Quantification of Metabolic Fluxes for Metabolic Engineering of Plant Products

ABSTRACT Unabated mining and utilisation of petroleum and petroleum resources and their conversion to essential fuels and chemicals have drastic environmental consequences, contributing to global warming and climate change. In addition, fossil fuels are finite resources, with a fast-approaching shortage. Accordingly, research efforts are increasingly focusing on developing sustainable alternatives for chemicals and fuels production. In this context, bioprocesses, relying on microorganisms, have gained particular interest. For example, acetogens use the Wood-Ljungdahl pathway to grow on single carbon C1-gases (CO2 and CO) as their sole carbon source and produce valuable products such as acetate or ethanol. These autotrophs can, therefore, be exploited for large-scale fermentation processes to produce industrially relevant chemicals from abundant greenhouse gases. In addition, genetic tools have recently been developed to improve these chassis organisms through synthetic biology approaches. This review will focus on the challenges of genetically and metabolically modifying acetogens. It will first discuss the physical and biochemical obstacles complicating successful DNA transfer in these organisms. Current genetic tools developed for several acetogens, crucial for strain engineering to consolidate and expand their catalogue of products, will then be described. Recent tool applications for metabolic engineering purposes to allow redirection of metabolic fluxes or production of non-native compounds will lastly be covered.

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Central metabolic fluxes in the endosperm of developing maize seeds and their implications for metabolic engineering

Metabolic Engineering ◽

10.1016/j.ymben.2010.10.002 ◽

2011 ◽

Vol 13 (1) ◽

pp. 96-107 ◽

Cited By ~ 70

Author(s):

Ana P. Alonso ◽

Dale L. Val ◽

Yair Shachar-Hill

Keyword(s):

Metabolic Engineering ◽

Metabolic Fluxes ◽

Maize Seeds

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Metabolic Fluxes and Metabolic Engineering

Metabolic Engineering ◽

10.1006/mben.1998.0101 ◽

1999 ◽

Vol 1 (1) ◽

pp. 1-11 ◽

Cited By ~ 378

Author(s):

Gregory Stephanopoulos

Keyword(s):

Metabolic Engineering ◽

Metabolic Fluxes

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Advancement of Metabolic Engineering Assisted by Synthetic Biology

Catalysts ◽

10.3390/catal8120619 ◽

2018 ◽

Vol 8 (12) ◽

pp. 619 ◽

Cited By ~ 5

Author(s):

Hyang-Mi Lee ◽

Phuong Vo ◽

Dokyun Na

Keyword(s):

Metabolic Engineering ◽

Synthetic Biology ◽

Computational Methods ◽

Metabolic Pathways ◽

Cell Factory ◽

Microbial Cell Factory ◽

Optimal Regulation ◽

Metabolic Fluxes ◽

Metabolic Systems ◽

Micro Organisms

Synthetic biology has undergone dramatic advancements for over a decade, during which it has expanded our understanding on the systems of life and opened new avenues for microbial engineering. Many biotechnological and computational methods have been developed for the construction of synthetic systems. Achievements in synthetic biology have been widely adopted in metabolic engineering, a field aimed at engineering micro-organisms to produce substances of interest. However, the engineering of metabolic systems requires dynamic redistribution of cellular resources, the creation of novel metabolic pathways, and optimal regulation of the pathways to achieve higher production titers. Thus, the design principles and tools developed in synthetic biology have been employed to create novel and flexible metabolic pathways and to optimize metabolic fluxes to increase the cells’ capability to act as production factories. In this review, we introduce synthetic biology tools and their applications to microbial cell factory constructions.

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Metabolic engineering of CHO cells to prepare glycoproteins

Emerging Topics in Life Sciences ◽

10.1042/etls20180056 ◽

2018 ◽

Vol 2 (3) ◽

pp. 433-442 ◽

Cited By ~ 1

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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