Although CRISPR-Cas9/Cpf1 have been employed as powerful genome engineering tools, heterologous CRISPR-Cas9/Cpf1 are often difficult to introduce into bacteria and archaea due to their severe ...toxicity. Since most prokaryotes harbor native CRISPR-Cas systems, genome engineering can be achieved by harnessing these endogenous immune systems. Here, we report the exploitation of Type I-B CRISPR-Cas of Clostridium tyrobutyricum for genome engineering. In silico CRISPR array analysis and plasmid interference assay revealed that TCA or TCG at the 5′-end of the protospacer was the functional protospacer adjacent motif (PAM) for CRISPR targeting. With a lactose inducible promoter for CRISPR array expression, we significantly decreased the toxicity of CRISPR-Cas and enhanced the transformation efficiency, and successfully deleted spo0A with an editing efficiency of 100%. We further evaluated effects of the spacer length on genome editing efficiency. Interestingly, spacers ≤ 20 nt led to unsuccessful transformation consistently, likely due to severe off-target effects; while a spacer of 30–38 nt is most appropriate to ensure successful transformation and high genome editing efficiency. Moreover, multiplex genome editing for the deletion of spo0A and pyrF was achieved in a single transformation, with an editing efficiency of up to 100%. Finally, with the integration of the alcohol dehydrogenase gene (adhE1 or adhE2) to replace cat1 (the key gene responsible for butyrate production and previously could not be deleted), two mutants were created for n-butanol production, with the butanol titer reached historically record high of 26.2 g/L in a batch fermentation. Altogether, our results demonstrated the easy programmability and high efficiency of endogenous CRISPR-Cas. The developed protocol herein has a broader applicability to other prokaryotes containing endogenous CRISPR-Cas systems. C. tyrobutyricum could be employed as an excellent platform to be engineered for biofuel and biochemical production using the CRISPR-Cas based genome engineering toolkit.
•The native Type I-B CRISPR-Cas of C. tyrobutyricum was harnessed for genome editing.•Off-target effects were revealed, and appropriate spacer length was suggested.•Single and multiplex genome editing were achieved with efficiencies of up to 100%.•Carbon flow was redirected to butanol by replacement of cat1 gene with adhE1/adhE2.•High level butanol (26.2 g/L) was obtained in batch fermentation at low temperature.
Repeated fed‐batch fermentation of glucose by Clostridium tyrobutyricum immobilized in a fibrous bed bioreactor (FBB) was successfully employed to produce butyric acid at a high final concentration ...as well as to adapt a butyric‐acid‐tolerant strain. At the end of the eighth fed‐batch fermentation, the butyric acid concentration reached 86.9 ± 2.17 g/L, which to our knowledge is the highest butyric acid concentration ever produced in the traditional fermentation process. To understand the mechanism and factors contributing to the improved butyric acid production and enhanced acid tolerance, adapted strains were harvested from the FBB and characterized for their physiological properties, including specific growth rate, acid‐forming enzymes, intracellular pH, membrane‐bound ATPase and cell morphology. Compared with the original culture used to seed the bioreactor, the adapted culture showed significantly reduced inhibition effects of butyric acid on specific growth rate, cellular activities of butyric‐acid‐forming enzyme phosphotransbutyrylase (PTB) and ATPase, together with elevated intracellular pH, and elongated rod morphology. Biotechnol. Bioeng. 2011; 108:31-40.
Butyl butyrate has broad applications in foods, cosmetics, solvents, and biofuels. Microbial synthesis of bio-based butyl butyrate has been regarded as a promising approach recently. Herein, we ...engineered Clostridium tyrobutyricum ATCC 25755 to achieve de novo biosynthesis of butyl butyrate from fermentable sugars. Through introducing the butanol synthetic pathway (enzyme AdhE2), screening alcohol acyltransferases (AATs), adjusting transcription of VAAT and adhE2 (i.e., optimizing promoter), and efficient supplying butyryl-CoA, an excellent engineered strain, named MUV3, was obtained with ability to produce 4.58 g/L butyl butyrate at 25 °C with glucose in serum bottles. More NADH is needed for butyl butyrate synthesis, thus mannitol (the more reduced substrate) was employed to produce butyl butyrate. Ultimately, 62.59 g/L butyl butyrate with a selectivity of 95.97%, and a yield of 0.21 mol/mol was obtained under mannitol with fed-batch fermentation in a 5 L bioreactor, which is the highest butyl butyrate titer reported so far. Altogether, this study presents an anaerobic fermentative platform for de novo biosynthesis of butyl butyrate in one step, which lays the foundation for butyl butyrate biosynthesis from renewable biomass feedstocks.
•Development of an upp/5-FU counter-selection system for efficient iterative genome-editing in C. tyrobutyricum.•Establishment of the de novo biosynthetic pathway of butyl butyrate from fermentable sugars in C. tyrobutyricum.•Mannitol as substrate for efficiency butyl butyrate production at a low temperature of 25°C.•Performed fermentation strategy with fed-batch fermentation in 5 L bioreacter for high level of butyl butyrate production.
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•C. tyrobutyricum was engineered to overexpress xylose catabolism genes.•The mutant used xylose and glucose simultaneously for butyrate production.•Undetoxified hydrolysates of ...lignocellulosic biomass were used as substrates.•42.6g/L butyrate at a productivity of 0.56g/L·h and yield of 0.36g/g was obtained.•This provides a sustainable process for cost-effective production of butyric acid.
