Furfural-tolerant Zymomonas mobilis derived from error-prone PCR-based whole genome shuffling and their tolerant mechanism

Huang, Suzhen; Xue, Tingli; Wang, Zhiquan; Ma, Yuanyuan; He, Xiaomei; Hong, Jiefang; Zou, Shaolan; Song, Hao; Zhang, Minhua

doi:10.1007/s00253-018-8817-8

Cited by 21 publications

(22 citation statements)

References 46 publications

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“…Errorprone PCR of the RpoD gene (global transcription factor) in Z. mobilis ZM4 resulted in a mutant, ZM4-MF2, which tolerated 3.0 g/L furfural [13]. Mutants F211 and F27, resulting from error-prone PCR-based wholegenome shuffling of Z. mobilis CP4, also survived in 3.0 g/L furfural [14].…”

Section: Introductionmentioning

confidence: 99%

Genome shuffling enhances stress tolerance of Zymomonas mobilis to two inhibitors

Wang

Han

et al. 2019

Biotechnol Biofuels

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Background: Furfural and acetic acid are the two major inhibitors generated during lignocellulose pretreatment and hydrolysis, would severely inhibit the cell growth, metabolism, and ethanol fermentation efficiency of Zymomonas mobilis. Effective genome shuffling mediated by protoplast electrofusion was developed and then applied to Z. mobilis. Results: After two rounds of genome shuffling, 10 different mutants with improved cell growth and ethanol yield in the presence of 5.0 g/L acetic acid and 3.0 g/L furfural were obtained. The two most prominent genome-shuffled strains, 532 and 533, were further investigated along with parental strains in the presence of 7.0 g/L acetic acid and 3.0 g/L furfural. The results showed that mutants 532 and 533 were superior to the parental strain AQ8-1 in the presence of 7.0 g/L acetic acid, with a shorter fermentation time (30 h) and higher productivity than AQ8-1. Mutant 533 exhibited subtle differences from parental strain F34 in the presence of 3.0 g/L furfural. Mutations present in 10 genome-shuffled strains were identified via whole-genome resequencing, and the source of each mutation was identified as either de novo mutation or recombination of the parent genes. Conclusions: These results indicate that genome shuffling is an efficient method for enhancing stress tolerance in Z. mobilis. The engineered strains generated in this study could be potential cellulosic ethanol producers in the future.

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Section: Introductionmentioning

confidence: 99%

Genome shuffling enhances stress tolerance of Zymomonas mobilis to two inhibitors

Wang

Han

et al. 2019

Biotechnol Biofuels

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“…Furfural is another key inhibitor in the cellulosic hydrolysate. The highest concentration of furfural tolerated by Z. mobilis was 3.0 g/L in previous studies [16,31]. Therefore, four different gradient concentrations (1.0, 2.0, 3.0, and 4.0 g/L) of furfural were employed in this study.…”

Section: Biochar Facilitated Ethanol Production Under Furfural Stressmentioning

confidence: 99%

“…Apart from removing inhibitors before fermentation, creating mutants capable of tolerating furfural and acetic acid is efficient for the economic production of cellulosic biofuels [14,15]. However, although several efforts have been applied to improve these inhibitors' tolerance in Z. mobilis, including forward and reverse genetics, so far, the reported highest concentrations of furfural and acetic acid that Z. mobilis could tolerate were 3.0 g/L and 8.0 g/L [10,16], respectively. But further efforts are still needed to meet the requirement of practical lignocellulose biorefinery with high inhibitor contents in the pretreated feedstock, and due to the complex mechanism of furfural and acetic acid stress in Z. mobilis, developing a robust strain will also be a difficult challenge [13][14][15].…”

Section: Introductionmentioning

confidence: 99%

Biochar-mediated enhanced ethanol fermentation (BMEEF) in Zymomonas mobilis under furfural and acetic acid stress

Wang

Dai

et al. 2020

Biotechnol Biofuels

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Background: Pretreatment of lignocellulosic biomass generates different types of inhibitors (e.g., furfural and acetic acid), which could remarkably inhibit subsequent ethanol fermentation. Here, biochar as an additive in the fermentation broth was first applied to enhance ethanol production by Z. mobilis wild-type strain ZM4 in the presence of typical inhibitors. Results:This study showed that the biochar-mediated tolerance to furfural and acetic acid for the strain Z. mobilis ZM4 was the highest reported level, resulting in much higher ethanol productivity under stress conditions than that in non-treated conditions. Further analysis showed that adsorptive detoxification was not the controlling factor for enhanced ethanol production under stress conditions, attributed to its low removal of furfural (< 20%) and incapability of acetic acid removal. When biochar was filtered from the biochar-treated inhibitor-containing broth, it still showed enhanced ethanol production. Furthermore, Z. mobilis immobilized on biochar was also observed. Thus, biochar extracts in the fermentation broth and cell immobilization on biochar might be the controlling factors for enhanced ethanol production under stress conditions. Conclusions:These results indicate that biochar-mediated enhanced ethanol fermentation (BMEEF) might be a promising strategy for ethanol production from lignocellulosic biomass.

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“…For improving Z. mobilis cells against external abiotic stress, several studies adopted top-down or forward approaches. These includes an error-prone PCR based mutagenesis [20], an adaptive laboratory evolution method [21][22][23], genome shu ing [24] and a transposon approach [13].…”

Section: Introductionmentioning

confidence: 99%

Increased salt tolerance in Zymomonas mobilis strain generated by adaptative evolution

Fuchino¹,

Bruheim²

2020

Preprint

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Background Ethanologenic alphaproteobacterium Zymomonas mobilis has been acknowledged as a promising biofuel producer. There have been numerous efforts to engineer this species applicable for an industrial-scale bioethanol production. Although Z. mobilis is robustly resilient to certain abiotic stress such as ethanol, the species is known to be sensitive to saline stress at a mild concentration, which hampers its industrial use as an efficient biocatalyst. To overcome this issue, we implemented a laboratory adaptive evolution approach to obtain salt tolerant Z. mobilis strain.Results During an adaptive evolution, we biased selection by cell morphology to exclude stressed cells. The evolved strains significantly improved growth and ethanol production in the medium supplemented with 0.225 M NaCl. Furthermore, comparative metabolomics revealed that the evolved strains did not accumulate prototypical osmolytes, such as proline, to counter the stress during their growth. The sequenced genomes of the studied strains suggest that the disruption of ZZ6_1149 encoding carboxyl-terminal protease was likely responsible for the improved phenotype.Conclusions The present work successfully generated strains able to grow and ferment glucose under the saline condition that severely perturbs parental strain physiology. Our approach to generate strains, cell shape-based diagnosis and selection, might be applicable to other kinds of strain engineering in Z. mobilis.

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Furfural-tolerant Zymomonas mobilis derived from error-prone PCR-based whole genome shuffling and their tolerant mechanism

Cited by 21 publications

References 46 publications

Genome shuffling enhances stress tolerance of Zymomonas mobilis to two inhibitors

Genome shuffling enhances stress tolerance of Zymomonas mobilis to two inhibitors

Biochar-mediated enhanced ethanol fermentation (BMEEF) in Zymomonas mobilis under furfural and acetic acid stress

Increased salt tolerance in Zymomonas mobilis strain generated by adaptative evolution

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