Metabolic engineering of<i>Zymomonas mobilis</i>for continuous co-production of bioethanol and poly-3-hydroxybutyrate (PHB)

Yang, Li; Wang, Ying; Wang, Ruxiang; Yan, Xiongying; Wang, Jiewen; Wang, Xia; Chen, Shouwen; Bai, Feng‐Wu; He, Qiaoning; Yang, Shihui

doi:10.1039/d1gc04522a

Cited by 24 publications

(15 citation statements)

References 60 publications

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“…Gibson assembly method as described before (Li et al, 2022) was utilized for donor construction. Donor sequences including extra ~800 bp upstream sequence and downstream sequence of the candidate gene were amplified using Primer STAR polymerase (Takara, Japan) from the genomic DNA of Z. mobilis ZM4.…”

Section: Genetic Manipulation and Recombinant Strain Constructionmentioning

confidence: 99%

“…Z. mobilis strains naturally use the anaerobic Entner-Doudoroff (ED) pathway efficiently to consume glucose for the production of ethanol and other biochemicals such as gluconic acid, levan, and sorbitol (Erzinger and Vitolo, 1996;Silbir et al, 2014). In addition, gene editing tools have been continuously developed in Z. mobilis including exogenous and native CRISPR-Cas genome editing tools (Shen et al, 2019;Zheng et al, 2019), and Z. mobilis has become a promising chassis for the economic production of lignocellulosic biofuels and biochemicals such as 2,3-butanediol, isobutanol, and poly-3-hydroxybutyrate (PHB) (Yang et al, 2016;Qiu et al, 2020;Li et al, 2022).…”

Section: Introductionmentioning

confidence: 99%

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A plasmid-free Zymomonas mobilis mutant strain reducing reactive oxygen species for efficient bioethanol production using industrial effluent of xylose mother liquor

Geng

Liu

Chen

et al. 2022

Front. Bioeng. Biotechnol.

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Genome minimization is an effective way for industrial chassis development. In this study, Zymomonas mobilis ZMNP, a plasmid-free mutant strain of Z. mobilis ZM4 with four native plasmids deleted, was constructed using native type I-F CRISPR-Cas system. Cell growth of ZMNP under different temperatures and industrial effluent of xylose mother liquor were examined to investigate the impact of native plasmid removal. Despite ZMNP grew similarly as ZM4 under different temperatures, ZMNP had better xylose mother liquor utilization than ZM4. In addition, genomic, transcriptomic, and proteomic analyses were applied to unravel the molecular changes between ZM4 and ZMNP. Whole-genome resequencing result indicated that an S267P mutation in the C-terminal of OxyR, a peroxide-sensing transcriptional regulator, probably alters the transcription initiation of antioxidant genes for stress responses. Transcriptomic and proteomic studies illustrated that the reason that ZMNP utilized the toxic xylose mother liquor better than ZM4 was probably due to the upregulation of genes in ZMNP involving in stress responses as well as cysteine biosynthesis to accelerate the intracellular ROS detoxification and nucleic acid damage repair. This was further confirmed by lower ROS levels in ZMNP compared to ZM4 in different media supplemented with furfural or ethanol. The upregulation of stress response genes due to the OxyR mutation to accelerate ROS detoxification and DNA/RNA repair not only illustrates the underlying mechanism of the robustness of ZMNP in the toxic xylose mother liquor, but also provides an idea for the rational design of synthetic inhibitor-tolerant microorganisms for economic lignocellulosic biochemical production.

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Section: Genetic Manipulation and Recombinant Strain Constructionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A plasmid-free Zymomonas mobilis mutant strain reducing reactive oxygen species for efficient bioethanol production using industrial effluent of xylose mother liquor

Geng

Liu

Chen

et al. 2022

Front. Bioeng. Biotechnol.

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“…Zymomonas mobilis is a natural ethanologenic bacterium with desirable industrial characteristics such as generally regarded as safe (GRAS), high sugar uptake efficiency and conversion rate, low by-product, phage free and no need to control aeration during fermentation ( Yang Q. et al, 2020a ; Yang et al, 2021 ). Various native and heterologous metabolic pathways have been enhanced or constructed using the established CRISPR-Cas genome editing toolkits ( Shen et al, 2019 ; Zheng et al, 2019 ; Yang Y. et al, 2020b ) for desirable bioproducts, such as isobutanol, 2,3-butanediol, polyhydroxybutyrate (PHB) and lactate ( Yang et al, 2016 ; Liu et al, 2020 ; Yang et al, 2021 ; Li et al, 2022 ). In addition to pure sugars, lignocellulosic hydrolysates can also be utilized as carbon sources by Z. mobilis ( Yang et al, 2016 ; Wang et al, 2018 ; Todhanakasem et al, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

Metabolic engineering of Zymomonas mobilis for co-production of D-lactic acid and ethanol using waste feedstocks of molasses and corncob residue hydrolysate

Bao²,

Peng³

et al. 2023

Front. Bioeng. Biotechnol.

