Parageobacillus Thermoglucosidasius Research Articles

Computer‐Aided Protein Surface Modification Strategy to Improve the Thermostability of α‐Amylase

AbstractStability under high‐temperature environments is crucial for amylase to function in starch‐based industries. This study develops a method of modifying protein surfaces by combining computer‐aided tools including FoldX, PoPMuSiC, Discovery Studio, and I‐Mutant 2.0 with conserved sequence analysis. A truncated α‐amylase ∆AmyPTG from Parageobacillus thermoglucosidasius DSMZ 2542 is rationally designed through this method to improve its thermostability. Seven single‐site variants are constructed and five of them displayed enhanced thermostability. Next, three double‐site variants are constructed with one particularly successful double‐site variant N31RT213R, exhibiting a 4.3‐fold longer half‐life at 80 °C. Notably, the specific activity of N31RT213R reaches 10 567.16 U mg−1, higher than ∆AmyPTG (6645.43 U mg−1). When applied to the corn starch liquefaction reaction, the mutant N31RT213R gets a higher yield of product concentration of about 255.70 µg mL−1, compared to 190.72 µg mL−1 for ∆AmyPTG. Intramolecular forces analysis and surface electrostatic charges analysis are conducted to determine possible causes for the improvement. Also, molecular dynamics simulation is used to analyze the flexibility shifts of the entire protein. This innovative rational engineering approach has proven to be a successful strategy for the selection of hot spots for protein thermostability evolution and has the potential to be applied to other enzymes.

Ethanologenic fermentation by Parageobacillus thermoglucosidasius with continuous hot microbubble gas-stripping

The increased use of biofuels in place of fossil fuels is one strategy to support the transition to net-zero carbon emissions, particularly in transport applications. However, expansion of the use of 1st generation crops as feedstocks is unsustainable due to the conflict with food use. The use of the lignocellulosic fractions from plants and/or co-products from food production including food wastes could satisfy the demand for biofuels without affecting the use of land and the availability of food, but organisms which can readily ferment all the carbohydrates present in these feedstocks often suffer from more severe bioethanol inhibition effects than yeast. This paper demonstrates the potential of hot gas microbubbles to strip ethanol from a thermophilic fermentation process using Parageobacillus thermoglucosidasius TM333, thereby reducing product inhibition and allowing production to continue beyond the nominal toxic ethanol concentrations of ≤ 2% v/v. Using an experimental rig in which cells were grown in fed-batch cultures on sugars derived from waste bread, and the broth continuously cycled through a purpose-built microbubble stripping unit, it was shown that non/low-inhibitory dissolved ethanol concentrations could be maintained throughout, despite reaching productivities equivalent to 4.7% v/v dissolved ethanol. Ethanol recovered in the condensate was at a concentration appropriate for dewatering to be cost effective and not prohibitively energy intensive. This suggests that hot microbubble stripping could be a valuable technology for the continuous production of bioethanol from fermentation processes which suffer from product inhibition before reaching economically viable titres, which is typical of most thermophilic ethanologenic bacteria.

A highly efficient method for genomic deletion across diverse lengths in thermophilic Parageobacillus thermoglucosidasius

Parageobacillus thermoglucosidasius is emerging as a highly promising thermophilic organism for metabolic engineering. The utilization of CRISPR-Cas technologies has facilitated programmable genetic manipulation in P. thermoglucosidasius. However, the absence of thermostable NHEJ enzymes limited the capability of the endogenous type I CRISPR-Cas system to generate a variety of extensive genomic deletions. Here, two thermophilic NHEJ enzymes were identified and combined with the endogenous type I CRISPR-Cas system to develop a genetic manipulation tool that can achieve long-range genomic deletion across various lengths. By optimizing this tool—through adjusting the expression level of NHEJ enzymes and leveraging our discovery of a negative correlation between GC content of the guide RNA (gRNA) and deletion efficacy—we streamlined a comprehensive gRNA selection manual for whole-genome editing, achieving a 100 % success rate in randomly selecting gRNAs. Notably, using just one gRNA, we achieved genomic deletions spanning diverse length, exceeding 200 kilobases. This tool will facilitate the genomic manipulation of P. thermoglucosidasius for both fundamental research and applied engineering studies, further unlocking its potential as a thermophilic cell factory.

