Progress 07/01/19 to 03/31/23
Outputs
Target Audience:Scientists, engineers, industries who are interested in agricultural fiber production, microbial fermentation, etc. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Through this project, I have trained four graduate students (Jingyao Li, Juya Jeon, Alden Filko, and Xinyuan Chang) and two technicians (John Jaeger and Han Yu). These training activities have increased their knowledge and skills in protein engineering, microbial genetic engineering, metabolic engineering, as well as synthetic biology. How have the results been disseminated to communities of interest?The PI has been invited to present results from this project at national conferences and department seminars, these activities include: Synthetic Biology for High-Performance Protein-Based Materials Invited seminar, The Henry L. Pierce Seminar Series, MIT, Mar 2023. 2. Understanding and Engineering Metabolic Heterogeneity for Enhanced Bioproduction Invited seminar, Center for Synthetic and Systems Biology, University of Edinburgh, Edinburgh, UK, Sept 2022 3. Synthetic Biology for High-Performance Materials Invited seminar, Guangdong Technion-Israel Institute of Technology, Virtual, May 2022 4. Using Synthetic Biology to Produce High-Performance Biopolymers Keynote Speaker, ACS Rubber Division Spring Meeting, Cleveland, OH, Apr, 2022 5. Understanding and Engineering Metabolic Heterogeneity for Enhanced Bioproduction Invited seminar, School of Biological and Health Systems Engineering, Arizona State University, Feb 2022 6. Synthetic Biology for Protein-based High-performance Materials Invited talk, Amy Collaborative Center for Biological Engineering, Virtual, Jul 2020. 7. Engineering Bacteria Phospholipid Pathways for Diverse Fatty Acid Profiles. Invited talk, AIChE 15C Bioengineering Division, Virtual, Nov 2020. 8. Synthetic Biology for Advanced Chemicals and Materials. Invited seminar, Tsinghua Forum on Industrial Biocatalysis (one of only two invited speakers), Tsinghua University, May, 2020 9. Synthetic Biology for Structurally-defined Chemicals and Protein-based Materials. Invited seminar, Department of Chemical and Biomolecular Engineering, Rice University, Feb, 2020 10. Understanding and controlling microbial metabolite dynamics and heterogeneity, Invited seminar, Program of Bioinformatics and Computational Biology, Saint Louis University, Oct, 2019. Other conference presentations presented by the PI include: Engineering Protein-based Materials via Synthetic biology Protein Society Meeting, San Francisco, Jul 2022 2. Microbial production of tough artificial muscle fibers via synthetic biology 2022 ACS Annual Spring Meeting, San Diego, Mar 2022 3. Microbial production of renewable fibers stronger and tougher than spider silk 2022 ACS Annual Spring Meeting, San Diego, Mar 2022 4. Synthetic biology for High-performance protein-based materials 2020 ACS Annual Meeting, BioT, Virtual, Aug, 2020 Additionally, trainees have been given presentations at national conferences: Li J., and Zhang F., Microbially Synthesized Polymeric Amyloid Fiber Promotes β-Nanocrystal Formation and Displays Gigapascal Tensile Strength. Oral Presentation, AIChE Annual Meeting, Phoenix, AZ, Nov 2022. CJ Sargent, CH Bowen, A Wang, Y Zhu, X Chang, J Li, X Mu, JM Galazka, YS Jun, S Keten, F Zhang. Synthetic biology enables the microbial polymerization of titin proteins and production of fibers with exceptional mechanical properties. Oral. Central Synthetic Biology Workshop, Chicago, IL. Sep 9. 2021 CJ Sargent, CH Bowen, A Wang, Y Zhu, X Chang, J Li, X Mu, JM Galazka, YS Jun, S Keten, F Zhang.Microbial synthesis of megadalton titin protein enables the production of fibers with exceptional mechanical properties and a unique molecular structure. Poster. Biochemistry, Biophysics & Structural Biology Program Retreat, St. Louis, MO. Oct 29, 2021 Li J., F Zhang. Macroscopic fibers made of polymeric amyloid proteins display gigapascal tensile strength. Poster. Central Synthetic Biology Workshop, Chicago, IL, Sept 9 2021. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
