Development of bifunctional biosensors for sensing and dynamic control of glycolysis flux in metabolic engineering

2021 ◽  
Author(s):  
Yuan Zhu ◽  
Ying Li ◽  
Ya Xu ◽  
Jian Zhang ◽  
Linlin Ma ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Dynamic Control

scholarly journals Dynamic control of pathway expression with riboregulated switchable feedback promoters

2019 ◽  
Author(s):  
Cameron J. Glasscock ◽  
John T. Lazar ◽  
Bradley W. Biggs ◽  
Jack H. Arnold ◽  
Min Kyoung Kang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Engineering ◽  
Stress Response ◽  
Dynamic Control ◽  
Transcriptional Networks ◽  
Cytochrome P450 Enzyme ◽  
Control Of Gene Expression ◽  
System Productivity ◽  
P450 Enzyme ◽  
Pathway Regulation

AbstractDynamic pathway regulation has emerged as a promising strategy in metabolic engineering for improved system productivity and yield, and continues to grow in sophistication. Bacterial stress-response promoters allow dynamic gene regulation using the host’s natural transcriptional networks, but lack the flexibility to control the expression timing and overall magnitude of pathway genes. Here, we report a strategy that uses RNA transcriptional regulators to introduce another layer of control over the output of natural stress-response promoters. This new class of gene expression cassette, called a riboregulated switchable feedback promoter (rSFP), can be modularly activated using a variety of mechanisms, from manual induction to quorum sensing. We develop and apply rSFPs to regulate a toxic cytochrome P450 enzyme in the context of a Taxol precursor biosynthesis pathway and show this leads to 2.4x fold higher titers than from the best reported strain. We envision that rSFPs will become a valuable tool for flexible and dynamic control of gene expression in metabolic engineering, protein and biologic production, and many other applications.

Design and Characterization of Rapid Optogenetic Circuits for Dynamic Control in Yeast Metabolic Engineering

2020 ◽  
Vol 9 (12) ◽  
pp. 3254-3266 ◽  
Author(s):  
Evan M. Zhao ◽  
Makoto A. Lalwani ◽  
Robert J. Lovelett ◽  
Sergio A. García-Echauri ◽  
Shannon M. Hoffman ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Dynamic Control

Current and future modalities of dynamic control in metabolic engineering

2018 ◽  
Vol 52 ◽  
pp. 56-65 ◽  
Author(s):  
Makoto A Lalwani ◽  
Evan M Zhao ◽  
José L Avalos
Keyword(s):  
Metabolic Engineering ◽  
Dynamic Control

scholarly journals Pgas, a Low-pH-Induced Promoter, as a Tool for Dynamic Control of Gene Expression for Metabolic Engineering of Aspergillus niger

2017 ◽  
Vol 83 (6) ◽  
Author(s):  
Xian Yin ◽  
Hyun-Dong Shin ◽  
Jianghua Li ◽  
Guocheng Du ◽  
Long Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Engineering ◽  
Cell Growth ◽  
Aspergillus Niger ◽  
Organic Acids ◽  
Dynamic Control ◽  
Low Ph ◽  
Transcriptional Analysis ◽  
Control Of Gene Expression ◽  
Content Type

ABSTRACT The dynamic control of gene expression is important for adjusting fluxes in order to obtain desired products and achieve appropriate cell growth, particularly when the synthesis of a desired product drains metabolites required for cell growth. For dynamic gene expression, a promoter responsive to a particular environmental stressor is vital. Here, we report a low-pH-inducible promoter, Pgas, which promotes minimal gene expression at pH values above 5.0 but functions efficiently at low pHs, such as pH 2.0. First, we performed a transcriptional analysis of Aspergillus niger, an excellent platform for the production of organic acids, and we found that the promoter Pgas may act efficiently at low pH. Then, a gene for synthetic green fluorescent protein (sGFP) was successfully expressed by Pgas at pH 2.0, verifying the results of the transcriptional analysis. Next, Pgas was used to express the cis-aconitate decarboxylase (cad) gene of Aspergillus terreus in A. niger, allowing the production of itaconic acid at a titer of 4.92 g/liter. Finally, we found that Pgas strength was independent of acid type and acid ion concentration, showing dependence on pH only. IMPORTANCE The promoter Pgas can be used for the dynamic control of gene expression in A. niger for metabolic engineering to produce organic acids. This promoter may also be a candidate tool for genetic engineering.

scholarly journals Dynamic control in metabolic engineering: Theories, tools, and applications

2020 ◽  
Author(s):  
Christopher J. Hartline ◽  
Alexander C. Schmitz ◽  
Yichao Han ◽  
Fuzhong Zhang
Keyword(s):  
Metabolic Engineering ◽  
Dynamic Control

scholarly journals Optogenetic Amplification Circuits for Light-Induced Metabolic Control

2020 ◽  
Author(s):  
Evan M. Zhao ◽  
Makoto A. Lalwani ◽  
Jhong-Min Chen ◽  
Paulina Orillac ◽  
Jared E. Toettcher ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Dynamic Control ◽  
Transcriptional Response ◽  
Attractive Alternative ◽  
Chemical Production ◽  
Chemical Inducers ◽  
High Tunability ◽  
Three Phase ◽  
Fold Induction ◽  
Cell Densities

AbstractDynamic control of microbial metabolism is an effective strategy to improve chemical production in fermentations. While dynamic control is most often implemented using chemical inducers, optogenetics offers an attractive alternative due to the high tunability and reversibility afforded by light. However, a major concern of applying optogenetics in metabolic engineering is the risk of insufficient light penetration at high cell densities, especially in large bioreactors. Here, we present a new series of optogenetic circuits we call OptoAMP, which amplify the transcriptional response to blue light by as much as 21.8-fold compared to the basal circuit (OptoEXP). These circuits show as much as a 41-fold induction between dark and light conditions, efficient activation at light doses as low as ~1%, and strong homogeneous light-induction in bioreactors of at least 5L, with limited illumination at cell densities above 40 OD600. We demonstrate the ability of OptoAMP circuits to control engineered metabolic pathways in novel three-phase fermentations using different light schedules to control enzyme expression and improve production of lactic acid, isobutanol, and naringenin. These circuits expand the applicability of optogenetics to metabolic engineering.

Metabolic engineering of CHO cells to prepare glycoproteins

2018 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Qiong Wang ◽  
Michael J. Betenbaugh
Keyword(s):  
Metabolic Engineering ◽  
Cho Cells ◽  
Genetic Manipulation ◽  
Chinese Hamster ◽  
Post Translational Modification ◽  
Effector Functions ◽  
Recombinant Glycoproteins ◽  
Production Platform ◽  
Chemical Inhibitors ◽  
Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

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