The effects of synthesis gas feedstocks and oxygen perturbation on hydrogen production by Parageobacillus thermoglucosidasius

BackgroundThe facultatively anaerobic thermophile Parageobacillus thermoglucosidasius is able to produce hydrogen gas (H2) through the water–gas shift (WGS) reaction. To date this process has been evaluated under controlled conditions, with gas feedstocks comprising carbon monoxide and variable proportions of air, nitrogen and hydrogen. Ultimately, an economically viable hydrogenogenic system would make use of industrial waste/synthesis gases that contain high levels of carbon monoxide, but which may also contain contaminants such as H2, oxygen (O2) and other impurities, which may be toxic to P. thermoglucosidasius.ResultsWe evaluated the effects of synthesis gas (syngas) mimetic feedstocks on WGS reaction-driven H2 gas production by P. thermoglucosidasius DSM 6285 in small-scale fermentations. Improved H2 gas production yields and faster onset towards hydrogen production were observed when anaerobic synthetic syngas feedstocks were used, at the expense of biomass accumulation. Furthermore, as the WGS reaction is an anoxygenic process, we evaluated the influence of O2 perturbation on P. thermoglucosidasius hydrogenogenesis. O2 supplementation improved biomass accumulation, but reduced hydrogen yields in accordance with the level of oxygen supplied. However, H2 gas production was observed at low O2 levels. Supplementation also induced rapid acetate consumption, likely to sustain growth.ConclusionThe utilisation of anaerobic syngas mimetic gas feedstocks to produce H2 and the relative flexibility of the P. thermoglucosidasius WGS reaction system following O2 perturbation further supports its applicability towards more robust and continuous hydrogenogenic operation.

Parageobacillus thermoglucosidasius Strain Engineering Using a Theophylline Responsive RiboCas for Controlled Gene Expression.

The relentless increase in atmospheric greenhouse gas concentrations as a consequence of the exploitation of fossil resources compels the adoption of sustainable routes to chemical and fuel manufacture based on biological fermentation processes. The use of thermophilic chassis in such processes is an attractive proposition; however, their effective exploitation will require improved genome editing tools. In the case of the industrially relevant chassis Parageobacillus thermoglucosidasius, CRISPR/Cas9-based gene editing has been demonstrated. The constitutive promoter used, however, accentuates the deleterious nature of Cas9, causing decreased transformation and low editing efficiencies, together with an increased likelihood of off-target effects or alternative mutations. Here, we rectify this issue by controlling the expression of Cas9 through the use of a synthetic riboswitch that is dependent on the nonmetabolized, nontoxic, and cheap inducer, theophylline. We demonstrate that the riboswitches are dose-dependent, allowing for controlled expression of the target gene. Through their use, we were then able to address the deleterious nature of Cas9 and produce an inducible system, RiboCas93. The benefits of RiboCas93 were demonstrated by increased transformation efficiency of the editing vectors, improved efficiency in mutant generation (100%), and a reduction of Cas9 toxicity, as indicated by a reduction in the number of single nucleotide polymorphisms (SNPs) observed. This new system provides a quick and efficient way to produce mutants in P. thermoglucosidasius.

Draft genome of Parageobacillus thermoglucosidasius, a member of hydrogenogenic carbon monoxide utilizers, isolated from a freshwater lake sediment.

Parageobacillus thermoglucosidasius is a facultatively anaerobic thermophile and possesses carbon monoxide dehydrogenase and hydrogenase for carbon monoxide (CO) oxidation and hydrogen production, respectively. In this study, we report a draft genome of P. thermoglucosidasius isolated from a freshwater sediment, expanding our knowledge on the distribution of CO utilizers.

A thermostable type I-B CRISPR-Cas system for orthogonal and multiplexed genetic engineering

Thermophilic cell factories have remarkably broad potential for industrial applications, but are limited by a lack of genetic manipulation tools and recalcitrance to transformation. Here, we identify a thermophilic type I-B CRISPR-Cas system from Parageobacillus thermoglucosidasius and find it displays highly efficient transcriptional repression or DNA cleavage activity that can be switched by adjusting crRNA length to less than or greater than 26 bp, respectively, without ablating Cas3 nuclease. We then develop an orthogonal tool for genome editing and transcriptional repression using this type I-B system in both thermophile and mesophile hosts. Empowered by this tool, we design a strategy to screen the genome-scale targets involved in transformation efficiency and established dynamically controlled supercompetent P. thermoglucosidasius cells with high efficiency ( ~ 108 CFU/μg DNA) by temporal multiplexed repression. We also demonstrate the construction of thermophilic riboflavin cell factory with hitherto highest titers in high temperature fermentation by genome-scale identification and combinatorial manipulation of multiple targets. This work enables diverse high-efficiency genetic manipulation in P. thermoglucosidasius and facilitates the engineering of thermophilic cell factories.

