Photosynthetic conversion of CO2 to farnesyl diphosphate-derived phytochemicals (amorpha-4,11-diene and squalene) by engineered cyanobacteria.
BACKGROUND
Metabolic engineering of cyanobacteria has enabled photosynthetic conversion of CO2 to value-added chemicals as bio-solar cell factories. However, the production levels of isoprenoids in engineered cyanobacteria were quite low, compared to other microbial hosts. Therefore, modular optimization of multiple gene expressions for metabolic engineering of cyanobacteria is required for the production of farnesyl diphosphate-derived isoprenoids from CO2.


RESULTS
Here, we engineered Synechococcus elongatus PCC 7942 with modular metabolic pathways consisting of the methylerythritol phosphate pathway enzymes and the amorphadiene synthase for production of amorpha-4,11-diene, resulting in significantly increased levels (23-fold) of amorpha-4,11-diene (19.8 mg/L) in the best strain relative to a parental strain. Replacing amorphadiene synthase with squalene synthase led to the synthesis of a high amount of squalene (4.98 mg/L/OD730). Overexpression of farnesyl diphosphate synthase is the most critical factor for the significant production, whereas overexpression of 1-deoxy-d-xylulose 5-phosphate reductase is detrimental to the cell growth and the production. Additionally, the cyanobacterial growth inhibition was alleviated by expressing a terpene synthase in S. elongatus PCC 7942 strain with the optimized MEP pathway only (SeHL33).


CONCLUSIONS
This is the first demonstration of photosynthetic production of amorpha-4,11-diene from CO2 in cyanobacteria and production of squalene in S. elongatus PCC 7942. Our optimized modular OverMEP strain (SeHL33) with either co-expression of ADS or SQS demonstrated the highest production levels of amorpha-4,11-diene and squalene, which could expand the list of farnesyl diphosphate-derived isoprenoids from CO2 as bio-solar cell factories.
Clean Energy Research Center, Korea Institute of Science and Technology, Hwarangro 14-gil 5, Seongbuk-gu, Seoul, 02792 Republic of Korea.|Green School (Graduate School of Energy and Environment), Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841 Republic of Korea.
Sun Young
SY
Choi
Clean Energy Research Center, Korea Institute of Science and Technology, Hwarangro 14-gil 5, Seongbuk-gu, Seoul, 02792 Republic of Korea.
Hyun Jeong
HJ
Lee
Clean Energy Research Center, Korea Institute of Science and Technology, Hwarangro 14-gil 5, Seongbuk-gu, Seoul, 02792 Republic of Korea.
Jaeyeon
J
Choi
Clean Energy Research Center, Korea Institute of Science and Technology, Hwarangro 14-gil 5, Seongbuk-gu, Seoul, 02792 Republic of Korea.|Department of Chemistry, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841 Republic of Korea.
Jiye
J
Kim
Green School (Graduate School of Energy and Environment), Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841 Republic of Korea.|Department of Chemical and Biological Engineering, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841 Republic of Korea.
Sang Jun
SJ
Sim
Clean Energy Research Center, Korea Institute of Science and Technology, Hwarangro 14-gil 5, Seongbuk-gu, Seoul, 02792 Republic of Korea.
Youngsoon
Y
Um
Clean Energy Research Center, Korea Institute of Science and Technology, Hwarangro 14-gil 5, Seongbuk-gu, Seoul, 02792 Republic of Korea.
Yunje
Y
Kim
Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608 USA.|Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 USA.
Taek Soon
TS
Lee
Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608 USA.|Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 USA.|Department of Bioengineering, University of California, Berkeley, CA 94720 USA.|Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720 USA.
Jay D
JD
Keasling
Department of Food Science and Biotechnology, Sungkyunkwan University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon, 16419 Republic of Korea.
