Source: UNIVERSITY OF WYOMING submitted to
PHOTOOXIDATIVE DAMAGE IN THE GREEN ALGA CHLAMYDOMONAS AND ITS REDUCTION BY HETEROLOGOUS PROTEIN EXPRESSION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
1002840
Grant No.
(N/A)
Project No.
WYO-518-14
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Apr 1, 2014
Project End Date
Feb 25, 2016
Grant Year
(N/A)
Project Director
Herbert, ST, K..
Recipient Organization
UNIVERSITY OF WYOMING
1000 E UNIVERSITY AVE DEPARTMENT 3434
LARAMIE,WY 82071-2000
Performing Department
Plant Sciences
Non Technical Summary
Farmed microalgae can be a source of valuable organic compounds, including renewable liquid fuels. These products can be priced competitively with petroleum-derived alternatives but key technological problems must be solved first. One is the loss of productivity that occurs as oxygen from photosynthesis accumulates in large-scale algal cultures when they are exposed to full sun. Under these photo-oxidative conditions, strong light damages proteins in the photosynthetic membranes of algal cells while excessive levels of oxygen slow repair of the damage by inhibiting chloroplast protein synthesis. To test strategies for improving the growth of algae in large-scale cultures, we will genetically modify the green alga Chlamydomonas reinhardtii to express proteins that may slow damage under high light, high oxygen conditions or accelerate repair. Candidate proteins will include antioxidant proteins that prevent damage to the photosynthetic membranes. This research will contribute to the development of biofuels and biobased products, which is a current priority for USDA-funded research.
Animal Health Component
0%
Research Effort Categories
Basic
45%
Applied
45%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20340991020100%
Goals / Objectives
To help domesticate algae for production of petroleum replacements, the following objectives will be pursued.Objective 1: Sensitivity of Chlamydomonas mutants to defined oxidative stresses will be determined. These data will indicate mechanisms of stress tolerance that could be enhanced by heterologous proteins expression.Objective 2: Based upon findings from Objective 1, strategies for heterologous protein expression in Chlamydomonas will be developed and tested for reduction of photooxidative damage. Initial strategies will include heterologous expression of antioxidant enzymes.
Project Methods
The following approaches will be taken to pursue Objectives 1 and 2.Sensitivity to oxidative stressors - Growth under defined oxidative stresses will be tested in 50 ml liquid batch cultures in 125 ml flasks on an orbital shaker set to 150 rpm. Other culture conditions will be as described below. Liquid cultures for growth experiments will be started from agar plate cultures and grown to saturation under unstressed conditions (108 cells/ml or more). The cultures will then be diluted to approximately 0.5 x 106 cells/ml with fresh medium and returned to unstressed conditions. After 24 h of adjustment to the dilution, different doses of oxidative stressors will be applied and cell densities measured at 24 h intervals over 72 h by apparent absorbance at 750 nm (A750), which is proportional to light scattering and linear with cells/ml. Growth rates for the different tubes will be calculated as hours required for cell doubling averaged over 72 h.These experiments will generate dose response curves for growth versus oxidative stress as imposed by different stressors, e.g. methyl viologen or transition metals such as copper. The curves will be used to determine the EC50 for growth, which is the "Effective Concentration" of a stressor that inhibits growth by 50% relative to unstressed controls. EC50 for growth will be determined from non-linear regression curves fitted to growth data from 5-7 experimental replicates at 5 to 7 stress doses. In most cases, we expect our dose response data to fit a steep log-logistic curve. To fit curves, non-linear regressions will be applied to the dose response data and the fit of the regression curve to the log-logistic equation will be F-tested. Other parameters can be calculated from the regression curve, including EC90, EC10, and the slope of the curve as it passes through the EC50 value.Physiological analyses will be designed from dose response results to define processes of oxidative stress that could be slowed by heterologous protein expression. For example, metabolite profiles that quantify free amino acids and other metabolites can be acquired from mutants and wild types before and after high light treatment. Metabolite profiles will be acquired by GC-MS analysis of derivatized (tri-methylsilylated) methanol extracts spiked with ribitol for normalization. Several recent studies present newly refined methods for metabolite profiling of algae by GC-MS. Patterns of free amino acids will allow determination of whether specific biosynthetic pathways are affected photo-oxidative stress and which enzymes might be involved.Heterologous protein expression in Chlamydomonas - The cell wall-deficient CC-400 cw15 mt+ strain of C. reinhardtii will be used as genetic background for transformations. Cultures will be maintained on liquid and solid (1.5% agar) tris-acetate-phosphate (TAP) growth medium (Gorman & Levine 1965) under continuous 60 µEm-2s-1 cool white fluorescent light at 25°C. Liquid cultures for physiological analyses of transformants will be grown under the same conditions except that lighting will be on a diurnal cycle of 16 h light, 8 h dark, which is more consistent with outdoor growing conditions than is continuous light.Standard methods for generating recombinant DNA expression vectors and expressing heterologous proteins in C. reinhardti will be employed. C. reinhardtii codon bias will be accounted for by adapting heterologous open reading frames (ORFs). Artificial genes with 5' and 3' restriction sites and C-terminal 3X FLAG tags will be synthesized using a service provider such as GenScript. Restriction sites will be used to ligate and clone the synthesized ORF and fusion sequences into the C. reinhardtii expression vector pChlamy_1, which is available from Life Technologies. The pChlamy_1 vector systems harbor effective selection markers and are optimized for transgene expression in C. reinhardtii. Public domain expression vectors based on the pSl105 system are also available for C. reinhardtii and will be utilized for later stages of this project. The C-terminal FLAG tag/epitope will allow screening of transformants and analysis of protein expression by standard Western blot using commercially available anti-FLAG M2 primary antibody (Sigma-Aldrich). Chromogenic detection of primary antibody will be mediated by an anti-mouse alkaline phosphatase-conjugated secondary antibody and NBT-BCIP substrate. Transformations will be done according to the nuclear glass bead method. We have used similar methods to express a cell adhesion gene from the colonial green alga Volvox in C. reinhardtii with the goal of making it autoflocculate for easier harvesting from culture water in large-scale cultures. RT-PCR analyses and an apparent clumping phenotype of our transformants indicate that we have succeeded in expressing the Volvox gene in C. reinhardtii.It should be noted that expression of transgenes from the nuclear genome of C. reinhardtii is often unstable. If more stable expression is needed, C. reinhardtii strains that have lost their tendency to silence expression of nucleus-encoded transgenes can be utilized. These strains exhibit a relatively uniform high expression of heterologous proteins.Successful transformants will be tested for their sensitivity photo-oxidative stress. For example, rates of PS II photoinhibition and repair will be determined for each with wild type controls under different oxidative stresses.

