Explore the vast range of titles available.

Abstract Lignin valorization can be obtained through cleavage of selected bonds by microbial enzymes, in which lignin is segregated from cellulose and hemicellulose and abundant phenolic compounds can be provided. In this study, Pseudomonas sp. Q18, previously isolated from rotten wood in China, was used to degrade alkali lignin and raw lignocellulosic material. Gel-permeation chromatography, field-emission scanning electron microscope, and GC–MS were combined to investigate the degradation process. The GC–MS results revealed that the quantities of aromatic compounds with phenol ring from lignin increased significantly after incubation with Pseudomonas sp. Q18, which indicated the degradation of lignin. According to the lignin-derived metabolite analysis, it was proposed that a DyP-type peroxidase (PmDyP) might exist in strain Q18. Thereafter, the gene of PmDyP was cloned and expressed, after which the recombinant PmDyP was purified and the enzymatic kinetics of PmDyP were assayed. According to results, PmDyP showed promising characteristics for lignocellulosic biodegradation in biorefinery.

Abstract Anaerobic gut fungi are biomass degraders that form syntrophic associations with other microbes in their native rumen environment. Here, RNA-Seq was used to track and quantify carbohydrate active enzyme (CAZyme) transcription in a synthetic consortium composed of the anaerobic fungus Anaeromyces robustus with methanogen Methanobacterium bryantii. Approximately 5% of total A. robustus genes were differentially regulated in co-culture with M. bryantii relative to cultivation of A. robustus alone. We found that 105 CAZymes (12% of the total predicted CAZymes of A. robustus) were upregulated while 29 were downregulated. Upregulated genes encode putative proteins with a wide array of cellulolytic, xylanolytic, and carbohydrate transport activities; 75% were fused to fungal dockerin domains, associated with a carbohydrate binding module, or both. Collectively, this analysis suggests that co-culture of A. robustus with M. bryantii remodels the transcriptional landscape of CAZymes and associated metabolic pathways in the fungus to aid in lignocellulose breakdown.

Abstract In this study, we constructed a coculture consortium comprising engineered Pseudomonas putida KT2440 and Escherichia coli MG1655. Provision of “related” carbon sources and synthesis of medium-chain-length polyhydroxyalkanoates (mcl-PHAs) were separately assigned to these strains via a modular construction strategy. To avoid growth competition, a preference for the use of a carbon source was constructed. Further, the main intermediate metabolite acetate played an important role in constructing the expected “nutrition supply–detoxification” relationship between these strains. The coculture consortium showed a remarkable increase in the mcl-PHA titer (0.541 g/L) with a glucose–xylose mixture (1:1). Subsequently, the titer of mcl-PHA produced by the coculture consortium when tested with actual lignocellulosic hydrolysate (0.434 g/L) was similar to that achieved with laboratory sugars’ mixture (0.469 g/L). These results indicate a competitive potential of the engineered E. coli–P. putida coculture consortium for mcl-PHA production with lignocellulosic hydrolysate.

Acetate esters and higher alcohols greatly influence the quality and flavor profiles of Chinese Baijiu (Chinese liquor). Various mutants have been constructed to investigate the interactions of ATF1 overexpression, IAH1 deletion, and BAT2 deletion on the production of acetate esters and higher alcohols. The results showed that the overexpression of ATF1 under the control of the PGK1 promoter with BAT2 and IAH1 double-gene deletion led to a higher production of acetate esters and a lower production of higher alcohols than the overexpression of ATF1 with IAH1 deletion or overexpression of ATF1 with BAT2 deletion. Moreover, deletion of IAH1 in ATF1 overexpression strains effectively increased the production of isobutyl acetate and isoamyl acetate by reducing the hydrolysis of acetate esters. The decline in the production of higher alcohol by the ATF1 overexpression strains with BAT2 deletion is due to the interaction of ATF1 overexpression and BAT2 deletion. Mutants with varying abilities of producing acetate esters and higher alcohols were developed by genetic engineering. These strains have great potential for industrial application.

Abstract A major barrier to both metabolic engineering and fundamental biological studies is the lack of genetic tools in most microorganisms. One example is Clostridium thermocellum ATCC 27405T, where genetic tools are not available to help validate decades of hypotheses. A significant barrier to DNA transformation is restriction–modification systems, which defend against foreign DNA methylated differently than the host. To determine the active restriction–modification systems in this strain, we performed complete methylome analysis via single-molecule, real-time sequencing to detect 6-methyladenine and 4-methylcytosine and the rarely used whole-genome bisulfite sequencing to detect 5-methylcytosine. Multiple active systems were identified, and corresponding DNA methyltransferases were expressed from the Escherichia coli chromosome to mimic the C. thermocellum methylome. Plasmid methylation was experimentally validated and successfully electroporated into C. thermocellum ATCC 27405. This combined approach enabled genetic modification of the C. thermocellum-type strain and acts as a blueprint for transformation of other non-model microorganisms.

Xylitol is commercially used in chewing gum and dental care products as a low calorie sweetener having medicinal properties. Industrial yeast strain of S. cerevisiae was genetically modified to overexpress an endogenous aldose reductase gene GRE3 and a xylose transporter gene SUT1 for the production of xylitol. The recombinant strain (XP-RTK) carried the expression cassettes of both the genes and the G418 resistance marker cassette KanMX integrated into the genome of S. cerevisiae . Short segments from the 5′ and 3′ delta regions of the Ty1 retrotransposons were used as homology regions for integration of the cassettes. Xylitol production by the industrial recombinant strain was evaluated using hemicellulosic hydrolysate of the corn cob with glucose as the cosubstrate. The recombinant strain XP-RTK showed significantly higher xylitol productivity (212 mg L −1  h −1 ) over the control strain XP (81 mg L −1  h −1 ). Glucose was successfully replaced by glycerol as a co-substrate for xylitol production by S. cerevisiae . Strain XP-RTK showed the highest xylitol productivity of 318.6 mg L −1  h −1 and titre of 47 g L −1 of xylitol at 12 g L −1 initial DCW using glycerol as cosubstrate. The amount of glycerol consumed per amount of xylitol produced (0.47 mol mol −1 ) was significantly lower than glucose (23.7 mol mol −1 ). Fermentation strategies such as cell recycle and use of the industrial nitrogen sources were demonstrated using hemicellulosic hydrolysate for xylitol production.

Abstract Levan is a fructose polymer with diverse applications in the food and medical industries. In this study, levansucrase from Rahnella aquatilis (RaLsrA) was hyper-secreted using a Saccharomyces cerevisiae protein secretion system. An optimal secretion signal, a translation fusion partner (TFP) containing an N-terminal 98 amino acid domain from a mitochondrial inner membrane protein, UTH1, was employed to secrete approximately 50 U/mL of bioactive RaLsrA into culture media with 63% secretion efficiency by fed-batch fermentation. Although the purified RaLsrA was useful for enzymatic conversion of high-molecular-weight levan of approximately 3.75 × 106 Da, recombinant yeast secreting RaLsrA could produce levan more efficiently by microbial fermentation. In a 50-L scale fermenter, 76-g/L levan was directly converted from 191-g/L sucrose by recombinant yeast cells, attaining an 80% conversion yield and 3.17-g/L/h productivity. Thus, we developed a cost-effective and industrially applicable production system for food-grade levan.

2-Phenylethanol (2-PE) is widely used in food, perfume and pharmaceutical industry, but lower production in microbes and less known regulatory mechanisms of 2-PE make further study necessary. In this study, crucial genes like ARO8 and ARO10 of Ehrlich pathway for 2-PE synthesis and key transcription factor ARO80 in Saccharomyces cerevisiae were re-regulated using constitutive promoter; in the meantime, the effect of nitrogen source in synthetic complete (SC) medium with l -phenylalanine ( l -Phe) on Aro8/Aro9 and Aro10 was investigated. The results showed that aromatic aminotransferase activities of ARO8 over-expressing strains were seriously inhibited by ammonia sulfate in SC + Phe medium. Flask fermentation test demonstrated that over-expressing ARO8 or ARO10 led to about 42 % increase in 2-PE production when compared with the control strain. Furthermore, influence of transcription factors Cat8 and Mig1 on 2-PE biosynthesis was explored. CAT8 over-expression or MIG1 deletion increased in the transcription of ARO9 and ARO10 . 2-PE production of CAT8 over-expressing strain was 62 % higher than that of control strain. Deletion of MIG1 also led to 2-PE biosynthesis enhancement. The strain of CAT8 over-expression and MIG1 deletion was most effective in regulating expression of ARO9 and ARO10 . Analysis of mRNA levels and enzyme activities indicates that transaminase in Ehrlich pathway is the crucial target of Nitrogen Catabolize Repression (NCR). Among the engineering strains, the higher 3.73 g/L 2-PE production in CAT8 over-expressing strain without in situ product recovery suggests that the robust strain has potentiality for commercial exploitation.

ε-Poly- l -lysine (ε-PL) is a natural food preservative, which exhibits antimicrobial activity against a wide spectra of microorganisms. The production of ε-PL was significantly enhanced by pH shock in our previous study, but the underlying mechanism is poorly understood. According to transcriptional and physiological analyses in this study, the mprA/B and pepD signal transduction system was first proved to be presented and activated in Streptomyces albulus M-Z18 by pH shock, which positively regulated the transcription of ε-PL synthetase (Pls) gene and enhanced the Pls activity during fermentation. Furthermore, pH shock changed the ratio of unsaturation to saturation fatty acid in the membrane through up-regulating the transcription of fatty acid desaturase genes (SAZ_RS14940, SAZ_RS14945). In addition, pH shock also enhanced the transcription of cytochrome c oxidase (SAZ_RS15070, SAZ_RS15075), ferredoxin reductase (SAZ_RS34975) and iron sulfur protein (SAZ_RS31410) genes, and finally resulted in the improvement of cell respiratory activity. As a result, pH shock was considered to influence a wide range of proteins including regulators, fatty acid desaturase, respiratory chain component, and ATP-binding cassette transporter during fermentation. These combined influences might contribute to enhanced ε-PL productivity with pH shock.

Abstract Sophorolipids (SLs) are surface-active molecules produced by the non-pathogenic yeast Starmerella bombicola CGMCC 1576. Several genes involved in the synthesis of SLs have been identified. However, the regulation mechanism of the synthesis pathway for SLs has not been investigated. We recently discovered a protein in S. bombicola, which is structurally related to Yarrowia lipolytica YlBro1. To identify the function of the protein SbBro1 in S. bombicola, the deletion, overexpression, and complementary mutant strains were constructed. We found that the deletion mutant no longer produced SLs. Transcriptome analysis indicated that the expression levels of the key enzyme genes of SLs biosynthetic pathway were significantly down-regulated in the Δbro1, especially the expression level of cyp52m1 encoding the first rate-limiting enzyme in SL synthesis pathway was down-regulated 13-folds and the expression of fatty acid β-oxidation-related enzymes was also down-regulated. This study can give insight into the regulation of SL synthesis.