Enhanced production of ectoine from methane using metabolically engineered Methylomicrobium alcaliphilum 20Z

Background Ectoine (1,3,4,5-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is an attractive compatible solute because of its wide industrial applications. Previous studies on the microbial production of ectoine have focused on sugar fermentation. Alternatively, methane can be used as an inexpensive and abundant resource for ectoine production by using the halophilic methanotroph, Methylomicrobium alcaliphilum 20Z. However, there are some limitations, including the low production of ectoine from methane and the limited tools for the genetic manipulation of methanotrophs to facilitate their use as industrial strains. Results We constructed M. alcaliphilum 20ZDP with a high conjugation efficiency and stability of the episomal plasmid by the removal of its native plasmid. To improve the ectoine production in M. alcaliphilum 20Z from methane, the ectD (encoding ectoine hydroxylase) and ectR (transcription repressor of the ectABC-ask operon) were deleted to reduce the formation of by-products (such as hydroxyectoine) and induce ectoine production. When the double mutant was batch cultured with methane, ectoine production was enhanced 1.6-fold compared to that obtained with M. alcaliphilum 20ZDP (45.58 mg/L vs. 27.26 mg/L) without growth inhibition. Notably, a maximum titer of 142.32 mg/L was reached by the use of an optimized medium for ectoine production containing 6% NaCl and 0.05 μM of tungsten without hydroxyectoine production. This result demonstrates the highest ectoine production from methane to date. Conclusions Ectoine production was significantly enhanced by the disruption of the ectD and ectR genes in M. alcaliphilum 20Z under optimized conditions favoring ectoine accumulation. We demonstrated effective genetic engineering in a methanotrophic bacterium, with enhanced production of ectoine from methane as the sole carbon source. This study suggests a potentially transformational path to commercial sugar-based ectoine production. Graphical Abstract

Transcriptomic Responses of Cordyceps militaris to Salt Treatment During Cordycepins Production

Cordycepin is a major bioactive compound found in Cordyceps militaris (C. militaris) that exhibits a broad spectrum of biological activities. Hence, it is potentially a bioactive ingredient of pharmaceutical and cosmetic products. However, overexploitation and low productivity of natural C. militaris is a barrier to commercialization, which leads to insufficient supply to meet its existing market demands. In this study, a preliminary study of distinct concentrations of salt treatments toward C. militaris was conducted. Although the growth of C. militaris was inhibited by different salt treatments, the cordycepin production increased significantly accompanied by the increment of salt concentration. Among them, the content of cordycepin in the 7% salt-treated group was five-fold higher than that of the control group. Further transcriptome analysis of samples with four salt concentrations, coupled with Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, several differentially expressed genes (DEGs) were found. Finally, dynamic changes of the expression patterns of four genes involved in the cordycepin biosynthesis pathway were observed by the quantitative real-time PCR. Taken together, our study provides a global transcriptome characterization of the salt treatment adaptation process in C. militaris and facilitates the construction of industrial strains with a high cordycepin production and salt tolerance.

Redirected Stress Responses in a Genome-Minimized ‘midi Bacillus ’ Strain with Enhanced Capacity for Protein Secretion

Our present study showcases a genome-minimized nonpathogenic bacterium, the so-called midi Bacillus , as a chassis for the development of future industrial strains that serve in the production of high-value difficult-to-produce proteins. In particular, we explain how midi Bacillus , which lacks about one-third of the original genome, effectively secretes a protein of the major human pathogen Staphylococcus aureus that cannot be produced by the parental Bacillus subtilis strain.

Decoding antibacterial and antibiofilm properties of cinnamon and cardamom essential oils: a combined molecular docking and experimental study

This study sets out to compare the antibacterial and antibiofilm profiles of Ci/Ca EOs alone and in combination together against infectious bacterial strains. MIC assay was carried out to survey the effectiveness of prepared EOs by two-fold serial dilution method and MTT evaluation. Synergic antibacterial properties of EOs against target strains were studied by using checkerboard titration method. Biofilm growth and development were evaluated using CV and XTT reduction assays. Antibacterial activity was observed for EOs against both bacterial strains with stronger activity for CiEO against both bacteria. The synergistic antibacterial effect was observed only against B. subtilis. Based on the FIC index, combinations could not inhibit the growth of E. coli. The pure EOs and their combination inhibited cell attachment for both studied bacteria with stronger effect on E. coli. CV and XTT reduction assays results showed that Ci EO and its combination with CaEO had the highest antibiofilm activity at lowest MIC value 0.08% and 0.04/0.02% against biofilm formed by E. coli and B. subtilis respectively, indicating a high antibiofilm potential. Computational docking analyses also postulated that the active constituents of evaluated EOs have the potential to interact with different bacterial targets, suggested binding mode of action of EOs metabolites. By and large, synergistic anti-biofilm properties of EOs may provide further options for developing novel formula to inhibit a variety of infectious clinical and industrial strains without (or less) toxicity effects on human body. Graphical Abstract

Decoding Antibacterial and Antibiofilm Properties of Cinnamon and Cardamom Essential Oils: A Combined Molecular Docking and Experimental Study

