Metabolic engineering of folate and its precursors in Mexican common bean (Phaseolus vulgaris L.)

Abstract

SummaryFolate (vitamin B9) deficiency causes several health problems globally. However, folate biofortification of major staple crops is one alternative that can be used to improve vitamin intakes in populations at risk. We increased the folate levels in common bean by engineering the pteridine branch required for their biosynthesis. GTP cyclohydrolase I from Arabidopsis (AtGchI) was stably introduced into three common bean Pinto cultivars by particle bombardment. Seed‐specific overexpression of AtGCHI caused significant increases of up to 150‐fold in biosynthetic pteridines in the transformed lines. The pteridine boost enhanced folate levels in raw desiccated seeds by up to threefold (325 μg in a 100 g portion), which would represent 81% of the adult recommended daily allowance. Unexpectedly, the engineering also triggered a general increase in PABA levels, the other folate precursor. This was not observed in previous engineering studies and was probably caused by a feedforward mechanism that remains to be elucidated. Results from this work also show that common bean grains accumulate considerable amounts of oxidized pteridines that might represent products of folate degradation in desiccating seeds. Our study uncovers a probable different regulation of folate homoeostasis in these legume grains than that observed in other engineering works. Legumes are good sources of folates, and this work shows that they can be engineered to accumulate even greater amounts of folate that, when consumed, can improve folate status. Biofortification of common bean with folates and other micronutrients represents a promising strategy to improve the nutritional status of populations around the world.

Genetic Engineering for Global Food Security: Photosynthesis and Biofortification

“…Over-expression (OE) of the GTP cyclohydrolase I (GCHI) ( Figure 4B), the first committed step in the pteridine pathway [167], in Arabidopsis resulted in a >1000-fold increase in pterins and only a 2-to 4-fold increase in folates (Table 3) [166]. Similar results were also observed following Seed-specific over-expression of GCHI in common Mexican bean [168]. These results were supported by similar work carried out by Diaz de la Gaza [164] who demonstrated that the OE of GCHI in tomato fruit resulted in an up to 140-fold increase in pterins and increased fruit folate content by an average of 2-fold [164].…”

“…In tomato fruit, OE of GCHI was also shown to result in the depletion of the chloroplast synthesized PABA, which suggested that PABA represents a further limiting step in folate biosynthesis in transgenic tomato fruit (Table 3). However, this depletion in PABA is in contrast with an increase observed following the OE of GCHI in Mexican bean [168]. These authors demonstrated that feeding fruit PABA though the fruit stalk could increase folate by up to 10-fold [164].…”

Expression Levels of the γ-Glutamyl Hydrolase I Gene Predict Vitamin B9 Content in Potato Tubers

“…Folate determination and quantification were performed as previously described [43,44] with minor modifications. 100 mg of freeze-dried potatoes were ground in liquid nitrogen and extracted in 10 mL of folate extraction buffer (50 mM HEPES, 50 mM CHES, 10 mM β-mercaptoethanol, 2% ascorbic acid, 4 mM CaCl 2 , pH 7.85) and boiled for 10 min for folate release.…”

Induction of competent cells for Agrobacterium tumefaciens-mediated stable transformation of common bean (Phaseolus vulgaris L.)

“…Genetic engineering is a powerful tool to introduce genes from sources that are inaccessible through sexual hybridization [12][13][14]. Thus, substantial efforts have been made to develop reliable transformation methods for engineering common beans with various traits [13,[15][16][17][18][19][20][21]. To date, no genetically engineered (GE) common bean has been commercialized, despite of the regulatory approval for the transgenic "Embrapa 5.1" common bean for golden mosaic virus (BGMV) resistance in Brazil in 2011 [22][23][24].…”

“…Stable transformation of common bean at low frequencies (< 1%) has been achieved using particle bombardment-mediated transformation of meristematic tissues for different goals [11,27,28]. Agrobacterium tumefaciens-mediated transformation is desirable for common bean transformation because of its accessibility and tendency to produce low-or single-copy insertion(s) of the transgene [18].…”