Metabolic engineering of Escherichia coli for the production of isoprenoids

Ward, Valerie C.A.; Chatzivasileiou, Alkiviadis Orfefs; Stephanopoulos, Gregory

doi:10.1093/femsle/fny079

Cited by 65 publications

(41 citation statements)

References 90 publications

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“…While isoprenoids are produced in all organisms, many of the compounds of greatest interest are made in small quantities in plants (Vickers et al, ). Due to the high cost of plant procurement and recovery from plants, high‐level production of isoprenoids through microbial metabolic engineering is highly desirable and significant progress has been made toward this goal with several challenges remaining, such as strict regulation (Y. Chen, Zhou, Siewers, & Nielsen, ; Ward, Chatzivasileiou, & Stephanopoulos, ) toxicity caused by over‐accumulation of pathway intermediates (George et al, ) and low productivities.…”

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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“…The biosynthesis of isoprenoids illustrates both the extraordinary diversity of biology and the inherent limitations of native metabolism. Isoprenoids, comprising more than 50,000 structures found in all kingdoms of life (2), play a wide range of ecological, physiological, and structural roles and have been exploited for applications ranging from high-value pharmaceuticals to commodity chemicals and fuels (2)(3)(4)(5). In all known organisms, isoprenoids are derived from the 5-carbon diphosphates IPP and DMAPP.…”

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confidence: 99%

“…S1A) (2) with the recent discovery of a 5′-methylthioadenosine-isoprenoid pathway also providing a unique MEP shunt linking methionine salvage to isoprenoid biosynthesis (6). Although these pathways have been engineered for isoprenoid production in a variety of organisms, their inherent inefficiencies, complex chemistry, and intrinsic regulatory mechanisms (Table 1) present significant challenges that have limited product titers and yields to well below theoretical maxima (3,5).…”

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confidence: 99%

The isoprenoid alcohol pathway, a synthetic route for isoprenoid biosynthesis

Clomburg

Qian

Tan

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

134

117

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The more than 50,000 isoprenoids found in nature are all derived from the 5-carbon diphosphates isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Natively, IPP and DMAPP are generated by the mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways, which have been engineered to produce compounds with numerous applications. However, as these pathways are inherently constrained by carbon, energy inefficiencies, and their roles in native metabolism, engineering for isoprenoid biosynthesis at high flux, titer, and yield remains a challenge. To overcome these limitations, here we develop an alternative synthetic pathway termed the isoprenoid alcohol (IPA) pathway that centers around the synthesis and subsequent phosphorylation of IPAs. We first established a lower IPA pathway for the conversion of IPAs to isoprenoid pyrophosphate intermediates that enabled the production of greater than 2 g/L geraniol from prenol as well as limonene, farnesol, diaponeurosporene, and lycopene. We then designed upper IPA pathways for the generation of (iso)prenol from central carbon metabolites with the development of a route to prenol enabling its synthesis at more than 2 g/L. Using prenol as the linking intermediate further facilitated an integrated IPA pathway that resulted in the production of nearly 0.6 g/L total monoterpenoids from glycerol as the sole carbon source. The IPA pathway provides an alternative route to isoprenoids that is more energy efficient than native pathways and can serve as a platform for targeting a repertoire of isoprenoid compounds with application as high-value pharmaceuticals, commodity chemicals, and fuels.

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“…Acetyl‐CoA is involved in various biological processes and it serves as a platform chemical for producing various high‐value products, such as 1‐butanol (Dong et al, 2017; Ohtake et al, 2017), 3‐hydroxypropionate (Cheng, Jiang, Wu, Li, & Ye, 2016; Liu et al, 2017), polyhydroxyalkanoates (Zheng, Yuan, Yang, & Ma, 2017) and isoprenoids. Isoprenoids may be used for flavorings, biofuels, pharmaceuticals, vitamins (Wang, Zada, Wei, & Kim, 2017; Ward, Chatzivasileiou, & Stephanopoulos, 2018). Acetyl‐CoA is the most important precursor for mevalonate production.…”

Section: Discussionmentioning

confidence: 99%

Metabolic engineering of E. coli for improving mevalonate production to promote NADPH regeneration and enhance acetyl‐CoA supply

Satowa

Fujiwara

Uchio

et al. 2020

Biotech & Bioengineering

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Microbial production of mevalonate from renewable feedstock is a promising and sustainable approach for the production of value‐added chemicals. We describe the metabolic engineering of Escherichia coli to enhance mevalonate production from glucose and cellobiose. First, the mevalonate‐producing pathway was introduced into E. coli and the expression of the gene atoB, which encodes the gene for acetoacetyl‐CoA synthetase, was increased. Then, the deletion of the pgi gene, which encodes phosphoglucose isomerase, increased the NADPH/NADP+ ratio in the cells but did not improve mevalonate production. Alternatively, to reduce flux toward the tricarboxylic acid cycle, gltA, which encodes citrate synthetase, was disrupted. The resultant strain, MGΔgltA‐MV, increased levels of intracellular acetyl‐CoA up to sevenfold higher than the wild‐type strain. This strain produced 8.0 g/L of mevalonate from 20 g/L of glucose. We also engineered the sugar supply by displaying β‐glucosidase (BGL) on the cell surface. When cellobiose was used as carbon source, the strain lacking gnd displaying BGL efficiently consumed cellobiose and produced mevalonate at 5.7 g/L. The yield of mevalonate was 0.25 g/g glucose (1 g of cellobiose corresponds to 1.1 g of glucose). These results demonstrate the feasibility of producing mevalonate from cellobiose or cellooligosaccharides using an engineered E. coli strain.

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Metabolic engineering of Escherichia coli for the production of isoprenoids

Cited by 65 publications

References 90 publications

Cell free biosynthesis of isoprenoids from isopentenol

Cell free biosynthesis of isoprenoids from isopentenol

The isoprenoid alcohol pathway, a synthetic route for isoprenoid biosynthesis

Metabolic engineering of E. coli for improving mevalonate production to promote NADPH regeneration and enhance acetyl‐CoA supply

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