Kamar Zekhnini
email: kamar.zekhnini@gmail.com

Anass Cherrafi
email: a.cherrafi@ensam.umi.ac.ma

Imane Bouhaddou

Abla Chaouni Benabdellah
email: benabdellah.abla@gmail.com

Suppliers selection ontology for viable digital supply chain performance

Keywords: Suppliers Selection, Viable Digital Supply Chain, Resilience, Sustainability. Ontology, Knowledge Management

Unprecedented challenges have confronted the contemporary era with serious negative effect on supply chain management performance. As known, organizations performance is dependent on their suppliers. For this reason, organizations have to improve their practices in suppliers' selection process considering resilience, sustainability and digitalization capabilities to retain competitive and ensure viable performance. In this context, this paper aims to propose an ontology-based model for suppliers' selection criteria with consideration of digitalization, sustainability and resilience capabilities. The incorporation of those paradigms while selecting appropriate suppliers enables the exploration of viable supply chain performance in regard to disruptions. The proposed method is useful to both academics and professionals because it addresses the prominent criteria taxonomy for an effective suppliers' selection in the disruption and digital era.

Introduction

With the spread of globalization and the evolution of industries, there is a crucial need for effective and reliable supply chain systems [START_REF] Zekhnini | Supply chain management 4.0: a literature review and research framework[END_REF]. A more interconnected environment certainly holds both benefits and risks [START_REF] Zekhnini | Supply Chain 4.0 Risk Management: Bibliometric Analysis and a Proposed Framework[END_REF][START_REF] Zekhnini | Supply chain 4.0 risk management: an interpretive structural modelling approach[END_REF]. For instance, disruptions affect the daily flow of goods or services within a system. Besides, organizations today are more exposed to increased and various disruptive events (unexpected exchange rates, supply fluctuations, dynamic markets, geopolitical tensions, volatile demand, and natural disasters) that have a direct impact on SC performance [START_REF] Zekhnini | Analytic Hierarchy Process (AHP) for Supply Chain 4.0 Risks Management[END_REF]. More clearly, this implies that there is an increased need for enhanced supply chain viability practice and study. One of the supply chain management (SCM) challenges that have piqued the interest of many researchers is supplier selection [START_REF] Mesmer | Enabling supplier discovery through a part-focused manufacturing process ontology[END_REF]. In other words, the supplier is an important actor in the end-to-end value chain [START_REF] Jaskó | Development of manufacturing execution systems in accordance with Industry 4.0 requirements: A review of standard-and ontology-based methodologies and tools[END_REF]. Because one of the most important factors to achieve an organizations' performance is the selection of relevant suppliers. Therefore, there has been an increased interest in the selection of reliable and robust suppliers [START_REF] Zekhnini | Supplier selection for smart supply chain: An adaptive fuzzy-neuro approach 8[END_REF]. In this context, to remain competitive and improve their performance, organizations need to consider and include the viable capability in suppliers' selection criteria. More clearly, the selected suppliers should have the capacity to adapt to the difficulties and function in a changing world. Besides, they should "react agilely to positive changes, be resilient to absorb negative events and re-cover after the disruptions and survive at the times of long-term" [8]. This means that organizations should take into consideration the 'digitalization', 'resilience', and, 'sustainability criteria in the supplier's selection.

Several studies focused on the suppliers' selection process methods and techniques [START_REF] Parmar | A clustering algorithm for supplier base management[END_REF][START_REF] Che | A genetic algorithm-based model for solving multi-period supplier selection problem with assembly sequence[END_REF][START_REF] Ghadimi | A multi-agent systems approach for sustainable supplier selection and order allocation in a partnership supply chain[END_REF]. However, little consideration is given to determining and selecting the right evaluation criteria. Whereas, choosing appropriate criteria determines the quality and accuracy of the developed model [START_REF] Konys | Green Supplier Selection Criteria: From a Literature Review to a Comprehensive Knowledge Base[END_REF]. For this reason, it is widely recommended to exploit and develop suppliers' selection knowledge assets. Especially that spreading knowledge in supplier selection while considering resilience, sustainability, and digitalization capabilities, as well as a lack of proper knowledge about criteria describing them, create an evident necessity to have a comprehensive solution for gathering and sharing knowledge, and underlining the interrelationships between them. To overcome this research challenge, the capitalization of knowledge in an ontology for supporting supplier selection and assessment criteria is recommended. In this respect, this paper aims to provide an ontology-based model for supporting suppliers' selection for viable supply chain (VSC) performance. It analyses and organizes knowledge about suppliers' selection criteria including so-called digitalization and resilience capabilities. Moreover, it helps to share information about currently used supplier selection criteria to allow semantics-driven knowledge processing.

