Understanding high ε-poly- l -lysine production by Streptomyces albulus using pH shock strategy in the level of transcriptomics Abstract 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.

Microbial production of O -methylated flavanones from methylated phenylpropanoic acids in engineered Escherichia coli Abstract Methylated flavonoids possess improved bioactivities compared to their unmethylated counterparts. In this study, for the efficient production of O-methylated flavonoids from simple methylated phenylpropanoic acids, a recombinant Escherichia coli strain expressing 4-coumarate:coenzyme A ligase (4CL) from Oryza sativa and chalcone synthase (CHS) from Hordeum vulgare was constructed; this strain produced significant amount of homoeriodictyol (~ 52 mg/L) as well as a few amount of hesperetin (0.4 mg/L), respectively, from ferulic acid and 4-methylcaffeic acid. This demonstrates, for the first time, that the scarce but valuable methylated flavanones can be successfully produced from methylated phenylpropanoic acids in a microbial host via an artificial biosynthetic pathway consisting of 4CL and CHS that can accept O-methylated precursors.

Beneficial mutations for carotenoid production identified from laboratory-evolved Saccharomyces cerevisiae Abstract Adaptive laboratory evolution (ALE) is a powerful tool used to increase strain fitness in the presence of environmental stressors. If production and strain fitness can be coupled, ALE can be used to increase product formation. In earlier work, carotenoids hyperproducing mutants were obtained using an ALE strategy. Here, de novo mutations were identified in hyperproducers, and reconstructed mutants were explored to determine the exact impact of each mutation on production and tolerance. A single mutation in YMRCTy1-3 conferred increased carotenoid production, and when combined with other beneficial mutations led to further increased β-carotene production. Findings also suggest that the ALE strategy selected for mutations that confer increased carotenoid production as primary phenotype. Raman spectroscopy analysis and total lipid quantification revealed positive correlation between increased lipid content and increased β-carotene production. Finally, we demonstrated that the best combinations of mutations identified for β-carotene production were also beneficial for production of lycopene.

Introduction to Special Issue on “Frontiers in Industrial Microbiology and Biotechnology 2019”

Towards sustainable bioplastic production using the photoautotrophic bacterium Rhodopseudomonas palustris TIE-1 Abstract Bacterial synthesis of polyhydroxybutyrates (PHBs) is a potential approach for producing biodegradable plastics. This study assessed the ability of Rhodopseudomonas palustris TIE-1 to produce PHBs under various conditions. We focused on photoautotrophy using a poised electrode (photoelectroautotrophy) or ferrous iron (photoferroautotrophy) as electron donors. Growth conditions were tested with either ammonium chloride or dinitrogen gas as the nitrogen source. Although TIE-1’s capacity to produce PHBs varied fairly under different conditions, photoelectroautotrophy and photoferroautotrophy showed the highest PHB electron yield and the highest specific PHB productivity, respectively. Gene expression analysis showed that there was no differential expression in PHB biosynthesis genes. This suggests that the variations in PHB accumulation might be post-transcriptionally regulated. This is the first study to systematically quantify the amount of PHB produced by a microbe via photoelectroautotrophy and photoferroautotrophy. This work could lead to sustainable bioproduction using abundant resources such as light, electricity, iron, and carbon dioxide.

Effect of temperature on methane oxidation and community composition in landfill cover soil Abstract Municipal solid waste (MSW) landfills are the third largest anthropogenic source of methane (CH4) emissions in the United States. The majority of CH4 generated in landfills is converted to carbon dioxide (CO2) by CH4-oxidizing bacteria (MOB) present in the landfill cover soil, whose activity is controlled by various environmental factors including temperature. As landfill temperature can fluctuate substantially seasonally, rates of CH4 oxidation can also vary, and this could lead to incomplete oxidation. This study aims at analyzing the effect of temperature on CH4 oxidation potential and microbial community structure of methanotrophs in laboratory-based studies of landfill cover soil and cultivated consortia. Soil and enrichment cultures were incubated at temperatures ranging from 6 to 70 °C, and rates of CH4 oxidation were measured, and the microbial community structure was analyzed using 16S rRNA gene amplicon sequencing and shotgun metagenome sequencing. CH4 oxidation occurred at temperatures from 6 to 50 °C in soil microcosm tests, and 6–40 °C in enrichment culture batch tests; maximum rates of oxidation were obtained at 30 °C. A corresponding shift in the soil microbiota was observed, with a transition from putative psychrophilic to thermophilic methanotrophs with increasing incubation temperature. A strong shift in methanotrophic community structure was observed above 30 °C. At temperatures up to 30 °C, methanotrophs from the genus Methylobacter were dominant in soils and enrichment cultures; at a temperature of 40 °C, putative thermophilic methanotrophs from the genus Methylocaldum become dominant. Maximum rate measurements of nearly 195 μg CH4 g−1 day−1 were observed in soil incubations, while observed maximum rates in enrichments were significantly lower, likely as a result of diffusion limitations. This study demonstrates that temperature is a critical factor affecting rates of landfill soil CH4 oxidation in vitro and that changing rates of CH4 oxidation are in part driven by changes in methylotroph community structure.