IMPACTS: Through this project, we have developed novel and efficient processes to convert agricultural waste biomass into high-performance silk fibers using engineered microbes. Current agricultural production of fibers such as plant-derived cotton, hemp, flax and animal-derived silk and wool requires vast amounts of arable land, water, fertilizers, herbicides, pesticides, facilities, and human resources. The laborious production process, low profit margins, and environmental concerns related to traditional agricultural fiber production have demanded the development of new processes to convert abundant and otherwise wasted agricultural biomass (e.g. corn stover, wheat straw, etc.) into such fibers. In this project, we have developed simple microbial processes that produced novel engineered silk proteins in high titers and yields from microbial fermentation. These proteins were then processed to fibers that displayed a combination of high tensile strength and high toughness for a wide range of applications (e.g. textile). These fibers can be used to replace current fibers derived from petroleum or produced using complicated agricultural processes. OUTCOMES: Accomplishment #1. Engineer strength-enhanced spider-silk-amyloid hybrid fibers. We hypothesized that engineering the amino acid sequence of spider silk protein can create new silk fibers with higher strength and toughness than those of natural spider silk fibers. To test this hypothesis, we synthesized multiple spider-silk-amyloid hybrid proteins where the polyaniline sequences of natural spider silk protein were replaced by amyloid peptides that form β-sheet crystals more readily. Using amyloid peptides from different structural groups, we created silk-amyloid hybrid proteins using engineered microbes and wet-spun into macroscopic fibers. Structural analyses using synchrotron-based wide-angle X-ray diffraction (WAXD) unveil the presence of β-nanocrystals in all silk-amyloid fibers. These β-nanocrystals resemble the cross-β structure of amyloid nanofibrils and have sizes greater than those observed in recombinant spider silk fibers. Further crystallinity of silk-amyloid fibers ranges from 15% to 20%, drastically higher than that of recombinant spider silk (4.2%). All engineered silk-amyloid fibers have displayed attractive ultimate tensile-strength and toughness, 2-3 fold greater than those of recombinant spider silk fibers of similar molecular weight. Additionally, a strong molecular-weight-dependent of fiber tensile strength was observed. Fibers made of one silk-amyloid protein containing 128 repeats of the FGAILSS sequence displayed an average ultimate tensile strength of 0.98 ± 0.08 GPa and an average toughness of 161 ± 26 MJ/m3, reaching our final target on mechanical properties. Excitingly, the strength and toughness of our new hybrid fibers have surpassed most recombinant protein fibers and even some natural spider silk fibers. The design strategy and the biosynthetic approach can be expanded to create numerous novel materials, and the macroscopic amyloid fibers will enable a wide range of mechanically-demanding applications. These results were published in the journal of ACS Nano 2021. Accomplishment #2. Engineer solubility-enhanced, high-strength spidroins. The objective of this task is to identify sequence modifications that can enhance water solubility of silk-amyloid hybrid proteins and fiber mechanical properties, thus creating simplified fiber production processes with enhanced fiber yields. Using genetic engineering approaches, we modified the sequence of spidroins to improve the protein's water solubility as well as fiber mechanical properties. We found that biterminal fusion of an intrinsically disordered Mussel Foot Protein 5 (Mfp) fragments to silk-amyloid repeats significantly improved both fiber mechanical properties and protein water solubility. Specifically, we split Mfp5 (sMfp) in the middle into two halves with similar number of residues on each segment. sMfp5 was then genetically fused to the termini of a 16-repeat silk-amyloid protein 16xFGA, resulting in a new fusion protein NM-16xFGA-CM. Standard tensile tests showed that sMfp5 fusion significantly enhanced both tensile strength and toughness of the resulting fiber. The ultimate tensile strength increased by 210% reaching 481±31 MPa, and toughness increased by 303% reaching 179±39 MJ*m-3. We further showed that sMfp5 fusion is a general method for promoting mechanical properties of other protein fibers. We fused sMfp5 to three types of fiber proteins, including a recombinant spider silk (polyalanine), another amyloid-silk hybrid protein 16xKLV, and a titin fragment Ig67-70. Fusion to sMfp5 enhanced fiber ultimate strength by 65.5-139% for all three proteins. Polarized Raman spectrometry showed increased IY/X peak intensity ratios for all three sMfp5-fused fibers, confirming enhanced β-sheet alignment along the fiber axis for all three types of protein materials. Additionally, we tested the mechanical performance of these engineered fibers in water or in wet conditions. Spider silk is well known for its super-contraction, where fibers contract when treated with water or placed in high humidity environments. Super-contraction is undesirable for many textile and