Thermostable homologues of the periplasmic siderophore-binding protein CeuE from Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius.

Siderophore-binding proteins from two thermophilic bacteria, Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius, were identified from a search of sequence databases, cloned and overexpressed. They are homologues of the well characterized protein CjCeuE from Campylobacter jejuni. The iron-binding histidine and tyrosine residues are conserved in both thermophiles. Crystal structures were determined of the apo proteins and of their complexes with iron(III)-azotochelin and its analogue iron(III)-5-LICAM. The thermostability of both homologues was shown to be about 20°C higher than that of CjCeuE. Similarly, the tolerance of the homologues to the organic solvent dimethylformamide (DMF) was enhanced, as reflected by the respective binding constants for these ligands measured in aqueous buffer at pH 7.5 in the absence and presence of 10% and 20% DMF. Consequently, these thermophilic homologues offer advantages in the development of artificial metalloenzymes using the CeuE family.

Novel leaderless bacteriocin geobacillin 6 from thermophilic bacterium Parageobacillus thermoglucosidasius.

Bacterial resistance to conventional antibiotics has urged us to develop alternative strategies against bacterial pathogens. Moreover, a demand for food products containing no chemical preservatives has led us to search for new alternative technologies for food preservation. Bacteriocins - ribosomally synthesized antimicrobial peptides - have been proposed as a new alternative to conventional antibiotics or chemicals for food preservation. This study describes biosynthesis and characterization of a novel leaderless bacteriocin, geobacillin 6, which was identified in the thermophilic bacterium Parageobacillus thermoglucosidasius. Its amino acid sequence shows low similarity to other bacteriocins and it is the first leaderless-type bacteriocin identified in thermophilic bacteria. Based on structure assessment, the bacteriocin forms a multi-helix bundle. Geobacillin 6 exhibits a relatively narrow antimicrobial spectrum, it is active in the μM range and against Gram-positive bacteria, mostly thermophilic species closely related to the producer strain. Bacteriocin demonstrates stability over pH 3-11 and is highly thermostable, retaining 100% of its activity after incubation at 95°C for 6 h. Geobacillin 6 has potential in the food industry and biotechnological processes where contamination with thermophilic bacteria is undesirable.

An improved integrative GFP-based vector for genetic engineering of Parageobacillus thermoglucosidasius facilitates the identification of a key sporulation regulator

Parageobacillus thermoglucosidasius is a thermophilic Gram-positive bacterium, which is a promising host organism for sustainable bio-based production processes. However, to take full advantage of the potential of P. thermoglucosidasius, more efficient tools for genetic engineering are required. The present study describes an improved shuttle vector, which speeds up recombination-based genomic modification by incorporating a thermostable sfGFP variant into the vector backbone. This additional selection marker allows for easier identification of recombinants, thereby removing the need for several culturing steps. The novel GFP-based shuttle is therefore capable of facilitating faster metabolic engineering of P. thermoglucosidasius through genomic deletion, integration, or exchange. To demonstrate the efficiency of the new system, the GFP-based vector was utilised for deletion of the spo0A gene in P. thermoglucosidasius DSM2542. This gene is known to be a key regulator of sporulation in Bacillus subtilis, and it was therefore hypothesised that the deletion of spo0A in P. thermoglucosiadius would produce an analogous sporulation-inhibited phenotype. Subsequent analyses of cell morphology and culture heat resistance suggests that the P. thermoglucosidasius ∆spo0A strain is sporulation-deficient. This strain may be an excellent starting point for future cell factory engineering of P. thermoglucosidasius, as the formation of endospores is normally not a desired trait in large-scale production.

