Mini-review: In vitro Metabolic Engineering for Biomanufacturing of High-value Products

Guo, Weihua; Sheng, Jiayuan; Feng, Xueyang

doi:10.1016/j.csbj.2017.01.006

Cited by 48 publications

(31 citation statements)

References 84 publications

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“…Finally, cell‐free systems can also be used to study pathway kinetics and elucidate rate‐limiting steps using a systematic approach to guide the optimization of metabolic pathways in vivo (Galloway, Laimins, Division, & Hutchinson, ; Guo, Sheng, & Feng, ). They can identify the most significant factors that control the overall flux of the pathway or to identify pathway regulatory mechanisms at the protein level (Guo et al, ; Zhu et al, ). The MVA pathway starting at MVA and acetyl‐CoA has been well studied using this method for the production of amorphadiene (X. Chen, Zhang, Zou, Stephanopoulos, & Too, ) and farnesene (Zhu et al, ).…”

Section: Introductionmentioning

confidence: 99%

Cell free biosynthesis of isoprenoids from isopentenol

Ward

Chatzivasileiou

Stephanopoulos

2019

Biotech & Bioengineering

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Cell-free systems are growing in importance for the biosynthesis of complex molecules. These systems combine the precision of traditional chemistry with the versatility of biology in creating superior overall processes. Recently, a new synthetic pathway for the biosynthesis of isoprenoids using the substrate isopentenol, dubbed the isopentenol utilization pathway (IUP), was demonstrated to be a promising alternative to the native 2C-methyl-D-erythritol-4-phosphate (MEP) and mevalonate (MVA) pathways. This simplified pathway, which contains a minimum of four enzymes to produce basic monoterpenes and only depends on ATP and isopentenol as substrates, allows for a highly flexible approach to the commercial synthesis of isoprenoid products. In this work, we use metabolic reconstitution to characterize this new pathway in vitro and demonstrate its use for the cell-free synthesis of mono-, sesquit-, and diterpenoids. Kinetic modeling and sensitivity analysis were also used to identify the most significant parameters for taxadiene productivity, and metabolic control analysis was employed to elucidate protein-level interactions within this pathway, which demonstrated that the IUP enzymatic system is primarily controlled by the concentration and kinetics of choline kinase (CK) and not regulated by any pathway intermediates. This is a significant advantage over the natural MEP or MVA pathways as it greatly simplifies future metabolic engineering efforts, both in vitro and in vivo, aiming at improving the kinetics of CK. Finally, we used the insights gathered to demonstrate an in vitro IUP system that can produce 220 mg/L of the diterpene taxadiene, in 9 hr, almost 3-fold faster than any system reported thus far. K E Y W O R D Sbiocatalysis, enzyme kinetics, isoprenoids, metabolic control analysis, taxadiene

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Section: Introductionmentioning

confidence: 99%

Cell free biosynthesis of isoprenoids from isopentenol

Ward

Chatzivasileiou

Stephanopoulos

2019

Biotech & Bioengineering

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“…Cell free systems have gained wide interest in metabolic engineering applications, primarily to circumvent the significant barriers in traditional in vivo processes [88]. Cell free metabolic engineering (CFME) has been practiced in both purified and crude cell extracts.…”

Section: Applications Of Cell Free Technologiesmentioning

confidence: 99%

The Evolution of Cell Free Biomanufacturing

Vilkhovoy¹,

Adhikari²,

Vadhin³

et al. 2020

Preprint

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Cell free systems are a widely used research tool in systems and synthetic biology and a promising platform for manufacturing of proteins and chemicals. In the past, cell free biology was primarily used to better understand fundamental biochemical processes. Notably, E. coli cell free extracts were used in the 1960s to decipher the sequencing of the genetic code. Since then, the transcription and translation capabilities of cell free systems have been repeatedly optimized to improve energy efficiency and protein yield. Today, cell free systems, in combination with the rise of synthetic biology, have taken on a new role as a promising technology for just in time manufacturing of therapeutically important biologics and high-value small molecules. They have also been implemented in an industrial scale for the production of antibodies and cytokines. In this review, we discuss the evolution of cell free systems, advancements in cell free protein synthesis, and cell free metabolic engineering, and conclude with discussing the importance and feasibility of mathematical modeling in cell free systems.

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“…Although in vitro synthetic biology is largely ignored compared to in vivo synthetic biology, it has made great and rapid progress [ 7 , [16] , [17] , [18] , [19] ]. These in vitro synthetic biology systems can be based on cell extracts [ 20 ] or purified enzymes [ 21 , 22 ] or their combinations.…”

Section: Introductionmentioning

confidence: 99%

An in vitro synthetic biology platform for emerging industrial biomanufacturing: Bottom-up pathway design

Shi

Han

You

et al. 2018

Synthetic and Systems Biotechnology

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Although most in vitro (cell-free) synthetic biology projects are usually used for the purposes of fundamental research or the formation of high-value products, in vitro synthetic biology platform, which can implement complicated biochemical reactions by the in vitro assembly of numerous enzymes and coenzymes, has been proposed for low-cost biomanufacturing of bioenergy, food, biochemicals, and nutraceuticals. In addition to the most important advantage-high product yield, in vitro synthetic biology platform features several other biomanufacturing advantages, such as fast reaction rate, easy product separation, open process control, broad reaction condition, tolerance to toxic substrates or products, and so on. In this article, we present the basic bottom-up design principles of in vitro synthetic pathway from basic building blocks-BioBricks (thermoenzymes and/or immobilized enzymes) to building modules (e.g., enzyme complexes or multiple enzymes as a module) with specific functions. With development in thermostable building blocks-BioBricks and modules, the in vitro synthetic biology platform would open a new biomanufacturing age for the cost-competitive production of biocommodities.

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Mini-review: In vitro Metabolic Engineering for Biomanufacturing of High-value Products

Cited by 48 publications

References 84 publications

Cell free biosynthesis of isoprenoids from isopentenol

Cell free biosynthesis of isoprenoids from isopentenol

The Evolution of Cell Free Biomanufacturing

An in vitro synthetic biology platform for emerging industrial biomanufacturing: Bottom-up pathway design

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