technical fiber applications. For example, natural spider silk and recombinant silk fibers contract by approximately 20% in their length when placed under 75% relative humidity. We found that many of our engineered protein fibers have significantly reduced super-contraction. Notably, one of the sMfp5-fused silk-amyloid fibers NM-16xFGA-CM (KRtoS) has only 1% contraction in length under 75% relative humidity, largely reducing this undeniable behavior of protein fiber. More excitingly, the engineered silk sequence and improved strain genetic stability have allowed us to produce the sMfp-fused silk-amyloid protein in high titers. The NM-16xFGA-CM (KRtoS) protein was produced at 8.0±0.7 g/L from a 2 L fermenter, demonstrating the potential for large-scale production of microbial fibers in an economically competitive way. These results were published in the journal of Nature Communications 2023. Accomplishment #3. Engineering P. putida to produce strength-enhanced spidroin fiber from lignin. The objective of this task is to engineer P. putida to produce the strength- and solubility-enhanced spidroin protein identified from previous tasks. Task 3 aims to prove the concept of producing strong silk fibers in P. putida rather than fully establish a production system, which requires more time and personnel efforts. We have used and engineered several expression systems for protein production in P. putida. Two of the engineered silk-amyloid proteins, 16xKLV and 16xFGA, were successfully expressed in P. putida using an arabinose-inducible promoter. Multiple expression conditions were tested, and the highest yield was obtained when cells were induced at OD600nm of 0.7 with 0.3% arabinose added. Under this optimal condition, 16xKLV was expressed as one of the most abundant proteins in the proteome of engineered P. putida. In general, this work has proved the concept that engineered high-performing silk-amyloid proteins can be expressed in industrially-compatible P. putida strain.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Li J., Jiang B., Chang X., Yu H., Han Y., Zhang F.*, Bi-terminal Fusion of Intrinsically Disordered Mussel Foot Protein Fragments Boosts Mechanical Strength for a Wide Range of Protein Fibers. Nat Commun, 14, 2127, 1-12, https://doi.org/10.1038/s41467-023-37563-0, (2023).
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Jeon J., Venkatesh S., Lee K.S., Jiang B., Zhang F.*, Microbial Synthesis of High Molecular Weight Highly Repetitive Protein Polymers Int J Mol Sci. 24 (7), 6416 (2023).
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Li J., Zhu Y., Yu H., Dai B., Jun Y., Zhang F.* Macroscopic fibers made of polymeric amyloid proteins display gigapascal tensile strength. ACS Nano 15, (7), 11843-11853, doi.org/10.1021/acsnano.1c02944 (2021)
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Li. J., Zhang F., Amyloids as Building Blocks for Macroscopic Functional Materials: Designs, Applications and Challenges. Int J Mol Sci 22, (19), 10698, doi.org/10.3390/ijms221910698. (2021)
|
Progress 07/01/21 to 06/30/22
Outputs
Target Audience:Scientists, engineers, industries who are interested in agricultural fiber production, microbial fermentation, etc. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In this reporting period, I have trained two graduate students (Jingyao Li and Xinyuan Chang) and two technicians (John Jaeger and Han Yu) through this project. These training activities have increased their knowledge and skills in protein engineering, microbial genetic engineering, metabolic engineering, as well as synthetic biology. How have the results been disseminated to communities of interest?The PI has presented conferences, workshop, and seminars discussing this project, including: Invited talk, 72nd SIMB Annual Meeting, San Francisco, Aug 2022 Invited seminar, Guangdong Technion-Israel Institute of Technology, Virtual, May 2022 Keynote Speaker, ACS Rubber Division Spring Meeting, Cleveland, OH, Apr, 2022 Invited seminar, School of Biological and Health Systems Engineering, Arizona State University, Feb 2022 Engineering Protein-based Materials via Synthetic biology Protein Society Meeting, San Francisco, Jul 2022 Microbial production of tough artificial muscle fibers via synthetic biology 2022 ACS Annual Spring Meeting, San Diego, Mar 2022 Microbial production of renewable fibers stronger and tougher than spider silk 2022 ACS Annual Spring Meeting, San Diego, Mar 2022 What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we expect to complete this project by completing Tasks 3. Specifically, we will further engineer P. putida to enhance the production of strength-enhanced spidroin fiber from lignin.
