Engineering sorghum for higher 4-hydroxybenzoic acid content

Chien-Yuan Lin; Yang Tian; Kimberly Nelson-Vasilchik; Joel Hague; Ramu Kakumanu; Mi Yeon Lee; Venkataramana R Pidatala; Jessica Trinh; Christopher M De Ben; Jutta Dalton; Trent R Northen; Edward E K Baidoo; Blake A Simmons; John M Gladden; Corinne D Scown; Daniel H Putnam; Albert P Kausch; Henrik V Scheller; Aymerick Eudes

doi:10.1016/j.mec.2022.e00207

Engineering sorghum for higher 4-hydroxybenzoic acid content

Metab Eng Commun. 2022 Sep 21:15:e00207. doi: 10.1016/j.mec.2022.e00207. eCollection 2022 Dec.

Authors

Chien-Yuan Lin^{1

2}, Yang Tian^{1

2}, Kimberly Nelson-Vasilchik³, Joel Hague³, Ramu Kakumanu^{1

4}, Mi Yeon Lee^{1

2}, Venkataramana R Pidatala^{1

4}, Jessica Trinh^{1

4}, Christopher M De Ben⁵, Jutta Dalton^{1

2}, Trent R Northen^{1

2}, Edward E K Baidoo^{1

4}, Blake A Simmons^{1

4}, John M Gladden^{1

6}, Corinne D Scown^{1

4

7

8}, Daniel H Putnam^{1

5}, Albert P Kausch³, Henrik V Scheller^{1

2

9}, Aymerick Eudes^{1

2}

Affiliations

¹ Joint BioEnergy Institute, Emeryville, CA, 94608, USA.
² Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA.
³ Department of Cell and Molecular Biology, University of Rhode Island, Rhode Island, RI, 02892, USA.
⁴ Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA.
⁵ Department of Plant Sciences, University of California-Davis, Davis, CA, 95616, USA.
⁶ Department of Biomaterials and Biomanufacturing, Sandia National Laboratories, Livermore, CA, 94551, USA.
⁷ Energy & Biosciences Institute, University of California-Berkeley, Berkeley, CA, 94720, USA.
⁸ Energy Analysis and Environmental Impacts Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA.
⁹ Department of Plant and Microbial Biology, University of California, Berkeley, CA, 94720, USA.

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.