SEED-BASED LIPASE AND PHOSPHOLIPASE PRODUCTION FOR ENZYMATIC BIODIESEL

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: SMALL BUSINESS GRANT
• Reporting Frequency: Annual
• Accession No.: 1009477
• Grant No.: 2016-33610-25495
• Cumulative Award Amt.: $100,000.00
• Proposal No.: 2016-01176
• Project Start Date: Aug 1, 2016
• Project End Date: Mar 31, 2018
• Grant Year: 2016
• Program Code: [8.8]- Biofuels and Biobased Products

Recipient Organization
Infinite Energy, LLC dba Infinite Enzymes
826 Sherwood Oaks Lane
Jonesboro,AR 72404

Performing Department
(N/A)

Non Technical Summary
The manufacture of large volumes of inexpensive biological tools (enzymes) for the production of biodiesel provides a market opportunity for the plant production system. Biodiesel predominantly is commercially manufactured through treatment of oil or fat with alcohols and chemical catalysts such as sodium hydroxide. Although this method works, it can be commercially profitable only when the used oils are inexpensive, such as those from restaurants, and contain few contaminants. However, used cooking oils are high in contaminants and these compounds must be removed prior to making biodiesel. Using enzymes, our biological tools, for manufacture of biodiesel resolves these contamination issues, increasing yield and lowering unit operations. Lipase and phospholipase are the preferred biodiesel synthetic enzymes and are too expensive today for widespread use. We hypothesize that lipase and phospholipase can be expressed at high enough levels in the corn kernel bio-production system to make them cost-effective for biodiesel manufacture. The Phase I objective is to determine the potential success of maize manufacture of the enzymes through transient expression of their genes in maize kernel tissues. Facilitating biodiesel manufacture using enzymes will promote better quality biofuel and lower environmental impact of the process.

Animal Health Component

90%

Research Effort Categories

Basic

(N/A)

Applied

90%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
511	1510	1040	80%
511	2499	1030	20%

Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
1510 - Corn; 2499 - Plant research, general;

Field Of Science
1040 - Molecular biology; 1030 - Cellular biology;

Keywords

biodiesel

lipase

maize

phosphlipase

recombinant

Goals / Objectives
The manufacture of large volumes of inexpensive enzymes for the production of biodiesel provides a market opportunity for the plant production system. Biodiesel predominantly is commercially manufactured through treatment of feedstock oil or fat with alcohols and chemical catalysts such as sodium hydroxide. Although this method works, it can be commercially profitable only when the feedstock oils are inexpensive, less than 2% free fatty acids (FFA), very low in phospholipids. Used cooking oils are high in FFA and phospholipids and these compounds must be removed prior to making biodiesel. Using enzymes for manufacture of biodiesel resolves these contamination issues,increasing yield and lowering unit operations. Lipase and phospholipase are the preferred biodiesel synthetic enzymes and are too expensive today for widespread use. We hypothesize that lipase and phospholipase can be expressed at high enough levels in the corn bio-production system to make them cost-effective for enzymatic biodiesel manufacture. The Phase I objective is to determine the potential success of maize expression of the enzymes through transient expression of their genes in maize tissues. The specific tasks to accomplish this objective are: 1) Synthesize enzyme genes with the first 40 amino acid codons optimized for maize. 2) Target each gene to embryo and endosperm,and to 3 subcellular locations. 3) Do transient expression in maize tissues to choose constructs for stably transforming into corn. 4) Develop assays for each of the enzymes. Facilitating biodiesel manufacture using enzymes will promote better quality biofuel and lower environmental impact of the process.

