Engineering sorghum for higher 4-hydroxybenzoic acid content

Abstract

Engineering bioenergy crops to accumulate coproducts in planta can increase the value of lignocellulosic biomass and enable a sustainable bioeconomy. In this study, we engineered sorghum with a bacterial gene encoding a chorismate pyruvate-lyase (ubiC) to reroute the plastidial pool of chorismate from the shikimate pathway into the valuable compound 4-hydroxybenzoic acid (4-HBA). A gene encoding a feedback-resistant version of 3-deoxy-d-arabino-heptulonate-7-phosphate synthase (aroG) was also introduced in an attempt to increase the carbon flux through the shikimate pathway. At the full maturity and senesced stage, two independent lines that co-express ubiC and aroG produced 1.5 and 1.7 dw% of 4-HBA in biomass, which represents 36- and 40-fold increases compared to the titer measured in wildtype. The two transgenic lines showed no obvious phenotypes, growth defects, nor alteration of cell wall polysaccharide content when cultivated under controlled conditions. In the field, when harvested before grain maturity, transgenic lines contained 0.8 and 1.2 dw% of 4-HBA, which represent economically relevant titers based on recent technoeconomic analysis. Only a slight reduction (11-15%) in biomass yield was observed in transgenics grown under natural environment. This work provides the first metabolic engineering steps toward 4-HBA overproduction in the bioenergy crop sorghum to improve the economics of biorefineries by accumulating a value-added coproduct that can be recovered from biomass and provide an additional revenue stream.

Keywords: 4-HBA, 4-hydroxybenzoic acid; 4-Hydroxybenzoic acid; Bioenergy crop; Bioproduct; CWR, cell wall residue; CaMV, cauliflower mosaic virus; DAHP, 3-deoxy-D-arabino-heptulosonate; HPLC-ESI-TOF-MS, high performance liquid chromatography electrospray ionization and time-of-flight mass spectrometry; RT-qPCR, reverse transcription quantitative PCR; RuBisCo, ribulose-1,5- bisphosphate carboxylase; Shikimate; Sorghum.

Fig. 1

Engineering strategy for 4-HBA overproduction in sorghum. (a) Diagram of the shikimate pathway and engineering approach. The two E. coli enzymes targeted to plastids are 3-deoxy-D-arabino-heptulosonate (DAHP) synthase with L175Q mutation (AroG^L175Q) and chorismate pyruvate-lyase (UbiC). Abbreviations: AAA: aromatic amino acids; E4P, erythrose 4-phosphate; PEP, phosphoenolpyruvate. (b) DNA construct used for sorghum transformation. Schl1 and schl2 encode transit peptides from pea and maize ribulose-1,5-bisphosphate carboxylase (RuBisCo) small subunits, respectively. pZmCesa10 and pRubi2 designate the promoters of maize cellulose synthase10 and rice polyubiquitin2 genes. p2x35S is the enhanced 35S promoter from cauliflower mosaic virus (CaMV). T_35S, T_RBCS, and T_NOS are the terminators from CaMV 35S, Arabidopsis RuBisCo small subunit, and Agrobacterium nopaline synthase genes, respectively. HygR denotes the aminoglycoside phosphotransferase marker gene used for plant selection. See also Supplementary Table S1. (c) Transgene expression in two independent engineered lines (Eng-1 and Eng-2). AroG (left panel) and ubiC (right panel) transcripts were detected by RT-qPCR using mRNA obtained from the bottom part of the main stem at three different developmental stages. Transcript abundance relative to that of the PP2A sorghum gene is shown. Wild-type segregants were used as negative controls. Values are means ± SE of four biological replicates (n = 4 plants). GDP: growth differentiation point stage.

Fig. 2

Metabolite analysis of engineered sorghum. (a) Titers of 4-HBA and its glucose conjugates extracted from of Eng-1 and Eng-2 using aqueous methanol. nd, not detected. (b) Total extractable 4-HBA content in Eng-1 and Eng-2 after acid-hydrolysis of the methanolic extracts. The stover from fully mature senesced dry plants in the T2 generation was analyzed. Values are means ± SE of five biological replicates (n = 5 plants). DW, dry weight.

Fig. 3

4-HBA analysis in engineered sorghum across different developmental stages. Titers of 4-HBA after acid-hydrolysis of methanolic extracts are shown. The stover from oven-dried plants in the T3 generation was analyzed. Values are means ± SE of five biological replicates (n = 5 plants). Abbreviation: DW, dry weight; GDP, growth differentiation point stage.

Fig. 4

Field testing of engineered sorghum. (a) 4-HBA titers and (b) stover biomass yields are shown. The stover from plants in the T3 generation grown until the soft dough stage was analyzed. Values are means ± SE of four biological replicates (n = 4 plots). ‘WT’ is conventional sorghum (variety BTx430) and ‘WT segregant’ is a pool of WT-1 and WT-2 segregants. The asterisk indicates a significant difference compared to the wildtypes using the unpaired Student's t-test (*P < 0.05). DW, dry weight.