Cell‐free metabolic engineering: Biomanufacturing beyond the cell

Abstract

Industrial biotechnology and microbial metabolic engineering are poised to help meet the growing demand for sustainable, low-cost commodity chemicals and natural products, yet the fraction of biochemicals amenable to commercial production remains limited. Common problems afflicting the current state-of-the-art include low volumetric productivities, build-up of toxic intermediates or products, and byproduct losses via competing pathways. To overcome these limitations, cell-free metabolic engineering (CFME) is expanding the scope of the traditional bioengineering model by using in vitro ensembles of catalytic proteins prepared from purified enzymes or crude lysates of cells for the production of target products. In recent years, the unprecedented level of control and freedom of design, relative to in vivo systems, has inspired the development of engineering foundations for cell-free systems. These efforts have led to activation of long enzymatic pathways (>8 enzymes), near theoretical conversion yields, productivities greater than 100 mg L−1 hr−1, reaction scales of >100L, and new directions in protein purification, spatial organization and enzyme stability. In the coming years, CFME will offer exciting opportunities to (i) debug and optimize biosynthetic pathways, (ii) carry out design-build-test iterations without re-engineering organisms, and (iii) perform molecular transformations when bioconversion yields, productivities, or cellular toxicity limit commercial feasibility.

Synthetic biology devices for in vitro and in vivo diagnostics

“…In this approach, commercially available cell-free transcription/translation systems (bacterial or mammalian) are freeze-dried onto paper or other porous substrates to create poised genetic regulatory networks that are stable for long-term storage at room temperature and are activated by rehydration. This work follows on other efforts in cell-free synthetic biology, which have provided important dynamic and mechanistic insight on gene regulatory networks and allowed for rapid "build-test" cycles for prototyping engineered gene circuits and biomanufacturing pathways (36)(37)(38)(39)(40)(41). The in vitro nature of these systems resolves the challenge faced by cell-based approaches of importing biomolecular components into the intracellular space, making these cell-free environments easily modified and excellent platforms for engineering.…”

Editorial: Methods and Advances – Biotech progress for science and our daily lives

“…While genetically modified food are still waiting to receive public acceptance, also life stock can be genetically modified [2]. More accepted is the biotech progress on biofuels and chemical production, i.e., processing raw materials, generating completely new chemical reactions [3], as well as economically viable and environmentally sustainable processes. One example is the production of biodiesel, where lipase is used for the transesterification reaction between fatty acids and shortchain alcohol.…”

“…The use of metabolic engineering to further improve reaction cascades has opened completely new possibilities for discovery in the field of biocatalysis. Dudley et al [3] review recent developments in cellfree metabolic engineering to overcome certain limitations of microbial metabolic engineering, they give an impressive insight how synthetic biology and metabolic engineering contribute to further development in this field. Microbial metabolic engineering research [3] has been blossoming over the recent years towards the goal of establishing sustainable bio-based chemical industry.…”

“…Dudley et al [3] review recent developments in cellfree metabolic engineering to overcome certain limitations of microbial metabolic engineering, they give an impressive insight how synthetic biology and metabolic engineering contribute to further development in this field. Microbial metabolic engineering research [3] has been blossoming over the recent years towards the goal of establishing sustainable bio-based chemical industry. In addition to its value for optimizing biosynthetic pathways, one of the promising applications includes bioconversion to produce such products that cause cellular toxicity [3].…”

“…Microbial metabolic engineering research [3] has been blossoming over the recent years towards the goal of establishing sustainable bio-based chemical industry. In addition to its value for optimizing biosynthetic pathways, one of the promising applications includes bioconversion to produce such products that cause cellular toxicity [3].…”