Plant cell walls and biomass conversion
 |
Field emission scanning electron micrograph of a maize stem cross section. Large
deep cells are vessel elements. Thick walled cells are cortical sclerenchyma whose
role is primarily support.
|
I have several reasons to study plant cell walls. First, cell walls are one of the
most complex organelles of the plant cell. Even though the basic nature of the wall is known in that the classes of
components have been defined, each tissue and cell type has a wall with unique composition and assembly of those components.
Second, by understanding how walls are put together, it will be much easier to take them apart. Third, my past several years
of industry experience put me in a unique position to apply a host of techniques that include plant transformation, gene
expression, protein accumulation, microscopy, biochemistry and genetics to study this problem.
Fossilized hydrocarbon-based energy sources, such as coal, petroleum and natural gas,
provide a limited, non-renewable resource pool. The transportation sector is a vital segment of our national economy, and
it depends almost entirely on fossil petroleum reserves for fuel. Petroleum reserves are probably the most limiting fossil
reserve and are likely to be in short supply within the next 50-100 years (http://tonto.eia.doe.gov/FTPROOT/forecasting/0383(2001).pdf).
The U.S. transportation sector alone consumes over 100 billion gallons of gasoline per year, and about 60% of the oil used
in the US today is imported (see the 2003 Annual Energy Review, available online at
http://www.eia.doe.gov/emeu/aer/contents.html). This creates a somewhat precarious situation in today’s political
climate, because supply disruptions are highly likely and could cripple the ability of our economy to function. This is
regarded as a national security issue.
Renewable, plant biomass-derived energy resources make
economic, environmental and political sense, and we should investigate and implement models for moving the economy
toward these new energy sources (http://www.eere.energy.gov/industry/forest/).
In 2004 the U.S. manufactured approximately 3.4 billion gallons of ethanol from corn grain-derived starch (http://www.ksgrains.com/ethanol/useth.html),
and this is expected to rise further in 2005. Even if all corn grain grown in the U.S. could be used for ethanol
production, it would not meet the projected demand for this fuel (over 10 billion gallons/year at today’s usage
rate) assuming only a 10% blend with gasoline (Sheehan, 2001). The demand for ethanol will increase greatly as it is
adopted for use as an 85% blend with gasoline (i.e., E85). To meet the increasing demand for ethanol, the lowest
cost and largest reservoir of plant-based renewable resources lies in the recovery of 5 and 6 carbon sugars from
plant cell walls. Residues from crops, such as corn, provide a ready supply of material that could make a
significant impact on the transportation fuel supply system.
The focus of my work is to test a select cadre of cell wall
degrading enzymes on corn stover to determine the collective ability of the enzymes to deconstruct corn cell walls
into their basic component building blocks -specifically, fermentable sugars. This work is designed to address two
needs in biomass conversion: provide a low cost supply of enzymes for use by a biomass conversion industry, and
replace or attenuate the need for thermochemical pretreatment through enzyme treatment. The target enzymes (i.e,
cellulases, ligninases and proteases) have recently been individually expressed in maize at high titer (see Hood et
al., 2003; Woodard et al., 2003; Clough et al., submitted). This approach to enzyme production could provide a
supply of large volumes of low cost enzymes without the need for large capital investments to build fermentation
facilities to service the biomass industry. If pretreatment severity can be significantly reduced by this
biochemical approach to biomass deconstruction, capital expense can also be avoided in the pretreatment area of the
process. In addition, the generation of sugar degradation products (i.e., yield loss) such as furfural and
hydroxymethylfurfural would probably be avoided. These thermochemical degradation compounds also inhibit downstream
fermentation organisms, and their avoidance would benefit the performance of the fermentation area of the process
and tend to increase process yield as well.
Hood, E.E., M.R. Bailey, K. Beifuss, M. Horn, M. Magallanes-Lundback, C.
Drees, D. E. Delaney, R. Clough and J. A. Howard 2003 Criteria for high-level expression of a fungal laccase gene in
transgenic maize Plant Biotechnology Journal. 1, 129-140
Bailey, M.R., S.L. Woodard, E. Callaway, K Beifuss, D. Delaney, M. Magallanes-Lundback, J. Lane, M.E. Horn, M. Ward, F. Van Gastel,
J.A. Howard, E.E. Hood 2004 Improved recovery of active recombinant laccase from maize seed Applied
Microbiology and Biotechnology 63(4):390-7, (2003 Epub)
Woodard, S.L., J.M. Mayor, M.R. Bailey, D.K. Barker, R.T. Love,
J.R. Lane, D.E. Delaney, J.M. McComas-Wagner, H.D. Mallubhotla, E.E. Hood, L.J. Dangott, S.E. Tichy and J.A. Howard.
