HCHL expression in hairy roots of Beta vulgaris yields a high accumulation of p-hydroxybenzoic acid (pHBA) glucose ester, and linkage of pHBA into cell walls

Abstract

As part of a study to explore the potential for new or modified bio-product formation, Beta vulgaris (sugar beet) has been genetically modified to express in root-organ culture a bacterial gene of phenylpropanoid catabolism. The HCHL gene, encoding p-hydroxycinnamoyl-CoA hydratase/lyase , was introduced into Beta vulgaris under the control of a CaMV 35S promoter, using Agrobacterium rhizogenes LBA 9402. Hairy root clones expressing the HCHL gene, together with non-expressing clones, were analyzed and revealed that one expression-positive clone accumulated the glucose ester of p-hydroxybenzoic acid (pHBA) at about 14 % on a dry weight basis. This is the best yield achieved in plant systems so far. Determination of cell-wall components liberated by alkaline hydrolysis confirmed that the ratio of pHBA to ferulic acid was considerably higher in the HCHL-expressing clones, whereas only ferulic acid was detected in a non-expressing clone. The change in cell-wall components also resulted in a decrease in tensile strength in the HCHL-expressing clones.

Vanillin production in metabolically engineered Beta vulgaris hairy roots through heterologous expression of Pseudomonas fluorescens HCHL gene

“…In the present paper we are reporting successful production of vanillin from the aforesaid B. vulgaris hairy root clones (Rahman et al, 2009) that reached up to the level of 0.1% of vanillin, which is quite significant being comparable to that of the natural vanillin from Vanilla planifolia pods. The present findings indeed facilitated addressing two major concerns: (i) does the HCHL transgene expression in hairy roots really need an intermediate time gap for stabilization of its proper function through long term cultivation as elucidated earlier (Peebles et al, 2008); and (ii) does the availability of precursor and/or other chemical parameters resuscitate the proper function of the HCHL gene?…”

“…On the other hand, insertion and expression of this HCHL gene in Datura stramonium hairy roots resulted in the accumulation of glucoside/glucose ester of 4-hydroxybenzoic acid and trace amount of vanillic acid with no vanillin production, which has been interpreted to might have occurred due to lesser availability of feruloyl CoA as its substrate (Mitra et al, 2002). Similarly, on introduction of this gene in Beta vulgaris resulted into the production of p-hydroxy benzaldehyde as the end product and no trace of vanillin was noted (Rahman et al, 2009). The mechanism of action of HCHL was also analysed in Sugarcane (McQualter et al, 2005) and in tobacco plants (Mayer et al, 2001) in which HCHL converts 4-coumaroyl-CoA to 4-hydroxybenzaldehyde through retro-aldol cleavage reaction of the phenyl propanoid intermediates.…”

“…The hairy root lines of B. Vulgaris, induced through Agrobacterium rhizogene LBA 4404 harbouring pBin19 vector containing pmHCHL under the control of 35S promoter were established and maintained, as described earlier (Rahman et al, 2009). Two superior hairy root lines i.e.…”

“…In the background of these information, the present study was undertaken with the two already established HCHL expressing B. vulgaris hairy root lines (Rahman et al, 2009) to streamline the deciding parameter in converting the naturally occurring phenylpropanoid precursor (ferulic acid) into vanillin (if so occur), through (i) optimization of critical parameters, such as media combination, concentration of carbon source, pH of the media and harvesting time and (ii) enrichment of the availability of precursor by feeding with ferulic acid. In the present paper we are reporting successful production of vanillin from the aforesaid B. vulgaris hairy root clones (Rahman et al, 2009) that reached up to the level of 0.1% of vanillin, which is quite significant being comparable to that of the natural vanillin from Vanilla planifolia pods.…”

Improving Secondary Metabolite Production in Tissue Cultures

“…Hairy root lines producing valuable phytochemicals have been developed from various plant species (Dehghan et al 2012 ;Cardillo et al 2013 ). Recently, have been used to hairy root cultures to improve secondary metabolism compounds in Hyoscyamus niger (Zhang et al 2004 ) and p-hydroxybenzoic acid (pHBA) glucose ester production in hairy roots of Beta vulgaris (Rahman et al 2009 ), express foreign proteins or vaccine in tobacco (Shadwick and Doran 2007 ). Several TIAs' biosynthesis genes have also been overexpressed in C. roseus hairy root cultures (Zhao et al 2012a, b ).…”