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Stoichiometric conversion of cellulosic biomass by in vitro synthetic enzymatic biosystems for biomanufacturing Dongdong Meng, Xinlei Wei, Yi-Heng P. Job Zhang, Zhiguang Zhu, Chun You, and Yanhe Ma ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b02473 • Publication Date (Web): 05 Sep 2018 Downloaded from http://pubs.acs.org on September 5, 2018
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ACS Catalysis
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Stoichiometric Conversion of Cellulosic Biomass by in Vitro Synthetic Enzymatic
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Biosystems for Biomanufacturing
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Dongdong Meng, Xinlei Wei, Yi-Heng P. Job Zhang, Zhiguang Zhu, Chun You*, Yanhe Ma
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Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th
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Avenue, Tianjin Airport Economic Area, Tianjin 300308, People’s Republic of China
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*Corresponding author: Chun You, Tianjin Institute of Industrial Biotechnology, Chinese
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Academy
of
Sciences,
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[email protected] Tianjin
300308,
China.
Tel.:
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E-mail:
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Abstract:
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Cellulosic biomass is the earth's most abundant renewable resource, which is considered to
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be a promising feedstock for manufacturing biofuels and biochemicals. In this study,
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stoichiometric enzymatic phosphorolysis of cellulosic biomass for manufacturing
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biochemicals or biofuels by in vitro synthetic enzymatic biosystems was designed. Three
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cascade phosphorolytic enzymes, cellodextrin phosphorylase, cellobiose phosphorylase and
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polyphosphate-dependent glucokinase, were used for the biotransformation of cellodextrins
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to high-energy phosphorylated sugars (that is, glucose 1-phosphate and glucose 6-phosphate).
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A series of downstream exergonic reactions then converted these high-energy phosphorylated
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sugars into myo-inositol resulted in a near-stoichiometric conversion of cellodextrins with a
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high product yield of 98% (wt/wt). Moreover, this enzymatic biosystem can even work for
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the acid-treated biomass hydrolysate containing microorganism-toxic compounds. The
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construction of this in vitro synthetic enzymatic biosystem provided an alternative method for
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the utilization of cellulosic biomass rather than cellulolytic enzyme hydrolysis to
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fermentative monomeric sugars followed by microorganism fermentation, showing potentials
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in the production of biocommodities like hydrogen, rare sugars, and electricity from
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cellulosic biomass.
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Keywords: cellulosic biomass; cellodextrin; phosphorolysis; myo-inositol; in vitro synthetic
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enzymatic biosystem
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Introduction
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In vitro synthetic enzymatic biosystems are constituted of numerous purified/partially
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purified enzymes and/or coenzymes to implement complicated biotransformations, and has
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been regarded as the next-generation biomanufacturing platform.1-13 Compared with
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microbial fermentation which is the currently dominant biomanufacturing platform, in vitro
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synthetic enzymatic biosystems exhibit several advantages, including high product yield,14
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fast reaction rate,13 great engineering flexibility in terms of enzyme choices and reaction
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conditions,15 high tolerance to microorganism-toxic environments,16 and easy scale up.11, 17
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Many in vitro enzymatic synthetic biosystems have been demonstrated with near theoretical
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yields, such as hydrogen and bioelectricity generation from various sugars,5,
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1,3-propandiol19 and lactate20 production from glycerol, α-ketoglutarate production from
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D-glucuronate,1 fructose 1,6-phosphate production from starch and pyrophosphate,9 and
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myo-inositol production from starch.11
12-13, 18
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Cellulose, a natural biopolymer comprised of anhydroglucose units, is the most abundant
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renewable bioresource. It is available from domestic waste, agricultural and forestry residues
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to energy plants.21 Cellulose has been suggested as a sustainable resource for the production
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of biochemicals.22-23 Over the past several decades, many studies have focused on the
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production of biofuels like ethanol from cellulosic materials through a series of process steps,
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which include: (1) biomass pretreatment to break up the recalcitrance of biomass; (2)
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enzymatic hydrolysis of pretreated cellulose to glucose by cellulolytic enzymes including
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endoglucanase (EC 3.2.1.4), cellobiohydrolase (EC 3.2.1.91), and β-glucosidase (EC
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3.2.1.21); (3) fermentation of glucose by engineered microorganisms.22 The utilization of 3
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pretreated cellulose is the combination of in vitro enzymatic biotransformation and in vivo
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microbial fermentation. Similar to cellulose, starch, which is the second abundant glucose
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polymer linked by alpha-1,4-glycosidic bonds and alpha-1,6-glycosidic bonds, can also be
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hydrolyzed to glucose by α-amylase (EC 3.2.1.1), pullulanase (EC 3.2.1.41), as well as
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glucoamylase (EC 3.2.1.3), and then undergo glucose fermentation.24 Alternatively, starch
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can be directly used as the starting feedstock in many in vitro synthetic enzymatic biosystems,
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in which the first step is starch phosphorolysis by α-glucan phosphorylase (EC 2.4.1.1) to
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glucose 1-phosphate (G1P).5, 12-13, 18 The production of myo-inositol from starch through an in
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vitro synthetic enzymatic pathway has been implemented on an industrial scale of 20,000
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liters.11 Because the annual resource of cellulosic materials is ∼40 times greater than starch
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produced by crops cultivated for food and feed, effective utilization cellulose for
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biomanufacturing via an in vitro synthetic enzymatic biosystem would be of significant
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importance.