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Lactate is the precursor for polylactide. In this study, a lactate producer of Z. mobilis was constructed by replacing ZMO0038 with LmldhA gene driven by a strong promoter PadhB, replacing ZMO1650 with native pdc gene driven by Ptet, and replacing native pdc with another copy of LmldhA driven by PadhB to divert carbon from ethanol to D-lactate. The resultant strain ZML-pdc-ldh produced 13.8 ± 0.2 g/L lactate and 16.9 ± 0.3 g/L ethanol using 48 g/L glucose. Lactate production of ZML-pdc-ldh was further investigated after fermentation optimization in pH-controlled fermenters. ZML-pdc-ldh produced 24.2 ± 0.6 g/L lactate and 12.9 ± 0.8 g/L ethanol as well as 36.2 ± 1.0 g/L lactate and 40.3 ± 0.3 g/L ethanol, resulting in total carbon conversion rate of 98.3% ± 2.5% and 96.2% ± 0.1% with final product productivity of 1.9 ± 0.0 g/L/h and 2.2 ± 0.0 g/L/h in RMG5 and RMG12, respectively. Moreover, ZML-pdc-ldh produced 32.9 ± 0.1 g/L D-lactate and 27.7 ± 0.2 g/L ethanol as well as 42.8 ± 0.0 g/L D-lactate and 53.1 ± 0.7 g/L ethanol with 97.1% ± 0.0% and 99.1% ± 0.8% carbon conversion rate using 20% molasses or corncob residue hydrolysate, respectively. Our study thus demonstrated that it is effective for lactate production by fermentation condition optimization and metabolic engineering to strengthen heterologous ldh expression while reducing the native ethanol production pathway. The capability of recombinant lactate-producer of Z. mobilis for efficient waste feedstock conversion makes it a promising biorefinery platform for carbon-neutral biochemical production.

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“…Z. mobilis has been acknowledged as a chassis that can be used for engineering as a biorefinery to produce commodities, including biofuels and bio-based chemicals. − …”

Section: Introductionmentioning

confidence: 99%

Electrochemical Control of Cell Metabolism Improves Ethanol Production of Zymomonas mobilis in an Electro-Fermentation System

Zhang

et al. 2023

ACS Sustainable Chem. Eng.

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This study investigated ethanol fermentation by Zymomonas mobilis in an electro-fermentation system (EFS) for efficient alcohol production, which increased the ethanol titer by 12.8% compared to fermentation without electricity input. The underlying mechanism was revealed by exploring the correlation among intracellular redox parameters such as NAD(P)H/NAD-(P) + ratio, total antioxidant capacity, and cell membrane permeability. Transcriptome analysis further investigated that genes related to glucose uptake, ethanol, and succinic acid synthesis were upregulated in the cathode chamber, which promoted ethanol production. Two strategies to enhance EFS were proposed. First, the supplementation of electron shuttles (methylene blue, HNQ) can facilitate electron transport between electrodes and cells. Second, the manipulation of essential electricity-sensing genes (ZMO1211, ZMO1753) amplified the metabolic change. This study showed the possible use of electro-regulation for the efficient production of alcohol in Z. mobilis, which also provided insights into other microbial electrochemical processes in the future.

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Metabolic engineering ofZymomonas mobilisfor continuous co-production of bioethanol and poly-3-hydroxybutyrate (PHB)

Abstract: Zymomonas mobilis is a native ethanologen with the unique anaerobic Entner Doudoroff pathway. In this study, a heterologous poly-3-hydroxybutyrate (PHB) pathway was engineered into Z. mobilis. Genetic strategies were applied...

Cited by 24 publications

References 60 publications

A plasmid-free Zymomonas mobilis mutant strain reducing reactive oxygen species for efficient bioethanol production using industrial effluent of xylose mother liquor

A plasmid-free Zymomonas mobilis mutant strain reducing reactive oxygen species for efficient bioethanol production using industrial effluent of xylose mother liquor

Metabolic engineering of Zymomonas mobilis for co-production of D-lactic acid and ethanol using waste feedstocks of molasses and corncob residue hydrolysate

Electrochemical Control of Cell Metabolism Improves Ethanol Production of Zymomonas mobilis in an Electro-Fermentation System

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