Project Methods
Task 1. Synthesize the lipase and phospholipase genes with the first 40 amino acid codons optimized for maize. Experimental: The genes of choice in this instance will be synthesized because growing the organisms for PCR is impractical and optimizing codons for maize is desired. The barley alpha amylase signal sequence (BAASS 25 aa) or the barley aleurain sequence will be substituted for the native signal sequence in each case for localization. Each of these signal sequences will be added using PCR after the gene is synthesized and cloned. The first fifteen codons of the gene will be optimized because we have shown that having 40 optimized codons is sufficient for high translatability, and with the signal sequence, 15 from the gene will be sufficient. We have used GenScript and Integrated DNA Technologies for oligo and gene synthesis. We will proceed with the company providing the best price. Each gene will be flanked with 5' NcoI and 3'PacI restriction sites to allow us to clone it downstream of the appropriate promoter.Task 2. Target each gene to 2 tissues--embryo and endosperm, and to 3 subcellular locations. Experimental: Each gene will be cloned downstream of the globulin-1 extended promoter for embryo expression and the a-zein promoter for endosperm expression. In addition, each tissue will have targeting sequences for the protein that localize it to the ER, the vacuole or the apoplast. The BAASS will target the protein to the apoplast (cell wall) by default. This same construct with a 3' KDEL sequence will retain the protein in the ER. For vacuolar expression, the barley aleurain sequence will be appended to the 5' end of the synthetic gene. Standard cloning vectors for our expression system are available in our laboratory (Egelkrout et al., 2013). These vectors already contain the selectable marker, phosphinothricin acetyltransferase (ppt) driven by the CaMV 35S promoter and the left and right border sequences for Agrobacterium tumefaciens transformation. The entire expression cassette will be built within this approximately 10 kb plasmid, then moved into A. tumefaciens LBA4404 to form the cointegrate with the super binary (SB), then the cointegrated SB will be isolated and electroporated into A. tumefaciens EHA101.Task 3. Do transient expression in maize embryo and endosperm samples, then stable corn transformation. Experimental: We maintain Hi II corn plants (Armstrong et al., 1991) in the greenhouse that have been planted 2X per week. These staged plants are self-pollinated and ears harvested for embryo isolation at 15 days after pollination. At that time the embryos will be approximately 4 mm in length. Although this is too large for stable transformation, it is an excellent size for transient expression of the proteins of interest. A. tumefaciens EHA101 with the constructs of interest are added to the embryos, sonicated for 30 seconds, and co-cultivated on solid MS medium for 5 days at 18oC. For transient endosperm expression, the endosperm must be past the liquid stage so that it will have structure for gene expression outcomes. Thus ears will be harvested at 17-20 days post pollination and endosperm pulled away from the embryos. This tissue will be co-cultivated with appropriate A. tumefaciens strains and sonicated as above for the embryos. Tissue will be harvested and assayed at 4 days post infection. Embryo or endosperm samples are homogenized in buffer (as in Task 4) and analyzed for enzyme activity.Task 4. Develop assays for each of the enzymes. Experimental: Extraction of embryo and endosperm tissues from the transient assays will be done with our usual buffer--50 mM sodium acetate, pH 5. However, because of the biphasic nature of lipases, we may require different buffers to solubilize the enzyme, ones including a dilute surfactant or organic solvent. We will test the following assays for lipase and phospholipase. A kit is available from Sigma Chemical Co. for analyzing lipase activity (MAK046), but its specificity for the Rhizomucor and Bacillus enzymes is unknown. A second method we will test will detect either enzyme and is also colorimetric (Rawyler and Siegenthaler, 1980). Commercial enzyme will be purchased that is from these or closely related organisms and the assays tested with pure enzyme and enzyme spiked into corn flour extract. We will also test antibodies from various sources to analyze their cross-reactivity with the chosen enzymes.

Progress 08/01/16 to 03/31/18

Outputs
Target Audience: Enzymatic production of biodiesel is the process of choice because of all the benefits: excellent quality biodiesel, ability to utilize free fatty acids and phospholipids in the mix, ambient conditions for reactions, less water and methanol, so fewer impacts on the environment. These are technical, social and environmental benefits. The current market for lipases is approximately $40 million and is expected to rise to $60 million by 2017 (BBC Research, Global Markets and Technologies for Biofuel Enzymes). The current cost of biodiesel utilizing enzymes is not feasible because of the high cost of enzymes. However, based on modeling studies (Howard et al., 2011) it appears that plant-made enzymes can be highly cost-competitive with fermentation-derived enzymes without considering the added cost of infrastructure. Thus, the investment in product creation through SBIR funding is highly beneficial. TransBiodiesel, Inc., is an Israeli company that produces enzymatic biodiesel and buys their enzymes on the open market. They immobilize the enzymes using their proprietary technology allowing the enzymes to last for up to a year, assisting with cost-efficiency. Nevertheless, low cost of the enzymes is a critical consideration because feedstock cost is usually high and any other savings contributes to better margins. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We hired an undergraduate student from Arkansas State University College of Agriculture and trained him in bacterial work, enzyme assays, and plant transformation. Although he is gone over the summer months, he will be returning to help complete this project in the fall. 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?As stated above, with the field season looming, we applied for and received a no-cost extension for the project which will allow us to finish the transient assays and the analysis in the fall in preparation for a Phase II submission.

