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Open Access Publications from the University of California

Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2016)

The goal of tissue engineering is to create the functional engineered tissue to replace or repair the damaged one in human body. The complex multi-phase collagen hydrogel systems are more and more popular in the tissue engineering field where they are used as scaffolds because of biodegradability and biocompatibility. The properties of collagen hydrogel are very important because they can lead to different cellular behavior. My study employed the non-invasive technologies to probe the properties of collagen-based materials (optical, microstructural and mechanical properties) and fibroblast cells’ behavior within collagen hydrogels.

We identified that the ion types and ionic strength, initial pH and self-assembly temperature all affect the microstructure of collagen hydrogels, which was detected with the Second Harmonic Generation (SHG) images. Besides that, initial collagen concentration can affect the mechanical properties of collagen hydrogels, which was determined with rheology (storage modulus of 4 g/l collagen hydrogels is five times higher compared to 2 g/l collagen hydrogels).

Collagen hydrogel needs to be crosslinked with cross-linkers to improve its mechanical strength since it is generally too soft to support cellular behavior, such as, cell proliferation and migration. According to our experience, the 3D collagen hydrogel collapsed into film after 2 days cell culture. Zero-length cross-linker (EDC) and non-zero-length cross-linker (Genipin) were employed to crosslink collagen hydrogel. Genipin can introduce very strong fluorescence into the collagen hydrogels and weaken their SHG signal. Genipin crosslinked collagen hydrogel has higher cross-linking degree (~85%) compared to EDC cross-linked collagen hydrogel (~10%). The storage modulus of genipin cross-linked collagen hydrogel is more than two times of EDC cross-linked collagen hydrogel.

Different “aged” genipin solution can result in different properties of collagen hydrogel. For the optical properties, older genipin solution can increase the fluorescence signal in the green channel (470 nm – 550 nm) and weaken fluorescence signal in the red channel (570 nm – 610 nm). For the microstructural properties, older genipin can result in a thinner collagen fiber within a hydrogel. For the mechanical properties, the storage modulus of genipin cross-linked collagen hydrogel dropped down along with the age increasing of genipin solutions. The embryonic BALB/3T3 (clone A31) fibroblasts cultured on collagen hydrogels cross-linked with stored genipin reagent are disproportionately more round compared to cells cultured on unmodified collagen hydrogels and collagen hydrogels stabilized with fresh genipin solutions. The best correlation of cellular extension is with morphological properties of synthesized hydrogels and no correlation is identified with mechanical properties of materials.

    Cover page: The Study of the Properties of Collagen Hydrogel and 3T3 Fibroblast Cells’ Behavior Within Collagen Hydrogel by Multiphoton Microscopy (MPM)
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2020)
    Kluyveromyces marxianus is an emerging host for metabolic engineering. This thermotolerant yeast is the fastest growing eukaryote, has high flux through the TCA cycle, and can metabolize a broad range of C5, C6, and C12 carbon sources. In comparison to the common host Saccharomyces cerevisiae, this non-conventional yeast suffers from a lack of metabolic engineering tools to control gene expression over a wide transcriptional range. To address this issue, we designed a library of 25 native-derived promoters from K. marxanius CBS6556 that spans 87-fold transcriptional strength under glucose metabolism. Six promoters from the library were further characterized in both glucose and xylose as well as across various temperatures from 30 to 45 ​°C. The temperature study revealed that in most cases EGFP expression decreased with elevating temperature; however, two promoters, P SSA3 and P ADH1 , increased expression above 40 ​°C in both xylose and glucose. The six-promoter set was also validated in xylose for triacetic acid lactone (TAL) production. By controlling the expression level of heterologous 2-pyrone synthase (2-PS), the specific TAL titer increased over 8-fold at 37 ​°C. Cultures at 41 ​°C exhibited a similar TAL biosynthesis capability, while at 30 ​°C TAL levels were lower. Taken together, these results advance the metabolic engineering tool set in K. marxianus and further develop this new host for chemical biosynthesis.
      Cover page: Developing a broad-range promoter set for metabolic engineering in the thermotolerant yeast Kluyveromyces marxianus.
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2021)
      BACKGROUND: 2-phenylethanol (2-PE) is a rose-scented flavor and fragrance compound that is used in food, beverages, and personal care products. Compatibility with gasoline also makes it a potential biofuel or fuel additive. A biochemical process converting glucose or other fermentable sugars to 2-PE can potentially provide a more sustainable and economical production route than current methods that use chemical synthesis and/or isolation from plant material. RESULTS: We work toward this goal by engineering the Shikimate and Ehrlich pathways in the stress-tolerant yeast Kluyveromyces marxianus. First, we develop a multigene integration tool that uses CRISPR-Cas9 induced breaks on the genome as a selection for the one-step integration of an insert that encodes one, two, or three gene expression cassettes. Integration of a 5-kbp insert containing three overexpression cassettes successfully occurs with an efficiency of 51 ± 9% at the ABZ1 locus and was used to create a library of K. marxianus CBS 6556 strains with refactored Shikimate pathway genes. The 33-factorial library includes all combinations of KmARO4, KmARO7, and KmPHA2, each driven by three different promoters that span a wide expression range. Analysis of the refactored pathway library reveals that high expression of the tyrosine-deregulated KmARO4K221L and native KmPHA2, with the medium expression of feedback insensitive KmARO7G141S, results in the highest increase in 2-PE biosynthesis, producing 684 ± 73 mg/L. Ehrlich pathway engineering by overexpression of KmARO10 and disruption of KmEAT1 further increases 2-PE production to 766 ± 6 mg/L. The best strain achieves 1943 ± 63 mg/L 2-PE after 120 h fed-batch operation in shake flask cultures. CONCLUSIONS: The CRISPR-mediated multigene integration system expands the genome-editing toolset for K. marxianus, a promising multi-stress tolerant host for the biosynthesis of 2-PE and other aromatic compounds derived from the Shikimate pathway.
        Cover page: CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus.
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