Title: 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, coexpression 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 agriculturalmore » 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.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.« less

Authors:

Welner, Ditte Hededam ;

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Shin, David; Tomaleri, Giovani P.; DeGiovanni, Andy M.; Tsai, Alex Yi-Lin; Tran, Huu M.; Hansen, Sara Fasmer; Green, Derek T.; Scheller, Henrik V.; Adams, Paul D.; Hofmann, ed., Andreas

Publication Date:

Fri Jun 09 00:00:00 EDT 2017

Research Org.:

Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:

1372595

Alternate Identifier(s):

OSTI ID: 1379873

Grant/Contract Number:  

AC02-05CH11231

Resource Type:

Journal Article: Published Article

Journal Name:

PLoS ONE

Additional Journal Information:

Journal Name: PLoS ONE Journal Volume: 12 Journal Issue: 6; Journal ID: ISSN 1932-6203

Publisher:

Public Library of Science (PLoS)

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES