LOW COST APPROACH FOR PRODUCTION OF SWEET PROTEINS AS SUGAR SUBSTITUTES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: SMALL BUSINESS GRANT
• Reporting Frequency: Annual
• Accession No.: 1002828
• Grant No.: 2014-33610-21936
• Cumulative Award Amt.: $99,973.00
• Proposal No.: 2014-00575
• Project Start Date: Jun 1, 2014
• Project End Date: Jan 31, 2015
• Grant Year: 2014
• Program Code: [8.5]- Food Science & Nutrition

Recipient Organization
ABZYME THERAPEUTICS, LLC
271 GREAT VALLEY PKWY
MALVERN,PA 193551326

Performing Department
(N/A)

Non Technical Summary
In a large portion of the population the strong desire to eat sweet foods is a major contributing factor leading to overweightness and obesity, and the concomitant predisposition to diabetes diseases. Low calorie sweeteners like Saccharin, Aspartame, Cyclamate and Acesulfame K are popular with patients affected by diseases caused by the consumption of sugar, e.g. diabetes, hyperlipemia, caries, obesity etc. Unfortunately, these sweeteners are linked to serious side effects such as psychological problems, mental disorders, bladder cancer, heart failure and brain tumors. Naturally occurring sweet and taste modifying proteins that are a thousand times sweeter than sucrose have been identified in several exotic fruits and are seen as potential replacements for the currently available artificial low calorie sweeteners. Hence, the project goal is to develop a low cost approach to produce a sweet protein. During phase I, probiotic yeast will be engineered for high yield production of either secreted protein or protein displayed on the yeast cell surface that are accessible to taste receptors. The sweetening activity of purified sweet protein and yeast with surface displayed sweet protein will be validated by a sensory panel. This work will be the foundation for full product development (Phase II) and eventual commercialization that will be completed within a 2-year period. The unique value proposition of the product lies in low cost production of heat stable sweet proteins by probiotic microorganism to replace sugar in sugary foods and drinks. The products will have a highly beneficial impact on the reduction of carbohydrate consumption without compromising the sweet taste. Sweet protein food additives will have great potential application in soft drinks and the food industry.

Animal Health Component

50%

Research Effort Categories

Basic

20%

Applied

50%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
502	4020	1010	100%

Knowledge Area
502 - New and Improved Food Products;

Subject Of Investigation
4020 - Fungi;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

beverage

food

low calorie

sugar substitute

sweet proteins

thaumatin

yeast

Goals / Objectives
The major goal of this project is to develop an approach for low cost production of sweet proteins that can serve as sugar substitutes.To achieve that major goal, following objectives will be obtained:(Month 1) Gene encoding heat stable, pH-resistant sweet protein, Thaumatin, that is 100,000 times sweeter than sugar will be synthesized. Probiotic and nonpathogenic yeast Saccharomyces will be engineered for the use for Thaumatin expression and production.(Month 2) Thaumatin will be cloned and expressed under strong galactose-inducible promoter Gal1/10: Three Thaumatin expression constructs will be obtained: (i) Thaumatin is fused with mating factor secretory signal; (ii) Thaumatin is fused with mating factor secretory signal and SUMO chaperone. The latter is to enhance protein expression; (iii) Thaumatin is fused with mating factor secretory signal at the N-terminal and surface display anchor AGA2 in C-terminal, so Thaumatin will be displayed on yeast cell surface.(Month 3-5) Galactose-induced Thaumatin expression and secretion levels in the construct (i) and construct (ii) will be evaluated and compared. If the protein expression is high, Thaumatin will be purified.(Month 3) Yeast biomass with Thaumatin expressed and displayed on cell surface will be obtained by growing in media with galactose as a sole carbon source.(Month 5-6) The sweetening activity of purified sweet protein and yeast biomass with surface displayed sweet protein will be validated by a fruit fly Drosophila melanogaster sensory panel in comparison with other sweetners.(Month 6) The decision to use either secretion or display approach for Phase II sweet protein production will be made.

Project Methods
The main hurdle in the commercialization of sweetener protein to replace sugar is the development of a viable commercial manufacturing process.To produce sweet protein economically, we propose the following approaches/methods:1. To use probiotic microorganisms Saccharomyces as sweet protein producers and delivery systems.2. To use the proprietary SUMO protein expression system coupled with a yeast strong promoter (galactose-inducible Gal1/10 promoter) to enhance protein expression and secretion. The end product will be purified sweet protein3. To use our Surface Display System to produce yeast biomass with a high content of surface displayed sweetener protein accessible to taste receptors. We envision that the end product will consist of de-activated yeast powder with high sweet protein content displayed on cell surface accessible to taste receptors. Typically, each yeast cell displays 1 x 104 to 1x 105 copies of the AGA2-fusion protein.4. The sweetening activity of purified sweet protein and yeast with surface displayed sweet protein will be validated by a Drosophila melanogaster sensory panel compared with other sweetners.5. Selection of pure Thaumatin or Thaumatin display on yeast cell surface for the future development and commercialization is made based on the protein sweetening activity and yield/cost of protein production.

