First report of Pseudomonas nitroreducens cultured from the lungs of a patient with pneumonia.

A man in his 50s with neutropenic fever and multifocal lung opacities was diagnosed with a viral pneumonia. A small number of bacteria grown from bronchoalveolar lavage fluid collected during a repeat bronchoscopy were initially identified as Pseudomonas aeruginosa by VITEK-2 and mass spectrometry platforms. Whole-genome sequencing, however, subsequently demonstrated that the bacteria were Pseudomonas nitroreducens, representing the first known case of P. nitroreducens cultured from human lungs.

An extra copy of the ß-glucosidase gene improved the cellobiose fermentation capability of an engineered Saccharomyces cerevisiae strain.

In a previously engineered Saccharomyces cerevisiae recombinant, the cellobiose fermentation rate was significantly lower than the glucose fermentation rate. Thus, we implemented a genome-wide perturbation library to find gene targets for improving the cellobiose fermentation capability of the yeast strain. Unexpectedly, we discovered a transformant that contained an additional ß-glucosidase gene (gh1-1), possibly through homologous recombination between the plasmids. The additional ß-glucosidase led to the fastest cellobiose fermentation activity among all the transformants evaluated, and the strain demonstrated significantly higher ß-glucosidase activity than the control strain, especially during the initial exponential growth phase. The present work revealed the benefit of the extra gh1-1 copy for efficient cellobiose fermentation in the engineered S. cerevisiae strain.

Direct conversion of cellulose into ethanol and ethyl-ß-d-glucoside via engineered Saccharomyces cerevisiae.

Simultaneous saccharification and fermentation (SSF) of cellulose via engineered Saccharomyces cerevisiae is a sustainable solution to valorize cellulose into fuels and chemicals. In this study, we demonstrate the feasibility of direct conversion of cellulose into ethanol and a biodegradable surfactant, ethyl-ß-d-glucoside, via an engineered yeast strain (i.e., strain EJ2) expressing heterologous cellodextrin transporter (CDT-1) and intracellular ß-glucosidase (GH1-1) originating from Neurospora crassa. We identified the formation of ethyl-ß-d-glucoside in SSF of cellulose by the EJ2 strain owing to transglycosylation activity of GH1-1. The EJ2 strain coproduced 0.34 ± 0.03 g ethanol/g cellulose and 0.06 ± 0.00 g ethyl-ß-d-glucoside/g cellulose at a rate of 0.30 ± 0.02 g·L-1 ·h-1 and 0.09 ± 01 g·L-1 ·h-1 , respectively, during the SSF of Avicel PH-101 cellulose, supplemented only with Celluclast 1.5 L. Herein, we report a possible coproduction of a value-added chemical (alkyl-glucosides) during SSF of cellulose exploiting the transglycosylation activity of GH1-1 in engineered S. cerevisiae. This coproduction could have a substantial effect on the overall technoeconomic feasibility of theSSF of cellulose.

Expression of Gre2p improves tolerance of engineered xylose-fermenting Saccharomyces cerevisiae to glycolaldehyde under xylose metabolism.

Engineered S. cerevisiae employing the xylose reductase pathway enables efficient xylose valorization to fuels and chemicals. However, toxicity of thermochemically pretreated biomass hydrolysate on S. cerevisiae is one of the key technical challenges to upgrade biomass-derived sugars including xylose and glucose into high-value products. We investigated the effect of glycolaldehyde, one of the biomass-derived highly toxic aldehyde compounds, and its combinatorial inhibitory effect with other major fermentation inhibitors commonly found in plant hydrolysate such as methylglyoxal, 5-HMF, furfural, vanillin, and acetic acid on engineered xylose-fermenting S. cerevisiae in xylose and/or glucose media. We elucidated that glycolaldehyde and methylglyoxal are the key inhibitory short-aliphatic aldehydes on engineered xylose-fermenting S. cerevisiae in xylose-containing medium. Indeed, the degree of toxicity of these tested fermentation inhibitors varies with the sole carbon source of the medium. We demonstrate that genome integration of an extra copy of autologous GRE2 with its native promotor substantially improved the toxic tolerance of engineered xylose-fermenting S. cerevisiae to major inhibitory compounds including glycolaldehyde in the xylose-containing medium, and xylose-rich, lignocellulosic hydrolysate derived from Miscanthus giganteus, and concurrently improved the ethanol fermentation profile. Outcomes of this study will aid the development of next-generation robust S. cerevisiae strains for efficient fermentation of hexose and pentose sugars found in biomass hydrolysate.

Phenotypic evaluation and characterization of 21 industrial Saccharomyces cerevisiae yeast strains.

Microorganisms have been studied and used extensively to produce value-added fuels and chemicals. Yeasts, specifically Saccharomyces cerevisiae, receive industrial attention because of their well-known ability to ferment glucose and produce ethanol. Thousands of natural or genetically modified S. cerevisiae have been found in industrial environments for various purposes. These industrial strains are isolated from industrial fermentation sites, and they are considered as potential host strains for superior fermentation processes. In many cases, industrial yeast strains have higher thermotolerance, increased resistances towards fermentation inhibitors and increased glucose fermentation rates under anaerobic conditions when compared with laboratory yeast strains. Despite the advantages of industrial strains, they are often not well characterized. Through screening and phenotypic characterization of commercially available industrial yeast strains, industrial fermentation processes requiring specific environmental conditions may be able to select an ideal starting yeast strain to be further engineered. Here, we have characterized and compared 21 industrial S. cerevisiae strains under multiple conditions, including their tolerance to varying pH conditions, resistance to fermentation inhibitors, sporulation efficiency and ability to ferment lignocellulosic sugars. These data may be useful for the selection of a parental strain for specific biotechnological applications of engineered yeast.

Pyrosequencing analysis of microbiota reveals that lactic acid bacteria are dominant in Korean flat fish fermented food, gajami-sikhae.

The gajami-sikhae, traditional Korean cuisine fermented with flat fish, samples were collected from eight different manufacturers (GS1-GS8). We employed pyrosequencing method to analyze the bacterial communities of the gajami-sikhae samples. Family- and genus-level analyses indicated that the bacterial community compositions of GS3 and GS6 were distinct from those of the rest. The species-level structures of bacterial communities of the gajami-sikhae samples except for GS3 and GS6 featured Lactobacillus sakei as the most abundant species. Leuconostoc mesenteroides was revealed as the most dominant species among the bacterial community of GS6 and the bacterial community of GS3 was composed of various lactic acid bacteria. We employed a culture-based method to isolate beneficial strains from the gajami-sikhae samples. However, most of the 47 selected colonies were identified as Bacillus subtilis and Bacillus amyloliquefaciens. This study indicated that gajami-sikhae was mainly composed of many beneficial lactic acid bacteria.

Combinatorial genetic perturbation to refine metabolic circuits for producing biofuels and biochemicals.

Recent advances in metabolic engineering have enabled microbial factories to compete with conventional processes for producing fuels and chemicals. Both rational and combinatorial approaches coupled with synthetic and systematic tools play central roles in metabolic engineering to create and improve a selected microbial phenotype. Compared to knowledge-based rational approaches, combinatorial approaches exploiting biological diversity and high-throughput screening have been demonstrated as more effective tools for improving various phenotypes of interest. In particular, identification of unprecedented targets to rewire metabolic circuits for maximizing yield and productivity of a target chemical has been made possible. This review highlights general principles and the features of the combinatorial approaches using various libraries to implement desired phenotypes for strain improvement. In addition, recent applications that harnessed the combinatorial approaches to produce biofuels and biochemicals will be discussed.