skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Attention:

The NSF Public Access Repository (PAR) system and access will be unavailable from 11:00 PM ET on Friday, July 11 until 2:00 AM ET on Saturday, July 12 due to maintenance. We apologize for the inconvenience.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


This content will become publicly available on May 24, 2026

Title: Balancing Doses of EL222 and Light Improves Optogenetic Induction of Protein Production in Komagataella phaffii
ABSTRACT Komagataella phaffii, also known asPichia pastoris, is a powerful host for recombinant protein production, in part due to its exceptionally strong and tightly controlled PAOX1promoter. MostK. phaffiibioprocesses for recombinant protein production rely on PAOX1to achieve dynamic control in two‐phase processes. Cells are first grown under conditions that repress PAOX1(growth phase), followed by methanol‐induced recombinant protein expression (production phase). In this study, we propose a methanol‐free approach for dynamic metabolic control inK. phaffiiusing optogenetics, which can help enhance input tunability and flexibility in process optimization and control. The light‐responsive transcription factor EL222 fromErythrobacter litoralisis used to regulate protein production from the PC120promoter inK. phaffiiwith blue light. We used two system designs to explore the advantages and disadvantages of coupling or decoupling EL222 integration with that of the gene of interest. We investigate the relationship between EL222 gene copy number and light dosage to improve production efficiency for intracellular and secreted proteins. Experiments in lab‐scale bioreactors demonstrate the feasibility of the outlined optogenetic systems as potential alternatives to conventional methanol‐inducible bioprocesses usingK. phaffii.  more » « less
Award ID(s):
2300239
PAR ID:
10595683
Author(s) / Creator(s):
; ; ; ;
Publisher / Repository:
Wiley
Date Published:
Journal Name:
Biotechnology and Bioengineering
ISSN:
0006-3592
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; (, mSphere)
    Tringe, Susannah Green (Ed.)
    ABSTRACT Methanotrophic bacteria play a vital role in the biogeochemical carbon cycle due to their unique ability to use CH4as a carbon and energy source. Evidence suggests that some methanotrophs, includingMethylococcus capsulatus, can also use CO2as a carbon source, making these bacteria promising candidates for developing biotechnologies targeting greenhouse gas capture and mitigation. However, a deeper understanding of the dual CH4and CO2metabolism is needed to guide methanotroph strain improvements and realize their industrial utility. In this study, we show thatM. capsulatusexpresses five carbonic anhydrase (CA) isoforms, one α-CA, one γ-CA, and three β-CAs, that play a role in its inorganic carbon metabolism and CO2-dependent growth. The CA isoforms are differentially expressed, and transcription of all isoform genes is induced in response to CO2limitation. CA null mutant strains exhibited markedly impaired growth compared to an isogenic wild-type control, suggesting that the CA isoforms have independent, non-redundant roles inM. capsulatusmetabolism and physiology. Overexpression of some, but not all, CA isoforms improved bacterial growth kinetics and decreased CO2evolution from CH4-consuming cultures. Notably, we developed an engineered methanotrophic biocatalyst overexpressing the native α-CA and β-CA with a 2.5-fold improvement in the conversion of CH4to biomass. Given that product yield is a significant cost driver of methanotroph-based bioprocesses, the engineered strain developed here could improve the economics of CH4biocatalysis, including the production of single-cell protein from natural gas or anaerobic digestion-derived biogas.IMPORTANCEMethanotrophs transform CH4into CO2and multi-carbon compounds, so they play a critical role in the global carbon cycle and are of interest for biotechnology applications. Some methanotrophs, includingMethylococcus capsulatus, can also use CO2as a carbon source, but this dual one-carbon metabolism is incompletely understood. In this study, we show thatM. capsulatuscarbonic anhydrases are critical for this bacterium to optimally utilize CO2. We developed an engineered strain with improved CO2utilization capacity that increased the overall carbon conversion to cell biomass. The improvements to methanotroph-based product yields observed here are expected to reduce costs associated with CH4conversion bioprocesses. 
    more » « less
  2. ; ; (, Microbial Cell Factories)
    Abstract BackgroundSilk proteins have emerged as versatile biomaterials with unique chemical and physical properties, making them appealing for various applications. Among them, spider silk, known for its exceptional mechanical strength, has attracted considerable attention. Recombinant production of spider silk represents the most promising route towards its scaled production; however, challenges persist within the upstream optimization of host organisms, including toxicity and low yields. The high cost of downstream cell lysis and protein purification is an additional barrier preventing the widespread production and use of spider silk proteins. Gram-positive bacteria represent an attractive, but underexplored, microbial chassis that may enable a reduction in the cost and difficulty of recombinant silk production through attributes that include, superior secretory capabilities, frequent GRAS status, and previously established use in industry. ResultsIn this study, we explore the potential of gram-positive hosts by engineering the first production and secretion of recombinant