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Long-term strain improvements accumulate mutations in regulatory elements responsible for hyper-production of cellulolytic enzymes.

ABSTRACT: Long-term strain improvements through repeated mutagenesis and screening have generated a hyper-producer of cellulases and hemicellulases from Penicillium decumbens 114 which was isolated 30 years ago. Here, the genome of the hyper-producer P. decumbens JU-A10-T was sequenced and compared with that of the wild-type strain 114-2. Further, the transcriptomes and secretomes were compared between the strains. Selective hyper-production of cellulases and hemicellulases but not all the secreted proteins was observed in the mutant, making it a more specific producer of lignocellulolytic enzymes. Functional analysis identified that changes in several transcriptional regulatory elements played crucial roles in the cellulase hyper-producing characteristics of the mutant. Additionally, the mutant showed enhanced supply of amino acids and decreased synthesis of secondary metabolites compared with the wild-type. The results clearly point out that we can target gene regulators and promoters with minimal alterations of the genetic content but maximal effects in genetic engineering.

SUBMITTER: Liu G

PROVIDER: S-EPMC3610096 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Long-term strain improvements accumulate mutations in regulatory elements responsible for hyper-production of cellulolytic enzymes.

Liu Guodong G Zhang Lei L Qin Yuqi Y Zou Gen G Li Zhonghai Z Yan Xing X Wei Xiaomin X Chen Mei M Chen Ling L Zheng Kai K Zhang Jun J Ma Liang L Li Jie J Liu Rui R Xu Hai H Bao Xiaoming X Fang Xu X Wang Lushan L Zhong Yaohua Y Liu Weifeng W Zheng Huajun H Wang Shengyue S Wang Chengshu C Xun Luying L Zhao Guo-Ping GP Wang Tianhong T Zhou Zhihua Z Qu Yinbo Y

Scientific reports 20130101

Long-term strain improvements through repeated mutagenesis and screening have generated a hyper-producer of cellulases and hemicellulases from Penicillium decumbens 114 which was isolated 30 years ago. Here, the genome of the hyper-producer P. decumbens JU-A10-T was sequenced and compared with that of the wild-type strain 114-2. Further, the transcriptomes and secretomes were compared between the strains. Selective hyper-production of cellulases and hemicellulases but not all the secreted protei ...[more]

PMID: 23535838

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Project description:BACKGROUND:The transition from a petroleum-based economy towards more sustainable bioprocesses for the production of fuels and chemicals (circular economy) is necessary to alleviate the impact of anthropic activities on the global ecosystem. Lignocellulosic biomass-derived sugars are suitable alternative feedstocks that can be fermented or biochemically converted to value-added products. An example is lactic acid, which is an essential chemical for the production of polylactic acid, a biodegradable bioplastic. However, lactic acid is still mainly produced by Lactobacillus species via fermentation of starch-containing materials, the use of which competes with the supply of food and feed. RESULTS:A thermophilic and cellulolytic lactic acid producer was isolated from bean processing waste and was identified as a new strain of Bacillus coagulans, named MA-13. This bacterium fermented lignocellulose-derived sugars to lactic acid at 55 °C and pH 5.5. Moreover, it was found to be a robust strain able to tolerate high concentrations of hydrolysate obtained from wheat straw pre-treated by acid-catalysed (pre-)hydrolysis and steam explosion, especially when cultivated in controlled bioreactor conditions. Indeed, unlike what was observed in microscale cultivations (complete growth inhibition at hydrolysate concentrations above 50%), B. coagulans MA-13 was able to grow and ferment in 95% hydrolysate-containing bioreactor fermentations. This bacterium was also found to secrete soluble thermophilic cellulases, which could be produced at low temperature (37 °C), still retaining an optimal operational activity at 50 °C. CONCLUSIONS:The above-mentioned features make B. coagulans MA-13 an appealing starting point for future development of a consolidated bioprocess for production of lactic acid from lignocellulosic biomass, after further strain development by genetic and evolutionary engineering. Its optimal temperature and pH of growth match with the operational conditions of fungal enzymes hitherto employed for the depolymerisation of lignocellulosic biomasses to fermentable sugars. Moreover, the robustness of B. coagulans MA-13 is a desirable trait, given the presence of microbial growth inhibitors in the pre-treated biomass hydrolysate.

Project description:BackgroundThe major challenge of facing the efficient utilization of biomass is the high cost of cellulolytic enzyme, while the Trichoderma longibrachiatum plays an essential role in the production of industrial enzymes and biomass recycling.ResultsThe cellulase hyper‑producing mutants of LC-M4 and LC-M16 derived from the wild type T. longibrachiatum LC strain through heavy ion mutagenesis exhibited the high-efficiency secretion ability of cellulase and hemicellulose. The FPase activities of LC-M4 (4.51 IU/mL) and LC-M16 (4.16 IU/mL) mutants increased by 46.91% and 35.5% when compared to the LC strain, respectively. Moreover, these two cellulase hyper-producing mutants showed faster growth rate on the cellulosic substrates (Avicel and CMC-Na) plate than that of LC strain. Therefore, an integrative transcriptome and proteome profiling analysis of T. longibrachiatum LC and its cellulase hyper‑producing mutant LC-M4 and LC-M16 were employed to reveal the key genes involved in cellulolytic enzymes regulation. It was showed that the transcriptome and proteome profiles changed dramatically between the wild strain and mutant strains. Notably, the overlapped genes obtained from integrative analysis identified that the protein processing in ER involved in protein secretory pathway, starch and sucrose metabolism pathway and N-glycan biosynthesis pathway were significantly changed both in cellulase hyper-producing mutants and thereby improving the enzyme secretion efficiency, which maybe the main reason of cellulase hyper-production in LC-M4 and LC-M16 mutants. In addition, the three DEGs/DEPs (PDI, Sec61, VIP36) related with protein secretion in ER and two DEGs/DEPs (OST, MOGS) related with N-glycan biosynthesis were identified as key candidate genes participating in enzyme protein biosynthesis and secretion.ConclusionsIn this study, a hypothetical secretory model of cellulase protein in filamentous fungi was established on the basis of DEGs/DEPs and key genes identified from the omics analysis, which were of great guidance on the rational genetic engineering and/or breeding of filamentous fungi for improving cellulase production.

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Long-term strain improvements accumulate mutations in regulatory elements responsible for hyper-production of cellulolytic enzymes.

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Long-term strain improvements accumulate mutations in regulatory elements responsible for hyper-production of cellulolytic enzymes.

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