Establishment of strigolactone-producing bacterium-yeast consortium

Abstract

Strigolactones (SLs) are a class of phytohormones playing diverse roles in plant growth and development, yet the limited access to SLs is largely impeding SL-based foundational investigations and applications. Here, we developed Escherichia coli-Saccharomyces cerevisiae consortia to establish a microbial biosynthetic platform for the synthesis of various SLs, including carlactone, carlactonoic acid, 5-deoxystrigol (5DS; 6.65 ± 1.71 g/liter), 4-deoxyorobanchol (3.46 ± 0.28 g/liter), and orobanchol (OB; 19.36 ± 5.20 g/liter). The SL-producing platform enabled us to conduct functional identification of CYP722Cs from various plants as either OB or 5DS synthase. It also allowed us to quantitatively compare known variants of plant SL biosynthetic enzymes in the microbial system. The titer of 5DS was further enhanced through pathway engineering to 47.3 g/liter. This work provides a unique platform for investigating SL biosynthesis and evolution and lays the foundation for developing SL microbial production process.

Structure Elucidation and Biosynthesis of Orobanchol

“…Recently, it was reported that in a co-culture system of Escherichia coli and Saccharomyces cerevisiae co-expressing SL biosynthesis genes, orobanchol is generated by the co-expression of VuCYP722C with the upstream SL biosynthesis genes. However, the production of its diastereomer has not been described ( Wu et al, 2021 ). Therefore, a more detailed functional analysis of the CYP722C subfamily is necessary.…”

A Unique Sulfotransferase-Involving Strigolactone Biosynthetic Route in Sorghum

“…For CL production, XY medium [13.3 g/l monopotassium phosphate (KH 2 PO 4 ), 4 g/l diammonium phosphate [(NH 4 ) 2 HPO 4 ], 1.7 g/l citric acid, 0.0025 g/l cobalt(II) chloride (CoCl 2 ), 0.015 g/l manganese(II) chloride (MnCl 2 ), 0.0015 g/l copper(II) chloride (CuCl 2 ), 0.003 g/l boric acid (H 3 BO 3 ), 0.0025 g/l sodium molybdate (Na 2 MoO 4 ), 0.008 g/l zinc acetate [Zn(CH 3 COO) 2 ], 0.06 g/l iron(III) citrate, 0.0045 g/l thiamine, 1.3 g/l magnesium sulfate (MgSO 4 ), 5 g/l yeast extract, and 40 g/l xylose, pH 7.0] was prepped and used as previously described ( Wu et al, 2021 ). For yeast ectopic expression, synthetic dropout (SD) medium (SDM) was used [0.425 g yeast nitrogen base (YNB) (BD Biosciences, San Jose, CA, United States), 1.25 g ammonium sulfate [(NH 4 ) 2 SO 4 ] dissolved in 200 ml distilled water (dH 2 O), autoclave at 121°C for 20 min.…”

“…The E. coli strain ECL for CL production ( Supplementary Table 3 ) was prepared as described previously ( Wu et al, 2021 ). Single colony was grown overnight at 37°C in 1 ml Luria-Bertani (LB) containing 25 μg/ml chloramphenicol, 50 μg/ml spectinomycin, and 100 μg/ml ampicillin.…”

“…Most CYP711As encoded by monocot plants remain to be characterized. The other major group of SL-synthesizing CYPs, CYP722C subfamily, catalyzes the conversion of CLA toward either OB or 5DS ( Wakabayashi et al, 2019 , 2020 ; Wu et al, 2021 ). Currently, there are two known routes toward the synthesis of ( O )-type SLs catalyzed by either group I CYP722C (e.g., VuCYP722C) or OsCYP711A2 ( Zhang et al, 2014 ; Wakabayashi et al, 2019 ), while the only known 5DS biosynthetic route is through group II CYP722C (e.g., GaCYP722C) ( Wakabayashi et al, 2020 ).…”

“…In this study, harnessing the recently developed SL-producing microbial consortia ( Wu et al, 2021 ; Supplementary Figure 2 ), we investigated SL biosynthesis in Sorghum bicolor , which turns out to be distinct from that in rice ( Zhang et al, 2014 ). We identified SbMAX1a as a unique CYP that catalyzes up to four oxidation steps converting CL to 18-hydroxy-CLA and a small amount of OB.…”