The presence of lignocellulose-derived microbial inhibitory compounds (LDMICs) in lignocellulosic biomass (LB) hydrolysates is a barrier to efficient conversion of LB hydrolysates to fuels and chemicals by fermenting microorganisms. Results from this study provide convincing evidence regarding the effectiveness of metabolically engineered C. beijerinckii NCIMB 8052 for the fermentation of LB-derived hydrolysates to acetone–butanol–ethanol (ABE). The engineered microbial strain ( C. beijerinckii _SDR) was produced by the integration of an additional copy of a short-chain dehydrogenase/reductase (SDR) gene ( Cbei_ 3904) into the chromosome of C. beijerinckii NCIMB 8052 wildtype, where it is controlled by the constitutive thiolase promoter. The C. beijerinckii _SDR and C. beijerinckii NCIMB 8052 wildtype were used for comparative fermentation of non-detoxified and detoxified hydrothermolysis-pretreated switchgrass hydrolysates (SHs) with and without (NH 4 ) 2 CO 3 supplementation. In the absence of (NH 4 ) 2 CO 3 , fermentation of non-detoxified SH with C. beijerinckii _SDR resulted in the production of 3.13- and 2.25-fold greater quantities of butanol (11.21 g/L) and total ABE (20.24 g/L), respectively, than the 3.58 g/L butanol and 8.98 g/L ABE produced by C. beijerinckii _wildtype. When the non-detoxified SH was supplemented with (NH 4 ) 2 CO 3 , concentrations were similar for butanol (9.5 compared with 9.2 g/L) and ABE (14.2 compared with 13.5 g/L) produced by C. beijerinckii _SDR and C. beijerinckii _wildtype, respectively. Furthermore, when C. beijerinckii _SDR and C. beijerinckii _wildtype were cultured in detoxified SH medium, C. beijerinckii _SDR produced 1.11- and 1.18-fold greater quantities of butanol and ABE, respectively, than when there was culturing with C. beijerinckii _wildtype. When the combined results of the present study are considered, conclusions are that the microbial strain and medium modifications of the fermentation milieu resulted in greater production of fuels and chemicals from non-detoxified LB hydrolysates.
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Metabolically engineered Caldicellulosiruptor bescii as a platform for producing acetone and hydrogen from lignocellulose
The production of volatile industrial chemicals utilizing metabolically engineered extreme thermophiles offers the potential for processes with simultaneous fermentation and product separation. An excellent target chemical for such a process is acetone (
- NSF-PAR ID:
- 10235995
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biotechnology and Bioengineering
- Volume:
- 117
- Issue:
- 12
- ISSN:
- 0006-3592
- Page Range / eLocation ID:
- p. 3799-3808
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract The extreme thermophile
Caldicellulosiruptor bescii solubilizes and metabolizes the carbohydrate content of lignocellulose, a process that ultimately ceases because of biomass recalcitrance, accumulation of fermentation products, inhibition by lignin moieties, and reduction of metabolic activity. Deconstruction of low loadings of lignocellulose (5 g/L), either natural or transgenic, whether unpretreated or subjected to hydrothermal processing, byC. bescii typically results in less than 40% carbohydrate solubilization. Mild alkali pretreatment (up to 0.09 g NaOH/g biomass) improved switchgrass carbohydrate solubilization byC. bescii to over 70% compared to less than 30% for no pretreatment, with two‐thirds of the carbohydrate content in the treated switchgrass converted to acetate and lactate.C. bescii grown on high loadings of unpretreated switchgrass (50 g/L) retained in a pH‐controlled bioreactor slowly purged (τ = 80 hr) with growth media without a carbon source improved carbohydrate solubilization to over 40% compared to batch culture at 29%. But more significant was the doubling of solubilized carbohydrate conversion to fermentation products, which increased from 40% in batch to over 80% in the purged system, an improvement attributed to maintaining the bioreactor culture in a metabolically active state. This strategy should be considered for optimizing solubilization and conversion of lignocellulose byC. bescii and other lignocellulolytic microorganisms. -
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Saccharomyces cerevisiae is the most highly domesticated yeast, strain dependency in biotechnological processes still remains as a common, yet poorly understood phenomenon. To investigate this, the entrance to the aromatic amino acid biosynthetic pathway was compared in four commonly usedS. cerevisiae laboratory strains. The strains were engineered to accumulate shikimate by overexpressing a mutant version of the pentafunctional ARO1 enzyme with disrupted activity in the shikimate kinase subunit. Carbon tracing and13C metabolic flux analysis combined with quantitative PCR, revealed that precursor availability and shikimate production were dramatically different in the four equally engineered strains, which were found to be correlated with the strains’ capacity to deal with protein overexpression burden. By implementing a strain‐dependent approach, the genetic platform was reformulated, leading to an increase in yield and titer in all strains. The highest producing strain, INVSc1‐SA3, produced 358 mg L−1of shikimate with a yield of 17.9 mg g−1glucose.These results underline the importance of strain selection in developing biological manufacturing processes, demonstrate the first case of high production of shikimate in yeast, and provide an appropriate platform for strain selection for future production of aromatic compounds. Biotechnol. Bioeng. 2016;113: 2676–2685. © 2016 Wiley Periodicals, Inc. -
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Abstract Efficient co‐utilization of mixed sugar feedstocks remains a biomanufacturing challenge, thus motivating ongoing efforts to engineer microbes for improved conversion of glucose−xylose mixtures. This study focuses on enhancing phenylalanine production by engineering
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