Impacts
What was accomplished under these goals? Twelve constructs have been generated, 6 for lipase and 6 for phospholipase. Each of the constructs has been moved into Agrobacterium tumefaciens strain EHA101 and archived. Six of them have been used so far in transient transformation experiments and are frozen pending the development of the assay. One vector with the phospholipase gene has been transformed stably into corn with recovery of a number of lines. With the field season looming, we applied for and received a no-cost extension for the project which will allow us to finish the transient assays and the analysis in the fall in preparation for a Phase II submission.

Publications

Progress 08/01/16 to 07/31/17

Outputs
Target Audience:The target audience for this project is biodiesel manufacturers. Although many manufacturers are producing biodiesel, the vast majority are using chemical catalysts rather than enzymes because of the high cost of the enzymes. Our goal is to produce low cost enzymes so that biodiesel can be manufactured without chemical catalysts and with higher yields. Changes/Problems:We have had some challenges moving these plasmids into Agrobacterium, but the methods are fine and recent results show trouble shooting has yielded positive progress on this aspect of the project. We got a very late start on the work because of delays in obtaining funding. What opportunities for training and professional development has the project provided?The PI, Kendall Hood, is being trained in molecular biology and Agrobacterial techniques. His previous expertise is in protein purification and he is now becoming proficient in these new technologies. Further training has begun on transient transformation technologies. 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 plan to finish moving the constructed expression plasmids into A. tumefaciens and do transient expression in maize embryos (pr25 promoter constructs) or endosperm (pr37 promoter constructs). The resulting plant material will be analyzed for protein accumulation and activity. Assay kits are on order. The best constructs for each enzyme will be chosen for stable transformations in Phase II.

Impacts
What was accomplished under these goals? Synthesize lipase and phospholipase genes with the first 40 amino acid codons optimized for maize. Target each gene to 2 tissues--using embryo and endosperm promoters, and to 3 subcellular locations, the vacuole, the endoplasmic reticulum, and the apoplast. Expression plasmids from each of the two genes were constructed based on vector backbones from previous expression studies (Hood et al., 2007). These vectors contain a spectinomycin resistance gene, an E. coli origin of replication, a cos site for integration in Agrobacterium tumefaciens, a plant herbicide selection marker and the left and right borders of T-DNA. The following two genes were synthesized with partial codon-optimization for expression in the maize system. The details of the gene expression constructs are listed in Table 1. 1) Rhizomucor miehei lipase construct analysis and details (Boel et al., 1988): -Lipase sequence same as Genbank accession A34959 (from Rhizomucor miehei) without the signal sequence -5'UTR start in pr25 defined in Genbank accession L22344.1 (based on primer extension of globulin-1 promoter/pr2) -5'UTR start in pr37 defined in Genbank accession X05911 (maize 19 kDa alpha-zein promoter) 2) Bacillus cereus Phospholipase C construct analysis and details (Tan et al., 1997): Bacilllus cereus PLC (Genbank NP_830483) -5'UTR start in pr25 defined in Genbank accession L22344.1 (based on primer extension of globulin-1 promoter/pr2) -5'UTR start in pr37 defined in Genbank accession X05911 (maize 19 kDa alpha-zein promoter) Table 1: Vectors planned and constructed for expression of lipase and phospholipase in maize transient