Graphical abstract Highlights ► Metagenomic microbial community characterization. ► Extreme environment microbiology. ► High and low temperature adaptation.
A wide range of bioactive compounds is produced by enzymes and enzymatic complexes encoded in biosynthetic gene clusters (BGCs). These BGCs can be identified and functionally annotated based on their ...DNA sequence. Candidates for further research and development may be prioritized based on properties such as their functional annotation, (dis)similarity to known BGCs, and bioactivity assays. Production of the target compound in the native strain is often not achievable, rendering heterologous expression in an optimized host strain as a promising alternative. Genome-scale metabolic models are frequently used to guide strain development, but large-scale incorporation and testing of heterologous production of complex natural products in this framework is hampered by the amount of manual work required to translate annotated BGCs to metabolic pathways. To this end, we have developed a pipeline for an automated reconstruction of BGC associated metabolic pathways responsible for the synthesis of non-ribosomal peptides and polyketides, two of the dominant classes of bioactive compounds.
The developed pipeline correctly predicts 72.8% of the metabolic reactions in a detailed evaluation of 8 different BGCs comprising 228 functional domains. By introducing the reconstructed pathways into a genome-scale metabolic model we demonstrate that this level of accuracy is sufficient to make reliable in silico predictions with respect to production rate and gene knockout targets. Furthermore, we apply the pipeline to a large BGC database and reconstruct 943 metabolic pathways. We identify 17 enzymatic reactions using high-throughput assessment of potential knockout targets for increasing the production of any of the associated compounds. However, the targets only provide a relative increase of up to 6% compared to wild-type production rates.
With this pipeline we pave the way for an extended use of genome-scale metabolic models in strain design of heterologous expression hosts. In this context, we identified generic knockout targets for the increased production of heterologous compounds. However, as the predicted increase is minor for any of the single-reaction knockout targets, these results indicate that more sophisticated strain-engineering strategies are necessary for the development of efficient BGC expression hosts.
Acinetobacter sp. strain DSM 17874 is capable of utilizing n-alkanes with chain lengths ranging from that of decane (C₁₀H₂₂) to that of tetracontane (C₄₀H₈₂) as a sole carbon source. Two genes ...encoding AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, have been shown to be involved in the degradation of n-alkanes with chain lengths of from 10 to 20 C atoms in this strain. Here, we describe a novel high-throughput screening method and the screening of a transposon mutant library to identify genes involved in the degradation of n-alkanes with C chain lengths longer than 20, which are solid at 30°C, the optimal growth temperature for Acinetobacter sp. strain DSM 17874. A library consisting of approximately 6,800 Acinetobacter sp. strain DSM 17874 transposon mutants was constructed and screened for mutants unable to grow on dotriacontane (C₃₂H₆₆) while simultaneously showing wild-type growth characteristics on shorter-chain n-alkanes. For 23 such mutants isolated, the genes inactivated by transposon insertion were identified. Targeted inactivation and complementation studies of one of these genes, designated almA and encoding a putative flavin-binding monooxygenase, confirmed its involvement in the strain's metabolism of long-chain n-alkanes. To our knowledge, almA represents the first cloned gene shown to be involved in the bacterial degradation of long-chain n-alkanes of 32 C's and longer. Genes encoding AlmA homologues were also identified in other long-chain n-alkane-degrading Acinetobacter strains.
Apart from their archetypic use in anaerobic digestion (AD) methanogenic archaea are targeted for a wide range of applications. Using different methanogenic archaea for one specific application ...requires the optimization of culture media to enable the growth of different strains under identical environmental conditions, e.g., in microbial electrochemical technologies (MET) for (bio)electromethanation. Here we present a new culture medium (BFS01) adapted from the DSM-120 medium by omitting resazurin, yeast extract, casitone, and using a low salt concentration, that was optimized for
,
and
. The aim was to provide a medium for follow-up co-culture studies using specific methanogens and
spp. dominated biofilm anodes. All three methanogens showed growth and activity in the BFS01 medium. This was demonstrated by estimating the specific growth rates (
) and doubling times (
) of each methanogen. The
and
based on methane accumulation in the headspace showed values consistent with literature values for
and
. However,
and
based on methane accumulation in the headspace differed from literature data for
but still allowed sufficient growth. The lowered salt concentration and the omission of chemically complex organic components in the medium may have led to the observed deviation from
and
for
as well as the changed morphology. 16S rRNA gene-based amplicon sequencing and whole genome nanopore sequencing further confirmed purity and species identity.
Biofilms are resistant to many traditional antibiotics, which has led to search for new antimicrobials from different and unique sources. To harness the potential of aquatic microbial resources, we ...analyzed the meta-omics datasets of microalgae-bacteria communities and mined them for potential antimicrobial and quorum quenching enzymes. One of the most interesting candidates (Dlh3), a dienelactone hydrolase, is a α/β-protein with predicted eight α-helices and eight β-sheets. When it was applied to one of the major fish pathogens, Edwardsiella anguillarum, the biofilm development was reproducibly inhibited by up to 54.5%. The transcriptome dataset in presence of Dlh3 showed an upregulation in functions related to self-defense like active genes for export mechanisms and transport systems. The most interesting point regarding the biotechnological potential for aquaculture applications of Dlh3 are clear evidence of biofilm inhibition and that health and division of a relevant fish cell model (CHSE-214) was not impaired by the enzyme.
