Cell-free metabolic engineering enables selective biotransformation of fatty acids to value-added chemicals

Liu, Yushi; Liu, Wanqiu; Huang, San Yuan; Xu, Huiling; Lu, Haofan; Wu, Changzhu; Li, Jian

doi:10.1016/j.mec.2022.e00217

Cited by 12 publications

(7 citation statements)

References 44 publications

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“…After in vitro reconstitution of the full biosynthetic pathway, cell-free reaction conditions could be rationally optimized by adjusting key parameters such as supplementation of enzyme cofactors to increase the biosynthesis performance, thus giving rise to high productivity. Looking forward, we envision that cell-free biosynthetic platforms will open the door for rapid biosynthesis of valuable compounds as reported recently, including, for example, industrial chemicals, pharmaceutical intermediates, and complex natural products. − ,− …”

Section: Discussionmentioning

confidence: 99%

Cell-Free Biosynthesis of Lysine-Derived Unnatural Amino Acids with Chloro, Alkene, and Alkyne Groups

et al. 2023

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Crude extract-based cell-free expression systems have been used to produce natural products by reconstitution of their biosynthetic pathways in vitro. However, the chemical scope of cell-free synthesized natural compounds is still limited, which is partially due to the length of biosynthetic gene clusters. To expand the product scope, here, we report cell-free biosynthesis of several lysine-derived unnatural amino acids with functional moieties such as chloro, alkene, and alkyne groups. Specifically, five related enzymes (i.e., halogenase, oxidase, lyase, ligase, and hydroxylase) involved in β-ethynylserine biosynthesis are selected for cell-free expression. These enzymes can be expressed in single, in pairs, or in trios to synthesize different compounds, including, for instance, 4-Cl−L-lysine, 4-Cl−allyl-L-glycine, and L-propargylglycine. The final product of γ-L-glutamyl−L-β-ethynylserine (a dipeptide with an alkyne group) can also be synthesized by cell-free expression of the full biosynthetic pathway (i.e., five enzymes). Our results demonstrate the flexibility of cell-free systems, enabling easy regulation and rational optimization for target compound formation. Overall, this work expands not only the type of enzymes (e.g., halogenase) but also the scope of natural products (e.g., terminal-alkyne amino acid) that can be rapidly produced in cell-free systems. With the development of cell-free biotechnology, we envision that cell-free strategies will create a new frontier for natural product biosynthesis.

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Section: Discussionmentioning

confidence: 99%

Cell-Free Biosynthesis of Lysine-Derived Unnatural Amino Acids with Chloro, Alkene, and Alkyne Groups

et al. 2023

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“…Codon-optimized gene sequences of Pal2 and Car are shown in Table S1. pET15b-Sfp was constructed in our previous report . The gene of YjgB (also named as Ahr aldehyde reductase, GenBank: NP_418690.4) was PCR amplified and cloned into pET28a, generating pET28a-YjgB (primers used for PCR: forward, 5′-TGCCGCGCGGCAGC CATATG TCGATGATAAAAAGCTATGCCGC-3′; reverse, 5′-GCGGCCGCAAGCTT GTCGAC TCAAAAATCGGCTTTCAACACCACGC-3′; the restriction sites of Nde I and Sal I are underlined in the two primers, respectively).…”

Section: Materials and Methodsmentioning

confidence: 99%

“…It provides an open environment for in vitro transcription and translation with efficient utilization of substrates, energy, and cofactors, allowing for the high-level production of various proteins. − CFPS also significantly reduces the expression time (in hours) from genes (plasmids or linear DNA templates) to functional enzymes when compared to in vivo expression (in days). With the above features, CFPS-based biotransformation has become a promising means for the production of a wide range of valuable chemicals, such as aromatic alcohol, L-4-nitrotryptophan, fatty aldehydes, terminal-alkyne amino acids, L-theanine, and valinomycin …”

