Biosynthesis Of Acetoin By Thermophilic Enzymatic Hydrolysis Of Natural Xylan

Xylan,the most abundant hemicellulosic constituent of plant cell wall,has been considered as an abundant source of fermentable pentose(mainly xylose and arabinose)for biofuel and bio-chemical production by biorefinery.Acetoin(3-hydroxy-2-butanone)is a promising bio-based platform chemical with wide applications in food,chemical synthesis and multifunctional material.In present study,the enzymatic hydrolysis model of the natural biomass utilizing extremely thermophilic anaerobic bacterium Caldicellulosiruptor lactoaceticus 6A(C.lac)for natural xylan degradation was resolved,and the in vitro thermophilic enzymatic system for natural xylan degradation was constructed thereafter.Thermophilic Bacillus subtilis which can convert pentose and hexose simultaneously to acetoin was selected,and production of acetoin from natural biomass was realized through thermophilic simultaneous saccharification and fermentation(SSF).In addition,the cell-free enzyme catalytic biosystems for acetoin synthesis were constructed in vitro based on the metabolic pathway and recombinant enzymes.The results were summarized in the following three parts:1.The thermophilic enzymatic hydrolysis model of extremely thermophilic bacterium C.lac on natural xylan degradation was resolved:(1)Characterization of the synergy of intracellular xylanolytic glycoside hydrolases(GHs)and reconstruction of enzyme hydrolytic system for natural degradation in vitro:The intracellular xylanolytic GHs,GH10 ?-1,4-endo-xylanase(Xyn10A),GH51 ?-L-arabinofuranosidase(Abf51A)and GH67 a-glucuronidase(Agu67A),were cloned,heterologously expressed,and characterized.Natural xylan was degraded by the synergistic enzyme system under the optimal activity condition at 75-80? and pH 5.5-6.5.Xyn10A acted on the backbone of xylan polymer,while Abf51A and Agu67A removed the L-arabinofuranosyl and 4-O-methyl-glucuronic acid side-chains of xylan,respectively.Therefore,synergistic application of these three enzymes significantly improved the amounts of xylose,xylobiose,small molecular oligosaccharides and arabinose in the end products of xylan.(2)Characterization of the molecular mechanism of extracellular multidomain?-1,4-endo-xylanase:The extracellular GH10 ?-1,4-endo-xylanase(Xyn10B)contained CBM22/GH10/CBM9/SLH architecture.The truncated derivatives of Xyn10B with different module assembly modes displayed notable variation in thermostability,substrate specificity,and hydrolytic activity.The results indicated that CBM22s especially CBM22c promoted both thermostability and catalytic efficiency,while these positive effects were weakened with the presence of CBM9.2.Selection of thermotolerant acetoin producer,and bioconverting biomass to acetoin through thermophilic SSF process:(1)Selection of thermotolerant acetoin producer and optimization of fermentation process:A thermotolerant(up to 52?)acetoin producer B.subtilis IPE5-4-UD-4 was isolated by compound mutagenesis.By single factor and orthogonal optimization of the medium components and fermentation conditions,the mutant produced 26.09 ± 1.01 g/L acetoin at 37? with glucose as feedstock in shake flask fermentation.When fermenting at 37? in a 5-L bioreactor,the acetoin concentration reached the maximum of 48.09 ± 0.63 g/L with feeding 400 g glucose at a flow rate of 8 g/h.(2)Study of the fermentation capacity of strain IPE5-4-UD-4 at high temperature:Using the resting cells,a concentration of 2.70±0.02 mM acetoin was generated from 10 mM glucose at 50? for 24 h.Besides,the acetoin concentration of 17.78 ±0.19 g/L was achieved by IPE5-4-UD-4 in shake flask at 50? with the mixture of glucose and xylose as substrate,and the acetoin yield and productivity came to 0.38 g/g mixed sugar and 0.25 g/L/h,respectively.When fermented at 50? in a 5-L bioreactor with glucose as feedstock by IPE5-4-UD-4,the acetoin concentration reached 28.83 ±0.65g/L with the acetoin yield and productivity of 0.34 g/g glucose and 0.60 g/L/h,respectively.