Han Min
0000-0002-8797-0477
HM
Woo
Cyanobacteria; Isoprenoids; Metabolic engineering; Synthetic biology
J Plant Physiol. 2011 May 15;168(8):848-52
21185107
Biotechnol Appl Biochem. 2009 Mar;52(Pt 3):199-207
18564056
Proc Natl Acad Sci U S A. 2013 Jul 9;110(28):11290-5
23798438
Metab Eng. 2009 Jan;11(1):13-9
18775787
Metab Eng. 2013 Sep;19:33-41
23727191
J Mol Biol. 1983 Jun 5;166(4):557-80
6345791
Nature. 2009 Aug 13;460(7257):894-8
19633652
Nat Commun. 2013;4:1409
23361000
Photosynth Res. 2016 Dec;130(1-3):123-135
26895437
Metab Eng. 2005 Jan;7(1):18-26
15721807
Mol Plant. 2014 Jan;7(1):71-86
24157609
Nat Biotechnol. 2015 Apr;33(4):377-83
25558867
Plant Biotechnol J. 2016 Aug;14(8):1768-76
26879003
J Bacteriol. 2008 Sep;190(18):6084-96
18658271
Appl Environ Microbiol. 2010 May;76(10):3097-105
20348305
ACS Synth Biol. 2014 Jul 18;3(7):466-75
24679043
Front Bioeng Biotechnol. 2014 Jun 19;2:21
25152894
Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8915-20
11427737
Bioorg Chem. 2014 Dec;57:171-85
24998420
J Exp Bot. 2003 Dec;54(393):2635-42
14585822
Proc Natl Acad Sci U S A. 2013 Jan 22;110(4):1249-54
23297225
Nucleic Acids Res. 2014 Feb;42(4):2646-59
24234441
Curr Opin Biotechnol. 2015 Jun;33:8-14
25305544
Biotechnol Bioeng. 2001 Feb 20;72(4):408-15
11180061
Metab Eng. 2015 Nov;32:116-24
26410450
PLoS One. 2014 Mar 13;9(3):e90270
24625633
Arch Biochem Biophys. 1993 Jul;304(1):133-43
8323279
Nature. 2006 Apr 13;440(7086):940-3
16612385
Plant J. 2008 Oct;56(2):228-38
18643974
Nat Biotechnol. 2009 Dec;27(12):1177-80
19915552
Nat Commun. 2011 Sep 27;2:483
21952217
Metab Eng. 2011 Mar;13(2):194-203
21215324
Cell Syst. 2016 May 25;2(5):335-46
27211860
J Biotechnol. 2014 Sep 20;185:1-7
24905149
Nat Biotechnol. 2005 May;23(5):612-6
15821729
Planta. 2014 Aug;240(2):309-24
24838596
Metab Eng. 2013 Jul;18:53-9
23608473
Trends Plant Sci. 2005 Dec;10(12):594-602
16290212
J Biosci Bioeng. 2015 Feb;119(2):165-71
25282635
J Biosci Bioeng. 2014 Jul;118(1):20-8
24507902
Metab Eng. 2006 Jan;8(1):79-90
16257556
Biotechnol Bioeng. 2016 Aug;113(8):1755-63
26804325
FEBS Lett. 2000 Sep 22;481(3):221-6
11007968
J Biol Eng. 2011 Sep 20;5:12
21933410
Methods Enzymol. 1987;153:215-31
3123881
Front Plant Sci. 2014 Feb 25;5:67
24616731
Metab Eng. 2007 Jul;9(4):337-47
17509919
Nat Biotechnol. 2013 Nov;31(11):1039-46
24142050
Biotechnol Bioeng. 1999 Jan 20;62(2):235-41
10099534
Proc Natl Acad Sci U S A. 2012 Jan 17;109(3):E111-8
22247290
Biomol Eng. 2003 Jul;20(4-6):177-82
12919795
Nat Biotechnol. 2011 Dec 08;29(12):1072-4
22158354
Cell Res. 2010 Jun;20(6):688-700
20404857
Nat Biotechnol. 2003 Jul;21(7):796-802
12778056
Nat Commun. 2015 Feb 03;6:6143
25644758
Annu Rev Plant Biol. 2009;60:335-55
19575586