PRODUCTION OF INDUSTRIAL ENZYMES IN PLANTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0211869
• Project Start Date: Oct 1, 2007
• Project End Date: Sep 30, 2010
• Program Code: [(N/A)]- (N/A)

Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853

Performing Department
MOLECULAR BIOLOGY AND GENETICS

Non Technical Summary
Certain industrial enzymes and pharmaceutical proteins cannot be produced in plants because they are degraded by proteases. The purpose of this study is to learn how to prevent degradation of useful proteins when they are expressed in plants.

Animal Health Component

30%

Research Effort Categories

Basic

70%

Applied

30%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
204	1999	1040	50%
204	1999	1080	50%

Knowledge Area
204 - Plant Product Quality and Utility (Preharvest);

Subject Of Investigation
1999 - Tobacco, general/other;

Field Of Science
1040 - Molecular biology; 1080 - Genetics;

Keywords

chloroplast

foreign protein

degradation

protease

protein stability

transgenic plant

enzyme

Goals / Objectives
The main objective is to understand how protein stability affects the accumulation of foreign proteins in chloroplasts. Using tobacco as a model system, we will use genetic engineering methods to obtain plants with reduced amounts of chloroplast-localized proteases. We have identified a microbial cellulase which accumulates to undetectable levels when an encoding gene is introduced into the chloroplast, and have observed it is unstable when incubated with chloroplast extracts. We will use this enzyme as a basis for experiments to improve protein stability. We will synthesize a variety of modified versions of the microbial cellulase in E. coli and purify the enzymes. The enzymatic activity of the modified enzymes will be assessed in comparison to wild-type enzyme expressed in E. coli. We will test the stability of purified modified enzymes in extracts of wild-type and low-protease tobacco plants. Once a gene is identified that encodes an active stability-modified cellulase, it will be introduced in an appropriate chloroplast transformation vector into wild-type and low-protease tobacco plants. After homoplasmic plants are obtained, we will compare accumulation of the modified cellulase.

Project Methods
The chloroplast contains a number of stroma localized proteases that may attack foreign proteins. Nuclear genes encoding these proteases have been identified in genomic sequence databases. We will use this information to engineer plants that contain reduced amounts of chloroplast proteases. We will use RNA silencing methods to impair the expression of protease genes. We will test whether reduced protease expression improves the expression of a microbial cellulase that is expressed from the chloroplast genome, but does not accumulate in transplastomic plants. We will also assess the effect of inhibiting protease gene expression on production of another microbial cellulase we have observed to accumulate to 2 percent of soluble protein. We will make systematic alteration in the protein sequence of the unstable cellulase and express the modified versions in E. coli. We will add the purified modified enzyme to extracts of chloroplasts and observe whether the cellulase is stable over time. We will use both extracts of wild-type chloroplasts and chloroplasts from any reduced-protease plants we succeed in engineering. We will also determine the cellulose-digesting activity of the modified cellulases, in order to identify an active and stable enzyme. Among the modifications we will make will be N- and C-terminal fusions as possible means to prevent proteolytic attack. After identifying a coding region that encodes a protein stable in chloroplast extracts, we will introduce it into a chloroplast transformation vector. The vector will be bombarded into tobacco leaves and antibiotic selection will be used to obtain transplastomic plants. We will perform sufficient rounds of regeneration and selection to obtain homoplasmic plants in which all copies of the chloroplast genome carry the transgene. We will assess the level of accumulation of the cellulase and determine its enzymatic activity when expressed from the chloroplast genome.