assays. Allhave been delivered to Infinite Enzymes' labs in Arkansas from our collaborator, Applied Biotechnology Institute (ABI) in California. Pr37=the maize 19 kDa alpha zein promoter for endosperm expression and pr25=the maize extended globulin-1 promoter for embryo expression. Rmlipase = Rhizomucor miehei lipase; phlipase C = Bacillus cereus phospholipase C; BAASS = Barley alpha amylase signal sequence; KDEL = endosplamic reticulum retention sequence; vac = vacuolar targeting sequence from barley. The structure of this Table is not stable when converting to PDF. Construct Gene Clone Enzymes checked In E. coli In LBA4404 In EHA101 LIA pr37 BAASS rmlipase Done AgeI, NcoI Y Confirming LIB pr37 BAASS rmlipase KDEL Done AgeI+NcoI, and NcoI+PacI Y Confirming LIC pr37 vac rmlipase Done NcoI+PacI, SacII Y Confirming LID pr25 BAASS rmlipase Done NcoI, PacI, SacII Y Confirming LIE pr25 BAASS rmlipase KDEL Done NcoI+PacI, AgeI+PacI Y Done yes LIF pr25 vac rmlipase Done AgeI+NcoI, NcoI+PacI Y Done yes PLA pr37 BAASS phlipase C Done AgeI, NcoI, NdeI Y Done yes PLB pr37 BAASS phlipase C KDEL Done AgeI+NcoI, NdeI Y Confirming PLC pr37 vac phlipase C Done AgeI+NcoI, NdeI Y Confirming PLD pr25 BAASS phlipase C Done NcoI, PacI, HindIII, NheI Y Confirming PLE pr25 BAASS phlipase C KDEL NcoI+PacI, HindIII, NheI Y Confirming PLF pr25 vac phlipase C NcoI+PacI, HindIII, NheI Y Confirming As IE receives each of these DNAs from ABI, we immediately electroporate the DNA into DH5-a for archiving at -80oC. Each of the plasmids is then put into A. tumefaciens LBA4404 (pSB1), a strain licensed from Japan Tobacco, Inc. DNA is mobilized into LBA through either electroporation of competent cells or through a triparental mating. We are in the process of doing each of these to ensure prompt recovery of the LBA strain with a cointegrated pSB1 and the construct of interest from LIA to PLF. Strains are selected on a combination of spectinomycin (on the vectors from ABI) and rifampicin (resistance exhibited by the At bacterial chromosome). These cointegrates are being confirmed by DNA minipreps from the LBA colonies and the strains will be archived. The cointegrated plasmid will be electroporated into EHA101 for transformation of maize. We are training a new team member in microbiology of A. tumefaciens and have gotten off to a slow start with moving the plasmids into the agro strains. We have successfully prepared competent cells of E. coli and A. tumefaciens, but the selections of transformed A. tumefaciens have not revealed a positive outcome to date. We will continue to work closely with the new trainee to ensure a successful outcome. Dr. Elizabeth Hood, the co-PI and CEO of the company has many years of experience in this area and is taking an active role in moving this part of the project ahead. Do transient expression in maize embryo and endosperm samples to choose constructs for stably transforming into corn. Hi-II corn plants are being grown in the A-State greenhouse to have staged embryos and endosperm for transient assays. Embryos at 13 days after pollination (DAP) and endosperm at 18-20 DAP will be used to assess the activity of the enzymes from the constructs. Approximately 2 months are required to recover 10-20 DAP ears for harvest of embryos and endosperms for transient transformation experiments (Vicuna Requesens et al., 2010). We anticipate beginning these transformations within the next month and have staged Hi II corn plants growing to provide explants accordingly. Develop assays for each of the enzymes. We are in the process of determining the appropriate assays for each of these enzymes. We have determined previously that embryos at 5 days after co-cultivation and endosperm at 4 days after co-cultivation are ready for harvest and analysis. Tissue can be frozen until a later date if necessary to coordinate analyses.