Section: Introductionmentioning

confidence: 99%

“…8−12 CFPS also significantly reduces the expression time (in hours) from genes (plasmids or linear DNA templates) to functional enzymes when compared to in vivo expression (in days). With the above features, CFPS-based biotransformation has become a promising means for the production of a wide range of valuable chemicals, such as aromatic alcohol, 13 L-4-nitrotryptophan, 14 fatty aldehydes, 15 terminal-alkyne amino acids, 16 L-theanine, 17 and valinomycin. 18 Cinnamyl alcohol (cinOH) is a naturally occurring fragrance compound found in the bark of cinnamon trees, which is widely used in the flavor/fragrance and food industries 19 and as the precursor of pharmaceutical intermediates.…”

Section: ■ Introductionmentioning

confidence: 99%

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Combining Cell-Free Expression and Multifactor Optimization for Enhanced Biosynthesis of Cinnamyl Alcohol

Zhang

Liu

Ling

et al. 2023

J. Agric. Food Chem.

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Cell-free expression systems have emerged as a potent and promising platform for the biosynthesis of chemicals by reconstituting in vitro expressed enzymes. Here, we report cell-free biosynthesis of cinnamyl alcohol (cinOH) with enhanced productivity by using the Plackett−Burman experimental design for multifactor optimization. Initially, four enzymes were individually expressed in vitro and directly mixed to reconstitute a biosynthetic route for the synthesis of cinOH. Then, the Plackett−Burman experimental design was used to screen multiple reaction factors and found three crucial parameters (i.e., reaction temperature, reaction volume, and carboxylic acid reductase) for the cinOH production. With the optimum reaction conditions, approximately 300 μM of cinOH was synthesized after 10 h of cell-free biosynthesis. Extending the production time to 24 h also increased the production to a maximum yield of 807 μM, which is nearly 10 times higher than the initial yield without optimization. This study demonstrates that cell-free biosynthesis can be combined with other powerful optimization methodologies such as the Plackett−Burman experimental design for enhanced production of valuable chemicals.

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“…Establishment of the probiotic EcN-based CFPS system CFPS systems have been widely developed for in vitro protein synthesis with the advantages of time-saving, easily manipulable, microscale, high-throughput, and portable. [48] CFPS has also inspired the multidiscipline research fields, including genetic engineering, [49] metabolic engineering, [50][51][52][53] high-throughput screening, [54] pharmaceutical production, [55,56] and education. [57,58] Therefore, a powerful chassis is essential for establishment of CFPS system.…”

Section: 6mentioning

confidence: 99%

Expanding the toolbox of probiotic Escherichia coli Nissle 1917 for synthetic biology

Ba,

Zhang,

et al. 2023

Biotechnology Journal

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Escherichia coli Nissle 1917 (EcN) is a probiotic microbe that has the potential to be developed as a promising chassis for synthetic biology applications. However, the molecular tools and techniques for utilizing EcN remain to be further explored. To address this opportunity, the EcN‐based toolbox was systematically expanded, enabling EcN as a powerful platform for more applications. First, two EcN cryptic plasmids and other compatible plasmids were genetically engineered to enrich the manipulable plasmid toolbox for multiple gene coexpression. Next, two EcN‐based technologies were developed, including the conjugation strategy for DNA transfer, and quantification of protein expression capability. Finally, the EcN‐based applications were further expanded by developing EcN native integrase‐mediated genetic engineering and establishing an in vitro cell‐free protein synthesis (CFPS) system. Overall, this study expanded the toolbox for manipulating and making full use of EcN as a commonly used probiotic chassis, providing several simplified, dependable, and predictable strategies for researchers working in synthetic biology fields.

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Cell-free metabolic engineering enables selective biotransformation of fatty acids to value-added chemicals

Cited by 12 publications

References 44 publications

Cell-Free Biosynthesis of Lysine-Derived Unnatural Amino Acids with Chloro, Alkene, and Alkyne Groups

Cell-Free Biosynthesis of Lysine-Derived Unnatural Amino Acids with Chloro, Alkene, and Alkyne Groups

Combining Cell-Free Expression and Multifactor Optimization for Enhanced Biosynthesis of Cinnamyl Alcohol

Expanding the toolbox of probiotic Escherichia coli Nissle 1917 for synthetic biology

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