The production of concrete for construction purposes is a major source of anthropogenic CO2 emissions. One promising avenue towards a more sustainable construction industry is to make use of ...naturally occurring mineral-microbe interactions, such as microbial-induced carbonate precipitation (MICP), to produce solid materials. In this paper, we present a new process where calcium carbonate in the form of powdered limestone is transformed to a binder material (termed BioZEment) through microbial dissolution and recrystallization. For the dissolution step, a suitable bacterial strain, closely related to Bacillus pumilus, was isolated from soil near a limestone quarry. We show that this strain produces organic acids from glucose, inducing the dissolution of calcium carbonate in an aqueous slurry of powdered limestone. In the second step, the dissolved limestone solution is used as the calcium source for MICP in sand packed syringe moulds. The amounts of acid produced and calcium carbonate dissolved are shown to depend on the amount of available oxygen as well as the degree of mixing. Precipitation is induced through the pH increase caused by the hydrolysis of urea, mediated by the enzyme urease, which is produced in situ by the bacterium Sporosarcina pasteurii DSM33. The degree of successful consolidation of sand by BioZEment was found to depend on both the amount of urea and the amount of glucose available in the dissolution reaction.
The production of concrete is one of the most significant contributors to global greenhouse gas emissions. This work focuses on bio-cementation-based products and their potential to reduce global ...warming potential (GWP). In particular, we address a proposed bio-cementation method employing bacterial metabolism in a two-step process of limestone dissolution and recrystallisation (BioZEment). A scenario-based techno-economic analysis (TEA) is combined with a life cycle assessment (LCA), a market model and a literature review of consumers' willingness to pay, to compute the expected reduction of global GWP. Based on the LCA, the GWP of 1 ton of BioZEment is found to be 70-83% lower than conventional concrete. In the TEA, three scenarios are investigated: brick, precast and onsite production. The results indicate that brick production may be the easiest way to implement the products, but that due to high cost, the impact on global GWP will be marginal. For precast production the expected 10% higher material cost of BioZEment only produces a marginal increase in total cost. Thus, precast production has the potential to reduce global GWP from concrete production by 0-20%. Significant technological hurdles remain before BioZEment-based products can be used in onsite construction scenarios, but in this scenario, the potential GWP reduction ranges from 1 to 26%. While the potential to reduce global GWP is substantial, significant efforts need to be made both in regard to public acceptance and production methods for this potential to be unlocked.
Marine environments are home to an extensive number of microorganisms, many of which remain unexplored for taxonomic novelty and functional capabilities. In this study, a slow-growing
Streptomyces
...strain expressing unique genomic and phenotypic characteristics, P38-E01
T
, was described using a polyphasic taxonomic approach. This strain is part of a collection of over 8,000 marine Actinobacteria isolates collected in the Trondheim fjord of Norway by SINTEF Industry (Trondheim, Norway) and the Norwegian University of Science and Technology (NTNU, Trondheim, Norway). Strain P38-E01
T
was isolated from the sediments of the Trondheim fjord, and phylogenetic analyses affiliated this strain with the genus
Streptomyces
, but it was not closely affiliated with other described species. The closest related type strains were
Streptomyces daliensis
YIM 31724
T
(98.6%),
Streptomyces rimosus
subsp.
rimosus
ATCC 10970
T
(98.4%), and
Streptomyces sclerotialus
NRRL ISP-5269
T
(98.3%). Predominant fatty acids were C
16:0
iso, C
16:0
, and Summed Feature 3, and the predominant respiratory quinones were MK-10(H
6
), MK-10(H
4
), and MK9(H
4
). The main polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, and phosphoglycolipid. The whole-cell sugars were glucose, ribose, and in minor amounts, mannose. The cell wall peptidoglycan contained LL-diaminopimelic acid. The draft genome has a size of 6.16 Mb, with a %G + C content of 71.4% and is predicted to contain at least 19 biosynthetic gene clusters encoding diverse secondary metabolites. Strain P38-E01
T
was found to inhibit the growth of the pathogenic yeast
Candida albicans
ATCC 90028 and a number of Gram-positive bacterial human and plant pathogens. Metabolites extracted from cultures of P38-E01
T
were analyzed by mass spectrometry, and it was found that the isolate produced the antifungal compound candicidin. Phenotypic and chemotaxonomic signatures, along with phylogenetic analyses, distinguished isolate P38-E01
T
from its closest neighbors; thus, this isolate represents a novel species of the genus
Streptomyces
for which the name
Streptomyces tardus
sp. nov. (P38-E01
T
= CCM 9049
T
= DSM 111582
T
) is proposed.
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