Biomanufacturing by in vitro biosystems containing complex enzyme mixtures

Abstract

Microbial fermentation is the current predominant biomanufacturing platform. However, it suffers from low production yields, slow reaction rates, and scaling-up challenges. In vitro enzymatic biosystems are emerging to expand the traditional biotechnological mode by utilizing more than three enzymes for manufacturing the desired product from cheap substrate. In the past few years, numerous proofs of the concept of in vitro biosystems containing complex enzyme mixtures from different groups worldwide have inspired the development of these platforms for biomanufacturing, these biosystems show advantages such as near-theoretical product yields, faster reaction rates, reduced interference from toxic compounds, and unprecedented level of engineering. In this review, several examples of in vitro systems are presented to illustrate these advantages and possible solutions to overcome the remaining challenges are discussed. The continuing decrease in enzyme cost and improvements in enzyme engineering techniques will make in vitro biosystems a comparable biomanufacturing platform for microbial fermentation in the near future.

Conversion of d-glucose to l-lactate via pyruvate by an optimized cell-free enzymatic biosystem containing minimized reactions

“…At present, the l -lactate concentration and productivity of in vitro biosystem cannot be comparable with the fermentative routes. Yet with the development of enzyme engineering and enzyme immobilization, we believe that the production of l -lactate by in vitro synthetic enzymatic biosystem would have a bright prospect [ 3 ].…”

“…Cell-free synthetic enzymatic biosystem is an emerging biomanufacturing platform containing a number of enzymes and coenzymes in a single reaction vessel for the production of chemicals [ [1] , [2] , [3] ]. This biosystem has some obvious advantages compared with traditional microbial fermentation, including high product yields, fast reaction rates, broad reaction conditions, uncomplicated scale up, easy reaction control and optimization [ [4] , [5] , [6] ].…”

“…To date, a variety of products, such as alcohols [ 7 ], organic acids [ 8 ], carbohydrates [ 9 ], isoprene [ 10 ], fine chemicals [ 11 ], bioplastic [ 12 ], hydrogen [ 13 ], and electricity [ 14 ] have been produced by many cell-free synthetic enzymatic biosystems. The design principles for such cell-free synthetic enzymatic biosystems on an industrial scale include using thermophilic enzymes without any cofactor or with a closed cofactor balance at least, minimizing the number of enzymes, and making the last step of the biosystem irreversible for the high product yield [ 3 ].…”

Protein engineering of oxidoreductases utilizing nicotinamide-based coenzymes, with applications in synthetic biology

“…The pathway design principle of the in vitro synthetic biology platform requires balances between the coenzyme supply and consumption as well as their types [100] . The product cost of this platform is crucial for manufacturing biocommodities [12] , [26] , and can be reduced by the utilization of less costly coenzymes and the addition of coenzyme regeneration systems. NAD is preferable to NADP for in vitro synthetic biology because of its lower price [33] , [68] , higher stability [101] , and more regeneration methods [29] , [34] .…”

“…Strictly speaking, in vitro synthetic biology is slightly different from cell-free synthetic biology, where the former is based on the reconstitution of (purified) enzymes, coenzymes and/or other abiotic components (for example, using benzyl viologen as electron mediator for in vitro biohydrogen generation [25] ), and the latter is mainly based on the cell lysates of one or multiple cell cultures. The in vitro synthetic biology platform has some distinctive advantages, such as high product yield, fast reaction rate, highly engineering flexibility, and high tolerance in toxic environments [19] , [20] , [21] , [26] . Recently, the first industrial biomanufacturing example of the cost-effective production of myo-inositol from starch has been demonstrated in China [27] .…”