Progress 10/01/12 to 09/30/15
Outputs
Target Audience:- A presentation of our project was given in Animal Science to Prof. Xingen Lei's research group on October 30th 2013. - B.A. Ahner met with CEO and lead scientist of a large algae production facility in Hawaii. Discussed economic models and potential use of transgenic algae for co-product improvement. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The laboratory work done on this project was largely conducted by a post-doctoral researcher hired to work on this project.With respect to training activities, she had to learn a new set of laboratory techniques to work with algae and had to learn how to do chloroplast transformations.She was mentored to train undergraduate students in the laboratory and she helped them to develop independent projects.With respect to professional development, she attended the 3rd ISCGGE Chloroplast Biology meeting at Rutgers University in May of 2013. How have the results been disseminated to communities of interest?B.A. Ahner met with CEO and lead scientist of a large algae production facility in Hawaii. Discussed economic models and potential use of transgenic algae for co-product improvement. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The main goals of this project were: (1) to optimize the accumulation of valuable proteins in genetically modified algae strains to increase the value of protein byproducts that will be generated in algal biofuel production systems, and (2) to develop a strategy to transform an algae species that can accumulate high concentrations of lipids. The target proteins were the endoglucanase enzyme Cel6A, used during enzymatic cellulose hydrolysis in biodiesel industry, and the animal feed-additive enzyme phytase, used to increases phosphorous digestibility. We investigated the regulatory elements needed for high Cel6A and phytase accumulation in Chlamydomonas reinhardtii, a model algae species which is readily transformed but not particularly suited for biofuel production. Our main strategy to optimize protein accumulation was to find new components to build into expression cassettes for chloroplast transformation. While it was been shown that various native upstream and downstream DNA sequences (5'/ 3' UTRs, promoters) can be used to flank coding sequences for effective protein production, success rates are highly variable where some proteins accumulate to high levels and others do not. We identified four down-stream box (DB) candidates, a region of 10 to 15 codons inserted immediately after the start codon of the studied gene; two of which were proven effective in tobacco. Transplastomic C. reinhardtii algae were generated via Biolistic bombardment where gene fragments are physically delivered to cells, homologous recombination of the gene cassette occurs within the chloroplast and transformants are selected by antibiotic resistance. We developed C. reinhardtii transgenic strains using the cel6A gene, a cellulose degrading enzyme that we had used previously with different regulatory elements in tobacco transformants. We optimized PCR conditions for successful amplification of plastid DNA for insertion in the chloroplast genome, and developed a protein extraction protocol including a protein concentration step that resulted in detectable protein levels on immunoreactive detection blots. We confirmed that one specific downstream box enhances the level of Cel6A accumulation. We examined the effect of nitrogen starvation, a step used in biofuel production, on the level of Cel6A accumulation. Our findings are discussed in a manuscript currently in preparation for submission to the peer-reviewed journal Biotechnology and Bioengineering. (Optimization of Transgenes for Effective Production of Bacterial Cellulase in Chlamydomonas reinhardtii Chloroplast. LV. Richter, Yang H., Hanson MR. and Ahner BA.). Several rounds of transformation were attempted using the C. reinhardtii wild-type strain (C125) and plasmids harboring the phytase gene constructs. Despite having worked in the past, no mutants were generated. A cell-wall mutant line (CR 4349) was obtained and resistant colonies were selected for the algae transformants. We confirmed the proper homologous recombination for each of the five phytase-expressing transplastomic C. reinhardtii strains. Protein analyses of these strains did not yield measurable phytase levels on immunoreactive detection blots. To overcome the limited effectiveness of biolistic transformation with the wild-type strains we established a collaboration with Professor Hitomi Mikaibo, Chemical Engineering, University of Rochester, who is working on a new technique for efficient chloroplast transformation using plasmid-coated-microneedles. With her method, the plasmid is directly injected into the cells, which according to preliminary data, has proven to be more efficient than the conventional biolistic bombardment. We provided her lab with a highly concentrated cel6A-expressing plasmid to test the rates at which transformation can be effected and to compare them to those obtained with traditional methods. We submitted at least one grant proposal together to USDA but it was not funded. We worked toward developing an expression vector for transforming Chlorella vulgaris (C-27), an algae with better potentials for biofuel production. We extracted and purified DNA from this algae and developed multiple sets of primers for PCR amplification of chloroplast genome fragments. Primer sequences were based on a published C. vulgaris chloroplast genome sequence. We were successfully able to amplify and sequence fragments comprising the 5'/3' UTRs of the PsbA gene, but were unable to amplify any intergenic regions to be used as a site for plastid insertion of the foreign gene. We suspect the failure was due to a poor sequence quality in the published data base (the chloroplast sequencing was conducted in 1997 and no sequencing updates have been made). As part of a different project we sequenced the genome of a marine chlorophyte with excellent lipid accumulation. The chloroplast genome sequence is complete and available to determine homologous recombination sites for transformation in this organism. (Use of de novo Transcriptome Libraries to Characterize a Novel Oleaginous Marine Chlorella Species during the Accumulation of Triacylglycerols. CB. Mansfeldt, Richter LV., Ahner BA., Cochlan WP. and Richardson RE. PlosOne, in press).