spider silk in theBacillusgenus. Using an industrially relevantB. megateriumhost, it was found that the Sec secretion pathway enables secretory production of silk, however, the choice of signal sequence plays a vital role in successful secretion. Attempts at increasing secreted titers revealed that multiple translation initiation sites in tandem do not significantly impact silk production levels, contrary to previous findings for other gram-positive hosts and recombinant proteins. Notwithstanding, targeted amino acid supplementation in minimal media was found to increase production by 135% relative to both rich media and unaltered minimal media, yielding secretory titers of approximately 100 mg/L in flask cultures. ConclusionIt is hypothesized that the supplementation strategy addressed metabolic bottlenecks, specifically depletion of ATP and NADPH within the central metabolism, that were previously observed for anE. colihost producing the same recombinant silk construct. Furthermore, this study supports the hypothesis that secretion mitigates the toxicity of the produced silk protein on the host organism and enhances host performance in glucose-based minimal media. While promising, future research is warranted to understand metabolic changes more precisely in theBacillushost system in response to silk production, optimize signal sequences and promoter strengths, investigate the mechanisms behind the effect of tandem translation initiation sites, and evaluate the performance of this system within a bioreactor. 
    more » « less
  3. ; ; ; ; ; ; (, Proceedings of the National Academy of Sciences)
    Dynamic, multi-input gene regulatory networks (GRNs) are ubiquitous in nature. Multilayer CRISPR-based genetic circuits hold great promise for building GRNs akin to those found in naturally occurring biological systems. We develop an approach for creating high-performing activatable promoters that can be assembled into deep, wide, and multi-input CRISPR-activation and -interference (CRISPRa/i) GRNs. By integrating sequence-based design and in vivo screening, we engineer activatable promoters that achieve up to 1,000-fold dynamic range in anEscherichia coli-based cell-free system. These components enable CRISPRa GRNs that are six layers deep and four branches wide. We show the generalizability of the promoter engineering workflow by improving the dynamic range of the light-dependent EL222 optogenetic system from 6-fold to 34-fold. Additionally, high dynamic range promoters enable CRISPRa systems mediated by small molecules and protein–protein interactions. We apply these tools to build input-responsive CRISPRa/i GRNs, including feedback loops, logic gates, multilayer cascades, and dynamic pulse modulators. Our work provides a generalizable approach for the design of high dynamic range activatable promoters and enables classes of gene regulatory functions in cell-free systems. 
    more » « less
  4. ; ; ; ; (, Microbial Cell Factories)
    Abstract BackgroundThe increasing prevalence of plastic waste combined with the inefficiencies of mechanical recycling has inspired interest in processes that can convert these waste streams into value-added biomaterials. To date, the microbial conversion of plastic substrates into biomaterials has been predominantly limited to polyhydroxyalkanoates production. Expanding the capabilities of these microbial conversion platforms to include a greater diversity of products generated from plastic waste streams can serve to promote the adoption of these technologies at a larger scale and encourage a more sustainable materials economy. ResultsHerein, we report the development of a new strain ofPseudomonasbacteria capable of converting depolymerized 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 above 1 × 109 cfu/ml, 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 inPseudomonas 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 converted into silk protein byPseudomonas aeruginosaat titers of 11.3 ± 1.1 mg/L. ConclusionThis work demonstrates a microbial platform for the conversion of a both alkanes and plastic-derived substrates to recombinant, protein-based materials. The findings in this work can serve as a basis for future endeavors seeking to upcycle recalcitrant plastic wastes into value-added recombinant proteins. 
    more » « less
  5. ; ; ; (, Biotechnology and Bioengineering)
    Abstract Gene therapy is a promising therapeutic approach for genetic and acquired diseases nowadays. Among DNA delivery vectors, recombinant adeno‐associated virus (rAAV) is one of the most effective and safest vectors used in commercial drugs and clinical trials. However, the current yield of rAAV biomanufacturing lags behind the necessary dosages for clinical and commercial use, which embodies a concentrated reflection of low productivity of rAAV from host cells, difficult scalability of the rAAV‐producing bioprocess, and high levels of impurities materialized during production. Those issues directly impact the price of gene therapy medicine in the market, limiting most patients’ access to gene therapy. In this context, the current practices and several critical challenges associated with rAAV gene therapy bioprocesses are reviewed, followed by a discussion of recent advances in rAAV‐mediated gene therapy and other therapeutic biological fields that could improve biomanufacturing if these advances are integrated effectively into the current systems. This review aims to provide the current state‐of‐the‐art technology and perspectives to enhance the productivity of rAAV while reducing impurities during production of rAAV. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email