Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes

Abstract

Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, co-expression partners, purification methods, and optimization of protein solubility and stability. Hence researchers are presented with thousands of potential conditions to test. Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s) being studied. We have developed a rational approach to this process. We devise a pipeline comprising in silico selection of targets and construct design, and high-throughput expression screening, target enrichment, and hit identification. We have applied this pipeline to a test set of Arabidopsis thaliana cell wall glycosyltransferases known to be challenging to obtain in soluble form, as well as to a library of cell wall glycosyltransferases from other plants including agricultural and biofuel crops. The screening results suggest that recombinant cell wall glycosyltransferases in general have a very low soluble:insoluble ratio in lysates from heterologous expression cultures, and that co-expression of chaperones as well as lysis buffer optimization can increase this ratio. We have applied the identified preferred conditions to Reversibly Glycosylated Polypeptide 1 from Arabidopsis thaliana, and processed this enzyme to near-purity in unprecedented milligram amounts. The obtained preparation of Reversibly Glycosylated Polypeptide 1 has the expected arabinopyranose mutase and autoglycosylation activities.

Genome-wide comparative and evolutionary analysis of Calmodulin-binding Transcription Activator (CAMTA) family in Gossypium species

“…Previous studies demonstrated that the fiber strength commonly related to the strengthening of cell wall 59 . Interestingly, identification of cell wall related genes such as ABC transporter-like 47 , 60 , 61 , arabinogalactan peptide (AGP) 62 , 63 , alpha-1,4-glucan-protein synthase 64 , calcium-binding EF-hand protein 61 , cellulose synthase 63 , glutathione peroxidase 65 , glycoside hydrolase 66 , glycosyl transferase 67 – 69 , extensin 62 , UDP-glucuronosyl/UDP-glucosyltransferase 70 , small GTPase superfamily 71 , kinesin 72 , leucine-rich repeat 61 , thaumatin 73 , tubulin 74 , and WD40 repeat 75 in PCoEGs and NCoEGs, emphasizing their potential roles in regulating cell wall integrity and thus fiber quality (Supplementary Dataset S8). However, the detailed molecular investigation is needed further to establish a link between CAMTAs and fiber quality traits.…”

Microscale thermophoresis as a powerful tool for screening glycosyltransferases involved in cell wall biosynthesis

“…First, protein purification for biochemical activity assay has only been accomplished in few cases due to the labile nature and low abundance of these enzymes [ 12 , 13 ]. Heterologous expression in E. coli has been largely ineffective, likely limited by insufficient folding and post-translational modification in bacterial systems [ 14 – 16 ]. Heterologous expression in eukaryotic systems has been reported, but no generally successful system has been found.…”

Decoupling of recombinant protein production from Escherichia coli cell growth enhances functional expression of plant Leloir glycosyltransferases

“…Escherichia coli is most often used. In general, expression is low (≤10 mg/L; e.g., Arend, Warzecha, Hefner, & Stöckigt, 2001;Schmölzer, Gutmann, Diricks, Desmet, & Nidetzky, 2016) and poor (≤1 mg/L) in various instances (e.g., Cai et al, 2017;Welner et al, 2017). With notable exceptions (Arend et al, 2001;Dewitte et al, 2016;Priebe, Daschner, Schwab, & Weuster-Botz, 2018;Schmideder et al, 2016), the enzyme production has received relatively little attention for systematic process development.…”