Single cell mutant selection for metabolic engineering of actinomycetes

Actinomycetes are important producers of pharmaceuticals and industrial enzymes. However, wild type strains require laborious development prior to industrial usage. Here we present a generally applicable reporter-guided metabolic engineering tool based on random mutagenesis, selective pressure, and single-cell sorting. We developed fluorescence-activated cell sorting (FACS) methodology capable of reproducibly identifying high-performing individual cells from a mutant population directly from liquid cultures. Genome-mining based drug discovery is a promising source of bioactive compounds, which is complicated by the observation that target metabolic pathways may be silent under laboratory conditions. We demonstrate our technology for drug discovery by activating a silent mutaxanthene metabolic pathway in Amycolatopsis. We apply the method for industrial strain development and increase mutaxanthene yields 9-fold to 99 mg l-1 in a second round of mutant selection. Actinomycetes are an important source of catabolic enzymes, where product yields determine industrial viability. We demonstrate 5-fold yield improvement with an industrial cholesterol oxidase ChoD producer Streptomyces lavendulae to 20.4 U g-1 in three rounds. Strain development is traditionally followed by production medium optimization, which is a time-consuming multi-parameter problem that may require hard to source ingredients. Ultra-high throughput screening allowed us to circumvent medium optimization and we identified high ChoD yield production strains directly from mutant libraries grown under preset culture conditions. In summary, the ability to screen tens of millions of mutants in a single cell format offers broad applicability for metabolic engineering of actinomycetes for activation of silent metabolic pathways and to increase yields of proteins and natural products.

Bone Damage during Dental Implant Insertion: A Pilot Study Combining Strain Gauge and Histologic Analysis

Besides alveolar bone quality, the drilling protocol applied in conjunction with the design of an implant are the major determinants of primary implant stability. Surgical trauma and bone compression resulting from implant insertion may constitute one cause for marginal bone resorption. Inserting two current bone-level implant designs (Astra; Straumann; n = 5) in bovine ribs, primary stability, strain development on the buccal bone plate and histologic signs of bone damage were recorded. Besides comparing the implant designs (Welch t-tests), all measurement parameters were checked for potential correlations (Pearson product moment correlation coefficients) with the level of significance set at α = 0.05. Considerable numbers of crack formation and plastic deformation of bone were observed after implant insertion. Straumann implants showed slightly greater values for insertion torque (p = 0.772), strain development (p = 0.893) and implant stability (p = 0.642). Significantly greater bone to implant contact (cortical p = 0.014; trabecular p = 0.016) was observed in Straumann implants, while Astra implants caused a significantly greater number of microcracks in cortical bone (p = 0.020). In Straumann implants, insertion torque correlated with bone to implant contact in the cortical area (p = 0.029) and the number of macrocracks in trabecular bone correlated with bone to implant contact (p = 0.029). In Astra implants, insertion torque and bone to implant contact in the trabecular area correlated (p = 0.007) as well as the number of macrocracks in trabecular bone and implant stability (p = 0.016). Additionally, in the area of cortical bone, the number of macrocracks correlated with bone to implant contact (p = 0.019). Implant placement results in bone damage of varying magnitude, which is governed by the drill protocol, implant macrodesign and bone quality.

Investigation of the Influence of Moisture Content on Fatigue Behaviour of HPC by Using DMA and XRCT

High-performance concrete (HPC) is a topic of current research and construction projects, due to its outstanding compressive strength and durability. In particular, its behaviour under high-cycle fatigue loading is the focus of current investigations, to further pave the way to highly challenging long-lasting constructions; e.g., bridges or offshore buildings. In order to investigate the behaviour of HPC with different moisture contents in more detail, a mixture of silica sand and basalt aggregate with a maximum grain size of 8 mm was investigated with three different moisture contents. For this purpose, cyclic compressive fatigue tests at a loading frequency of 10 Hz and different maximum stress levels were performed. The main focus was the moisture influence on the number of cycles to failure and the development of concrete temperature and strain. In a further step, only the mortar matrix was investigated. For this purpose, the mixture was produced without basalt, and the moisture influence was investigated on smaller-sized test specimens using dynamic mechanical analysis (DMA) and X-ray computed tomography (XRCT). It was shown that the moisture content of HPC had a significant influence on the fatigue damage behaviour due to the number of cycles to failure decreasing significantly with increased moisture. In addition, there was also an influence on the temperature development, as well as on the strain development. It was shown that increasing moisture content was associated with an increase in strain development. XRCT scans, in the course of the damage phases, showed an increase in internal cracks, and made their size visible. With the help of DMA as a new research method in the field of concrete research, we were also able to measure damage development related to a decrease in sample stiffness. Both methods, XRCT and DMA, can be listed as nondestructive methods, and thus can complement the known destructive test methods, such as light microscopy.

Development and characterization of efficient xylose utilization strains of Zymomonas mobilis

