NOTE: Project dates (POP) changed; now 12/1/2021-11/30/2023 per F. Hernandez/ARC (Ed., 1/19/22)

The goal of this proposal is to develop methods to preserve, grow, and measure production of desired synthetic biology products from edible yeast, using the BioSensor platform. BioSensor automates yeast culture activation and monitors growth with light absorbance of specific wavelengths produced by light emitting diodes (LED). The project goal is to expand the capability of BioSensor to enable monitoring synthetic biology traits along with cell growth and metabolism. The central objectives are: 1) Develop methods to predict synthetic biology production traits, namely carotenoids and recombinant proteins, using multivariate statistical models based on three wavelength light absorbance. We anticipate that the BioSensor platform will need to be modified to replace one of the current wavelengths to a blue LED; 2) Yeast may be overly sensitive to deep space radiation, and we will engineer carotenoid producing strains to express a DNA damage protection protein from tardigrades; 3) Non-conventional, yeast species may be more efficient for recombinant protein expression in deep space conditions. We will engineer three edible species to produce a target protein that absorbs blue light, to enable monitoring of recombinant protein and carotenoids in the same BioSensor device; and 4) Determine strain and media storage conditions as well as test the multiwavelength light monitoring strategy to establish the requirements and methodology for a future lunar surface mission.

The project is expected to advance the remote sensing technology in the BioSensor platform. A future flight experiment is expected to develop fundamental knowledge on the effects of deep space on protein expression and metabolite production. In addition, the project directly addresses critical gaps to advance crewed missions in deep space exploration. The specific carotenoids, beta-carotene and zeaxanthin, are important micronutrients to prevent macular degeneration and have been identified as potential countermeasures to maintain vision in astronauts during deep space missions. Recombinant protein expression in non-conventional yeast will demonstrate feasibility for production of bioactive protein therapies in deep space missions. These synthetic biology approaches are critical to provide products that are sensitive to radiation and have short shelf lives in prepackaged foods and medicines.

This project made significance progress on all four specific aims as follows.

Aim 1) Develop methods to predict synthetic biology production traits, namely carotenoids and recombinant proteins, using multivariate statistical models based on three wavelength light absorbance.

We determined that optical density measurements at 465 nm and 850 nm can be used to detect beta-carotene production in microtiter plate formats. Mixing experiments with non-producing and carotenoid producing yeast strains showed high accuracy calibrations of 465 nm and 850 nm absorbance to dry cell weight equivalents of beta-carotene production levels. We also engineered baker’s yeast to express Enhanced Green-Cyan Fluorescent Protein (ECGFP) and determined that this protein does not absorb sufficient 465 nm light to gauge engineered protein production. These experiments provided evidence that the spectroscopy approaches planned for carotenoid detection in lunar surface BioSensor experiments as part of the LEIA mission are feasible.

Aim 2) Engineer carotenoid-expressing yeast strains that enhance sensitivity or resistance to expected lunar surface environment stressors.

We generated twelve yeast strains that are expected to alter sensitivity to abiotic stresses such as reactive oxygen species (ROS) or radiation. Four strains were confirmed to be more sensitive to long-term storage of dry cells in ambient, laboratory conditions. Five of the yeast strains tested expression of Dsup or CAHS genes from tardigrades, which have been shown to enhance radiation or desiccation tolerance in plants, animals, or yeast.

3) Engineer a non-conventional yeast species to express a blue light compatible marker for synthetic biology-enabled production.

We developed three single-locus beta-carotene biosynthesis pathway constructs that were integrated into multiple yeast species, including three non-conventional yeast species and baker’s yeast. Although the non-conventional yeast species produced carotenoids, desiccation tolerance testing showed that these species were not suitable for the LEIA lunar surface mission. As an alternate approach, we engineered gene disruptions of the rgt2 and snf3 loci in carotenoid-producing baker’s yeast to generate a strain that emulates Crabtree-negative yeast metabolism found in non-conventional yeast species. Preliminary testing of this strain showed higher levels of carotenoid production.

4) Use BioSensor microfluidics cards to test inoculum, desiccation, and spectroscopy methods to measure cell growth and product formation.

Initial tests with BioSensor microfluidics cards in ground support equipment (GSE) showed feasibility to detect beta-carotene production using visible light absorbance at 465 nm and NIR absorbance at 850 nm. However, our primary beta-carotene producing strain was not biocompatible with the media planned for the LEIA lunar surface mission. We engineered additional baker’s yeast strains in the BioSentinel genetic background with BioSensor flight heritage. These new carotenoid producing strains were found to be biocompatible with the LEIA flight media and produced sufficient carotenoids to be measurable with the optical assay planned for the lunar surface mission.