Publications

Progress 08/01/16 to 03/31/17

Outputs
Target Audience:The target audience for this project is biodiesel manufacturers. Although many manufacturers are producing biodiesel, the vast majority are using chemical catalysts rather than enzymes because of the high cost of the enzymes. Our goal is to produce low cost enzymes so that biodiesel can be manufactured without chemical catalysts and with higher yields. Changes/Problems:We have had some challenges moving these plasmids into Agrobacterium, but the methods are fine and recent results show trouble shooting has yielded positive progress on this aspect of the project. We got a very late start on the work because of delays in obtaining funding. What opportunities for training and professional development has the project provided?The PI, Kendall Hood, is being trained in molecular biology and Agrobacterial techniques. His previous expertise is in protein purification and he is now becoming proficient in these new technologies. Further training has begun on transient transformation technologies. 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 plan to finish moving the constructed expression plasmids into A. tumefaciens and do transient expression in maize embryos (pr25 promoter constructs) or endosperm (pr37 promoter constructs). The resulting plant material will be analyzed for protein accumulation and activity. Assay kits are on order. The best constructs for each enzyme will be chosen for stable transformations in Phase II.

Impacts
What was accomplished under these goals? Synthesize lipase and phospholipase genes with the first 40 amino acid codons optimized for maize. Target each gene to 2 tissues--using embryo and endosperm promoters, and to 3 subcellular locations, the vacuole, the endoplasmic reticulum, and the apoplast. Expression plasmids from each of the two genes were constructed based on vector backbones from previous expression studies (Hood et al., 2007). These vectors contain a spectinomycin resistance gene, an E. coli origin of replication, a cos site for integration in Agrobacterium tumefaciens, a plant herbicide selection marker and the left and right borders of T-DNA. The following two genes were synthesized with partial codon-optimization for expression in the maize system. The details of the gene expression constructs are listed in Table 1. 1) Rhizomucor miehei lipase construct analysis and details (Boel et al., 1988): -Lipase sequence same as Genbank accession A34959 (from Rhizomucor miehei) without the signal sequence -5'UTR start in pr25 defined in Genbank accession L22344.1 (based on primer extension of globulin-1 promoter/pr2) -5'UTR start in pr37 defined in Genbank accession X05911 (maize 19 kDa alpha-zein promoter) 2) Bacillus cereus Phospholipase C construct analysis and details (Tan et al., 1997): Bacilllus cereus PLC (Genbank NP_830483) -5'UTR start in pr25 defined in Genbank accession L22344.1 (based on primer extension of globulin-1 promoter/pr2) -5'UTR start in pr37 defined in Genbank accession X05911 (maize 19 kDa alpha-zein promoter) Table 1: Vectors planned and constructed for expression of lipase and phospholipase in maize transient assays. Allhave been delivered to Infinite Enzymes' labs in Arkansas from our collaborator, Applied Biotechnology Institute (ABI) in California. Pr37=the maize 19 kDa alpha zein promoter for endosperm expression and pr25=the maize extended globulin-1 promoter for embryo expression. Rmlipase = Rhizomucor miehei lipase; phlipase C = Bacillus cereus phospholipase C; BAASS = Barley alpha amylase signal sequence; KDEL = endosplamic reticulum retention sequence; vac = vacuolar targeting sequence from barley. The structure of this Table is not stable when converting to PDF. Construct Gene Clone Enzymes checked In E. coli In LBA4404 In EHA101 LIA pr37 BAASS rmlipase Done AgeI, NcoI Y Confirming LIB pr37 BAASS rmlipase KDEL Done AgeI+NcoI, and NcoI+PacI Y Confirming LIC pr37 vac rmlipase Done NcoI+PacI, SacII Y Confirming LID pr25 BAASS rmlipase Done NcoI, PacI, SacII Y Confirming LIE pr25 BAASS rmlipase KDEL Done NcoI+PacI, AgeI+PacI Y Done yes LIF pr25 vac rmlipase Done AgeI+NcoI, NcoI+PacI Y Done yes PLA pr37 BAASS phlipase C Done AgeI, NcoI, NdeI Y Done yes PLB pr37 BAASS phlipase C KDEL Done AgeI+NcoI, NdeI Y Confirming PLC pr37 vac phlipase C Done AgeI+NcoI, NdeI Y Confirming PLD pr25 BAASS phlipase C Done NcoI, PacI, HindIII, NheI Y Confirming PLE pr25 BAASS phlipase C KDEL NcoI+PacI, HindIII, NheI Y Confirming PLF pr25 vac phlipase C NcoI+PacI, HindIII, NheI Y Confirming As IE receives each of these DNAs from ABI, we immediately electroporate the DNA into DH5-a for archiving at -80oC. Each of the plasmids is then put into A. tumefaciens LBA4404 (pSB1), a strain licensed from Japan Tobacco, Inc. DNA is mobilized into LBA through either electroporation of competent cells or through a triparental mating. We are in the process of doing each of these to ensure prompt recovery of the LBA strain with a cointegrated pSB1 and the construct of interest from LIA to PLF. Strains are selected on a combination of spectinomycin (on the vectors from ABI) and rifampicin (resistance exhibited by the At bacterial chromosome). These cointegrates are being confirmed by DNA minipreps from the LBA colonies and the strains will be archived. The cointegrated plasmid will be electroporated into EHA101 for transformation of maize. We are training a new team member in microbiology of A. tumefaciens and have gotten off to a slow start with moving the plasmids into the agro strains. We have successfully prepared competent cells of E. coli and A. tumefaciens, but the selections of transformed A. tumefaciens have not revealed a positive outcome to date. We will continue to work closely with the new trainee to ensure a successful outcome. Dr. Elizabeth Hood, the co-PI and CEO of the company has many years of experience in this area and is taking an active role in moving this part of the project ahead. Do transient expression in maize embryo and endosperm samples to choose constructs for stably transforming into corn. Hi-II corn plants are being grown in the A-State greenhouse to have staged embryos and endosperm for transient assays. Embryos at 13 days after pollination (DAP) and endosperm at 18-20 DAP will be used to assess the activity of the enzymes from the constructs. Approximately 2 months are required to recover 10-20 DAP ears for harvest of embryos and endosperms for transient transformation experiments (Vicuna Requesens et al., 2010). We anticipate beginning these transformations within the next month and have staged Hi II corn plants growing to provide explants accordingly. Develop assays for each of the enzymes. We are in the process of determining the appropriate assays for each of these enzymes. We have determined previously that embryos at 5 days after co-cultivation and endosperm at 4 days after co-cultivation are ready for harvest and analysis. Tissue can be frozen until a later date if necessary to coordinate analyses.

Publications