Amino acid sequence of the Pinus taeda α-pinene synthase (GenBank : Q84KL3) MRRTLFGFSHELKAIHSTVPNLGMCRGGKSIAPSMSMSSTTSVSNEDGVPRRIAGHHSNLWDDDSIASLSTSYEAPSYRKRADKLIGEVKNIFDLMSVEDGVFTSPLSDLHHRLWM VDSVERLGIDRHFKDEINSALDHVYSYWTEKGIGRGRESGVTDLNSTALGLRTLRLHGYTVSSHVLDHFKNEKGQFTCSAIQTEGEIRDVLNLFRASLIAFPGEKIMEAAEIFSTM YLKDALQKIPPSGLSQEIEYLLEFGWHTNLPRMETRMYIDVFGEDTTFETPYLIREKLLELAKLEFNIFHSLVKRELQSLSRWWKDYGFPEITFSRHRHVEYYTLAACIANDPKHS AFRLGFGKISHMITILDDIYDTFGTMEELKLLTAAFKRWDPSSIECLPDYMKGVYMAVYDNINEMAREAQKIQGWDTVSYARKSWEAFIGAYIQEAKWISSGYLPTFDEYLENGKV SFGSRITTLEPMLTLGFPLPPRILQEIDFPSKFNDLICAILRLKGDTQCYKADRARGEEASAVSCYMKDHPGITEEDAVNQVNAMVDNLTKELNWELLRPDSGVPISYKKVAFDIC RVFHYGYKYRDGFSVASIEIKNLVTRTVVETVPL*


 S1) opened with the same enzymes. The resulting (Sp R /Sm R ,Cm S ) pCPS plasmid expresses the α-pinene synthase gene from the strong λ phage pR promoter (PR). The Sp R /Sm R gene is flanked by transcription terminators (TT) preventing readthrough of gene expression. S1). Second, the pTwist_NS-slr0168 plasmid was opened at its unique EcoRV restriction site flanked by the two 300 bp chromosomal DNA regions surrounding slr0168. Third, all three DNA cassettes (pR-FS, Km R and pTwist_NS-slr0168) were assembled by Gibson® cloning. The resulting pTwist_NS-slr0168-FS plasmid was transformed to Synechocystis to insert the pR-FS-Km R cassette in slr0168. Bisabolene (increasing concentrations) and trans-caryophyllene (fixed-concentration internal standard) were spiked in dodecane prior to GC-MS analysis. Due to the presence of several bisabolene isomers in the commercial standard, the contribution of (E)-α-bisabolene to the molarity was adjusted based on an attribution of 19.31  0.91% of total combined peak areas to the target molecules, as described (Wichmann et al., 2018). Farnesene (increasing concentrations) and trans-caryophyllene (fixed-concentration) were spiked in dodecane prior to GC-MS analysis. As the commercial standard contains several farnesene isomers, the contribution of α-farnesene to the molarity was adjusted based on an attribution of 7.78  0.37% of total combined peak areas to the target molecules, as described (Wichmann et al., 2018). Figure S3.G. Standard curve used to calculate the concentration of -santalene in the dodecane overlay of cultures of the Synechocystis strain harboring the pCSS plasmid. -santalene (increasing concentrations) and nerolidol (fixed-concentration) were spiked in dodecane prior to GC-MS analysis. As the commercial standard contains several santalene, bergamotene, santalol and bergamotol isomers, the contribution of α-santalene to the molarity was adjusted based on an attribution of only 0.270.02% of total combined peak areas to the target molecules, as described (Wichmann et al., 2018). were spiked in dodecane prior to GC-MS analysis. As the commercial standard contains several santalene, bergamotene, santalol and bergamotol isomers, the contribution of -exo-bergamotene to the molarity was adjusted based on an attribution of only 0.1% of total combined peak areas to the target molecules, as described (Wichmann et al., 2018). Figure S3.I. -Standard curve used to calculate the concentration of epi-β-santalene in the dodecane overlay of cultures of the Synechocystis strain harboring pCSS. epi-β-santalene (increasing concentrations) and nerolidol (fixed-concentration internal standard) were spiked in dodecane prior to GC-MS analysis. As the commercial standard contains several santalene, bergamotene, santalol and bergamotol isomers, the contribution of epi-β-santalene to the molarity was adjusted based on an attribution of only 0.39%0.02% of total combined peak areas to the target molecules, as described (Wichmann et al., 2018). 

