Novel insights into construct toxicity, strain optimization, and primary sequence design for producing recombinant silk fibroin and elastin-like peptide in E. coli

Connor, Alexander; Wigham, Caleb; Yang, Bai; Kumar, Manish; Nassif, Sebastian; Koffas, Mattheos; Zha, R. Helen

doi:10.1016/j.mec.2023.e00219

Cited by 12 publications

(23 citation statements)

References 104 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Both agmatinase and GTP cyclohydrolase 1 type 2 homolog (Ybg1) proteins have also been shown to have functions to protect against oxidative damage [ 37 ]. Interestingly, the speB gene was identified as one of forty-seven mutated genes in a MaSp1-hosting SoluBL21 strain generated through directed evolution [ 38 ]. This mutant strain produced the highest level of spidroins compared to nine other strains.…”

Section: Discussionmentioning

confidence: 99%

Quantitative Shotgun Proteomic Analysis of Bacteria after Overexpression of Recombinant Spider Miniature Spidroin, MaSp1

Randene,

Mendoza,

Ysit

et al. 2024

IJMS

View full text Add to dashboard Cite

Spider silk has extraordinary mechanical properties, displaying high tensile strength, elasticity, and toughness. Given the high performance of natural fibers, one of the long-term goals of the silk community is to manufacture large-scale synthetic spider silk. This process requires vast quantities of recombinant proteins for wet-spinning applications. Attempts to synthesize large amounts of native size recombinant spidroins in diverse cell types have been unsuccessful. In these studies, we design and express recombinant miniature black widow MaSp1 spidroins in bacteria that incorporate the N-terminal and C-terminal domain (NTD and CTD), along with varying numbers of codon-optimized internal block repeats. Following spidroin overexpression, we perform quantitative analysis of the bacterial proteome to identify proteins associated with spidroin synthesis. Liquid chromatography with tandem mass spectrometry (LC MS/MS) reveals a list of molecular targets that are differentially expressed after enforced mini-spidroin production. This list included proteins involved in energy management, proteostasis, translation, cell wall biosynthesis, and oxidative stress. Taken together, the purpose of this study was to identify genes within the genome of Escherichia coli for molecular targeting to overcome bottlenecks that throttle spidroin overexpression in microorganisms.

show abstract

Section: Discussionmentioning

confidence: 99%

Quantitative Shotgun Proteomic Analysis of Bacteria after Overexpression of Recombinant Spider Miniature Spidroin, MaSp1

Randene,

Mendoza,

Ysit

et al. 2024

IJMS

View full text Add to dashboard Cite

show abstract

“…Puri ed protein was then collected by passing 3-6 ml of elution buffer (300 mM imidazole, 20 mM Tris-HCl, pH 8) through the column. Titer of the A5 4mer protein was found using the ChemiDoc XRS + Image Lab software and was performed in an identical fashion to previously described methods 47 . All SDS PAGEs for both GFPuv and A5 4mer were performed using previously described methods 47 .…”

Section: Lysis and Titer Quanti Cationmentioning

confidence: 99%

Microbial Upcycling of Polyethylene into Recombinant Proteins

Connor

Lamb

Delferro

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Herein, we report the development of a new strain of Pseudomonas bacteria capable of upcycling deconstructed polyethylene into high value bespoke recombinant protein products. Using hexadecane, a proxy for depolymerized polyethylene, as a sole carbon nutrient source, we optimized media compositions that facilitate robust biomass growth, with results suggesting the benefits of lower hydrocarbon concentrations and the use of NH4Cl as a nitrogen source. We genomically integrated recombinant genes for green fluorescent protein and spider dragline-inspired silk protein, and we showed their expression in Pseudomonas aeruginosa, reaching titers of approximately 10 mg/L when hexadecane was used as the sole carbon source. Lastly, we demonstrated that chemically depolymerized polyethylene, comprised of a mixture of branched and unbranched alkanes, could be upcycled into silk protein by Pseudomonas aeruginosa at titers of 11.3 ± 1.1 mg/L. Thus, this work establishes a versatile microbial platform for valorizing recalcitrant plastic waste.

show abstract

“…Equation (1). σ strength describes the ability of promoters to be transcribed by the sigma factor; σ selectivity describes the preference of the promoter to one of the two factors.…”

Section: Fluorescence Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…In the early fermentation stage of E. coli, the extensive expression of genes encoding toxic proteins [1][2][3][4][5], membrane proteins [6][7][8], and other organic compounds [9][10][11][12][13][14][15] negatively impacts biomass accumulation. Delaying the expression of these genes to the stationary phase weakens this effect and increases the yield of the target product [1]. Chemical inducers, such as isopropyl β-D-1-thiogalactopyrano-side (IPTG) [16,17], are commonly used to regulate these promotors, but their extensive usage in factory fermentation increases production costs and is toxic to the host.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Enhancing the Activity of a Self-Inducible Promoter in Escherichia coli through Saturation Mutation and High-Throughput Screening

Tong

Amin

et al. 2023

Fermentation

View full text Add to dashboard Cite

The use of self-inducible promoters is a promising strategy to address metabolic imbalances caused by overexpression. However, the low activity of natural self-inducible promoters hinders their widespread application. To overcome this limitation, we selected the fic promoter as a model promoter to create an enhanced self-inducible promoter library using saturation mutations and high-throughput screening. Sequence analysis revealed that these promoters share certain characteristics, including semi-conservation in the −35 hexamer, highly conserved cytosine in the −17 motif (compared to −13 for other promoters), and moderate A+T content between positions −33 and −18 in the spacer region. Additionally, the discriminator region of these promotors features high A+T content in the first five bases. We identified PficI-17, PficII-33, and PficIII-14 promoters as the optional promoters in the −35 hexamer, spacer region, and discriminator mutation libraries, respectively. These promotors were used as representatives to measure the specific fluorescence and OD600 nm dynamics in different media and to confirm their effect on the expression of different proteins, including egfp (enhanced green fluorescence protein) and rfp (red fluorescence protein). Overall, our findings provide valuable guidance for modifying promoters and developing a promoter library suitable for regulating target genes.

show abstract

Novel insights into construct toxicity, strain optimization, and primary sequence design for producing recombinant silk fibroin and elastin-like peptide in E. coli

Cited by 12 publications

References 104 publications

Quantitative Shotgun Proteomic Analysis of Bacteria after Overexpression of Recombinant Spider Miniature Spidroin, MaSp1

Quantitative Shotgun Proteomic Analysis of Bacteria after Overexpression of Recombinant Spider Miniature Spidroin, MaSp1

Microbial Upcycling of Polyethylene into Recombinant Proteins

Enhancing the Activity of a Self-Inducible Promoter in Escherichia coli through Saturation Mutation and High-Throughput Screening

Contact Info

Product

Resources

About