NOTE: Project dates (POP) changed; now 12/1/2021-11/30/2023 per F. Hernandez/ARC (Ed., 1/19/22)

The goal of this proposal is to develop methods to preserve, grow, and measure production of desired synthetic biology products from edible yeast, using the BioSensor platform. BioSensor automates yeast culture activation and monitors growth with light absorbance of specific wavelengths produced by light emitting diodes (LED). The project goal is to expand the capability of BioSensor to enable monitoring synthetic biology traits along with cell growth and metabolism. The central objectives are: 1) Develop methods to predict synthetic biology production traits, namely carotenoids and recombinant proteins, using multivariate statistical models based on three wavelength light absorbance. We anticipate that the BioSensor platform will need to be modified to replace one of the current wavelengths to a blue LED; 2) Yeast may be overly sensitive to deep space radiation, and we will engineer carotenoid producing strains to express a DNA damage protection protein from tardigrades; 3) Non-conventional, yeast species may be more efficient for recombinant protein expression in deep space conditions. We will engineer three edible species to produce a target protein that absorbs blue light, to enable monitoring of recombinant protein and carotenoids in the same BioSensor device; and 4) Determine strain and media storage conditions as well as test the multiwavelength light monitoring strategy to establish the requirements and methodology for a future lunar surface mission.

The project is expected to advance the remote sensing technology in the BioSensor platform. A future flight experiment is expected to develop fundamental knowledge on the effects of deep space on protein expression and metabolite production. In addition, the project directly addresses critical gaps to advance crewed missions in deep space exploration. The specific carotenoids, beta-carotene and zeaxanthin, are important micronutrients to prevent macular degeneration and have been identified as potential countermeasures to maintain vision in astronauts during deep space missions. Recombinant protein expression in non-conventional yeast will demonstrate feasibility for production of bioactive protein therapies in deep space missions. These synthetic biology approaches are critical to provide products that are sensitive to radiation and have short shelf lives in prepackaged foods and medicines.

This project has made significance progress on all four specific aims as follows.

Aim 1) Develop methods to predict synthetic biology production traits, namely carotenoids and recombinant proteins, using multivariate statistical models based on three wavelength light absorbance.

We determined that optical density measurements at 465 nm and 850 nm can be used to detect beta-carotene production in microtiter plate formats. To measure protein content, we engineered baker’s yeast to express Enhanced Cyan-Green Fluorescent Protein (ECGFP). Although in year 1 of the project we found that florescence of ECGFP was predictive of ECGFP levels in stationary cultures, absorbance at 465 nm is not significant enough to detect ECGFP expression. These experiments provided evidence that the spectroscopy approaches planned for carotenoid detection in LEIA BioSensor experiments are feasible. Additional experiments with desiccated yeast strains are ongoing and will be used to calibrate 465 nm absorbance to beta-carotene production levels.

Aim 2) Engineer carotenoid-expressing yeast strains that enhance sensitivity or resistance to expected lunar surface environment stressors.

We generated twelve yeast strains that are expected to alter sensitivity to abiotic stresses, such as reactive oxygen species (ROS) or radiation. Four strains were confirmed to be more sensitive to long-term storage of dry cells in ambient, laboratory conditions. Five of the yeast strains tested expression of damage suppressor (Dsup) or cytoplasmic abundant heat soluble (CAHS) genes from tardigrades, which have been shown to enhance radiation or desiccation tolerance in plants, animals, or yeast.

3) Engineer a non-conventional yeast species to express a blue light compatible marker for synthetic biology-enabled production.

We developed a single-locus beta-carotene biosynthesis pathway construct that can be integrated into multiple yeast species. The first iteration of this construct produced low levels of beta-carotene in three baker’s yeast strains. We are currently revising the construct to improve beta-carotene production for eventual transfer into Crabtree-negative yeast.

4) Use BioSensor microfluidics cards to test inoculum, desiccation, and spectroscopy methods to measure cell growth and product formation.

We completed initial tests with BioSensor microfluidics cards in ground support equipment (GSE). These tests showed feasibility to detect beta-carotene production using visible light absorbance at 465 nm and Near-Infrared Spectroscopy (NIR) absorbance at 850 nm.

The goal of this proposal is to develop methods to preserve, grow, and measure production of desired synthetic biology products from edible yeast, using the BioSensor platform. BioSensor automates yeast culture activation and monitors growth with light absorbance of specific wavelengths produced by light emitting diodes (LED). The project goal is to expand the capability of BioSensor to enable monitoring synthetic biology traits along with cell growth and metabolism. The central objectives are: 1) Develop methods to predict synthetic biology production traits, namely carotenoids and recombinant proteins, using multivariate statistical models based on three wavelength light absorbance. We anticipate that the BioSensor platform will need to be modified to replace one of the current wavelengths to a blue LED; 2) Yeast may be overly sensitive to deep space radiation, and we will engineer carotenoid producing strains to express a DNA damage protection protein from tardigrades; 3) Non-conventional, yeast species may be more efficient for recombinant protein expression in deep space conditions. We will engineer three edible species to produce a target protein that absorbs blue light, to enable monitoring of recombinant protein and carotenoids in the same BioSensor device; and 4) Determine strain and media storage conditions as well as test the multiwavelength light monitoring strategy to establish the requirements and methodology for a future lunar surface mission.