Functional characterization of the phosphotransferase system in Parageobacillus thermoglucosidasius

Parageobacillus thermoglucosidasius is a thermophilic bacterium characterized by rapid growth, low nutrient requirements, and amenability to genetic manipulation. These characteristics along with its ability to ferment a broad range of carbohydrates make P. thermoglucosidasius a potential workhorse in whole-cell biocatalysis. The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) catalyzes the transport and phosphorylation of carbohydrates and sugar derivatives in bacteria, making it important for their physiological characterization. In this study, the role of PTS elements on the catabolism of PTS and non-PTS substrates was investigated for P. thermoglucosidasius DSM 2542. Knockout of the common enzyme I, part of all PTSs, showed that arbutin, cellobiose, fructose, glucose, glycerol, mannitol, mannose, N-acetylglucosamine, N-acetylmuramic acid, sorbitol, salicin, sucrose, and trehalose were PTS-dependent on translocation and coupled to phosphorylation. The role of each putative PTS was investigated and six PTS-deletion variants could not grow on arbutin, mannitol, N-acetylglucosamine, sorbitol, and trehalose as the main carbon source, or showed diminished growth on N-acetylmuramic acid. We concluded that PTS is a pivotal factor in the sugar metabolism of P. thermoglucosidasius and established six PTS variants important for the translocation of specific carbohydrates. This study lays the groundwork for engineering efforts with P. thermoglucosidasius towards efficient utilization of diverse carbon substrates for whole-cell biocatalysis.

Metabolic engineering for the production of acetoin and 2,3-butanediol at elevated temperature in Parageobacillus thermoglucosidasius NCIMB 11955.

The current climate crisis has emphasised the need to achieve global net-zero by 2050, with countries being urged to set considerable emission reduction targets by 2030. Exploitation of a fermentative process that uses a thermophilic chassis can represent a way to manufacture chemicals and fuels through more environmentally friendly routes with a net reduction in greenhouse gas emissions. In this study, the industrially relevant thermophile Parageobacillus thermoglucosidasius NCIMB 11955 was engineered to produce 3-hydroxybutanone (acetoin) and 2,3-butanediol (2,3-BDO), organic compounds with commercial applications. Using heterologous acetolactate synthase (ALS) and acetolactate decarboxylase (ALD) enzymes, a functional 2,3-BDO biosynthetic pathway was constructed. The formation of by-products was minimized by the deletion of competing pathways surrounding the pyruvate node. Redox imbalance was addressed through autonomous overexpression of the butanediol dehydrogenase and by investigating appropriate aeration levels. Through this, we were able to produce 2,3-BDO as the predominant fermentation metabolite, with up to 6.6g/L 2,3-BDO (0.33g/g glucose) representing 66% of the theoretical maximum at 50°C. In addition, the identification and subsequent deletion of a previously unreported thermophilic acetoin degradation gene (acoB1) resulted in enhanced acetoin production under aerobic conditions, producing 7.6g/L (0.38g/g glucose) representing 78% of the theoretical maximum. Furthermore, through the generation of a ΔacoB1 mutant and by testing the effect of glucose concentration on 2,3-BDO production, we were able to produce 15.6g/L of 2,3-BDO in media supplemented with 5% glucose, the highest titre of 2,3-BDO produced in Parageobacillus and Geobacillus species to date.

Characterization of a Novel One-Domain Halotolerant Laccase from Parageobacillus thermoglucosidasius and Its Application in Dye Decolorization.

Laccases are widespread multi-copper oxidases and generally classified into three-domain laccases and two-domain laccases. In this study, a novel laccase PthLac from Parageobacillus thermoglucosidasius harbored only one domain of Cu-oxidase_4 and showed no sequence relatedness or structure similarity to three-domain and two-domain laccases. PthLac was heterologously expressed in Escherichia coli, purified, and characterized. The optimum temperature and pH of PthLac on guaiacol were at 60 ℃ and pH 6, respectively. The effects of various metal ions on PthLac were analyzed. All the tested metal ions did not suppress the activity of PthLac, except for 10mM Cu2+, which increased the activity of PthLac to 316%, indicating that PthLac was activated by Cu2+. Meanwhile, PthLac kept 121% and 69% activity when incubated at concentrations of 2.5 and 3M NaCl for 9h, suggesting the long-term halotolerancy of this enzyme. In addition, PthLac showed resistance to the organic solvents and surfactants, and displayed dye decolorization capacity. This study enriched our knowledge about one-domain laccase and its potential industrial applications.