Progress 04/01/14 to 02/25/16

Outputs
Target Audience: Nothing Reported Changes/Problems:This PD is not employed by University of Wyoming so we must terminate the project early with no final report. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This PD is not employed by University of Wyoming so we must terminate the project early with no final report.

Publications


    Progress 04/01/14 to 09/30/14

    Outputs
    Target Audience: Target audience is the scientific community and biotechnology professionals interested in developing algal traits that cut the cost of their large scale production. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? PhD graduate student Levi Loweder included this preliminary result in a draft of his dissertation How have the results been disseminated to communities of interest? Not yet. They will be disseminated as a chapter in the dissertation when it is published. What do you plan to do during the next reporting period to accomplish the goals? Drafts of Mr. Lowder's dissertation will be reviewed and revised by his committee with an expected graduation date of December 2014 and publication of the dissertation after.

    Impacts
    What was accomplished under these goals? A transformation vector encoding a heterologous catalase enzyme from yeast was used to transform strains of the green alga Chlamydomonas reinhardtii. Approximately 20 marker-resistant transformants were isolated and initially screened for elevated catalase activity. One transformant exhibited an apparent 3-fold increase in catalase activity and an apparent change in the sensitivity of the activity to hydrogen peroxide concentration (Km). These data suggest expression of the yeast catalase in Chlamydomonas that could ultimately lead to resistance toward sodium peroxycarbonate-based algaecides. Much additional work is needed to confirm these initial findings, however.

    Publications

    • Type: Theses/Dissertations Status: Under Review Year Published: 2014 Citation: Genetic Engineering of Microalgae for Improved Biomass Production at Large Scales: Proof of Concept Studies for Advanced Harvesting and Pest-Management Strategies (Draft title)