The project is expected to advance the remote sensing technology in the BioSensor platform. A future flight experiment is expected to develop fundamental knowledge on the effects of deep space on protein expression and metabolite production. In addition, the project directly addresses critical gaps to advance crewed missions in deep space exploration. The specific carotenoids, beta-carotene and zeaxanthin, are important micronutrients to prevent macular degeneration and have been identified as potential countermeasures to maintain vision in astronauts during deep space missions. Recombinant protein expression in non-conventional yeast will demonstrate feasibility for production of bioactive protein therapies in deep space missions. These synthetic biology approaches are critical to provide products that are sensitive to radiation and have short shelf lives in prepackaged foods and medicines.

This project has made significance progress on all four specific aims as follows.

Aim 1) Develop methods to predict synthetic biology production traits, namely carotenoids and recombinant proteins, using multivariate statistical models based on three wavelength light absorbance. We determined that optical density measurements at 465 nm can be used to predict the relative level of carotenoids produced in stationary cultures. Mixing experiments with near isogenic wild-type and carotenoid-producing yeast strains were used to develop a linear regression model that can estimate relative carotenoid content to within 5% of maximum production level. To measure protein content, we engineered baker’s yeast to express Enhanced Green-Cyan Fluorescent Protein (ECGFP). Mixing experiments of stationary cultures showed a predictive correlation of relative ECGFP levels with fluorescence spectroscopy to within 0.5% of the maximum production level. These experiments provided evidence that the spectroscopy approaches planned for the BioSensor experiments are feasible. Additional experiments with desiccated yeast strains are ongoing and being used to define the biological parameters for BioSensor experiments and spectroscopy analysis strategies.

Aim 2) Engineer carotenoid-expressing yeast strains that enhance sensitivity or resistance to expected lunar surface environment stressors. We generated four yeast strains that are expected to increase sensitivity to reactive oxygen species (ROS) cellular damage. These strains were confirmed to be more sensitive to exogenous hydrogen peroxide, which is a toxic cellular by-product after exposure to high energy radiation. We also generated one strain that is mutated in the rad51 DNA damage repair gene. In addition, we generated a yeast strain expressing the Dsup gene from tardigrades. This gene is known to enhance radiation tolerance in plants and animals.

3) Engineer a non-conventional yeast species to express a blue light compatible marker for synthetic biology-enabled production. DNA constructs were developed to initiate this genetic engineering.

4) Use BioSensor microfluidics cards to test inoculum, desiccation, and spectroscopy methods to measure cell growth and product formation. The project team received training to work with BioSensor microfluidics cards in both plate readers. NASA Ames Research Center (Ames) engineers started development of ground support equipment to replicate the BioSensor optical measurements.

The goal of this proposal is to develop methods to preserve, grow, and measure production of desired synthetic biology products from edible yeast, using the BioSensor platform. BioSensor automates yeast culture activation and monitors growth with light absorbance of specific wavelengths produced by light emitting diodes (LED). The project goal is to expand the capability of BioSensor to enable monitoring synthetic biology traits along with cell growth and metabolism. The central objectives are: 1) Develop methods to predict synthetic biology production traits, namely carotenoids and recombinant proteins, using multivariate statistical models based on three wavelength light absorbance. We anticipate that the BioSensor platform will need to be modified to replace one of the current wavelengths to a blue LED; 2) Yeast may be overly sensitive to deep space radiation, and we will engineer carotenoid producing strains to express a DNA damage protection protein from tardigrades; 3) Non-conventional, yeast species may be more efficient for recombinant protein expression in deep space conditions. We will engineer three edible species to produce a target protein that absorbs blue light, to enable monitoring of recombinant protein and carotenoids in the same BioSensor device; and 4) Determine strain and media storage conditions as well as test the multiwavelength light monitoring strategy to establish the requirements and methodology for a future lunar surface mission.

The project is expected to advance the remote sensing technology in the BioSensor platform. A future flight experiment is expected to develop fundamental knowledge on the effects of deep space on protein expression and metabolite production. In addition, the project directly addresses critical gaps to advance crewed missions in deep space exploration. The specific carotenoids, beta-carotene and zeaxanthin, are important micronutrients to prevent macular degeneration and have been identified as potential countermeasures to maintain vision in astronauts during deep space missions. Recombinant protein expression in non-conventional yeast will demonstrate feasibility for production of bioactive protein therapies in deep space missions. These synthetic biology approaches are critical to provide products that are sensitive to radiation and have short shelf lives in prepackaged foods and medicines.