Figure

  Figure S1 Construction of plasmids for high-level expression of terpene synthase genes in Synechocystis. Fig. S1A. Construction of the pCPS plasmid for strong expression of the Pinus taeda α-pinene synthase gene adapted to the Synechocystis codon usage. All genes are represented by colored arrows pointing into the direction of their transcription. The α-pinene synthase gene of the pEX-A2-PS plasmid was cloned as a NdeI-EcoRI restriction fragment in the (Sp R /Sm R ,Cm R ) pC vector (TableS1) opened with the same enzymes. The resulting (Sp R /Sm R ,Cm S ) pCPS plasmid expresses the α-pinene synthase gene from the strong λ phage pR promoter (PR). The Sp R /Sm R gene is flanked by transcription terminators (TT) preventing readthrough of gene expression. 




  Fig. S1B. Relevant part of the nucleotide sequence of the pCPS plasmid. The NdeI (catATG) and EcoRI (gaattc) restriction sites were used for cloning the Pinus taeda α-pinene synthase gene downstream of the strong lambda-phage pR promoter (-35 box (TTGACT), -10 box (GATAAT) & transcription start site (A)) and associated ribosome binding site (AAGGAGG) of the pC vector. 




  Fig. S1C. Construction of the pCSS plasmid for strong expression of the Santalum album santalene synthase gene adapted to the Synechocystis codon usage. The santalene synthase gene of the pEX-A258-SS plasmid (TableS1) was cloned as a NdeI-EcoRI restriction fragment in the pC plasmid, yielding pCSS that expresses the santalene synthase gene from the strong pR promoter (PR box). TT indicates the transcription terminators flanking the Sp R /Sm R marker. 


Fig. S1E .Figure S1I .Figure S1J .

 S1ES1IS1J Fig.S1E. Construction of the pCBS plasmid for strong expression of the Abies grandis E-α-bisabolene synthase gene adapted to the Synechocystis codon usage. All genes are represented by colored arrows. The E-α-bisabolene synthase gene of the pEX-A258-BS plasmid (TableS1) was cloned as a NdeI-EcoRI restriction fragment in the pC vector, yielding the pCBS plasmid that expresses the E-α-bisabolene synthase gene from the strong λ phage pR promoter (PR box). The transcription terminators flanking the Sp R /Sm R gene are indicated as TT. 




  Fig. S1K. Nucleotide sequence of the pR-FS-Km R DNA cassette cloned in slr0168 chromosome neutral site. The pR-FS-Km R DNA cassette embedded in an EcoRV restriction site GAT/ATC flanked by DNA sequences upstream and downstream of slr0168, expressed the codonadapted α-farnesene synthase gene from the strong pR promoter (TTGACT, GATAAT & A) and associated ribosome binding site (AAGGAGG). 


Figure

  Fig. S1L. PCR verification showing that the pR-FS-Km R DNA cassette expressing the Picea abies farnesene synthase gene is properly integrated in the slr0168 site of all chromosome copies of Synechocystis PCC 6803 strains harboring or not the pFCS plasmid.Upper part: schematic representation of the genome of the Synechocystis strains expressing the farnesene synthase gene from the slr0168 chromosomal site alone (chrFS strain) or together with the pCFS plasmid (chrFS + pCFS strain). Cells are shown as green oval shapes representing their chromosome (orange line) arbitrarily showed as attached to the cell membrane to distinguish it from the pCFS plasmid. The strong λ phage pR promoter directing the expression of the FS gene is shown as the red triangle. 