Impacts
What was accomplished under these goals?
IMPACTS: The major impact of this project is to provide a novel and efficient process to convert agricultural waste biomass into high-performance silk fibers using engineered microbes. Current agricultural production of fibers such as plant-derived cotton, hemp, flax and animal-derived silk and wool requires vast amounts of arable land, water, fertilizers, herbicides, pesticides, facilities, and human resources. The laborious production process, low profit margins, and environmental concerns related to traditional agricultural fiber production have demanded the development of new processes to convert abundant and otherwise wasted agricultural biomass (e.g. corn stover, wheat straw, etc.) into such fibers. This project aims to develop a simple microbial process to produce novel, strength- and solubility-enhanced silk fibers from inexpensive agricultural waste biomass. This new process will offer high theoretical conversion yield and productivity, drastically reduced resource use, and tunable fiber mechanical performance. We have combined our capabilities in protein engineering, microbial genetic engineering, as well as metabolic engineering to synthesize a few high MW amyloid-silk hybrid proteins. Fibers made from these microbially-synthesized hybrid proteins have displayed high tensile strength and toughness, even higher than some natural spider silk fibers--the most tough natural materials in nature. Completing this project will dramatically improve both production efficiency and economic viability of recombinant spider silks, providing a novel process for the production of high performance agricultural fibers. Outcomes: In this reporting period (Jul 2021-June 2022), we have completed Task 2 and made significant progresses in Task 3 of the original proposal. Task 2. Engineer solubility-enhanced spidroins. The objective of this task is to systematically study how sequence modifications affect the water solubility of spider-silk-amyloid hybrid proteins and fiber mechanical properties. Using genetic engineering approaches, we modified the sequence of spidroins to improve the protein's water solubility as well as fiber mechanical properties. We found that fusion of an intrinsically disordered split-Mussel Foot Protein 5 (sMfp5) to the terminal regions of silk-amyloid repeats significantly improved both fiber mechanical properties and behavior in water. We split Mfp5 in the middle into two halves with similar number of residues and MW on each segment. sMfp5 was then genetically fused to the termini of a 16-repeat silk-amyloid protein 16xFGA, resulting in a new fusion protein NM-16xFGA-CM. Standard tensile tests showed that fibers of the sMfp5-fused protein have enhanced strength and toughness. The ultimate tensile strength increased by 77% from 230±34 MPa of 16xFGA fibers to 406±36 MPa of NM-16xFGA-CM fibers, and toughness increased by 100% from 59±17 MJ*m-3 of 16xFGA fibers to 118±21 MJ*m-3 of NM-16xFGA-CM. Next, we investigated the underlying mechanism and structural influence on fibers. Polarized Raman spectrometry was used to examine the amide I β-sheet peak (1670 cm−1) and compared the Raman spectra when fibers was oriented either parallel (X-axis) and perpendicular (Y-axis) to the direction of laser polarization.For 16xFGA fibers, their amide I β-sheet peak measured at perpendicular position (IY) is only slightly stronger than that at parallel position (IX). The peak intensity ratio between perpendicular and parallel positions IY/X was 1.13±0.04, suggesting that β-sheets in 16xFGA fibers are only weakly aligned to the fiber axes. In contrast, amide I β-sheet peak of NM-16xFGA-CM fibers are dramatically higher at the perpendicular position (IY) than at the parallel position (IX), indicating a substantially enhanced β-sheet alignment compared to 16xFGA fibers. The peak intensity ratio (IY/X) reached 1.68±0.06, significantly higher than that of previously reported high MW (556 kDa) recombinant spider silk. Upon demonstrating the enhanced mechanical property for 16xFGA fibers, we examined whether sMfp5 fusion can be a general method for promoting the mechanical properties of other protein fibers. We fused sMfp5 to three types of fiber proteins, including a recombinant spider silk 16xAAA (polyalanine), another amyloid-silk hybrid protein 16xKLV, and a titin fragment Ig67-70. Fusion to sMfp5 enhanced fiber ultimate strength by 65.5-139% for all