This study sets out to compare the antibacterial and antibiofilm profiles of Ci/Ca EOs alone and in combination together against infectious bacterial strains. MIC assay was carried out to survey the effectiveness of prepared EOs by two-fold serial dilution method and MTT evaluation. Synergic antibacterial properties of EOs against target strains were studied by using checkerboard titration method. Biofilm growth and development were evaluated using CV and XTT reduction assays. Antibacterial activity was observed for EOs against both bacterial strains with stronger activity for CiEO against both bacteria. The synergistic antibacterial effect was observed only against B. subtilis. Based on the FIC index, combinations could not inhibit the growth of E. coli. The pure EOs and their combination inhibited cell attachment for both studied bacteria with stronger effect on E. coli. CV and XTT reduction assays results showed that Ci EO and its combination with CaEO had the highest antibiofilm activity at lowest MIC value 0.08% and 0.04/0.02% against biofilm formed by E. coli and B. subtilis respectively, indicating a high antibiofilm potential. Computational docking analyses also postulated that the active constituents of evaluated EOs have the potential to interact with different bacterial targets, suggested binding mode of action of EOs metabolites. By and large, synergistic anti-biofilm properties of EOs may provide further options for developing novel formula to inhibit a variety of infectious clinical and industrial strains without (or less) toxicity effects on human body.

Ecological safety and prospects of development of low-waste technologies in the biotechnology industry

Environmental pollution with industrial waste is an urgent problem today. A special place in the list of pollutants belongs to waste from biotechnological enterprises and industries, whose activities are related to the production of various drugs. Russian Research Anti-Plague Institute «Microbe» is the only manufacturer of unique immunobiological drugs in the Russian Federation – bivalent chemical cholera vaccine and rabies immunoglobulin from horse blood serum (AIG). At present, the institute actively uses fibrin as a basis for nutrient media – a waste in the production of AIG; a technology for the regeneration of alcohol waste has been developed; biologically active substances were obtained from the production waste of specific components of the cholera vaccine. The aim of the work was to assess the prospects of using waste products from the production of specific components of cholera vaccine (cholerogen-toxoid – X-AT, and O-antigen – O-AG) – formalized detoxified microbial-free filtrate (FMF), as a nutrient medium for the cultivation of industrial strains of microorganisms. It has been shown that the best methods for reducing formalin concentration are autoclaving and chemical neutralization with aqueous ammonia. During low-volume cultivation of Vibrio cholerae 569B and V. cholerae M-41 strains on all variants of experimental media based on PBP, an increase in biomass was noted. The production of Vibrio cholerae antigens at a level comparable to that of growing on a control nutrient medium was recorded in a medium variant based on O-AG production waste. The use of FMF as a nutrient medium in the future will reduce the volume of waste generated and reduce the load on the treatment facilities of the Institute, which will increase the environmental safety of production.

Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria

Abstract4-Androstene-3,17-dione (4-AD), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD), which are important starting compounds for the synthesis of steroidal medicines, can be biosynthetically transformed from phytosterols by Mycobacterium strains. Genomic and metabolic analyses have revealed that currently available 4-AD-producing strains maintain the ability to convert 4-AD to ADD and 9OH-AD via 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase (Ksh), not only lowering the production yield of 4-AD but also hampering its purification refinement. Additionally, these 4-AD industrial strains are excellent model strains to construct ADD- and 9OH-AD-producing strains. We recently found that Mycobacterium neoaurum HGMS2, a 4-AD-producing strain, harbored fewer kstd and ksh genes through whole-genomic and enzymatic analyses, compared with other strains (Wang et al. in Microbial Cell Fact 19:187, 2020). In this study, we attempted to construct an efficient 4-AD-producing strain by knocking out the kstd and ksh genes from the M. neoaurum HGMS2 strain. Next, we used kstd- and ksh-default HGMS2 mutants as templates to construct ADD- and 9OH-AD-producing strains by knocking in active kstd and ksh genes, respectively. We found that after knocking out its endogenous kstd and ksh genes, one of these knockout mutants, HGMS2Δkstd211 + ΔkshB122, showed a 20% increase in the rate of phytosterol to 4-AD conversion, compared relative to the wild-type strain and an increase in 4-AD yield to 38.3 g/L in pilot-scale fermentation. Furthermore, we obtained the ADD- and 9OH-AD-producing strains, HGMS2kstd2 + Δkstd211+ΔkshB122 and HGMS2kshA51 + Δkstd211+ΔkshA226, by knocking in heterogenous active kstd and ksh genes to selected HGMS2 mutants, respectively. During pilot-scale fermentation, the conversion rates of the ADD- and 9OH-AD-producing mutants transforming phytosterol were 42.5 and 40.3%, respectively, and their yields reached 34.2 and 37.3 g/L, respectively. Overall, our study provides efficient strains for the production of 4-AD, ADD and 9OH-AD for the pharmaceutical industry and provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds.

Genome Editing in Filamentous Fungus Penicillium Verruculosum Using the CRISPR/Cas9 System

PurposeTo adapt CRISPR/Cas9 genome editing method for use in filamentous fungus Penicillium verruculosum, which is industrial producer of carbohydrases. ResultsFor the first time the CRISPR/Cas9 method was adapted for genome editing in the filamentous fungi Penicillium verrucullosum. Using the nitrate reductase gene (niaD) as a selective marker with the CRISPR/Cas9 system we performed double knockout of niaD and cellobiohydrolase 1 (cbh1) genes. The efficiency of double editing was 50%. At the same time, it was unexpected that the specific cellobiohydrolase activity rised after knockout of cbh1 gene due to the increase CBH2 expression. ConclusionWe developed effective method for genome editing in P. verruculosum, which can be used to improve qualities of industrial strains.