Fig. S3 .

 S3 Fig. S3.B. GC-MS analyses of the dodecane overlay of cultures of the Synechocystis strains harboring the pC or pCPS plasmids.Ion chromatograms (left panels) and corresponding mass spectra (right panels) of an α-pinene standard (upper panel) or dodecane samples (lower panel) of cultures of Synechocystis harboring either pC (negative control) or the pCPS (pinene production). S-(-)-limonene (retention time = 6.89 min) was used as the fixed-concentration (0.01 g.L -1 ) internal standard (IS) for quantification. 


Fig. S3 .

 S3 Fig. S3.E GC-MS analyses of the dodecane overlays of cultures of the Synechocystis strains harboring the plasmids pC, pCBS or pCFS. Ion chromatograms (left panels) and corresponding mass spectra (right panels) of farnesene-isomers standard (a) or dodecane samples from cultures of Synechocystis propagating pCFS (b, production of α-farnesene), pCBS (c, E-α-bisabolene production) or pC (d, negative control). β-caryophyllene (retention time = 17.3 min) was used as the fixed-concentration (0.01 g.L -1 ) internal standard (IS) for quantification. 


Figure

  Figure S3.J. GC-MS analyses of the dodecane overlays of cultures of the Synechocystis strain harboring the pCSS plasmid. m/z=93 ion chromatograms (upper panel) and mass spectra (lower panels) of the α-santalene (framed in mauve), epi-β-santalene (framed in purple) and α-exo-bergamotene (framed in pink) isomers were obtained from GC-MS analyses of a Synechocystis pCSS culture grown for 21 days. Nerolidol at 0.01 g.L -1 was used as the internal standard (IS) for quantification. 




  




  




  




  




  


' Amino acid sequence of the Santalum album santalene synthase (Genbank: ADO87000.1)