Characterization of a novel cold-adapted intracellular serine protease from the extremophile Planococcus halocryophilus Or1.

The enzymes of microorganisms that live in cold environments must be able to function at ambient temperatures. Cold-adapted enzymes generally have less ordered structures that convey a higher catalytic rate, but at the cost of lower thermodynamic stability. In this study, we characterized P355, a novel intracellular subtilisin protease (ISP) derived from the genome of Planococcus halocryophilus Or1, which is a bacterium metabolically active down to -25°C. P355's stability and activity at varying pH values, temperatures, and salt concentrations, as well as its temperature-dependent kinetics, were determined and compared to an uncharacterized thermophilic ISP (T0099) from Parageobacillus thermoglucosidasius, a previously characterized ISP (T0034) from Planococcus sp. AW02J18, and Subtilisin Carlsberg (SC). The results showed that P355 was the most heat-labile of these enzymes, closely followed by T0034. P355 and T0034 exhibited catalytic constants (k cat ) that were much higher than those of T0099 and SC. Thus, both P355 and T0034 demonstrate the characteristics of the stability-activity trade-off that has been widely observed in cold-adapted proteases.

Genetic and functional characterization of multiple thermophilic organosulfur-removal systems reveals desulfurization potentials for waste residue oil cleaning

Heavy oil and petroleum refining residues usually contain high concentrations of recalcitrant hazardous organosulfur compounds, causing long-term serious global environmental pollution during leakage and combustion. Research conducted here identified a unique thermophilic bacterium Parageobacillus thermoglucosidasius W-36 with the notable ability of waste residue oil desulfurization, utilization and tolerance of multiplex hazardous organosulfur pollutants. Genome information mining revealed multiple desulfurization systems in three organosulfur-utilizing gene clusters. Enzymatic characterization, phylogenetic relationships, transcriptional performance and structural prediction indicated four novel key monooxygenases for diverse organosulfur removal. Importantly, all monooxygenases shared obvious commonalities in the predicted tertiary structure backbone and catalytic characteristics of C-S bond cleavage, implying the potential of genetic engineering for broad-spectrum hazardous organosulfur removal. Therefore, this work demonstrated the important application potential of thermophilic bacteria as a promising alternative biodesulfurization way for waste residue oil cleaning.

Simultaneous saccharification and ethanologenic fermentation (SSF) of waste bread by an amylolytic Parageobacillus thermoglucosidasius strain TM333

The starch in waste bread (WB) from industrial sandwich production was directly converted to ethanol by an amylolytic, ethanologenic thermophile (Parageobacillus thermoglucosidasius strain TM333) under 5 different simultaneous saccharification and fermentation (SSF) regimes. Crude α-amylase from TM333 was used alone or in the presence of amyloglucosidase (AMG), a starch monomerizing enzyme used in industry, with/without prior gelatinisation/liquefaction treatments and P. thermoglucosidasius TM333 fermentation compared with Saccharomyces cerevisiae as a control. Results suggest that TM333 can ferment WB using SSF with yields of 94–100% of theoretical (based on all sugars in WB) in 48 h without the need for AMG addition or any form of heat pre-treatment. This indicates that TM333 can transport and ferment all of the malto-oligosaccharides generated by its α-amylase. In the yeast control experiments, addition of AMG together with the crude α-amylase was necessary for full fermentation over the same time period. This suggests that industrial fermentation of WB starch to bio-ethanol or other products using an enhanced amylolytic P. thermoglucosidasius strain could offer significant cost savings compared to alternatives requiring enzyme supplementation.