three proteins. Polarized Raman spectrometry showed increased IY/X peak intensity ratios for all three sMfp5-fused fibers, confirming enhanced β-sheet alignment along the fiber axis for all three types of protein materials. Additionally, we tested the mechanical performance of these engineered fibers in water or in wet conditions. Spider silk is well known for its super-contraction, where fibers contract when treated with water or placed in high humidity environments. Super-contraction is undesirable for many textile and technical fiber applications. For example, natural spider silk and recombinant silk fibers contract by approximately 20% in their length when placed under 75% relative humidity. We found that many of our engineered protein fibers have significantly reduced super-contraction. Notably, one of the sMfp5-fused silk-amyloid fibers NM-16xFGA-CM (KRtoS) has only 1% contraction in length under 75% relative humidity, largely reducing this undeniable behavior of protein fiber. Task 3. Engineering P. putida to produce strength-enhanced spidroin fiber from lignin. The objective of this task is to engineer P. putida to produce the strength- and solubility-enhanced spidroin protein identified from previous tasks. Task 3 aims to prove the concept of producing strong silk fibers in P. putida rather than fully establish a production system, which requires more time and personnel efforts. We have used and engineered several expression systems for protein production in P. putida. Two of the engineered silk-amyloid proteins, 16xKLV and 16xFGA, were successfully expressed in P. putida using an arabinose-inducible promoter. Multiple expression conditions were tested, and the highest yield was obtained when cells were induced at OD600nm of 0.7 with 0.3% arabinose added. Under this optimal condition, 16xKLV was expressed as one of the most abundant proteins in the proteome of engineered P. putida. Expression level of 16xFGA was lower compared to that of 16xKLV and will require further optimization. In general, this work has proved the concept that engineered high-performing silk-amyloid proteins can be expressed in industrially-compatible P. putida strain.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Li. J., Zhang F., Amyloids as Building Blocks for Macroscopic Functional Materials: Designs, Applications and Challenges. Int J Mol Sci 22, (19), 10698, doi.org/10.3390/ijms221910698. (2021)
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Li J., Zhu Y., Yu H., Dai B., Jun Y., Zhang F.* Macroscopic fibers made of polymeric amyloid proteins display gigapascal tensile strength. ACS Nano 15, (7), 11843-11853, doi.org/10.1021/acsnano.1c02944 (2021)
- Type:
Journal Articles
Status:
Under Review
Year Published:
2022
Citation:
Li J., Jiang B., Chang X., Yu H., Han Y., Zhang F.* Intrinsically Disordered Split Mussel Foot Protein Fusion Boosts Mechanical Strength for a Wide Range of Protein Fibers. Submitted
|
Progress 07/01/20 to 06/30/21
Outputs
Target Audience:Scientists, engineers, industries who are interested in agricultural fiber production, microbial fermentation, etc. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In this reporting period, I have trained two graduate students (Jingyao Li and Alden Filko) and two technicians (John Jaeger and Han Yu) through this project. These training activities have increased their knowledge and skills in protein engineering, microbial genetic engineering, metabolic engineering, as well as synthetic biology. Additionally, the PI has presented conferences, workshop, and seminars discussing this project, including: Invited presentation, AIChE 15C Bioengineering Division, Virtual, Nov 2020. Invited presentation, Amy Collaborative Center for Biological Engineering, Virtual, Jul 2020. How have the results been disseminated to communities of interest? Li J., Zhu Y., Yu H., Dai B., Jun Y., Zhang F.* Macroscopic fibers made of polymeric amyloid proteins display gigapascal tensile strength. Submitted (2021) What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we expect to continue this project according to the Project Timeline as described in the original proposal: to complete Tasks 2 and 3. Specifically, we will perform Subtask 2.3 "Combining water soluble amorphous sequences with amyloid crystalline forming sequences" and Task 3 "Engineering P. putida to produce strength-enhanced spidroin fiber from lignin".
Impacts
What was accomplished under these goals?