(3)Constructing the thermophilic SSF process for acetoin production from alkali pretreated corncob(APC):Thermophilic SSF process was optimized and constructed by using commercial cellulase and thermophilic xylan enzymatic system and strain IPE5-4-UD-4 with APC as feedstock at 50?.An acetoin concentration of 12.55 ±0.28 g/L was achieved in shake flask SSF,and the acetoin yield and productivity came to 0.25 g/g APC and 0.17 g/L/h,respectively.Meanwhile,the utilization of cellulose and hemicellulose in the SSF approach reached the maximum of 96.34%and 93.29%,respectively.When further fermented at 50? in a 5-L bioreactor for 60 h,22.76 ±1.16 g/L acetoin was obtained,the acetoin yield and productivity reached 0.46 g/g APC and 0.38 g/L/h,respectively.This was by far the highest acetoin yield from lignocellulosic biomass in SSF.3.Analysis of enzyme catalytic process of acetoin and(R)-acetoin at room or high temperature based on the microbial D-xylose and D-glucose metabolism,and construction of cell-free biosystems for acetoin and(R)-acetoin synthesis in vitro:(1)Thermophilic cell-free enzymatic synthesis of acetoin from pyruvate:Based on the metabolic pathway starting from pyruvate,a thermophilic cascade cell-free enzymatic reaction for acetoin synthesis was reconstructed in vitro.Two thermophilic catalytic elements were involved,and neither ATP nor coenzyme I was required.Recombinant thermophilic enzymes were cloned,heterologously expressed,and characterized from Caldicellulosiruptor owensensis OL and B.subtilis IPE5-4,respectively.Under optimal condition at 65? and pH 6.5,a maximum concentration of 3.36±0.26 mM acetoin corresponding to 67.80%of theoretical yield was obtained from 10 mM pyruvate within 24 h cell-free synthesis,and the acetoin productivity and yield reached 0.14 mM/h and 33.92%,respectively.(2)Cell-free enzymatic synthesis of(R)-acetoin from D-xylose:Based on the Dahms metabolic pathway starting from D-xylose,a NAD+-balanced synthetic pathway involving seven-step continuous reaction was constructed to produce(R)-acetoin from D-xylose in vitro.Recombinant enzymes were cloned,heterologously expressed,and characterized from Escherichia coli W3110,B.subtilis shaijiu32 and Caulobacter crescentus CB 2,respectively.Under optimal condition at 30? and pH 7.5,a maximum concentration of 3.17 ± 0.06 mM(R)-acetoin with enantiomeric excess of 99.07%was obtained from 10 mM D-xylose within 24 h cell-free synthesis,and the(R)-acetoin productivity and yield reached 0.13 mM/h and 18.75%,respectively.(3)Thermophilic cell-free enzymatic synthesis of acetoin from D-glucose:Based on the Embdem-Meyerhof-Pamas pathway starting from D-glucose,A NAD+-balanced synthetic pathway involving thirteen-step continuous reaction was also constructed to synthetize acetoin from D-glucose in vitro at high temperature.Recombinant enzymes were cloned,heterologously expressed,and purified from C.owensensis OL,Thermobifida fusca DSM 43792 and B.subtilis IPE5-4,respectively.Using this cell-free enzyme catalytic biosystem,trace amount of acetoin was detected using D-glucose or D-fructose 1,6-bisphosphate as the substrate.Besides,the crude enzyme catalyst of B.subtilis IPE5-4-UD-4 resting cells and the supernatant of crude enzyme catalysts converted D-glucose to acetoin at 50? in vitro,and a maximum concentration of 2.65 ± 0.59 mM acetoin was produced from 10 mM D-glucose by the crude enzyme catalyst of resting cells at 50? for 24 h.