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Met with CEO and lead scientist of a large algae production facility in Hawaii (Cellana LLC). Discussed economic models, laboratory results and RFP opportunities. Changes/Problems:
Nothing Reported
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? We will continue experimentation as described in our accomplishment report above and as described in the original proposal.
Impacts
What was accomplished under these goals?
The continued goal of this project is to determine whether we can modify and optimize the accumulation of valuable proteins in genetically modified algae strains to increase the value of protein that will be generated as a co-product in algal biofuel systems. As mentioned in last year's report our primary target enzyme is phytase, a feed-additive that increases phosphorous digestibility. Furthermore we are investigating the influence of several specific DNA regulatory elements on protein accumulation. To begin this component of the work we developed strains using a cellulase degrading enzyme (Cel6A) that we had used previously with different regulatory elements in tobacco transformants. Using these strains, we developed a protein extraction protocol including a protein concentration step that resulted in detectable protein levels on an immunoprotein detection blot. We confirmed that one specific downstream box enhances the level of Cel6A expression. We designed experiments to examine the possible effect of nitrogen starvation on the level of Cel6A expression. We have in hand a full suite of chloroplast transformed Chlamydomonas reinhardtii cells containing phytase with five distinct regulatory elements in place (described in previous report) and have confirmed the proper homologous recombination by various sets of PCR. Preliminary protein analyses of two of these strains did not yield measurable phytase. We have planned experiments to measure the level of phytase expression for all five strains using the protein extraction and concentration protocol we have recently established. Also as mentioned in the last report, all of the above strains were developed using the cell-wall mutant line (CR 4349) as we ran into trouble with the wild type strain (C125). Because of the limited effectiveness of biolistic transformation with the wild type strains we established a collaboration with Chemical engineering Professor Hitomi Mikaibo, University of Rochester, who is working on a new technique for efficient chloroplast transformation using plasmid-coated-microneedles. With her method, the plasmid is directly injected into the cells, which according to preliminary data, has proven to be more efficient than the conventional biolistic bombardment. We have provided her lab with a highly concentrated cel6A-expressing plasmid to test the rates at which transformation can be effected and to compare them to those obtained with traditional methods. As part of a different project we are sequencing the full genome of a marine chlorophyte with excellent lipid accumulation. We have generated a nearly complete chloroplast genome sequence (currently on a handful of contigs) and it is available to determine homologous recombination sites for transformation in this organism once we complete our work with the model algae species.
Publications
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The laboratory work being undertaken on this project is largely being done by a post-doctoral researcher hired to work on this project. With respect to training activities, she has had to learn a new set of laboratory techniques to work with algae and has had to learn how to do chloroplast transformations. She has also been mentored to train undergraduate students in the laboratory and she has helped them to develop independent projects. With respect to professional development, she attended the 3rd ISCGGE Chloroplast Biology meeting at Rutgers University in May of 2013. 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?
The main goal of this project is to determine whether we can modify the accumulation of valuable proteins in genetically modified algae strains to increase the value of protein byproducts that will be generated in algal biofuel production systems. The animal feed-additive enzyme phytase is our target protein and we have begun by using a model algae species which is readily transformed but not particularly suited for biofuel production. We have also taken steps toward our longer term goal to develop a strategy to transform an algae species that can accumulate high concentrations of lipids. Our main strategy to optimize protein accumulation is find new components to build into expression cassettes for chloroplast transformation. While it was been shown that various native upstream and downstream DNA sequences (5’ and 3’ UTRs, promoters) can be used to flank coding sequences for effective protein production, success rates are highly variable—some proteins accumulate to high levels whereas others do not. During this report period, we identified two new candidates for use in the down-stream box (DB)-- a region of 10 to 15 codons immediately following the start codon in the coding region of the gene. Together with two DB that were effective in tobacco and the native DB of the codon optimized phytase gene, a total of five transformation vectors were constructed to transform the model algae Chlamydomonas reinhardtii. Transplastomic algae were generated via published methods in which gene fragments are physically delivered to cells, homologous recombination of the gene cassette occurs within the chloroplast and resistance is screened for to find transformants. Several rounds of transformation were attempted with the wild type strain (C125) and despite having worked in the past, no mutants were generated. A cell-wall mutant line (CR 4349) was obtained and resistant colonies were obtained for each transformation vector. In preparation for screening of each strain, we have optimized PCR conditions for successful amplification of DNA from insertion in chloroplast genome. We also worked toward developing an expression vector for transforming Chlorella vulgaris (C-27), an algae with better potential for biofuel production. We extracted and purified DNA from this algae and developed multiple sets of primers for PCR amplification of chloroplast genome fragments. Primer sequences were based on a published chloroplast sequence. We were successfully able to amplify and sequence fragments comprising the 5’UTR and 3’ UTR of the PsbA gene, but were unable to amplify any intergenic regions to be used as a site for homologous recombination into the chloroplast. We do not know if our failure was due to poor DNA quality (we have since improved extractions protocols) or due to poor sequence quality in the published data base. We will test our primers at least once more on better quality DNA. As part of a different project we have begun genome sequencing of a marine chlorophyte with excellent lipid accumulation. We may turn our efforts toward transformation of this species rather than continue working with the freshwater chlorpohyte.
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