Clostridium tyrobutyricum can utilize glucose and xylose as carbon source for butyric acid production. However, xylose catabolism is inhibited by glucose, hampering butyric acid production from lignocellulosic biomass hydrolysates containing both glucose and xylose. In this study, an engineered strain of C. tyrobutyricum Ct-pTBA overexpressing heterologous xylose catabolism genes (xylT, xylA, and xylB) was investigated for co-utilizing glucose and xylose present in hydrolysates of plant biomass, including soybean hull, corn fiber, wheat straw, rice straw, and sugarcane bagasse. Compared to the wild-type strain, Ct-pTBA showed higher xylose utilization without significant glucose catabolite repression, achieving near 100% utilization of glucose and xylose present in lignocellulosic biomass hydrolysates in bioreactor at pH 6. About 42.6g/L butyrate at a productivity of 0.56g/L·h and yield of 0.36g/g was obtained in batch fermentation, demonstrating the potential of C. tyrobutyricum Ct-pTBA for butyric acid production from lignocellulosic biomass hydrolysates.
•FbFP protein Bs2 was used as a reporter for gene expression in C. tyrobutyricum.•It’s applied for confirming transformation and monitoring gene expression in vivo.•This is the first report of ...FbFP-based reporter system in C. tyrobutyricum.•This study also demonstrated the production of isopropanol in C. tyrobutyricum.
C. tyrobutyricum, an acidogenic Clostridium, has aroused increasing interest due to its potential to produce biofuel efficiently. However, construction of recombinant C. tyrobutyricum for enhanced biofuel production has been impeded by the limited genetic engineering tools. In this study, a flavin mononucleotide (FMN)-dependent fluorescent protein Bs2-based gene expression reporter system was developed to monitor transformation and explore in vivo strength and regulation of various promoters in C. tyrobutyricum and C. acetobutylicum. Unlike green fluorescent protein (GFP) and its variants, Bs2 can emit green light without oxygen, which makes it extremely suitable for promoter screening and transformation confirmation in organisms grown anaerobically. The expression levels of bs2 under thiolase promoters from C. tyrobutyricum and C. acetobutylicum were measured and compared based on fluorescence intensities. The capacities of the two promoters in driving secondary alcohol dehydrogenase (adh) gene for isopropanol production in C. tyrobutyricum were distinguished, confirming that this reporter system is a convenient, effective and reliable tool for promoter strength assay and real time monitoring in C. tyrobutyricum, while demonstrating the feasibility of producing isopropanol in C. tyrobutyricum for the first time.
Clostridium tyrobutyricum ATCC 25755, a butyric acid producing bacterium, has been engineered to overexpress aldehyde/alcohol dehydrogenase 2 (
adhE2, Genebank no.
AF321779) from
Clostridium ...acetobutylicum ATCC 824, which converts butyryl-CoA to butanol, under the control of native thiolase (
thl) promoter. Butanol titer of 1.1
g/L was obtained in
C. tyrobutyricum overexpressing
adhE2. The effects of inactivating acetate kinase (
ack) and phosphotransbutyrylase (
ptb) genes in the host on butanol production were then studied. A high C4/C2 product ratio of 10.6 (mol/mol) was obtained in
ack knockout mutant, whereas a low C4/C2 product ratio of 1.4 (mol/mol) was obtained in
ptb knockout mutant, confirming that
ack and
ptb genes play important roles in controlling metabolic flux distribution in
C. tyrobutyricum. The highest butanol titer of 10.0
g/L and butanol yield of 27.0% (w/w, 66% of theoretical yield) were achieved from glucose in the
ack knockout mutant overexpressing
adhE2. When a more reduced substrate mannitol was used, the butanol titer reached 16.0
g/L with 30.6% (w/w) yield (75% theoretical yield). Moreover,
C. tyrobutyricum showed good butanol tolerance, with >80% and ∼60% relative growth rate at 1.0% and 1.5% (v/v) butanol. These results suggest that
C. tyrobutyricum is a promising heterologous host for
n-butanol production from renewable biomass.
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•The effects of phenolic acids/aldehydes on butyrate fermentation were investigated.•Phenolic inhibitors were reduced to low toxicity alcohols/acids by C. tyrobutyricum.•Some ...reductase, HSP, and membrane transporter genes were induced by phenolic stress.•Accelerating the reduction of PCs could enhance the strain’s tolerance to PC stress.•Valuable insights for enhancing utilization of lignocellulosic biomass were provided.
Phenolic compounds (PCs) generated during pretreatment of lignocellulosic biomass severely hinder the biorefinery by Clostridia. As a hyperbutyrate-producing strain, Clostridium tyrobutyricum has excellent tolerance to PCs, but its tolerance mechanism is poorly understood. In this study, a comprehensive transcriptome analysis was applied to elucidate the response of C. tyrobutyricum to four typical PCs. The findings revealed that the expression levels of genes associated with PC reduction, HSPs, and membrane transport were significantly altered under PC stress. Due to PCs being reduced to low-toxicity alcohols/acids by C. tyrobutyricum, enhancing the reduction of PCs by overexpressing reductase genes could enhance the strain’s tolerance to PCs. Under 1.0 g/L p-coumaric acid stress, compared with the wild-type strain, ATCC 25755/sdr1 exhibited a 31.2 % increase in butyrate production and a 38.5 % increase in productivity. These insights contribute to the construction of PC-tolerant Clostridia, which holds promise for improving biofuel and chemical production from lignocellulosic biomass.
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