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In vitro multi-enzyme molecular machines that follow the designed multi-enzyme pathways, require the rational optimization and adaptation of several purified or partially purified enzyme components, in order to convert certain substrates into target compounds in vitro in an efficient manner. This type of molecular machine is component-based and modularized, so that its design, assembly, and regulation processes are highly flexible. Recently, the advantages of in vitro multi-enzyme molecular machines on the precise control of reaction process and the enhancement of product yield have suggested their great application potential in biomanufacturing. Studies on in vitro multi-enzyme molecular machines have become an important branch of synthetic biology, and are gaining increasing attentions. This article systematically reviews the enzyme component-/module-based construction strategy of in vitro multi-enzyme molecular machines, as well as the research progress on the improvement of compatibility among enzyme components/modules. The current challenges and future prospects of in vitro multi-enzyme molecular machines are also discussed.

(-)-vibo-Quercitol (VQ: 1L-1,2,4/3,5-cyclohexanepentol), a form of deoxyinositol, is an alternative chiral building block in the synthesis of bioactive compounds to control diabetes. In this study, an adenosine triphosphate-free in vitro synthetic enzymatic biosystem composed of five enzymes (including one enzyme for NADH regeneration) was constructed to produce VQ from maltodextrin in one-pot. After optimization of reaction conditions, 7.6 g/L VQ was produced from 10 g/L maltodextrin with a product yield (mol/mol) of 77%, and 25.3 g/L VQ with a purity of 87% was produced from 50 g/L maltodextrin through simple scaling up of this nonfermentative enzymatic biosystem. Therefore, this study provides an economical and environmentally friendly method for the envisioned quercitol biosynthesis., (© 2019 Wiley Periodicals, Inc.)

Many existing in vitro biosystems harness power from the chemical energy contained in substrates and co‐substrates, and light or electric energy provided from abiotic parts, leading to a compromise in atom economy, incompatibility between biological and abiotic parts, and most importantly, incapability to spatiotemporally co‐regenerate ATP and NADPH. In this study, we developed a light‐powered in vitro biosystem for poly(3‐hydroxybutyrate) (PHB) synthesis using natural thylakoid membranes (TMs) to regenerate ATP and NADPH for a five‐enzyme cascade. Through effective coupling of cofactor regeneration and mass conversion, 20 mM PHB was yielded from 50 mM sodium acetate with a molar conversion efficiency of carbon of 80.0 % and a light‐energy conversion efficiency of 3.04 %, which are much higher than the efficiencies of similar in vitro PHB synthesis biosystems. This suggests the promise of installing TMs as a green engine to drive more enzyme cascades. [ABSTRACT FROM AUTHOR]