Mutations in adaptively evolved Escherichia coli LGE2 facilitated the cost-effective upgrading of undetoxified bio-oil to bioethanol fuel

Levoglucosan is a promising sugar present in the lignocellulose pyrolysis bio-oil, which is a renewable and environment-friendly source for various value-added productions. Although many microbial catalysts have been engineered to produce biofuels and chemicals from levoglucosan, the demerits that these biocatalysts can only utilize pure levoglucosan while inhibited by the inhibitors co-existing with levoglucosan in the bio-oil have greatly limited the industrial-scale application of these biocatalysts in lignocellulose biorefinery. In this study, the previously engineered Escherichia coli LGE2 was evolved for enhanced inhibitor tolerance using long-term adaptive evolution under the stress of multiple inhibitors and finally, a stable mutant E. coli-H was obtained after ~ 374 generations’ evolution. In the bio-oil media with an extremely acidic pH of 3.1, E. coli-H with high inhibitor tolerance exhibited remarkable levoglucosan consumption and ethanol production abilities comparable to the control, while the growth of the non-evolved strain was completely blocked even when the pH was adjusted to 7.0. Finally, 8.4 g/L ethanol was achieved by E. coli-H in the undetoxified bio-oil media with ~ 2.0% (w/v) levoglucosan, reaching 82% of the theoretical yield. Whole-genome re-sequencing to monitor the acquisition of mutations identified 4 new mutations within the globally regulatory genes rssB, yqhA, and basR, and the − 10 box of the putative promoter of yqhD-dgkA operon. Especially, yqhA was the first time to be revealed as a gene responsible for inhibitor tolerance. The mutations were all responsible for improved fitness, while basR mutation greatly contributed to the fitness improvement of E. coli-H. This study, for the first time, generated an inhibitor-tolerant levoglucosan-utilizing strain that could produce cost-effective bioethanol from the toxic bio-oil without detoxification process, and provided important experimental evidence and valuable genetic/proteinic information for the development of other robust microbial platforms involved in lignocellulose biorefining processes.

Genome-Wide Transcriptome Profiling Provides Insight on Cholesterol and Lithocholate Degradation Mechanisms in Nocardioides simplex VKM Ac-2033D

Steroid microbial degradation plays a significant ecological role for biomass decomposition and removal/detoxification of steroid pollutants. In this study, the initial steps of cholesterol degradation and lithocholate bioconversion by a strain with enhanced 3-ketosteroid dehydrogenase (3-KSD) activity, Nocardioides simplex VKM Ac-2033D, were studied. Biochemical, transcriptomic, and bioinformatic approaches were used. Among the intermediates of sterol sidechain oxidation cholest-5-en-26-oic acid and 3-oxo-cholesta-1,4-dien-26-oic acid were identified as those that have not been earlier reported for N. simplex and related species. The transcriptomic approach revealed candidate genes of cholesterol and lithocholic acid (LCA) catabolism by the strain. A separate set of genes combined in cluster and additional 3-ketosteroid Δ1-dehydrogenase and 3-ketosteroid 9α-hydroxylases that might be involved in LCA catabolism were predicted. Bioinformatic calculations based on transcriptomic data showed the existence of a previously unknown transcription factor, which regulates cholate catabolism gene orthologs. The results contribute to the knowledge on diversity of steroid catabolism regulation in actinobacteria and might be used at the engineering of microbial catalysts for ecological and industrial biotechnology.

Biopurification of Oligosaccharides by Immobilized Kluyveromyces Lactis

Oligosaccharides with diverse and complex structures such as milk oligosaccharides have physiological functions including modulating intestinal microbiota or stimulating immune cell responses. However, milk carbohydrates include about 40–50% of lactose which requires a cost-effective method to separate. We developed a new method to purify the oligosaccharides from carbohydrate mixtures such as human milk oligosaccharides (HMOs) and galactooligosaccharides (GOSs) by exploiting immobilized Kluyveromyces lactis as microbial catalysts. Evaluation of media components exhibited no significant differences in the lactose removal efficiency when nutrient-rich media, minimal salt media, and distilled water without any media components were used. With the immobilization on alginate beads, the lactose removal efficiency was increased 3.4 fold compared to that of suspension culture. When the immobilized cells were reused to design a continuous process, 4 h of pre-activation enhanced the lactose eliminating performance 2.5 fold. Finally, immobilized K. lactis was used as microbial catalysts for the biopurification of HMOs and GOSs, and lactose was effectively removed without altering the overall distribution of oligosaccharides.

