Expression of a recombinant Lentinula edodes cellobiohydrolase by Pichia pastoris and its effects on in vitro ruminal fermentation of agricultural straws

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Abstract

An experiment was conducted to determine the effects of recombinant cellobiohydrolase on the hydrolysis and in vitro rumen microbial fermentation of agricultural straws including rice straw, wheat straw, and corn straw. The cellobiohydrolase from Lentinula edodes (LeCel7A) was produced in Pichia pastoris. The optimal temperature and pH for LeCel7A were 60 °C and 5.0, respectively. The recombinant protein enhanced the hydrolysis of three straws. During in vitro rumen fermentation of three straws, the fiber digestibility, concentration of acetate and total volatile fatty acids, and fermentation liquid microbial protein were increased by LeCel7A. High throughput sequencing and real-time PCR data showed that the effects of LeCel7A on ruminal microbial community depended on the fermentation substrates. The relative abundances of Prevotellaceae_UCG_003 and Saccharofermentans were increased by LeCel7A regardless of agricultural straws. With rice straw, LeCel7A increased the relative abundances of Desulfovibrio, Ruminococcaceae and its some genus. With wheat straw, LeCel7A increased the relative abundances of Succiniclasticum, Ruminococcus flavefaciens, and Ruminococcus albus. With corn straw, Succiniclasticum, Christensenellaceae_R_7_group and Desulfovibrio were increased by LeCel7A. This study demonstrates that LeCel7A could enhance the hydrolysis and in vitro ruminal fermentation of agricultural straws, showing the potential of LeCel7A for improving the utilization of agricultural straws in ruminants.

Introduction

Agricultural by-products, for instance rice straw, wheat straw, and maize straw, are generated in billions of tons per year all around the world [1]. These fibrous materials contain considerable quantities of cellulose and hemicellulose and have the potential to be a valuable feed source for ruminants. However, though these straws could be utilized by ruminants through the ruminal microorganisms [2], their nutritive feeding value is limited by their high-order molecular packing of lignocelluloses [3]. Consequently, the majority of these agricultural straws are as wastes either left in the field for natural decay or burnt adding to environmental pollution [4]. In past years, some physical and chemical technologies such as steaming, alkaline, and acidic treatments have been investigated to improve the nutritional value of agricultural straws [5,6]. However, the application of these methods presents many disadvantages, including high energy consumption [7] and high risk to the animal and environment, especially when alkali used [8]. Biological treatments, including the use of white rot fungi and their enzyme, have the potential to eliminate/reduce the problems associated with physicochemical methods and appear to be the most promising in improvement of straws digestibility [4,9].

As one of white-rot fungi, Lentinula edodes, commonly referred to as the Shiitake mushroom, produces all the core enzymes essential to the complete enzymatic hydrolysis of lignocellulose [10]. L. edodes can use its enzymatic machineries to break down lignocellulose and improve nutritive value of low quality feeds, such as rape straw, wheat straw, rice straw, corn stover, and sugarcane bagasse, but also resulted in the great losses of cellulose and hemicellulose during the degradation, which limits its practical use [4,11,12]. In addition, considering that the long incubation time of L. edodes and its cultivation products may contain toxic substances [6], crude enzyme extracts of L. edodes as feed additive may be also not an attractive option for treatment of straws. However, at least it means that L. edodes is a good source for fibrolytic enzymes gene pool. Using heterologous expression technology to obtain the enormous amount of interest enzyme protein within a short time may be an easy way worthy of consideration.

From the perspective of the best-studied enzyme systems, the heart of depolymerization of cellulose to glucose comprises cellobiohydrolases, endoglucanases and β-glucosidase [13]. Cellobiohydrolases (also called exoglucanases) hydrolyze the crystalline parts of the substrate by initiating their action from the reducing or non-reducing ends of the cellulose chains, producing primarily cellobiose and decreasing the substrate polymerization degree very slowly [14,15]. Cellobiohydrolases are key components in the multi-enzyme cellulose complexes. Many studies have been conducted to investigate the functional characterization of cellobiohydrolases and its potential synergistic role in the enzymatic hydrolysis of biomass. Some results showed that cellobiohydrolases from Trichoderma reesei displayed a high synergistic effect with cellulase and xylanase and enhanced the enzymatic hydrolysis of corn stover, rice straw, and wheat straw [16,17]. Within the rumen of ruminants, a complex group of anaerobic microorganisms, including bacteria, archaea and eukaryotes, produces a vast array of lingo-cellulolytic enzymes, which means that the rumen can provide an enzyme-containing environment for the synergistic action and boosting activity of cellobiohydrolases. Lee et al. cloned and characterized two cellobiohydrolases genes from L. edodes, one of which belonged to glycosyl hydrolase family 7 (LeCel7A) [10]. Based on the effects of L. edodes on agricultural straws degradation aforementioned and characteristics of rumen, we hypothesize that supplementation of LeCel7A in the rumen could enhance the hydrolysis and ruminal fermentation of agricultural straws, however, little information is available. Therefore, this study recombined, expressed and purified the LeCel7A by P. pastoris, investigated the effects of recombinant LeCel7A on the hydrolysis of agricultural straws, and evaluated the effects of recombinant LeCel7A on in vitro ruminal fermentation and microbial community of agricultural straws and consequently the application possibility of LeCel7A in the utilization of agricultural straws by ruminants.

Section snippets

Materials and methods

This study was approved by the Animal Care and Use Committee of the College of Animal Science and Technology of the Jiangxi Agricultural University.

Production and analysis of LeCel7A

For the production of LeCel7A, the LeCel7A gene from L. edodes was optimized for the preferred codon usage and expressed in P. pastoris in the present study. The native LeCel7A includes >19% codons, such as GGC (Gly), UGU (Cys), and AGC (Ser), which share <10‰ usage in P. pastoris. These rare codons were replaced by preferred ones which were more frequently used in P. pastoris. In addition, an algorithm (GenScript Nanjing Co., Ltd., Nanjing, China) with parameter settings was used to optimize

Conclusions

The LeCel7A was expressed and produced using P. pastoris and shown maximum activity at 60 °C and pH 5.0. The LeCel7A could improve the hydrolysis and in vitro ruminal fermentation of agricultural straws by increasing the digestion of fiber, changing the microbial community, stimulating the growth of fibrolytic bacteria and production of VFA, as well as increased rumen microbial protein synthesis. The LeCel7A might be useful for improving the utilization of agricultural straws as a feed additive

Funding

This work was supported by National Key Research and Development Program of China (2018YFD0501804), the National Natural Science Foundation of China (31760687), and the National Beef Cattle Industry Technology & System (CARS-Beef Cattle System: CARS-38).

Declaration of Competing Interest

The authors declare no conflict of interest.

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