2003 Maize-derived bovine trypsin: Characterization of the first large-scale, commercial protein product from
transgenic plants. Biotechnology and Applied Biochemistry 38:123-130
Hood, EE 2004 Bioindustrial and Biopharmaceutical Products from Transgenic Plants Online publication at 4th
ICSC, Brisbane Australia
http://www.cropscience.org.au/icsc2004/symposia/3/5/1955_hoode.htm
Howard, JA and Hood, EE. 2005 Bioindustrial and Biopharmaceutical Products Produced in Plants Adv in Agron
85:91-124
Clough, RC, Beifuss, K, Lane, J, Pappu, K, Thompson, K, Bailey, MR, Delaney, DE, Harkey, R, Drees, C, Howard, JA and
Hood, EE. 2004 Recombinant manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium is
enzymatically active and accumulates to high levels in transgenic corn seed. Plant Biotechnology Journal
Submitted
Control of Gene Expression
Control of gene expression is one of the most fundamental
questions in biology. In functional genomics, it is key. Gene expression is a very complex process compounded at
many levels by multiple factors. Levels of control are exerted in tissue type, cell type, subcellular location of
the gene product, timing, environment and molecular elements such as promoter strength and transcription factors.
 |
| View the entire slideshow here. |
Observations of transgenic plants
have generated many questions concerning the control of gene expression. Variation in transgene expression can be
see among individual seeds from the same plant, among clonal plants from the same transgenic event and among events
from the same vector. These variations cannot be totally explained by position effects and chromatin
structure—particularly for seed to seed and clonal plant variation. Through breeding and selection, individual
plants can be selected that increase in their expression by as much as 150 fold in 6-8 generations. This magnitude
of an increase will most likely not be through a simple mechanism, nor a subtle mechanism, but more probably through
coordinated multilevel action.
My hypothesis is that genetic
factors affect gene expression in a specific manner, and these factors are most likely the same for different genes
expressed from the same promoter when their products are targeted to the same location. However, they most likely
will differ with respect to promoter (which will reflect tissue and cell type differences) and subcellular location
of the gene product. Specific protein characteristics may have tertiary influences.
The question can be approached at
multiple levels. Initially, high and low expressing individual (clonal) plants will be identified from a single
independent event. Because of the nature of transgenic maize, the T0 plants already demonstrate variation and
provide a segregating population. Differentially expressed genes will be identified that co-segregate with the
high-expression phenotype. Candidate genes for factors influencing expression will be identified, cloned and tested
for phenotype singly and multiply in transgenic plants.
Hood, E.E., M.R. Bailey, K. Beifuss, M. Horn, M. Magallanes-Lundback, C.
Drees, D. E. Delaney, R. Clough and J. A. Howard 2003 Criteria for high-level expression of a fungal laccase gene in
transgenic maize Plant Biotechnology Journal. 1, 129-140
Woodard, S.L., J.M. Mayor, M.R. Bailey, D.K. Barker, R.T. Love,
J.R. Lane, D.E. Delaney, J.M. McComas-Wagner, H.D. Mallubhotla, E.E. Hood, L.J. Dangott, S.E. Tichy and J.A. Howard.
2003 Maize-derived bovine trypsin: Characterization of the first large-scale, commercial protein product from
transgenic plants. Biotechnology and Applied Biochemistry 38:123-130
Streatfield, S.J., M.E. Magallanes-Lundback, K.K. Beifuss, C.A.
Brooks, R.L. Harkey, R.T. Love, J. Bray, J.A. Howard, J.M. Jilka and E.E. Hood. 2004 Analysis of the maize
polyubiquitin-1 promoter heat shock elements and generation of promoter variants with modified expression
characteristics. Transgenic Research 13(4):299-312
Lamphear, BJ DK Barker, CA Brooks, DE Delaney, JR Lane, K Beifuss, R Love, K Thompson, J Mayor, R Clough, R Harkey,
M Poage, C Drees, ME Horn, SJ Streatfield, Z Nikolov, SL Woodard, EE Hood JM Jilka, and JA Howard. Expression of
the Sweet Protein Brazzein in Maize for Production of a New Commercial Sweetener Plant Biotechnology J
3:103-114
