Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

High (School) Throughput Screening of BAHD Transferases

Authors:

Justin F. Acheson1* (jacheson@wisc.edu), Ella C. Lodewyk1,2, Hailey R. Sieren1,2, Daniel H. Lee1,2, Ella R. Torkelson1, Melina N. Guestella1,2, Riley A. Malecki1,2, Varsha Kumar1,2, Lauryn D. Swenson1,2, Jaiden A. Mezera1, Craig A. Bingman1,3, Rebecca A. Smith3, Steven D. Karlen3, John Ralph1,3, Yuchen Pu3,4, Zimou Sun3,4, Shawn D. Mansfield3,4, Brian G. Fox1,3

Institutions:

1Department of Biochemistry, University of Wisconsin–Madison; 2Dane County Youth Apprentice Program, University of Wisconsin–Madison; 3Great Lakes Bioenergy Research Center; 4Department of Wood Science, University of British Columbia

Goals

BAHD acyltransferases represent a large family of enzymes typically found in plants. They use acyl-CoA donors (produced from acyl-CoA ligases) to form esters or amides with alcohol or amine acceptor molecules. The products of these reactions are incorporated into large polymers such as lignin and suberin or into small secondary metabolites including phenolic esters, antimicrobials, antifungals, or compounds that contribute to drought resistance. The goal is to elucidate the identities and functions of these enzymes and use them in conjunction with acyl-CoA ligases to precision engineer bioenergy crops (Chaudhury et al. 2023). Pairing specific acyl-CoAs and BAHD transferases can allow fine-tuning of lignin content for simple deconstruction (Zip-lignin) or by incorporation of useful aromatics that can then easily be “clipped-off” increasing the net value of the plant biomass.

Abstract

BAHD acyltransferases have the ability to produce valuable molecules in bioenergy crops. The discovery and characterization of a specific BAHD acyltransferases led to the creation of ‘Zip-lignin,’ in which introduction of ester-linked monolignols allows hydrolysis under milder conditions, avoiding harsher chemical treatments needed to remove lignin during bioenergy processing (Wilkerson et al. 2014). Further investigation showed that specific aromatics could be incorporated into terminal lignin positions, such as p-hydroxybenzoate, that can easily be clipped off due to their attachment via an ester linkage (de Vries et al. 2022). Thus, the ability to tune lignin composition not only allows for improved deconstruction but also positions lignin as an attractive source of energy-rich molecules.

By taking advantage of continually improving genomic data and tools, the team curated lists of high-potential target genes focusing on two priority bioenergy crops and a model plant (poplar, sorghum, Arabidopsis). Selected genes were synthesized into cell-free expression vectors by the DOE Joint Genome Institute and were then screened using a wheatgerm cell-free system (Cell Free Sciences) by a team of high school–student laboratory members. The expressed proteins were screened for potential activity and categorized by their preferred substrates. Active enzymes catalyzing interesting reactions were then introduced into Populus sp. to assess in vivo impacts (de Vries et al. 2022; Gonzales-Vigil et al. 2021; Smith et al. 2022), and cell-based expression systems such as Escherichia coli have been used to facilitate structural and biochemical characterization. The work presented here has given further understanding into the breadth of molecules this large family of enzymes can synthesize and how these molecules may be useful in producing more energy-efficient plants or providing engineered plant sources for fine specialty chemicals.

References

Chaudhury, D., et al. 2023. “Rapid Biocatalytic Synthesis of Aromatic Acid CoA Thioesters by Using Microbial Aromatic Acid CoA Ligases,” ChemBioChem 24, e202300001. DOI:10.1002/cbic.202300001.

de Vries, L., et al. 2022. “pHBMT1, a BAHD-Family Monolignol Acyltransferase, Mediates Lignin Acylation in Poplar,” Plant Physiology 188, 1014–27. DOI:10.1093/plphys/kiab546.

Gonzales-Vigil, E., et al. 2021. “Understanding the Role of Populus ECERIFERUM2-Likes in the Biosynthesis of Very-Long-Chain Fatty Acids for Cuticular Waxes,” Plant Cell Physiology 62, 827–38. DOI:10.1093/pcp/pcab040.

Smith, R. A., et al. 2022. “Identification and Characterization of a Set of Monocot BAHD Monolignol Transferases,” Plant Physiology 189, 37–48. DOI:10.1093/plphys/kiac035.

Wilkerson, C. G., et al. 2014. “Monolignol Ferulate Transferase Introduces Chemically Labile Linkages into the Lignin Backbone,” Science 344, 90–93. DOI:10.1126/science.1250161.

Funding Information

This work was supported by the U.S. DOE, Office of Science, Basic Energy Sciences under award no. DE-SC0020349 to B.G.F. C.A.B., R.A.S., S.D.K., S.D.M., and J.R. were also supported in part by the Great Lakes Bioenergy Research Center (GLBRC, DOE BER Office of Science DE-SC0018409). This research used resources of the Advanced Photon Source, a U.S. DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817). GM/CA @ APS has been funded by the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006, P30GM138396).