Biologically evolved dual-pathway catalytic pattern indicating an efficient bioremediation strategy for phenol removal

Bioremediation of phenol has been developed for years, however, its actual application is challenged by low efficiency and environmental intolerance. In addition, although multiple phenol degradation pathways have been reported to coexist in bacteria, the potential significance and value have not been clearly revealed. Here, highly efficient phenol catalytic patterns were identified, and multi-pathway catalytic mechanism was investigated. Parageobacillus thermoglucosidasius W-36 employing a dual-pathway pattern exhibited superior degradation performance than P. thermoglucosidasius W-2 that employed a single-pathway pattern. An exogenous copy of degradation pathway in dual-pathway pattern was obtained via horizontal gene transfer. Based on homology modeling and molecular docking, the three-dimensional structures and substrate-binding sites of exogenous key enzymes were distinct from those of isofunctional enzymes encoded by the chromosome. The dual-pathway pattern was expressed in response to substrate levels in a time-dependent manner, indicating that synergistic effect of degradation pathways conferred strain adaptability to high-concentration phenol. Evolutionary analysis showed that the artful single/dual-pathway patterns in this species may be a natural selection phenomenon caused by high-concentration phenol. Collectively, this naturally evolved pattern enlightens the new strategy and feasibility of introducing auxiliary metabolic pathways to improve degradation efficiency of pollutants.

Thermophilic Water Gas Shift Reaction at High Carbon Monoxide and Hydrogen Partial Pressures in Parageobacillus thermoglucosidasius KP1013

The facultatively anaerobic Parageobacillus thermoglucosidasius oxidizes carbon monoxide to produce hydrogen via the water gas shift (WGS) reaction. In the current work, we examined the influence of carbon monoxide (CO) and hydrogen (H2) on the WGS reaction in the thermophilic P. thermoglucosidasius by cultivating two hydrogenogenic strains under varying CO and H2 compositions. Microbial growth and dynamics of the WGS reaction were monitored by evaluating parameters such as pressure, headspace composition, metabolic intermediates, pH, and optical density. Our analyses revealed that compared to the previously studied P. thermoglucosidasius strains, the strain KP1013 demonstrated higher CO tolerance and improved WGS reaction kinetics. Under anaerobic conditions, the lag phase before the WGS reaction shortened to 8 h, with KP1013 showing no hydrogen-induced product inhibition at hydrogen partial pressures up to 1.25 bar. The observed lack of product inhibition and the reduced lag phase of the WGS reaction support the possibility of establishing an industrial process for biohydrogen production with P. thermoglucosidasius.

Removing carbon catabolite repression in Parageobacillus thermoglucosidasius DSM 2542.

Parageobacillus thermoglucosidasius is a thermophilic bacterium of interest for lignocellulosic biomass fermentation. However, carbon catabolite repression (CCR) hinders co-utilization of pentoses and hexoses in the biomass substrate. Hence, to optimize the fermentation process, it is critical to remove CCR in the fermentation strains with minimal fitness cost. In this study, we investigated whether CCR could be removed from P. thermoglucosidasius DSM 2542 by mutating the Ser46 regulatory sites on HPr and Crh to a non-reactive alanine residue. It was found that neither the ptsH1 (HPr-S46A) nor the crh1 (Crh-S46A) mutation individually eliminated CCR in P. thermoglucosidasius DSM 2542. However, it was not possible to generate a ptsH1 crh1 double mutant. While the Crh-S46A mutation had no obvious fitness effect in DSM 2542, the ptsH1 mutation had a negative impact on cell growth and sugar utilization under fermentative conditions. Under these conditions, the ptsH1 mutation was associated with the production of a brown pigment, believed to arise from methylglyoxal production, which is harmful to cells. Subsequently, a less directed adaptive evolution approach was employed, in which DSM 2542 was grown in a mixture of 2-deoxy-D-glucose(2-DG) and xylose. This successfully removed CCR from P. thermoglucosidasius DSM 2542. Two selection strategies were applied to optimize the phenotypes of evolved strains. Genome sequencing identified key mutations affecting the PTS components PtsI and PtsG, the ribose operon repressor RbsR and adenine phosphoribosyltransferase APRT. Genetic complementation and bioinformatics analysis revealed that the presence of wild type rbsR and apt inhibited xylose uptake or utilization, while ptsI and ptsG might play a role in the regulation of CCR in P. thermoglucosidasius DSM 2542.

PMTL60000: A modular plasmid vector series for Parageobacillus thermoglucosidasius strain engineering

Parageobacillus thermoglucosidasius is a promising chassis for producing chemicals and fuels. Here we designed, built and tested the pMTL60000 modular plasmids containing standardised Gram-positive and Gram-negative replicons, selectable markers and application-specific modules. The pMTL60000 modular plasmids were characterised with regard to transformation efficiency, segregational stability, copy number and compatibility.