This article is organized as follows: in the following section, we present the related works. Section 3 illustrates the research methodology. Section 4 presents the developed ontology supporting the viable-oriented suppliers' selection. And finally, section 5 presents the managerial and practical implications.

Related works

Previous research explored and analyzed supplier selection in the digital supply chain using a variety of methods such as Multi-Criteria Decision-Making Methods, Mathematical Programming models, AI approaches for the development of decision support systems [START_REF] Parmar | A clustering algorithm for supplier base management[END_REF][START_REF] Che | A genetic algorithm-based model for solving multi-period supplier selection problem with assembly sequence[END_REF][START_REF] Ghadimi | A multi-agent systems approach for sustainable supplier selection and order allocation in a partnership supply chain[END_REF][START_REF] Stević | Sustainable supplier selection in healthcare industries using a new MCDM method: Measurement of alternatives and ranking according to COmpromise solution (MARCOS)[END_REF]. However, every robust decision-making system requires a variety of context data to support the assessment process. To handle the massive amount of information in suppliers' selection process, it is valuable to use knowledge management tools [START_REF] Yu | An agent-based negotiation model for supplier selection of multiple products with synergy effect[END_REF]. Ontologies refer to a hierarchical conceptual model representing and capitalizing knowledge that provides definitions of relations and classes (concepts) that incorporate domain knowledge in an integrated manner [START_REF] Kundu | On development of supplier segmentation ontology using latent semantic analysis for supplier knowledge management in supply chain[END_REF][START_REF] Benabdellah | Design for the environment: An ontology-based knowledge management model for green product development[END_REF].

The reviewed literature also presented a range of supplier selection criteria approaches, evolving from standard data gathering methods for text analysis to more advanced semantic-matching-oriented methods based on ontologies [17]. In this context, some ontological models aimed at addressing suppliers' selection criteria have been developed. On the one side, [18,[START_REF] Achatbi | Advanced system based on ontology and multi agent technology to handle upstream supply chain: intelligent negotiation protocol for supplier and transportation provider selection[END_REF] suggest an ontology-based negotiating decision protocol to support the suppliers' selection. Reference [START_REF] Kundu | On development of supplier segmentation ontology using latent semantic analysis for supplier knowledge management in supply chain[END_REF] established a Supplier Segmentation Ontology based on knowledge derived from the literature on behavioral operations in the supply chain from 1934 to 2013. On the other side, references [START_REF] Konys | Green Supplier Selection Criteria: From a Literature Review to a Comprehensive Knowledge Base[END_REF][START_REF] Wątróbski | Ontology Supporting Green Supplier Selection Process[END_REF] provide a conceptual context for collecting and maintaining knowledge about green supplier selection criteria, as well as formal guidance for their proper acquisition.

These ontologies have proved to expand the knowledge, but they have a restricted scope. More clearly, these ontologies have covered the green aspect or the sustainable aspect of suppliers' selection criteria. And as far as we know, there is no paper dealing with developing an ontology-based model considering the resilience and digitalization capabilities. For this reason, this paper aims to cover the three capabilities namely resilience, digitalization, and sustainability in one ontology to ensure the SC viability performance.

3

Research methodology

Development of ontology-based model for supporting viable-oriented suppliers' selection 4.1 Knowledge identification and classification

Our ontology-based model is built on the identification and classification of knowledge via a structured approach to modeling the criteria required for supplier selection while considering three complex capabilities: "resilience", "sustainability," and "digitalization." Concepts and relations are the principal component of our model. Thus, the first step to be taken when defining the relations is to define the domain and range. For doing so, through the literature review, the unstructured data obtained from a wide variety of scientific articles have been reincorporated into semi-structured form. Besides, the collection of criteria and sub-criteria was conceptualized based on the given set of clusters and items allocated to them. More clearly, we defined five classes (Resilience Criteria, Sustainability Criteria, Digitalization Criteria, Primary Criteria, and Profile Criteria) representing the criteria ensuring the viable selection of suppliers. Besides, several sub-criteria were defined for each criterion considering also circular economy (use and recovery).

Knowledge capitalization

The analysis of literature and existing ontologies enables one to specifically define knowledge that is used and elaborated. With this analysis, we have written a series of knowledge which we have attributed to criteria. They were then verified and validated by an expert to finally can be capitalized on the taxonomy. We may build a list of knowledge that has been established as important for capitalization.

Knowledge typology and taxonomy

We generated an information classification to present a Knowledge classification for the supplier selection criteria. We also defined five Knowledge classes (Table 1).