Tools and systems for evolutionary engineering of biomolecules and microorganisms Abstract Evolutionary approaches have been providing solutions to various bioengineering challenges in an efficient manner. In addition to traditional adaptive laboratory evolution and directed evolution, recent advances in synthetic biology and fluidic systems have opened a new era of evolutionary engineering. Synthetic genetic circuits have been created to control mutagenesis and enable screening of various phenotypes, particularly metabolite production. Fluidic systems can be used for high-throughput screening and multiplexed continuous cultivation of microorganisms. Moreover, continuous directed evolution has been achieved by combining all the steps of evolutionary engineering. Overall, modern tools and systems for evolutionary engineering can be used to establish the artificial equivalent to natural evolution for various research applications.

Prebiotics: tools to manipulate the gut microbiome and metabolome Abstract The human gut is an ecosystem comprising trillions of microbes interacting with the host. The composition of the microbiota and their interactions play roles in different biological processes and in the development of human diseases. Close relationships between dietary modifications, microbiota composition and health status have been established. This review focuses on prebiotics, or compounds which selectively encourage the growth of beneficial bacteria, their mechanisms of action and benefits to human hosts. We also review advances in synthesis technology for human milk oligosaccharides, part of one of the most well-characterized prebiotic–probiotic relationships. Current and future research in this area points to greater use of prebiotics as tools to manipulate the microbial and metabolic diversity of the gut for the benefit of human health.

Heterologous expression of the diazaquinomycin biosynthetic gene cluster Abstract Members of the diazaquinomycin class of natural products have shown potent and selective activity against Mycobacterium tuberculosis. However, poor aqueous solubility has prevented extensive studies in animal models thus far. Our long-term goal is to harness knowledge regarding diazaquinomycin biosynthesis towards the generation of derivatives for structure–activity relationship studies. We have previously sequenced the genomes of two diazaquinomycin-producing, actinomycete bacteria and identified putative daq biosynthetic gene clusters. Here, we report the heterologous expression of the daq gene cluster from the marine Streptomyces sp. F001 in S. coelicolor M1152. In addition to serving as functional proof for gene cluster assignment, the heterologous expression system reported here is expected to facilitate investigations aimed at elucidating diazaquinomycin biosynthesis.

Genomic and physiological analyses reveal that extremely thermophilic Caldicellulosiruptor changbaiensis deploys uncommon cellulose attachment mechanisms Abstract The genus Caldicellulosiruptor is comprised of extremely thermophilic, heterotrophic anaerobes that degrade plant biomass using modular, multifunctional enzymes. Prior pangenome analyses determined that this genus is genetically diverse, with the current pangenome remaining open, meaning that new genes are expected with each additional genome sequence added. Given the high biodiversity observed among the genus Caldicellulosiruptor, we have sequenced and added a 14th species, Caldicellulosiruptor changbaiensis, to the pangenome. The pangenome now includes 3791 ortholog clusters, 120 of which are unique to C. changbaiensis and may be involved in plant biomass degradation. Comparisons between C. changbaiensis and Caldicellulosiruptor bescii on the basis of growth kinetics, cellulose solubilization and cell attachment to polysaccharides highlighted physiological differences between the two species which are supported by their respective gene inventories. Most significantly, these comparisons indicated that C. changbaiensis possesses uncommon cellulose attachment mechanisms not observed among the other strongly cellulolytic members of the genus Caldicellulosiruptor.