Stoichiometric Conversion of Cellulosic Biomass by in Vitro Synthetic Enzymatic Biosystems for Biomanufacturing

Abstract

Cellulosic biomass is the earth’s most abundant renewable resource, which is considered to be a promising feedstock for manufacturing biofuels and biochemicals. In this study, stoichiometric enzymatic phosphorolysis of cellulosic biomass for manufacturing biochemicals or biofuels by in vitro synthetic enzymatic biosystems was designed. Three cascade phosphorolytic enzymes, cellodextrin phosphorylase, cellobiose phosphorylase, and polyphosphate-dependent glucokinase, were used for the biotransformation of cellodextrins to high-energy phosphorylated sugars (that is, glucose 1-phosphate and glucose 6-phosphate). A series of downstream exergonic reactions then converted these high-energy phosphorylated sugars into myo-inositol, resulting in a near-stoichiometric conversion of cellodextrins with a high product yield of 98% (w/w). Moreover, this enzymatic biosystem can even work for the acid-treated biomass hydrolysate containing microorganism-toxic compounds. The construction of this in vitro synthetic enzymatic biosystem provided an alternative method for the utilization of cellulosic biomass rather than cellulolytic enzyme hydrolysis to fermentative monomeric sugars followed by microorganism fermentation, showing potentials in the production of biocommodities such as hydrogen, rare sugars, and electricity from cellulosic biomass.

Consolidated bioprocessing of lignocellulose for production of glucaric acid by an artificial microbial consortium

“…Production of D-glucaric acid from myo -inositol (marked with yellow arrows in Fig. 2A), however, is not a desirable approach because the latter is also an industrially value-added chemical 27, 28 . Moreover, so far there has not been relevant reports pertaining to D-glucaric acid production from lignocellulose, though it was considered as one of the top value-added chemicals from biomass 1 .…”

“…The higher titer of D-glucaric acid produced from glucose and myo -inositol than that from glucose suggests that myo -inositol availability is still the rate-liming step for D-glucaric acid production in the engineered S. cerevisiae . Furthermore, myo -inositol itself is a valuable chemical in industry 27, 28 . The production of D-glucaric acid from myo -inositol was not so economically competitive as the direct production from glucose.…”

Characterization of a hyperthermophilic phosphatase from Archaeoglobus fulgidus and its application in in vitro synthetic enzymatic biosystem

“…In vitro synthetic enzymatic biosystem is using several cascade enzymes to implement complicated biotransformation, emerging as a promising biomanufacturing platform for producing some products (such as inositol and rare sugars) better than microbial fermentation (Li et al 2011;Meng et al 2018;. This platform exhibits several advantages, such as high product yields (Opgenorth et al 2016;Zhu et al 2014), fast reaction rate (Karim and Jewett 2016), easy process control (Kim and Zhang 2016;Opgenorth et al 2017), tolerance of toxic compounds (Korman et al 2017), biosystem robustness (Zhu and Zhang 2017), and implementation of non-natural biosynthesis (You et al 2013).…”

Facile synthesis of (−)‐vibo‐quercitol from maltodextrin via an in vitro synthetic enzymatic biosystem

“…The in vitro (cell-free) synthetic enzymatic biosystem features several advantages over microbial fermentation, such as high product yield, fast reaction rate, high flexibility of control, easy product separation, broad reaction condition, and high tolerance to toxic substrates or products, and so on (Dudley et al, 2015;Zhang, 2015). High concentrations of phosphate can precipitate Mg 2+ , which is an essential activator for many enzymes (Meng et al, 2018). The production of VQ from D-glucose can also be implemented by an in vitro enzymatic biosystem containing HK, DOIS, and QUDH.…”

“…The use of ATP in this in vitro enzymatic biosystem is not only expensive but also likely to result in the accumulation of inorganic phosphate even if an ATP generation system is involved. High concentrations of phosphate can precipitate Mg 2+ , which is an essential activator for many enzymes (Meng et al, 2018).…”