Each class reflects a particular type of knowledge. Hence, we developed a taxonomy to organize the Knowledge. In other words, it provides a hierarchical classification of data entities based on the implied connections of the real-world entities that they represent. Besides, the grouping is based on the resemblance of the knowledge entities known as concepts. This taxonomy illustrates the layout of the necessary criteria for selecting viable suppliers (Fig. 2). More clearly, the suggested taxonomy aims to hierarchize knowledge about resilient-digital-sustainable suppliers' selection criteria that characterize viable suppliers. After identifying knowledge to capitalize, we have analyzed and studied existing ontologies that we can re-use. Among ontologies-based models for suppliers' selection, we have used [START_REF] Konys | Green Supplier Selection Criteria: From a Literature Review to a Comprehensive Knowledge Base[END_REF][START_REF] Singh | Enhancing supply chain resilience using ontology-based decision support system[END_REF][START_REF] Mohammed V University | Onto-Digital: An Ontology-Based Model for Digital Transformation's Knowledge[END_REF]. Some of the concepts presented aided us in conceptualization, especially concerning the primary criteria, profile criteria, and sustainability criteria. The [START_REF] Konys | Green Supplier Selection Criteria: From a Literature Review to a Comprehensive Knowledge Base[END_REF] ontology is veritable in selecting green suppliers. However, its concepts don't cover the resilience and digitalization capabilities. Therefore, using the presented ontologies above, we conceptualized the viable-oriented suppliers' selection criteria model and defined attributes and relationships between the concepts related to ensuring viability.

Table 1. Knowledge classification

Name of the knowledge type

Knowledge

ResilienceCriteria

Knowledge presenting the criteria related to ensuring resilience SustainabilityCriteria Knowledge related to sustainability criteria DigitalizationCriteria Knowledge presenting the suppliers digital abilities ProfileCriteria Knowledge defining the general information of suppliers PrimaryCriteria knowledge related to the basic criteria for suppliers selection

Knowledge concepts, relations and attributes

This step of the ontology consists of specifying concepts and relations of suppliers' selection criteria. More clearly, this phase consists of attributing a unique name for concepts, a concept ID, the inheritance ID, and a natural language definition of the concept (Table 2). Besides, it attributes potential relations name, the domain, and range (Table 3). To sum up, the proposed ontology has 399 concepts and 70 relations.

Viable-oriented Suppliers selection ontology implementation with Protégé

After extracting the concepts defining a set of criteria and sub-criteria, and, defining properties and relations between them, the core ontology model is formed. The main structure of the proposed ontology is modeled using Protégé software (see Fig. 2). Protégé software is a tool allowing to visualizing, validating and building the proposed ontology in the OWL language respecting W3C recommendations [23]. Each of the presented classes contains a set of sub-classes. We have five classes namely ResilienceCriteria, SustainabilityCriteria, DigitalizationCriteria, PrimaryCriteria, and ProfileCriteria, and 394 subclasses. Each class has many subclasses. For instance, the DigitalizationCriteria has the following sub-classes: Innovation, Process_Quality, Product_Quality, Ressources, Security, Technological_Capability, Technologies. For ResilienceCriteria class, it has 3 subclasses namely Actions, Performance, and Risks.

In a summary, our ontology offers a structured model for storing knowledge related to relevant criteria for selecting suitable suppliers considering the viability performance. Thanks to the proposed ontology, the domain definition of viableoriented supplier selection criteria, its attributes, and relations with other concepts may be queried, inferred, or interpreted using the formal, explicit, and rich semantics to enable efficient viable suppliers' selection.

Practical and managerial implications

The developed ontology reflects current knowledge of the viable-oriented supplier selection domain criteria. It allows many benefits. It helps to systematize knowledge of the selected set of criteria and to represent knowledge structurally. Besides, it offers the opportunity of transdisciplinary conditions to collaboration between systems. The ontology's hierarchical structure enables rigorous thinking regarding domain concepts. That is to say, that managers will have the ability to rebuild the model and replenish it with information systems data. Furthermore, it can provide a dynamic mechanism for building or integrating information and other componentbased information systems. It may also contribute to the development of information systems for the evaluation of the whole domain, enabling automatization, visibility, and transparency. The proposed ontology's practical implications involve efforts to implement a large amount of knowledge covering resilience, digitalization, and sustainability dynamic capabilities that define viable-oriented supplier selection criteria. In other words, it is referencing the literature source metadata. These criteria knowledge could be used to interoperate with any knowledge base or database, or information systems. Furthermore, the proposed ontology unifies the scattered knowledge regarding viable-oriented supplier selection criteria and categorizes them into a comprehensive hierarchy.

Conclusion

With an increasingly global consciousness of resilience, viable-oriented cooperation in end-to end supply chains has emerged as a key feature. Because of the international changes that have arisen as a result of market globalization, technological advancements, or natural disasters [START_REF] Zekhnini | Supplier selection for smart supply chain: An adaptive fuzzy-neuro approach 8[END_REF]. Therefore, a successful viable-oriented supplier selection strategy will help reduce risks and disruptions effect while increasing organizations' competitiveness. For doing so, several sets of criteria were established to strengthen this process. While there are numerous studies on supplier selection, there is no comprehensive study considering viability issue in the suppliers' selection process.

