The next step in microbial production of plant natural compounds – pathway optimization and stable integration

Glucosinolates (GLs) are specialized bioactive compounds characteristic of plants in the order Brassicales, including the model plant Arabidopsis thaliana. GLs are key players in the defense of these plants against herbivores and microorganisms. For humans, their biological functions range from health-promoting and flavor compounds to bio-pesticides. Particularly, glucoraphanin (GRN), the major glucosinolate in broccoli has been associated with cancer-preventive properties of broccoli [1].

In planta, the methionine-derived GRN is formed by a 13-step pathway that is partly compartmentalized between plastid and cytosol [2]. After successfully engineering the GRN pathway into the non-cruciferous plant species Nicotiana benthamiana by transient expression [3], we recently increased production of the major intermediate dihomomethionine (DHM) by 9-fold to 432 nmol/g fresh weight [4] using pathway optimization.

To ultimately establish sustainable GRN production in a microbial host organism we aim at stably integrating the pathway into several of our microbial host systems. This was already achieved for the simple, tryptophan-derived indole GLs by stable integration into the yeast genome [5]. Recently, we succeeded in producing up to 57 mg/L DHM, the precursor for GRN, in Escherichia coli [6]. We are currently implementing the full pathway into our expression systems to evaluate the most promising production host.

Keywords: Metabolic engineering, glucoraphanin, dihomomethionine, pathway optimization.

References:

[1] Juge N, Mithen RF, Traka M. Molecular basis for chemoprevention by sulforaphane: a comprehensive review. Cell Mol Life Sci 2007; 64: 1105 – 1127

[2] Sonderby IE, Geu-Flores F, Halkier BA. Biosynthesis of glucosinolates – gene discovery and beyond. Trends Plant Sci 2010; 15: 283 – 290

[3] Mikkelsen MD, Olsen CE, Halkier BA. Production of the cancer-preventive glucoraphanin in tobacco. Mol Plant 2010; 3: 751 – 759

[4] Crocoll C, Mirza N, Reichelt M, Gershenzon J, Halkier BA. Optimization of engineered production of the glucoraphanin precursor dihomo-methionine in Nicotiana benthamiana. Front Bioeng Biotechnol 2016; 4: doi: 10.3389/fbioe.2016.00014

[5] Mikkelsen MD, Buron LD, Salomonsen B, Olsen CE, Hansen BG, Mortensen UH, Halkier BA. Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in a versatile yeast expression platform. Metab Eng 2012; 14: 104 – 111

[6] Mirza N, Crocoll C, Olsen CE, Halkier BA. Engineering of methionine chain elongation part of glucoraphanin pathway in E. coli. Metab Eng 2016; 35: 31 – 37