Lignocellulose is the most abundant renewable biomass resource on Earth, and its efficient conversion is a part of the circular bioeconomy [1]. Most times, lignocellulose is hydrolyzed into glucose by a synergistic action of three major cellulases, i.e., cellobiohydrolases, endoglucanases, and β-glucosidases, followed by microbial fermentation to produce biofuels or value-added chemicals [2]. Another promising strategy employs cellobiose phosphorylase (CBP, cellobiose: orthophosphate α-D-glucosyl transferase, EC 2.4.1.20) to utilize cellobiose for the ATP-free generation of α-D-glucose-1-phosphate (G1P) that can be converted to starch [3], [4], myo-inositol [5], [6], [7], D-tagatose [8], D-allulose [9]. Therefore, CBP has an important role in the phosphorylation of cellobiose in anaerobic cellulolytic bacteria for ATP conservation [10], [11], [12]. This mechanism has been explored to construct non-cellulolytic ethanol-producing microbes with better cellobiose metabolism and more energetic advantages [13], [14], [15], [16].

In vitro, based on its reversible phosphorolysis activity, CBP has emerged as a key enzyme in in vitro BioTransformation (ivBT) - an emerging biomanufacturing platform for the production of value-added biochemicals and biocommodities (i.e., low-value and mass-scale biochemicals) [17], [18]. Representative applications of phosphorolysis include: high-yield hydrogen production from cellodextrins [19], [20] as well as synthesis of artificial starch [3], [4], [21], γ-cyclodextrin [22], and myo-inositol [6], [7]. Beyond phosphorolysis, CBP’s reverse synthesis capability enables cellobiose production from sucrose [23] or starch [24]. Furthermore, CBP exhibits the transglycosylation activity, facilitating the synthesis of β-glucosides [25] such as long-chain alkyl β-glucosides [26], [27], [28].

A few CBPs have been reported in several microorganisms [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45] and the biochemical properties of CBPs are shown in Table 1. However, many of CBPs have suffered from several limitations, including poor soluble expression, incompatibility between catalytic activity and stability, and inadequate industrial applicability. Furthermore, the majority demonstrated a narrow pH range, predominantly in the near-neutral region. Enzymes with high expression, high thermostability, high catalytic efficiency and high economic potential are highly desired for the ivBT platform [17]. Therefore, gene mining, characterization, and application of more novel CBPs are crucial to meet industrial requirements for this emerging biomanufacturing platform.

In this study, we identified a novel cellobiose phosphorylase from Thermoclostridium caenicola (TcCBP) and established its heterologous expression system in Escherichia coli BL21(DE3). The recombinant enzyme was subsequently purified and functionally characterized. Leveraging the biochemical properties, TcCBP as well as three other core enzymes, i.e., phosphoglucomutase (PGM), inositol 1-phosphate synthase (IPS), and inositol monophosphatase (IMP), and another auxiliary enzyme, i.e., polyphosphate glucokinase (PPGK), were mixed and applied to the biosynthesis of myo-inositol from cellobiose via a reported in vitro enzymatic biosystem [6] without step-by-step addition of enzymes.