHI4–4N RS , PSAD chloroplast target peptide (cTP shown as blue box), codon optimized casbene synthase ( CBS ) containing multiple copies of the Chlamydomonas RBCS2 intron 1 (i1) fused with a GS linker peptide (orange box) to Venus containing RBCS2 intron 2 (i2), the 3′ UTR was derived from CA1. (b) Casbene production in a Chlamydomonas transformant that expressed the casbene synthase transgene was assessed using Gas Chromatography Mass Spectrometry (GC-MS). A representative transformant was cultured in TAP media with a 10% n-dodecane overlay. Nine days postinoculation, the overlay was analyzed by GC-MS. Casbene captured by the n -dodecane overlay was detected at the expected retention time (black trace), thereby confirming that the casbene synthase enzyme fused to Venus fluorescent protein was functional. The GC-MS ion chromatogram ( m / z 121) shows metabolites carrying a mass-to-charge ratio ( m / z ) of 121 ± 0.5. Internal standard β-caryophyllene was detected at 12.32 min retention time (RT), while casbene was detected at 23.16 min RT. In agreement with previous reports, a selection of oxidized casbene molecules was also detectable between RT 25.6 and 26.7 RT. The detection of casbene and its oxidized derivatives demonstrates capacity of this transformant to produce casbene. When this transformant was cultured in media containing 10 μM thiamine (green trace), casbene was not detected, a result that highlighted the utility of the riboswitch for regulation of transgene expression.

Article Snippet: The amino acid sequences of the Jatropha curcas casbene synthase (JcCBS, NCBI Reference Sequence: NP_001292945.1) was codon optimized for expression from the Chlamydomonas nuclear genome and synthesized by GenScript Corporation (USA).

Techniques: Expressing, Derivative Assay, Gas Chromatography, Mass Spectrometry, Gas Chromatography-Mass Spectrometry, Cell Culture, Functional Assay, Selection