  
	5'-
	ggcgacgtgcgtcctcaagctgctcttgtgttaatggtttcttttttgtgctcatacgttaaatctatcaccgcaagggataaatatctaacaccgtgcgtgTTGACTattttacc
	tctggcggtGATAATggttgcAtgtactaaggaggtcatATGGACAGTTCTACAGCAACCGCGATGACTGCACCCTTTATTGACCCGACCGATCACGTAAACCTCAAAACTGATAC
	CGACGCTAGTGAGAATCGCAGAATGGGCAATTATAAGCCGTCAATTTGGAACTACGATTTTCTTCAGTCCTTGGCTACCCACCATAATATAGTTGAAGAAAGACATCTGAAGCTTG
	CTGAGAAACTCAAAGGCCAAGTGAAATTTATGTTTGGTGCACCCATGGAACCCCTGGCTAAATTGGAGTTAGTGGATGTGGTACAACGCCTAGGGCTAAATCATTTATTTGAGACG
	GAAATAAAAGAAGCCTTATTTTCAATTTACAAAGATGGGAGCAATGGCTGGTGGTTTGGACACCTACATGCTACTTCCCTGCGCTTTAGGCTCTTGCGCCAGTGTGGGCTATTTAT
	CCCACAAGATGTCTTCAAGACATTTCAAAACAAGACAGGAGAGTTCGATATGAAGCTTTGTGATAACGTAAAAGGTCTGTTGTCTTTGTACGAGGCAAGTTACTTGGGGTGGAAAG
	GTGAGAACATTTTGGACGAGGCGAAGGCGTTTACTACGAAATGCCTTAAGTCGGCATGGGAGAATATCTCTGAGAAATGGTTAGCCAAACGAGTTAAGCACGCGCTGGCGCTCCCG
	CTACACTGGAGAGTCCCCCGTATAGAGGCTCGCTGGTTTATTGAGGCCTACGAGCAAGAAGCGAACATGAACCCGACATTGCTCAAATTGGCCAAACTGGACTTCAACATGGTACA
	GAGTATCCATCAAAAGGAAATTGGAGAATTAGCCCGATGGTGGGTGACCACCGGTTTGGACAAACTAGCCTTTGCTCGTAATAACCTGCTGCAGAGCTACATGTGGTCATGTGCTA
	TTGCCTCCGATCCTAAGTTTAAGTTAGCCCGTGAAACTATTGTAGAAATTGGCTCTGTGTTGACGGTGGTAGATGACGGCTACGACGTTTACGGGTCCATCGATGAATTAGATTTG
	TATACTAGCAGTGTGGAACGTTGGAGCTGTGTCGAAATTGATAAATTACCTAATACCCTTAAATTGATCTTTATGAGTATGTTTAATAAAACCAATGAAGTGGGGTTACGGGTGCA
	ACACGAACGTGGCTATAACAGTATTCCTACCTTCATCAAAGCTTGGGTGGAACAGTGTAAATCGTATCAAAAAGAAGCCCGGTGGTTTCATGGGGGTCACACCCCCCCCCTGGAAG
	AATATAGCTTAAATGGCTTGGTCTCCATTGGTTTTCCCTTACTCCTTATCACCGGGTATGTTGCCATTGCCGAAAATGAAGCCGCCTTGGACAAAGTCCATCCGCTACCCGACCTC
	CTGCATTATTCTTCCCTTTTATCCCGGTTAATTAATGACATTGGAACCAGTCCAGATGAAATGGCCCGAGGTGATAACCTTAAATCCATCCATTGCTATATGAATGAAACCGGTGC
	CTCTGAAGAAGTGGCCCGGGAACATATCAAGGGGGTTATTGAAGAAAATTGGAAAATTCTCAATCAGTGTTGCTTTGATCAGTCCCAATTCCAAGAACCCTTCATTACATTTAATT
	TAAATAGTGTTCGGGGAAGCCATTTCTTTTATGAATTTGGTGATGGGTTCGGCGTGACTGACTCGTGGACGAAAGTGGATATGAAATCCGTGTTGATTGATCCAATTCCTTTAGGA
	GAAGAATAAtcgcgactcgagctagcggatccgatgaacgcgttcagctgaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccg
	ttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcc
	tatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgtttt
	caccatgggcaaat -3MDSSTATAMTAPFIDPTDHVNLKTDTDASENRRMGNYKPSIWNYDFLQSLATHHNIVEERHLKLAEKLKGQVKFMFGAPMEPLAKLELVDVVQRLGLNHLFETEIKEALFSIYKDG
	SNGWWFGHLHATSLRFRLLRQCGLFIPQDVFKTFQNKTGEFDMKLCDNVKGLLSLYEASYLGWKGENILDEAKAFTTKCLKSAWENISEKWLAKRVKHALALPLHWRVPRIEARWF
	IEAYEQEANMNPTLLKLAKLDFNMVQSIHQKEIGELARWWVTTGLDKLAFARNNLLQSYMWSCAIASDPKFKLARETIVEIGSVLTVVDDGYDVYGSIDELDLYTSSVERWSCVEI
	DKLPNTLKLIFMSMFNKTNEVGLRVQHERGYNSIPTFIKAWVEQCKSYQKEARWFHGGHTPPLEEYSLNGLVSIGFPLLLITGYVAIAENEAALDKVHPLPDLLHYSSLLSRLIND
	IGTSPDEMARGDNLKSIHCYMNETGASEEVAREHIKGVIEENWKILNQCCFDQSQFQEPFITFNLNSVRGSHFFYEFGDGFGVTDSWTKVDMKSVLIDPIPLGEE

168 y=0.3182x R 2 =0.9994 Figure S3.H -Standard curve used to calculate the concentration of

