Development of a Gene Knockout System Using Mobile Group II Introns (Targetron) and Genetic Disruption of Acid Production Pathways in Clostridium beijerinckii

Wang, Yi; Li, Xiangzhen; Milne, Caroline B.; Janssen, Holger; Lin, Wan-Ying; Phan, Gloria; Hu, Huiying; Liu, Jing Jing; Price, Nathan D.; Blaschek, Hans P.

doi:10.1128/aem.00971-13

Cited by 53 publications

(36 citation statements)

References 50 publications

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“…All the clostridial strains were grown in an anaerobic chamber (N 2 -CO 2 -H 2 with a volume ratio of 85:10:5) at 35 °C. Strains of C. tyrobutyricum, C. saccharoperbutylacetonicum and C. beijerinckii were cultivated using tryptone-glucose-yeast extract (TGY) medium 41 , while strains of C. pasteurianum were cultivated using 2×YTG medium 42 . When required, clarithromycin (Cla) or thiamphenicol (Tm) was supplemented into the medium at a final concentration of 30 μg/mL and 15 μg/mL, respectively.…”

Section: Methodsmentioning

confidence: 99%

Renewable Fatty Acid Ester Production inClostridium

Feng

Zhang

Wang

et al. 2020

Preprint

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44Endproduct toxicity is a key bottleneck for biofuel/biochemical production. Biosynthesis of 45 high-value derivatives that can easily separate would alleviate such toxicity and thus enhance 46 bioproduction efficiency. As a proof of principle, biosynthesis (along with in-line extractive 47 recovery) of fatty acid esters using clostridia was investigated in this study. We hypothesize 48 that solventogenic clostridia are excellent platforms for ester production, because they co-49 produce acyl-CoAs (acetyl-CoA and butyryl-CoA), acids (acetate and butyrate), and alcohols 50 (ethanol and butanol). Through rational screening for host strains and genes (encoding alcohol 51 acyl transferases and lipase), systematic metabolic engineering, and elimination of putative 52 prophages, we obtained strains that can produce 20.3 g/L butyl acetate and 1.6 g/L butyl 53 butyrate respectively, which were both historically highest levels in microbial hosts. Our 54 principle for engineering microorganisms to produce high-value and easy-recoverable 55 endproducts is highly applicable to other bioprocesses, and could lead to breakthroughs in 56 biofuel/biochemical production and general bioeconomy. 57 58 59 60 61 Keywords: clostridia, biofuels and biochemicals, fatty acid ester, butyl acetate, butyl butyrate, 62 ethyl acetate, CRISPR-Cas9, hydrolysates, prophages 63 64 4 Main 65 Although tremendous efforts have been invested for biofuel and biochemical research, it 66is still challenging to generate robust strains that can produce target products at desirable 67 levels 1 . One key bottleneck is the intrinsic toxicity of endproducts to host cells 2 . Therefore, the 68 production of high-value bioproducts which can be easily recovered from fermentation might 69 be a solution to tackle the bottleneck in bioproduction. Fatty acid esters, or mono-alkyl esters, 70 can be used as valuable fuels such as diesel components or specialty chemicals for food 71 flavoring, cosmetic and pharmaceutical industries 3 . It is projected that the US market demand 72 for fatty acid esters could reach $4.99 billion by 2025 4 . In addition, esters, with fatty acid and 73 alcohol moieties, are generally hydrophobic and can easily separate from fermentation; thus 74 the production of ester can help mitigate endproduct toxicity to host cells and efficient 75 bioproduction can be achieved. 76 Conventionally, esters are produced through Fischer esterification which involves high 77 temperature and inorganic catalysts 5,6 . The reaction consumes a large amount of energy and 78 generates tremendous wastes, and thus is not environmentally friendly 5 . On the other hand, 79 ester production through biological routes is becoming more and more attractive because it is 80 renewable and environmentally benign. There are two primary biological pathways for ester 81 production: one is through esterification of fatty acid and alcohol catalyzed by lipases 7 , and the 82 other is based on condensation of acyl-CoA and alcohol catalyzed by alcohol acyl transfer...

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Section: Methodsmentioning

confidence: 99%

Renewable Fatty Acid Ester Production inClostridium

Feng

Zhang

Wang

et al. 2020

Preprint

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“…Because FDCA can be used by whole-cell biocatalysts, it was interesting to determine whether elimination of the FDCA degradation pathway improves FDCA production. In this study, we used the TargeTron mobile group II intron gene insertional mutagenesis system to create an insertional mutant of dcaD, the gene encoding dicarboxylic acid decarboxylase (RTBF60-1), and thereby to reduce FDCA degradation (18,19). Successful insertion was confirmed by PCR with gene-and intron-specific primers and by Southern blotting (see Fig.…”

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confidence: 99%

Metabolic Engineering of Raoultella ornithinolytica BF60 for Production of 2,5-Furandicarboxylic Acid from 5-Hydroxymethylfurfural

Hossain

Yuan

et al. 2017

Appl Environ Microbiol

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2,5-Furandicarboxylic acid (FDCA) is an important renewable biotechnological building block because it serves as an environmentally friendly substitute for terephthalic acid in the production of polyesters. Currently, FDCA is produced mainly via chemical oxidation, which can cause severe environmental pollution. In this study, we developed an environmentally friendly process for the production of FDCA from 5-hydroxymethyl furfural (5-HMF) using a newly isolated strain, Raoultella ornithinolytica BF60. First, R. ornithinolytica BF60 was identified by screening and was isolated. Its maximal FDCA titer was 7.9 g/liter, and the maximal molar conversion ratio of 5-HMF to FDCA was 51.0% (mol/mol) under optimal conditions (100 mM 5-HMF, 45 g/liter whole-cell biocatalyst, 30°C, and 50 mM phosphate buffer [pH 8.0]). Next, dcaD, encoding dicarboxylic acid decarboxylase, was mutated to block FDCA degradation to furoic acid, thus increasing FDCA production to 9.2 g/liter. Subsequently, aldR, encoding aldehyde reductase, was mutated to prevent the catabolism of 5-HMF to HMF alcohol, further increasing the FDCA titer, to 11.3 g/liter. Finally, the gene encoding aldehyde dehydrogenase 1 was overexpressed. The FDCA titer increased to 13.9 g/liter, 1.7 times that of the wild-type strain, and the molar conversion ratio increased to 89.0%.IMPORTANCE In this work, we developed an ecofriendly bioprocess for green production of FDCA in engineered R. ornithinolytica. This report provides a starting point for further metabolic engineering aimed at a process for industrial production of FDCA using R. ornithinolytica.

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“…The fermentation solution consisted of the substrate (either biomass hydrolysates or synthetic substrate as control) supplemented with 1% of P2 stock solutions. The P2 medium contained the following compounds (in g/L): [26]. Prior to inoculation, the pH value of the fermentation broth was adjusted to 6.8-7.0 with filter-sterilized KOH solution.…”

Section: Fermentationmentioning

confidence: 99%