Biodegradation of Phenol

Aromatic compounds are widely distributed in nature. Free phenols are frequently liberated as metabolic intermediates during the degradation of plant materials. In recent years the natural supply of phenolic substances has been greatly increased due to the release of industrial byproducts into the environment. Phenolic compounds are hazardous pollutants that are toxic at relatively low concentration. Effluents from petrochemical, textile and coal industries contain phenolic compounds in very high concentration; therefore there is a necessity to remove phenolic compounds from the environment. Microorganisms capable of degrading phenol are common and include both aerobes and anaerobes. The use of microbial catalysts in the biodegradation of organic compounds has advanced significantly during the past three decades. The efficiency of biodegradation of organic compounds is influenced by the type of the organic pollutant, the nature of the organism, the enzyme involved, the mechanism of degradation and the nature of the influencing factors.

Porous nickel hollow fiber cathodes coated with CNTs for efficient microbial electrosynthesis of acetate from CO2 using Sporomusa ovata

Nickel porous hollow fibers coated with CNTs acted as both a gas transfer membrane for CO2 delivery and a cathode for providing electrons to microbial catalysts, achieving improved CO2 conversion to value-added products in microbial electrosynthesis.

Biodegradation of Phenol

Aromatic compounds are widely distributed in nature. Free phenols are frequently liberated as metabolic intermediates during the degradation of plant materials. In recent years the natural supply of phenolic substances has been greatly increased due to the release of industrial byproducts into the environment. Phenolic compounds are hazardous pollutants that are toxic at relatively low concentration. Effluents from petrochemical, textile and coal industries contain phenolic compounds in very high concentration; therefore there is a necessity to remove phenolic compounds from the environment. Microorganisms capable of degrading phenol are common and include both aerobes and anaerobes. The use of microbial catalysts in the biodegradation of organic compounds has advanced significantly during the past three decades. The efficiency of biodegradation of organic compounds is influenced by the type of the organic pollutant, the nature of the organism, the enzyme involved, the mechanism of degradation and the nature of the influencing factors.

Identification of Genes Conferring Tolerance to Lignocellulose-Derived Inhibitors by Functional Selections in Soil Metagenomes

ABSTRACTThe production of fuels or chemicals from lignocellulose currently requires thermochemical pretreatment to release fermentable sugars. These harsh conditions also generate numerous small-molecule inhibitors of microbial growth and fermentation, limiting production. We applied small-insert functional metagenomic selections to discover genes that confer microbial tolerance to these inhibitors, identifying both individual genes and general biological processes associated with tolerance to multiple inhibitory compounds. Having screened over 248 Gb of DNA cloned from 16 diverse soil metagenomes, we describe gain-of-function tolerance against acid, alcohol, and aldehyde inhibitors derived from hemicellulose and lignin, demonstrating that uncultured soil microbial communities hold tremendous genetic potential to address the toxicity of pretreated lignocellulose. We recovered genes previously known to confer tolerance to lignocellulosic inhibitors as well as novel genes that confer tolerance via unknown functions. For instance, we implicated galactose metabolism in overcoming the toxicity of lignin monomers and identified a decarboxylase that confers tolerance to ferulic acid; this enzyme has been shown to catalyze the production of 4-vinyl guaiacol, a valuable precursor to vanillin production. These metagenomic tolerance genes can enable the flexible design of hardy microbial catalysts, customized to withstand inhibitors abundant in specific bioprocessing applications.