  -exo-bergamotene in 


	Concentration of α-santalene (mg.L -1 ) in dodecane	0.07	0.13	0.27	0.53	1.33	2.00	2.67	4.00
		0	1621	360	1432	2848	4140	3443	4797
	Area α-santalene	947	1494	446	744	1671	4447	3497	4734
			363	395	755	2151	2444	5281	3671
	Area nerolidol at 10 mg.L -1 (IS) (area cis-nerolidol + trans-nerolidol)	43133 48969	55945 48677	3891 6699	7765 3369	5943 4260	6569 7206	3561 3915	3548 3451
			7268	4685	4571	5422	4056	6914	3425
		0	0.029	0.093	0.18	0.48	0.63	0.97	1.35
	Area α-santalene / Area nerolidol	0.019	0.031	0.067	0.22	0.39	0.62	0.89	1.37
			0.050	0.084	0.17	0.40	0.60	0.76	1.07
	Average	0.01	0.04	0.08	0.19	0.42	0.62	0.87	1.27
	Standard deviation	0.014	0.014	0.013	0.028	0.049	0.014	0.103	0.

the dodecane overlay of cultures of the Synechocystis strain harboring pCSS

  . -exo-bergamotene (increasing concentrations) and nerolidol (fixed-concentration internal standard) 



111 y=0.1771x R 2 =0.9961

  
	Concentration of epi-β-santalene (mg.L -1 ) in dodecane	0.20	0.39	0.78	1.95	2.93	3.91	5.86	7.82
		1592	281	1125	2142	3669	2975	4214	8173
	Area epi-β-santalene	1025	372	630	1453	4185	2852	3909	8012
		327	321	763	1893	2091	4833	3223	8173
		55945	3891	7765	5943	6569	3561	3548	6529
	Area nerolidol at 10 mg.L -1 (IS)	48677	6699	3369	4260	4646	3915	3451	6559
		7268	4685	4571	5422	4056	6914	3425	6529
		0.03	0.07	0.14	0.36	0.56	0.84	1.19	1.25
	Area epi-β-santalene / Area nerolidol	0.03	0.06	0.19	0.34	0.58	0.73	1.13	1.22
		0.04	0.07	0.17	0.35	0.52	0.70	0.94	1.43
	Average	0.032	0.065	0.166	0.350	0.553	0.754	1.087	1.300
	Standard deviation	0.012	0.009	0.021	0.010	0.034	0.072	0.130	0.

NS-slr0168-FS genomic region (ChrFS chromosome) 

y=0.6071x R 2 =0.9949
Amino acid sequence of the Pinus taeda α-pinene synthase (GenBank : Q84KL3) MRRTLFGFSHELKAIHSTVPNLGMCRGGKSIAPSMSMSSTTSVSNEDGVPRRIAGHHSNLWDDDSIASLSTSYEAPSYRKRADKLIGEVKNIFDLMSVEDGVFTSPLSDLHHRLWM VDSVERLGIDRHFKDEINSALDHVYSYWTEKGIGRGRESGVTDLNSTALGLRTLRLHGYTVSSHVLDHFKNEKGQFTCSAIQTEGEIRDVLNLFRASLIAFPGEKIMEAAEIFSTM YLKDALQKIPPSGLSQEIEYLLEFGWHTNLPRMETRMYIDVFGEDTTFETPYLIREKLLELAKLEFNIFHSLVKRELQSLSRWWKDYGFPEITFSRHRHVEYYTLAACIANDPKHS AFRLGFGKISHMITILDDIYDTFGTMEELKLLTAAFKRWDPSSIECLPDYMKGVYMAVYDNINEMAREAQKIQGWDTVSYARKSWEAFIGAYIQEAKWISSGYLPTFDEYLENGKV SFGSRITTLEPMLTLGFPLPPRILQEIDFPSKFNDLICAILRLKGDTQCYKADRARGEEASAVSCYMKDHPGITEEDAVNQVNAMVDNLTKELNWELLRPDSGVPISYKKVAFDIC RVFHYGYKYRDGFSVASIEIKNLVTRTVVETVPL*