Systematic Optimization of Limonene Production in Engineered <i>Escherichia coli</i>

Wu, Jun; Si, Cheng; Cao, Jiayu; Qiao, Jianjun; Zhao, Guang‐Rong

doi:10.1021/acs.jafc.9b01427

Cited by 67 publications

(56 citation statements)

References 48 publications

(79 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Thus, refining the TREE score (e.g., by weighting each factor) to better correlate in vitro and in vivo data will help identify the minimal amount of cellfree data needed to inform pathway design. Finally, our isoprenoid prototyping platform could be adapted for alternate pathway routes that utilize neryl pyrophosphate as a substrate for monoterpene synthases rather than GPP Ignea et al, 2019;Wu et al, 2019) or produce IPP and DMAPP using prenol or isoprenol as an intermediate Clomburg et al, 2019;Lund et al, 2019;Ward et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

Cell-free prototyping of limonene biosynthesis using cell-free protein synthesis

Dudley¹,

Karim²,

Nash³

et al. 2020

Preprint

View full text Add to dashboard Cite

Metabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-built-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 hours from 0.2 to 4.5 mM (23 to 610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. We anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design. Dudley, et al. -Cell-free prototyping of limonene biosynthesis 3 TOC Figure Highlights • Applied the iPROBE framework to build the nine-enzyme pathway to produce limonene • Assessed the impact of cofactors and 54 enzyme homologs on cell-free enzyme performance • Iteratively optimized the cell-free production of limonene by exploring more than 580 unique reactions • Extended pathway to biofuel precursors pinene and bisabolene Dudley, et al. -Cell-free prototyping of limonene biosynthesis 4

show abstract

Section: Discussionmentioning

confidence: 99%

Cell-free prototyping of limonene biosynthesis using cell-free protein synthesis

Dudley¹,

Karim²,

Nash³

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…It is well established that during (S)-(-)-perillyl alcohol production, titers can be increased by 100 mg/L using Escherichia coli [12]. Many terpenoids have been produced to high titers through metabolic engineering, including monoterpenes, sesquiterpenes, diterpenes and tetraterpenes [25][26][27][28][29][30][31][32][33][34]. In this study, P-cymene monoxygenase hydroxylase (CymAa) and P-cymene monoxygenase reductase (CymAb) derived from Pseudomonas putida were introduced into BL21 (DE3) [13,16].…”

Section: Characterization Of Perillyl Alcohol By Gc-msmentioning

confidence: 99%

“…Many terpenoids have been produced at high titers by metabolic engineering, including monoterpenes, sesquiterpenes, diterpenes and tetraterpenes [25][26][27][28][29][30][31][32][33][34].…”

Section: Introductionmentioning

confidence: 99%

Effectiveness of recombinant Escherichia coli on the production of (R)-(+)-perillyl alcohol

Sun

Dong

Zhang

et al. 2020

Preprint

View full text Add to dashboard Cite

Background : ( R )-(+)-perillyl alcohol is a naturally oxygenated monoterpene that is produced in perilla leaves, citrus, lemon and lavender. Perillyl alcohols have many uses, including as natural flavor additives, insecticides, jet fuels and anti-cancer therapies. The bioconversion of readily available monoterpene precursors, such as ( R )-(+)-limonene, are recognized as valuable oxygenated derivatives. However, as this natural product is present at low concentrations in plant oils, alternative microbial production methods are required for its extraction from natural plant sources. Results : We engineered Escherichia coli to possess a heterologous mevalonate (MVA) pathway, including limonene synthase , P-cymene monoxygenase hydroxylase and P-cymene monoxygenase reductase for the production of ( R )-(+)-perillyl alcohol. The concentration of ( R )-(+)-limonene (the monoterpene precursor to ( R )-(+)-perillyl alcohol) reached 27.3 mg/L from glucose. Enhanced ( R )-(+)-perillyl alcohol production was therefore achieved. The strain produced ( R )-(+)-perillyl alcohol at a titer of 45.7 mg/L in a 5 L bioreactor fed batch system. Conclusions : These data highlight the efficient production of ( R )-(+)-perillyl alcohol through the mevalonate pathway from glucose. This method serves as a platform for the future production of other monoterpenes.

show abstract

“…Recently, Wu et al (2019), optimized production of another potential monoterpene jet fuel feedstock, limonene, in E. coli cell factories using a modularization approach. The limonene pathway was distributed between three modules, with the upstream module expressing MvaE and MvaS genes, responsible for the conversion of acetyl-CoA to MVA, originating from Enterococcus faecalis.…”

Section: E Colimentioning

confidence: 99%

Sustainable Production of Microbial Isoprenoid Derived Advanced Biojet Fuels Using Different Generation Feedstocks: A Review

Walls

Rios‐Solis

2020

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

As the fastest mode of transport, the aircraft is a major driver for globalization and economic growth. The development of alternative advanced liquid fuels is critical to sustainable development within the sector. Such fuels should be compatible with existing infrastructure and derived from second generation feedstocks to avoid competition with food markets. With properties similar to petroleum based fuels, isoprenoid derived compounds such as limonene, bisabolane, farnesane, and pinene dimers are of increasing interest as "drop-in" replacement jet fuels. In this review potential isoprenoid derived jet fuels and progress toward their microbial production was discussed in detail. Although substantial advancements have been achieved, the use of first generation feedstocks remains ubiquitous. Lignocellulosic biomass is the most abundant raw material available for biofuel production, however, technological constraints associated with its pretreatment and saccharification hinder its economic feasibility for low-value commodity production. Non-conventional microbes with novel characteristics including cellulolytic bacteria and fungi capable of highly efficient lignocellulose degradation and xylose fermenting oleaginous yeast with enhanced ligninassociated inhibitor tolerance were investigated as alternatives to traditional model hosts. Finally, innovative bioprocessing methods including consolidated bioprocessing and sequential bioreactor approaches, with potential to capitalize on such unique natural capabilities were considered.

show abstract

Systematic Optimization of Limonene Production in Engineered Escherichia coli

Cited by 67 publications

References 48 publications

Cell-free prototyping of limonene biosynthesis using cell-free protein synthesis

Cell-free prototyping of limonene biosynthesis using cell-free protein synthesis

Effectiveness of recombinant Escherichia coli on the production of (R)-(+)-perillyl alcohol

Sustainable Production of Microbial Isoprenoid Derived Advanced Biojet Fuels Using Different Generation Feedstocks: A Review

Contact Info

Product

Resources

About