IMPACTS: The major impact of this project is to provide a novel and efficient process to convert agricultural waste biomass into high-performance silk fibers using engineered microbes. Current agricultural production of fibers such as plant-derived cotton, hemp, flax and animal-derived silk and wool requires vast amounts of arable land, water, fertilizers, herbicides, pesticides, facilities, and human resources. The laborious production process, low profit margins, and environmental concerns related to traditional agricultural fiber production have demanded the development of new processes to convert abundant and otherwise wasted agricultural biomass (e.g. corn stover, wheat straw, etc.) into such fibers. This project aims to develop a simple microbial process to produce novel, strength- and solubility-enhanced silk fibers from inexpensive agricultural waste biomass. This new process will offer high theoretical conversion yield and productivity, drastically reduced resource use, and tunable fiber mechanical performance. We have combined our capabilities in protein engineering, microbial genetic engineering, as well as metabolic engineering to synthesize a few high MW amyloid-silk hybrid proteins. Fibers made from these microbially-synthesized hybrid proteins have displayed high tensile strength and toughness, even higher than some natural spider silk fibers--the most tough natural materials in nature. Completing this project will dramatically improve both production efficiency and economic viability of recombinant spider silks, providing a novel process for the production of high performance agricultural fibers. Outcomes: In this reporting period (Jul 2020-June 2021), we have completed Task 1 and made significant progresses in Task 2 of the original proposal. Task 1. Engineer strength-enhanced spider-silk-amyloid hybrid fibers. The objective of this task is to explore an index of amyloid peptide sequences to synthesize hybrid fibers and screen their mechanical properties. From previous reporting period, we synthesized multiple spider-silk-amyloid hybrid proteins and characterized their mechanical properties. In this reporting period, we further studied how different amyloid sequences affect the structure of their fibers using synchrotron-based wide-angle X-ray diffraction (WAXD). We found that the spider-silk-amyloid fibers are semi-crystalline materials, containing both amorphous domains and nano crystallites. The crystalline domains maintain some characters of the cross-β structure unique to amyloids. The crystallite size along the inter-strand axis was estimated to be 3.7 nm, similar to that of 16x recombinant silk fibers. All spider-silk-amyloid fibers displayed a drastically higher crystallinity from 15-19%, compared to 4.2% in 16x recombinant silk fibers. The dramatically enhanced crystallinity in spider-silk-amyloid materials provides higher strengths and moduli to protein fibers under similar molecular weight (MW). We further synthesized high MW spider-silk-amyloid proteins. The best hybrid protein (namely 128xFGA) contains 128 repeating peptide sequences and has a MW of 378 kDa, both of which are 33% higher than those in the original proposal (proposed for 96mer, and 285 kDa). The 128xFGA protein was spun to fibers which display a tensile strength of 0.98 ± 0.08 GPa and a toughness of 161 ± 26 MJ/m3, reaching our target on mechanical properties. Excitingly, the strength and toughness of our new hybrid fibers have surpassed most recombinant protein fibers and even some natural spider silk fibers. The design strategy and the biosynthetic approach can be expanded to create numerous novel materials, and the macroscopic amyloid fibers will enable a wide range of mechanically-demanding applications. Task 2. Engineer solubility-enhanced spidroins. The objective of this task is to systematically study how sequence modifications affect the water solubility of spider-silk-amyloid hybrid proteins and fiber mechanical properties. Using genetic engineering approaches, we have modified the sequence of spidroins to improve the protein's water solubility. Thus far, we have identified multiple hybrid protein sequences with drastically enhanced water solubility, which significantly simplified the protein purification procedure. First, we found that the FGAILSS amyloid sequence enhances protein solubility. Further, compared to poly-alanine sequences in natural spidroin, sequence repetitiveness of the FGAILSS hybrid protein is drastically lower, thus facilitating its bacterial production. While recombinant silk protein with 128 repeats failed to express in engineered E. coli due to highly repetitive alanine sequences, our 128x FGAILSS protein can be overexpressed in our engineered E. coli host in relative high yield. Remarkably, these high MW proteins can be purified by a single affinity chromatography, instead of the laborious selective-precipitation required for high MW recombinant spidroin, thus greatly simplifying fabrication process and reducing production cost. Additionally, we also engineered a few hybrid proteins by inserting the solubility-promoting GPGQQ motif to the amorphous regions of the VQIVYK hybrid proteins. The resulting hybrid protein was expressed in soluble fraction of E. coli, and the purified protein can be dissolved in aqueous solution up to 50 mg/ml.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
Li J., Zhu Y., Yu H., Dai B., Jun Y., Zhang F.* Macroscopic fibers made of polymeric amyloid proteins display gigapascal tensile strength. Submitted (2021)
|
Progress 07/01/19 to 06/30/20
Outputs
Target Audience:Scientists, engineers, industries who are interested in agricultural fiber production, microbial fermentation, etc. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In this reporting period, I have trained two graduate students (Jingyao Li and Juya Jeon) working on this project, including knowledge and skills in protein engineering, microbial genetic engineering, metabolic engineering, as well as synthetic biology. Additionally, the PI has presented conferences, workshop, and seminars discussing this project, including: Invited seminar, Department of Chemical and Biomolecular Engineering, Rice University, Spring, 2020 Workshop, Cellular Engineering Program, Department of Mechanical Engineering & Materials Science, Washington University in St. Louis, Oct 2019. Invited seminar, Program of Bioinformatics and Computational Biology, Saint Louis University, Oct, 2019. 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?In the next reporting period, we expect to continue this project according to the Project Timeline as described in the original proposal: to complete Tasks 1 and 2. Specifically, we will perform Subtask 1.3 "Synthesizing high MW hybrid fibers", Subtask 2.2 "Studying the effect of mutations within the amorphous regions", and Subtask 2.3 "Combining water soluble amorphous sequences with amyloid crystalline forming sequences".