Progress 10/01/07 to 09/30/10

Outputs
OUTPUTS: The project director presented an invited lecture describing project results at the Annual Symposium of the Max-Planck Institute in Potsdam-Golm, Germany, in June, 2010, and visited with researchers there interested in expressing biomass-degrading enzymes and pharmaceutical proteins in plants. Likewise, project results were described to Agriculture Canada in London Ontario in 2009. A manuscript was submitted to the journal Plant Molecular Biology, accepted, and has appeared online. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Industrial concerns interested in enhancing efficiency of lignocellulosic ethanol production. Researchers in the biotechnology industry who wish to produce valuable proteins in plants and members of the basic research community interested in gene expression in chloroplasts. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Chloroplast transformation is an attractive method to cause plants to manufacture valuable proteins. Extremely high level expression of some proteins have been achieved, but other proteins are expressed at very low levels for unknown reasons. One possibility for poor yield of proteins in chloroplasts is protein or RNA degradation. The downstream box (DB) region, which is found immediately downstream of the start codon, can greatly affect protein accumulation from chloroplast transgenes over several orders of magnitude. β-glucosidase has been shown to improve the performance of lab-scale cellulosic ethanol production when added to commercial cellulases and thus may enhance glucose production in commercial-scale enterprises. Different DB regions were attached to the coding region for the enzyme a β-glucosidase (BglC) from the thermophilic bacterium Thermobifida fusca and inserted into the tobacco (Nicotiana tabacum) plastid genome. The accumulation of the BglC protein varied by two orders of magnitude, depending on which DB was encoded by the transgene. Unexpectedly, the most efficient DB for β-glucosidase was not the same one that previously had been found to be most effective for production of a cellulase. The transcripts of the transgene that resulted in the greatest accumulation of enzyme exhibited less 3' degradation than the transcripts of the less effective transgenes. The enzyme produced in the chloroplasts exhibited activity against cellobiose and tobacco lignocelluloses. Transgenic plants expressing the most effective DB-enzyme fusion accumulated enzyme to approximately 10% of total soluble protein. Protein accumulation was maintained in the leaves as the plants aged. These results demonstrate that proper engineering of chloroplast transgenes can result in high-level production of enzymes for conversion of cellulose and cellobiose into glucose for fermentation to ethanol.

Publications

• Gray BN, Yang H, Ahner BA, Hanson MR. 2011. An efficient downstream box fusion allows high-level accumulation of active bacterial beta-glucosidase in tobacco chloroplasts. Plant Mol Biol. Online Jan 30, in press.

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: The project director presented a lecture at Agriculture Canada in London Ontario that described project results and visited with researchers there interested in expressing biomass-degrading enzymes and pharmaceutical proteins in plants. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Researchers in the biotechnology industry who wish to produce valuable proteins in plants and members of the basic research community interested in gene expression in chloroplasts. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Chloroplast transformation is an attractive method to cause plants to manufacture valuable proteins. Extremely high level expression of some proteins have been achieved, but other proteins are expressed at very low levels for unknown reasons. One possibility for poor yield of proteins in chloroplasts is protein or RNA degradation. We have constructed 6 different chloroplast transgenic lines of tobacco that express either a cellulase or a sugar-converting enzyme at levels <0.1% to over 10% of total soluble protein. The proteins encoded by the 6 different genes differ only in the first 14 amino acids. We are investigating whether the difference in expression levels is caused by RNA or protein stability or by translation efficiency. In the case of the cellulase, analysis of translation revealed that the monocistronic transcript of the transgene that was most highly expressed appears to be present on polysomes in greater quantity than the transcripts of the more poorly expressed transgenes. In the case of the sugar-converting enzyme, the transcripts of the less well-expressed transgene exhibit more 3' degradation than the RNA derived from the highly-expressed transgene. Further experiments concerning the relative role of translation vs. protein stability are underway.

Publications

• No publications reported this period

Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: Presentations about the project have been made to prospective graduate students at Cornell University who are choosing laboratories for thesis research. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Researchers in the biotechnology industry who wish to produce valuable proteins in plants and members of the basic research community interested in gene expression in chloroplasts. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Chloroplast transformation is an attractive method to cause plants to manufacture valuable proteins. Extremely high level expression of some proteins have been achieved, but other proteins are expressed at very low levels for unknown reasons. One possibility for poor yield of proteins in chloroplasts is protein degradation. In order to examine the stability of proteins in chloroplasts, we have implemented an in vitro assay. Chloroplast extracts are incubated under various conditions in the presence of exogenously added foreign proteins. The amount of a protein known to be highly protease-resistant, green fluorescent protein, does not change during our incubation period, but Cel9A, a cellulase from Thermobifida fusca, exhibits degradation. Cel9A is poorly expressed in transgenic chloroplasts, in contrast to GFP. We have produced a number of clones with different N-terminal sequences on cellulase enzymes and expressed them in E. coli to assay their stability in bacteria vs. chloroplasts. We have made constructs to alter the construction of protease in chloroplasts to determine whether we can increase the abundance of foreign proteins that do not accumulate to desirable levels.

Publications

• No publications reported this period