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Title: Metabolic characterization of anaerobic fungi provides a path forward for bioprocessing of crude lignocellulose
Abstract   more » « less
NSF-PAR ID:
10049263
Author(s) / Creator(s):
 ;  ;  ;  ;  ;  ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Biotechnology and Bioengineering
Volume:
115
Issue:
4
ISSN:
0006-3592
Page Range / eLocation ID:
p. 874-884
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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  1. Anaerobic fungi are among the most active plant‐degrading microbes in nature. Increased insight into the mechanisms and environmental cues that regulate fungal hydrolysis would better inform bioprocessing strategies to depolymerize lignocellulose. Here, we compare the response of three strains of anaerobic fungi (Piromyces finnis,Anaeromyces robustus, andNeocallimastix californiae) to catabolite regulation by simple carbohydrates. Anaerobic fungi exhibited high enzymatic activity against crystalline cellulose, which was repressed upon incubation with free sugars. Cellulolytic degradation was also inhibited when fungi were exposed to sugars they did not metabolize, suggesting a general mode of catabolite repression. RNA‐Seq experiments in the presence of excess glucose confirmed repression of carbohydrate active enzymes during sugar uptake, and offer a path towards unmasking the function of co‐regulated genes that could be involved in biomass degradation. Overall, these results suggest that sugar‐rich hydrolysates tune the behavior of anaerobic fungi by dampening production of their biomass‐degrading enzymes. © 2018 American Institute of Chemical EngineersAIChE J, 64: 4263–4270, 2018

     
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  2. Abstract

    Anaerobic fungi and methanogenic archaea are two classes of microorganisms found in the rumen microbiome that metabolically interact during lignocellulose breakdown. Here, stable synthetic co-cultures of the anaerobic fungusCaecomyces churrovisand the methanogenMethanobacterium bryantii(not native to the rumen) were formed, demonstrating that microbes from different environments can be paired based on metabolic ties. Transcriptional and metabolic changes induced by methanogen co-culture were evaluated inC. churrovisacross a variety of substrates to identify mechanisms that impact biomass breakdown and sugar uptake. A high-quality genome ofC. churroviswas obtained and annotated, which is the first sequenced genome of a non-rhizoid-forming anaerobic fungus.C. churrovispossess an abundance of CAZymes and carbohydrate binding modules and, in agreement with previous studies of early-diverging fungal lineages, N6-methyldeoxyadenine (6mA) was associated with transcriptionally active genes. Co-culture with the methanogen increased overall transcription of CAZymes, carbohydrate binding modules, and dockerin domains in co-cultures grown on both lignocellulose and cellulose and caused upregulation of genes coding associated enzymatic machinery including carbohydrate binding modules in family 18 and dockerin domains across multiple growth substrates relative toC. churrovismonoculture. Two other fungal strains grown on a reed canary grass substrate in co-culture with the same methanogen also exhibited high log2-fold change values for upregulation of genes encoding carbohydrate binding modules in families 1 and 18. Transcriptional upregulation indicated that co-culture of theC. churrovisstrain with a methanogen may enhance pyruvate formate lyase (PFL) function for growth on xylan and fructose and production of bottleneck enzymes in sugar utilization pathways, further supporting the hypothesis that co-culture with a methanogen may enhance certain fungal metabolic functions. Upregulation of CBM18 may play a role in fungal–methanogen physical associations and fungal cell wall development and remodeling.

     
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  3. Abstract

    Development of the bioeconomy is driven by our ability to access the energy‐rich carbon trapped in recalcitrant plant materials. Current strategies to release this carbon rely on expensive enzyme cocktails and physicochemical pretreatment, producing inhibitory compounds that hinder subsequent microbial bioproduction. Anaerobic fungi are an appealing solution as they hydrolyze crude, untreated biomass at ambient conditions into sugars that can be converted into value‐added products by partner organisms. However, some carbon is lost to anaerobic fungal fermentation products. To improve efficiency and recapture this lost carbon, we built a two‐stage bioprocessing system pairing the anaerobic fungusPiromyces indianaewith the yeastKluyveromyces marxianus, which grows on a wide range of sugars and fermentation products. In doing so we produce fine and commodity chemicals directly from untreated lignocellulose.P.indianaeefficiently hydrolyzed substrates such as corn stover and poplar to generate sugars, fermentation acids, and ethanol, whichK.marxianusconsumed while producing 2.4 g/L ethyl acetate. An engineered strain ofK.marxianuswas also able to produce 550 mg/L 2‐phenylethanol and 150 mg/L isoamyl alcohol fromP.indianaehydrolyzed lignocellulosic biomass. Despite the use of crude untreated plant material, production yields were comparable to optimized rich yeast media due to the use of all available carbon including organic acids, which formed up to 97% of free carbon in the fungal hydrolysate. This work demonstrates that anaerobic fungal pretreatment of lignocellulose can sustain the production of fine chemicals at high efficiency by partnering organisms with broad substrate versatility.

     
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  4. na (Ed.)
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  5. Abstract

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    Key points

    IfCelS12A from an anaerobic alkaliphile Iocasia fronsfrigidae shows salt tolerance

    IfCelS12A in cocktails with other enzymes efficiently degrades cellulosic biomass

    IfCelS12A used with mobile enzyme sequestration platforms enhances hydrolysis

     
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