Background Efficient use of glucose and xylose is a key for the economic production of lignocellulosic biofuels and biochemicals, and different recombinant strains have been constructed for xylose utilization including those using Zymomonas mobilis as the host. However, the xylose utilization efficiency still needs to be improved. In this work, the strategy of combining metabolic engineering and adaptive laboratory evolution (ALE) was employed to develop recombinant Z. mobilis strains that can utilize xylose efficiently at high concentrations, and NGS-based genome resequencing and RNA-Seq transcriptomics were performed for strains evolved after serial transfers in different media to understand the impact of xylose and differences among strains with different xylose-utilization capabilities at molecular level. Results Heterologous genes encoding xylose isomerase and xylulokinase were evaluated, which were then introduced into xylose-utilizing strain Z. mobilis 8b to enhance its capacity of xylose utilization. The results demonstrated that the effect of three xylose isomerases on xylose utilization was different, and the increase of copy number of xylose metabolism genes can improve xylose utilization. Among various recombinant strains constructed, the xylose utilization capacity of the recombinant strain 8b-RsXI-xylB was the best, which was further improved through continuous adaption with 38 transfers over 100 days in 50 g/L xylose media. The fermentation performances of the parental strain 8b, the evolved 8b-S38 strain with the best xylose utilization capability, and the intermediate strain 8b-S8 in different media were compared, and the results showed that only 8b-S38 could completely consume xylose at 50 g/L and 100 g/L concentrations. In addition, the xylose consumption rate of 8b-S38 was faster than that of 8b at different xylose concentrations from 50 to 150 g/L, and the ethanol yield increased by 16 ~ 40%, respectively. The results of the mixed-sugar fermentation also demonstrated that 8b-S38 had a higher xylose consumption rate than 8b, and its maximum ethanol productivity was 1.2 ~ 1.4 times higher than that of 8b and 8b-S8. Whole-genome resequencing identified three common genetic changes in 8b-S38 compared with 8b and 8b-S8. RNA-Seq study demonstrated that the expression levels of genes encoding chaperone proteins, ATP-dependent proteases, phage shock proteins, ribosomal proteins, flagellar operons, and transcriptional regulators were significantly increased in xylose media in 8b-S38. The up-regulated expression of these genes may therefore contribute to the efficient xylose utilization of 8b-S38 by maintaining the normal cell metabolism and growth, repairing cellular damages, and rebalancing cellular energy to help cells resist the stressful environment. Conclusions This study provides gene candidates to improve xylose utilization, and the result of expressing an extra copy of xylose isomerase and xylulokinase improved xylose utilization also provides a direction for efficient xylose-utilization strain development in other microorganisms. In addition, this study demonstrated the necessity to combine metabolic engineering and ALE for industrial strain development. The recombinant strain 8b-S38 can efficiently metabolize xylose for ethanol fermentation at high xylose concentrations as well as in mixed sugars of glucose and xylose, which could be further developed as the microbial biocatalyst for the production of lignocellulosic biofuels and biochemicals.

Engineering of Streptoalloteichus tenebrarius 2444 for Sustainable Production of Tobramycin

Tobramycin is a broad-spectrum aminoglycoside antibiotic agent. The compound is obtained from the base-catalyzed hydrolysis of carbamoyltobramycin (CTB), which is naturally produced by the actinomycete Streptoalloteichus tenebrarius. However, the strain uses the same precursors to synthesize several structurally related aminoglycosides. Consequently, the production yields of tobramycin are low, and the compound’s purification is very challenging, costly, and time-consuming. In this study, the production of the main undesired product, apramycin, in the industrial isolate Streptoalloteichus tenebrarius 2444 was decreased by applying the fermentation media M10 and M11, which contained high concentrations of starch and dextrin. Furthermore, the strain was genetically engineered by the inactivation of the aprK gene (∆aprK), resulting in the abolishment of apramycin biosynthesis. In the next step of strain development, an additional copy of the tobramycin biosynthetic gene cluster (BGC) was introduced into the ∆aprK mutant. Fermentation by the engineered strain (∆aprK_1-17L) in M11 medium resulted in a 3- to 4-fold higher production than fermentation by the precursor strain (∆aprK). The phenotypic stability of the mutant without selection pressure was validated. The use of the engineered S. tenebrarius 2444 facilitates a step-saving, efficient, and, thus, more sustainable production of the valuable compound tobramycin on an industrial scale.

Microbial Cellulases: A Review on Strain Development, Purification, Characterization and their Industrial Applications

In this advance era, the enzymes are considered as a core kernel of white biotechnology and their demand is increasing day by day. According to report published in Research and Markets (ID: 5009185), the estimated global market for industrial enzymes were USD 10.0 billion in 2019, which is continuously increasing as it is expected to reach about USD 14.7 billion by 2022. Among all enzymes, cellulases are the major group of enzymes act synergistically in breakdown of cellulose, that facilitates its conversion to various value-added products and also offer several other important applications at industrial scale. The hyper production of cellulases are required to overcome their demand of global market. Cellulases production can be enhanced by strain improvement as well as using advance fermentation technology. In this review a detail studies of strategies to enhance production of cellulases and improve their physiochemical properties for industrial application have been described.

Structural behavior and shear bond capacity of composite slabs with high performance concrete

Many research works have been conducted on the behavior of composite slabs with profiled steel deck to study the longitudinal shear bond resistance using the m-k method. In this study, experimental investigations are conducted to evaluate the shear bond characeristics of composite slabs. 15 composite slabs are tested to study the effect of different high performance concrete (HPC) mixes namely engineered cementitious composites (ECC) and self-consolidating concrete (SCC), diverse profile sheets (with embossments or without embossments) and variable shear span on load-deflection characteristics, stress-strain development in concrete/steel, cracking/crack propagation and failure modes. The values of shear bond parameters (m and k) derived from the test results can be used for the design of composite slabs.

Structural behavior and shear bond capacity of composite slabs with high performance concrete

Many research works have been conducted on the behavior of composite slabs with profiled steel deck to study the longitudinal shear bond resistance using the m-k method. In this study, experimental investigations are conducted to evaluate the shear bond characeristics of composite slabs. 15 composite slabs are tested to study the effect of different high performance concrete (HPC) mixes namely engineered cementitious composites (ECC) and self-consolidating concrete (SCC), diverse profile sheets (with embossments or without embossments) and variable shear span on load-deflection characteristics, stress-strain development in concrete/steel, cracking/crack propagation and failure modes. The values of shear bond parameters (m and k) derived from the test results can be used for the design of composite slabs.