Progress 06/01/14 to 01/31/15

Outputs
Target Audience: Target Audience: Food industry segments where surgar and artificial sweetener are used. Efforts: Develop yeast strains to effectively express/produce or display sweet proteins Brazein and Thaumatin. 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? Cargill company (food and Nutrition company) has sent us a letter requesting discussion for potential collaboration. We are intended to discuss with them once Confidential agreement is signed. While it is final report and the phase I ended, as both of our company and Cargill are interested in this product, we plan to further optimize the yeast display system to make sweet protein accessible to the taste receptors. 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 major goal of this project isto develop an approach for low cost production of sweet proteins that can serve as sugar substitutes. In the process to achieve this major goal, following objectives have been obtained: Gene encoding heat stable, pH-resistant sweet protein, Thaumatin and Brazein have been synthesized. Yeast strain defective in proteases in pep4 and prb1 have been obtained. Protease-defficient yeast strain with Surface display capacity has been obtained. Genes encoding sweet proteins Brazein and Thaumatin have been cloned into yeast expression vectors under Gal1/10 galactose-inducible promoters for (i) expression and secretion from yeast cells into media and (ii) to display on a cell surface. Expression of sweet proteins have been characterized. The expression data showed that the yield of sweet protein is low and economically, it will be viable to produce purified proteins as surgar substitution. The display of sweet protein on cell surface has been characterized by Cytoflow analysis - fluorescence activation cell sorting or FACS. The FACS data show that the sweet proteins effectively display on yeast cells. Biomass of yeast strains with efficiently displayed sweet proteins have been produced. The sweetening activity of sweet yeast have been tested by taste-testing by PI and one another volunteer. Both have made a conclusion that yeast was not sweet. One potential explanation is that the linker between AGA2 (membrane anchor) and Sweet Protein was too short and the sweet protein was not accessible to taste receptors. New constructs with longer linkers between AGA2 and sweet proteins have been obtained. Constructs have been introduced into engineered yeast strains for surface display. Biomass cultured in galactose media to induce protein expression has been undergone tase-testing. Initial taste-testing by two indendent testers concludes that the yeast taste creamy and a little sweet. Heating yeast for 1 hours at 95oC eliminated that taste. We concluded that it is possible to optimize the yeast strains (testing various length of linkers) to make the sweet protein more accessible to taste receptors or test various methods to release sweet proteins make it more accessible to taste receptors.

Publications

Progress 06/01/14 to 01/31/15

Outputs
Target Audience: Target audiences: The target audiences of this project is the food industry, where surgar and artificial sweeteners are used. Effort: In this project, the main effort is to develop the yeast based approach to produce sweet protein Thaumatin and/or Brazein at low cost. 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? Cargill company (Food and Nutrition company) has contacted us requesting for discussion about potential collaboration. We are planning to discuss with Cargill after signing confidential agreement. What do you plan to do during the next reporting period to accomplish the goals? We would expect to have yeast strain which can display functional sweet proteins accessible to taste receptors.

Impacts
What was accomplished under these goals? Gene encoding heat stable, pH-resistant sweet protein, Thaumatin and Brazein that are 3,000 - 100,000 times sweeter than sugar have been synthesized. Yeast Saccharomyces yeast strains for expression of these sweet proteins have been obtained. Synthesized genes encoding Brazein and Thaumatin have been cloned and expressed under strong galactose-inducible promoter Gal1/10: Three sets of constructs for expressing sweet proteins have been obtained: (i) Protein is fused with mating factor secretory signal; (ii) Protein is fused with mating factor secretory signal and SUMO chaperone; (iii) Protein is fused with mating factor secretory signal at the N-terminal and surface display anchor AGA2 in C-terminal, so Thaumatin or Brazein is displayed on yeast cell surface. In all constructs, FLAG epitope tag was added to the C-terminal to facilitate protein detection. For expression constructs (i) and (ii) - in addition to FLAG tag, 6 xHistidine tags also have been added. Constructs have been introduced into yeast strains for expression and surface display by yeast transformation to Trp+. No protein secretion was observed with Sumo-fusion constructs. For constructs where protein is fused with the secretory signal, the level of protein expression in the supernatant (media) was not visible in SDS-PAGE gels, but could detected by Western blotting. All this indicated that the expression of sweet protein was low. We decided that production of purified sweet protein as sugar substitution would be not economically viable due to low protein yield. Thefore we have focused our effort on the option 2 - displaying sweet proteins on yeast cell surface and using total yeast biomass as sweet protein delivery. The constructs secretory signal-Sweet protein (Brazein or Thaumatin) -AGA2 (Surface display anchor) -FLAG tag (Tag for protein detection) have been introduced in to specially engineered yeast strains where AGA1 (partner of AGA2) in under Gal1/10 promoters. Display of sweet proteins have been induced in galactose media and the display efficiency has been avaluated by cytoflow analysis using anti-FLAG antibodies (FACS). FACS data showed that 40% to 80% yeast population displayed sweet protein on yeast cell surface. We did not use thefruit flyDrosophila melanogastersensory panel for testing as we decided not to pursue purification of sweet proteins due to low protein expression yield and economically not viable. As mentioned above, we pursued using yeast surface display approach for sweet protein production. We have grown yeast strains displaying sweet proteins on cell surface in a large scale and produced yeast biomass has been tested for sweet taste by PI and one volunteer. Both of us came to conclusion that yeast did not tasted sweet. There are two possible explanations why yeast displaying sweet proteins on cell surface did not taste sweet. (i) The first explanation is that sweet protein expressed in yeast maybe have been heavily glycosylated. Western blotting data confirmed that notion. How glycosylation can effect the sweet protein function is remain open question. We are in process to obtain yeast strain defective in glycosylation (OCH1 mutation) to reduce glycosylation. (ii) the second possible explanation is that the arm (linker between AGA2 and sweet protein) is not long enough, therefore the sweet protein is not accessible to taste receptors. We extended the linkers by inserting human IgG1 CH1 domain (130 aa) between AGA2 and sweet proteins. Yeast with new constructs (long arm) tasted creamy and a little sweet. Heating yeast at 95oC for 1 hours resulted in a loss of taste. The next step is to optimize the display constructs or test different methods to release the displayed proteins making them accessible to taste receptors.

Publications