Impacts
What was accomplished under these goals?
IMPACTS: The major impact of this project is to provide a novel and efficient process to convert agricultural waste biomass into high-performance silk fibers using engineered microbes. Current agricultural production of fibers such as plant-derived cotton, hemp, flax and animal-derived silk and wool requires vast amounts of arable land, water, fertilizers, herbicides, pesticides, facilities, and human resources. The laborious production process, low profit margins, and environmental concerns related to traditional agricultural fiber production have demanded the development of new processes to convert abundant and otherwise wasted agricultural biomass (e.g. corn stover, wheat straw, etc.) into such fibers. This project aims to develop a simple microbial process to produce novel, strength- and solubility-enhanced silk fibers from inexpensive agricultural waste biomass. This new process will offer high theoretical conversion yield and productivity, drastically reduced resource use, and tunable fiber mechanical performance. We have used a combination of protein engineering, microbial genetic engineering, metabolic engineering, as well as synthetic biology approaches and demonstrated that our new hybrid proteins indeed yielded fibers stronger than synthetic spider silk fibers at similar molecular weight. Completing this project will dramatically improve both production efficiency and economic viability of recombinant spider silks, providing a novel process for the production of high performance agricultural fibers. Outcomes: In this reporting period, we have made progress in Task 1 and 2 of the original proposal. The specific goal of Task 1 is to understand how different amyloid classes and sequences affect the strength of hybrid fibers. We have completed subtask 1.1 and are in the middle of completing subtask 1.2. In this reporting period, we have created 10 novel hybrid protein sequences with each sequence containing the flexible region of spider silk protein and a unique amyloid sequence with 16 repeating units (16x). These hybrid proteins have been synthesized using our synthetic biology method and purified by Ni-NTA affinity chromatography. Purified spidroins were dissolved in HFIP to form dopes (15-25% w/v) and spun into fibers using our self-constructed spinning apparatus. Fiber morphology were examined by light microscopy and SEM. Fiber mechanical properties (stress-strain curves) of 6 fibers were measured (fibers from the other 4 proteins contracted right after spinning). All hybrid fibers showed strength, modulus, elasticity, and toughness 2~3 fold higher than those of a synthetic spider silk protein with similar molecular weight. These results indicate that our amyloid-silk hybrid proteins indeed yielded fibers stronger than spider silk fiber. The specific goal of Task 2 is to engineer the water solubility of our hybrid proteins by adding water soluble GPGQQ motifs to amorphous regions, and by mutating hydrophobic residues in amorphous regions to hydrophilic residues. We have completed subtask 2.1 and started on subtask 2.2. In this reporting period, we have created 4 GPGQQ-containing proteins with increasing numbers of GPGQQ motifs to each repeat unit. Additionally, we have also created 2 hybrid protein sequences by mutating the hydrophobic leucine and tyrosine residues in amorphous regions to hydrophilic glutamine residues. These proteins were synthesized in our engineered microbial hosts and purified using our established method. So far, all the GPGQQ-containing proteins showed higher water solubility than the synthetic spider silk protein. This promising result could lead to significant simplification of the downstream protein purification process, which we are currently developing. We will also test the mechanical properties of fibers from these water-soluble proteins.
Publications
|