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Recently, cellulose has been converted to produce starch by an in vitro enzymatic synthetic
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biosystem plus microbial fermentation.25 In this in vitro enzymatic pathway, cellulose was
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hydrolyzed to cellobiose instead of glucose by the synergistic action of endoglucanase and
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cellobiohydrolase without β-glucosidase. Then, cellobiose was phosphorylated by cellobiose
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phosphorylase (CBP, EC 2.4.1.20) to generate an equimolar mixture of G1P and glucose in
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the presence of inorganic phosphate. G1P was taken up by α-glucan phosphorylase to
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elongate the chain of starch and to release inorganic phosphate, whereas glucose was
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assimilated by yeast for the production of ethanol to eliminate glucose inhibition. About 30%
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of the anhydroglucose units in cellulose were converted to starch. The design principle of this 4
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in vitro synthetic enzymatic biosystem can be applied to other in vitro biosystems utilizing
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cellulose for the production of hydrogen,26 bioelectricity,13 and biochemicals such as
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myo-inositol. However, these in vitro enzymatic biosystems suffer from low production
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yields because (i) it is difficult to efficiently hydrolyze cellulose to cellobiose by
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β-glucosidase-free cellulases,27 and (ii) a half of the glucose unit of cellobiose can be
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converted to G1P by CBP, whereas the other half of cellobiose was released as glucose.
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Although glucose can be converted to glucose 6-phosphate (G6P) by hexokinase (EC 2.7.1.1)
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or polyphosphate-dependent glucokinase (PPGK, EC 2.7.1.63), using ATP or polyphosphate
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as the phosphate donor,18 respectively, such high concentrations of polyphosphate or ATP
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used in in vitro biosystems would result in increased production costs and the accumulation
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of undesired phosphate ions in the reaction systems when producing phosphate-free
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products.28 In addition, high concentrations of released phosphate ions and polyphosphate
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itself can precipitate divalent ions like magnesium to decrease apparent activities of
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Mg2+-dependent enzymes.9, 29 Therefore, it is difficult to achieve the stoichiometric utilization
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of cellulose by in vitro enzymatic biosystems involving the hydrolysis of cellulose to
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cellobiose by cellulases.
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In this study, stoichiometric conversion of cellulose into myo-inositol (called inositol later)
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was implemented by an ATP-free non-natural in vitro synthetic enzymatic biosystem without
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using cellulases (Figure 1). Cellodextrins were prepared from cellulose by pretreatment and
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acid hydrolysis.30 Subsequently, three enzymes, cellodextrin phosphorylase (CDP, EC
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2.4.1.49), CBP, and PPGK, were used for the stoichiometric phosphorylation of cellodextrins
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to G1P and G6P. Three additional enzymes, phosphoglucomutase (PGM, EC 5.4.2.2), inositol 5
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1-phosphate synthase (IPS, EC 5.5.1.4), and inositol monophosphatase (IMP, EC 3.1.3.25),
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which catalyze a series of exergonic reactions, were introduced to convert all the G1P and
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G6P into inositol, resulting in the production of stoichiometric amounts of inositol from
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cellulose. In this biosystem, the longer degree of polymerization (DP) of cellodextrins allows
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more bonds to be conserved by CDP, resulting in higher theoretical yields of G1P and
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decreased use of CBP, PPGK, and polyphosphate. In this study, the weight ratio of released
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glucose to initial cellulose input was much lower than 50% based on the DP value of
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cellodextrins, resulting in a decreased requirement of high energy phosphate donor, a reduced
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amount of released phosphate ions, and less precipitation of divalent metal ions. In addition, a
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high concentration of cellodextrins in microorganism-toxic biomass hydrolysate31 were tested
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in this study for the production of inositol. This strategy for the phosphorolysis of cellulose
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provides an alternative strategy for the utilization of cellulosic biomass, showing great
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potentials in producing biocommodities like hydrogen, rare sugar, and electricity from this
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widespread material.
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Materials and Methods
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Chemicals
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All chemicals were reagent grade and purchased from Sinopharm (Shanghai, China) or
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Sigma-Aldrich (St. Louis, MO, USA), unless otherwise noted. Cello-oligosaccharides
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(cellotriose, G3; cellotetraose, G4; cellopentaose, G5; cellohexaose, G6) were purchased
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from Aladdin (Shanghai, China). Microcrystalline cellulose Avicel PH-105 was purchased
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from FMC (Philadelphia, PA, USA). Cellodextrins were prepared by mixed-acid hydrolysis 6
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from Avicel PH-105 as previous reported with a little modification.30 Twenty gram of Avicel
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PH-105 was mixed with 160 mL ice-cold HCl and 40 mL ice-cold H2SO4 for 4 hours under
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22°C, followed by adding 200 mL distilled water gently. After centrifugation at 10000 g for
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10 min, soluble cellodextrins in the supernatant was precipitated from the hydrolysate using
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1.8 L of cold acetone. After vacuum filtration, the filter cake was washed in 400 mL of
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acetone twice more. Dry precipitates were dissolved in distilled water and the supernatant
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was neutralized to pH 7 using Ba(OH)2 to obtain pure cellodextrins. Biomass hydrolysate was
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prepared from corn stover as described below. Corn stover was steam-exploded at 1.0 MPa
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for 10 min. Then, twenty gram of pretreated dry biomass (particle size >60 mesh and
