Developing Clostridium diolis as a biorefinery chassis by genetic manipulation

Li, Ang; Wen, Zhiqiang; Fang, Daqing; Lu, Minrui; Ma, Yong; Quan, Xie; Jin, Mingjie

doi:10.1016/j.biortech.2020.123066

Cited by 15 publications

(20 citation statements)

References 47 publications

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“…Taken together, total BA production in FJ-1201 was 20.3 g/L, which was the highest level that has ever been reported in a microbial host. It is 2400-fold higher than the highest level that has been previously reported 23 , and also 14.8-fold higher than that by the very recently reported C. diolis strain 25 .…”

Section: Resultscontrasting

confidence: 58%

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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: Resultscontrasting

confidence: 58%

“…C. acetobutylicum CaSAAT (with the overexpression of saat from Fragaria xananassa ) was reported to produce 8.37 mg/L BA 23 . While this manuscript was prepared, a newly engineered C. diolis strain was reported to produce 1.37 g/L BA 25 . The highest BB production of 0.3 g/L was observed in C. pasteurianum J-5 with the overexpression of eht1 .…”

Section: Resultsmentioning

confidence: 99%

Renewable Fatty Acid Ester Production inClostridium

Feng

Zhang

Wang

et al. 2020

Preprint

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“…Interestingly, butyl butyrate accounted for about 90% of the total esters, while butyl acetate accounted for a very small proportion. In two recent studies, Fang et al (2020) and Li et al (2020) expressed ATF1 in C. beijerinckii and Clostridium diolis , respectively; the generated strains produced 5.42 and 1.37 g/L of butyl acetate as the main ester products from glucose. The type difference in the main ester products in the above studies may be attributed to substrate selectivity of AATs from different sources ( Noh et al, 2018 ; Aleksander et al, 2019 ).…”

Section: Alcohol Acyltransferase-mediated Ester Synthesis In Clostridiamentioning

confidence: 99%

Developing Clostridia as Cell Factories for Short- and Medium-Chain Ester Production

Wang

Makishah

et al. 2021

Front. Bioeng. Biotechnol.

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Short- and medium-chain volatile esters with flavors and fruity fragrances, such as ethyl acetate, butyl acetate, and butyl butyrate, are usually value-added in brewing, food, and pharmacy. The esters can be naturally produced by some microorganisms. As ester-forming reactions are increasingly deeply understood, it is possible to produce esters in non-natural but more potential hosts. Clostridia are a group of important industrial microorganisms since they can produce a variety of volatile organic acids and alcohols with high titers, especially butanol and butyric acid through the CoA-dependent carbon chain elongation pathway. This implies sufficient supplies of acyl-CoA, organic acids, and alcohols in cells, which are precursors for ester production. Besides, some Clostridia could utilize lignocellulosic biomass, industrial off-gas, or crude glycerol to produce other branched or straight-chain alcohols and acids. Therefore, Clostridia offer great potential to be engineered to produce short- and medium-chain volatile esters. In the review, the efforts to produce esters from Clostridia via in vitro lipase-mediated catalysis and in vivo alcohol acyltransferase (AAT)-mediated reaction are comprehensively revisited. Besides, the advantageous characteristics of several Clostridia and clostridial consortia for bio-ester production and the driving force of synthetic biology to clostridial chassis development are also discussed. It is believed that synthetic biotechnology should enable the future development of more effective Clostridia for ester production.

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“…The high level of flux through acetyl-CoA is particularly important because it can serve as the precursor to the synthesis of many classes of compounds such as acetate, ethanol, lactate, butyrate, butanol, and acetone [32][33][34] . Cofeeding of substrates to solventogenic clostridium is a possible mechanism to control proportions of ATP, NADH, and acetyl-CoA 35 .…”

mentioning

confidence: 99%

“…Cofeeding of substrates to solventogenic clostridium is a possible mechanism to control proportions of ATP, NADH, and acetyl-CoA 35 . A barrier to co-feeding is carbon catabolite repression (CCR), which occurs widely in nature and prevents co-utilization if preferred substrates are present 33 . CCR occurs in the solventogenic clostridium were glucose, and in some cases, arabinose represses the utilization of less preferred carbohydrates [36][37][38] .…”

mentioning

confidence: 99%

Co-feeding glucose with either gluconate or galacturonate during clostridial fermentations provides metabolic fine-tuning capabilities

Liu

Gerlach

et al. 2021

Sci Rep

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Clostridiumacetobutylicum ATCC 824 effectively utilizes a wide range of substrates to produce commodity chemicals. When grown on substrates of different oxidation states, the organism exhibits different recycling needs of reduced intracellular electron carrying co-factors. Ratios of substrates with different oxidation states were used to modulate the need to balance electron carriers and demonstrate fine-tuned control of metabolic output. Three different oxidized substrates were first fed singularly, then in different ratios to three different strains of Clostridium sp. Growth was most robust when fed glucose in exclusive fermentations. However, the use of the other two more oxidized substrates was strain-dependent in exclusive feeds. In glucose-galacturonate mixed fermentation, the main products (acetate and butyrate) were dependant on the ratios of the substrates. Exclusive fermentation on galacturonate was nearly homoacetic. Co-utilization of galacturonate and glucose was observed from the onset of fermentation in growth conditions using both substrates combined, with the proportion of galacturonate present dictating the amount of acetate produced. For all three strains, increasing galacturonate content (%) in a mixture of galacturonate and glucose from 0 to 50, and 100, resulted in a corresponding increase in the amount of acetate produced. For example, C.acetobutylicum increased from ~ 10 mM to ~ 17 mM, and then ~ 23 mM. No co-utilization was observed when galacturonate was replaced with gluconate in the two substrate co-feed.

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Developing Clostridium diolis as a biorefinery chassis by genetic manipulation

Cited by 15 publications

References 47 publications

Renewable Fatty Acid Ester Production inClostridium

Renewable Fatty Acid Ester Production inClostridium

Developing Clostridia as Cell Factories for Short- and Medium-Chain Ester Production

Co-feeding glucose with either gluconate or galacturonate during clostridial fermentations provides metabolic fine-tuning capabilities

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