High (School)-Throughput Screening of BAHD Transferases
Justin F. Acheson1, Ella Lodewyk1,2, Hailey Sieren1,2, Daniel Lee1,2, Melina Guestella1,2, Craig A. Bingman1,3, Rebecca A. Smith1,2, Steven D. Karlen1,3, John Ralph1,3 Yuchen Pu3,4, Zimou Sun3,4, Shawn D. Mansfield3,4, and Brian G. Fox1,3
1Department of Biochemistry and 2Dane County Youth Apprentice Program, 3Great Lakes Bioenergy Research Center, University of Wisconsin−Madison; and 4University of British Columbia
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 biofuel precursors, 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. Pairing specific acyl-CoAs and BAHD transferases can allow fine tuning of lignin content either for simple deconstruction (Zip-lignin) or incorporation of useful aromatics that can then easily be clipped-off increasing the net value of the plant biomass (Karlen et al. 2016; de Vries et al. 2022).
BAHD acyltransferases have the ability to produce valuable molecules in bioenergy crops. The discovery and characterization of specific BAHD acyltransferases led to the creation of Zip-lignin, where introduction of ester-linked monolignols allows hydrolysis under mild conditions, avoiding harsher chemical treatments needed to remove lignin during bioenergy processing (Karlen et al. 2016). 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 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 poises lignin as an attractive source of energy rich molecules. By taking advantage of consistently 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 Joint Genome Institute (JGI) and were then screened using a wheat germ 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 preferred substrates. Active enzymes catalyzing interesting reactions were then incorporated into Populus sp. to assess in vivo impacts, 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 of the breadth of molecules this large family of enzymes can produce, and how these molecules may be useful in producing more energy efficient plants or provide engineered plant sources for fine chemicals.
Karlen, S. D., et al. 2016. “Monolignol Ferulate Conjugates are Naturally Incorporated into Plant Lignins.” Science Advances 2(10).
de Vries, L., et al. 2022. “pHBMT1, a BAHD-Family Monolignol Acyltransferase, Mediates Lignin Acylation in Poplar.” Plant Physiology 188(2).
This work was supported by the U.S. Department of Energy, 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. Department of Energy (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). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.