Exchanging and transparently sharing information and knowledge represents a challenge for organizations to remain viable. To meet this challenge, this paper aims to present a new ontology-based-model for supporting suppliers' selection considering the viability performance. More clearly, the viable-oriented suppliers' selection is based on resilience, digitalization, and sustainability capabilities criteria. Thus, acquiring these criteria knowledge in an ontology is cost-effective and flexible to have access to needed information. It allows also to capitalize knowledge related to suppliers selection which can be considered as an important contribution for viable suppliers selection knowledge performance. In this article, to implement the viableoriented suppliers' selection, the first step consisted of retrieving criteria from the literature. After that, we have identified, classified, and capitalized knowledge to draw up the taxonomy and define knowledge concepts, relations, and attributes. Then, we have implemented and visualized the proposed ontology using Protégé software.

This ontology-based-model supporting viable-oriented suppliers' selection can be pursued in further work. This ontology can promote the standardization and sharing of knowledge by qualified actor-assisted tools such as the Multi-Agent System. This paper has some limitations that provide the opportunity for potential research. In other words, there is always the potential of enhancing the veracity of data collection and enriching knowledge while implementing an ontology. Moreover, future ontologies can consider disruption phases (pre-disruption, post-disruption, and disruption) knowledge to enhance the supply chain viability. Furthermore, we can introduce fuzzy logic theory in the proposed ontology to handle the concept of the decision subjectivity.

Fig. 1 .

 1 Fig. 1. Research methodology Fig. 1 outlines the research methodology used in this study. It presents the conceptual steps used to address the study's aim. The proposed approach is divided into 3 interconnected phases. The first phase is about searching relevant documents and

Fig. 2 .

 2 Fig. 2. Viable-oriented suppliers selection ontology implementation

Table 2 .

 2 Extract from the original concepts table

	ConceptID	ParentID	Natural language definition
	Economic_Risks	Risks	Risks that may affect an investment
			or a business
	Technological_Capability DigitalizationCriteria	Capabilities	related	to	novel
			technologies
	Material_Cost	Cost	Costs of materials
	Risks_Identification	Risks_solutions	a step that identifies risks

Table 3 .

 3 Extract from the original relations table

	Relation	Relation ID	Domain	Range
	Avoid	Avoid	Cybersecurity	Itrisks
	Can Lead To	Can_Lead_To	Digitalizationcriterea	Resiliencecriterea
	Ensure	Ensure	AI	Forecastingcapabilities
	Has	For
	Challenge	Has_For_Challenge Information	Datasecurity
	Has For Issue	Has_For_Issue	Profilecriteria	Resiliencecriterea
	Improve	Improve	Digitalizationcriterea	Primarycriterea
	Prevent	Prevent	Technologies	Risks
	Secure	Secure	Blockchain	Communicationcapability


Margot Di Cesare

Aissatou Maty Diagne

Benjamin Bourgey

Jean-Michel Jault
email: jean-michel.jault@ibcp.fr

Cédric Orelle
email: cedric.orelle@ibcp.fr

Functional overexpression of membrane proteins in E. coli: the good, the bad and the ugly

Keywords: Membrane proteins, overexpression, E. coli, detergent, solubilisation, ABC transporter, T7

Overexpression of properly folded membrane proteins is a mandatory step for their functional and structural characterization. One of the most used expression systems for the production of proteins is Escherichia coli. Many advantageous strains combined with T7 expression systems have been developed over the years. Recently, we showed that the choice of the strain is critical for the functionality of membrane proteins, even when the proteins are successfully incorporated in the membrane (Mathieu et al., Sci Rep. 2019; 9(1):2654). Notably, the amount and/or activity of the T7-RNA polymerase, which drives the transcription of the genes of interest, may indirectly affect the folding and functionality of overexpressed membrane proteins. Moreover, we reported a general trend in which mild detergents mainly extract the population of active membrane proteins, whereas a harsher detergent like Fos-choline 12 could solubilize them irrespectively of their functionality. Based on these observations, we provide some guidelines to optimize the quality of membrane proteins overexpressed in E. coli.

Introduction

Membrane proteins represent between 20 and 30% of synthesized proteins in all organisms [1]. They play essential roles and are targeted by more than half of therapeutic drugs. Because native membrane proteins are generally insufficiently abundant to conduct biochemical and structural studies, they are often overexpressed in heterologous systems. The bacterium Escherichia coli is the most widely used organism for the overexpression of both soluble and membrane proteins [2,3]. This is due to multiple reasons: (i) development of effective genetic tools, including strains and plasmids, allowing high levels of overexpression in favorable cases; (ii) easy DNA transformation, fast growth and high cell density cultures; (iii) inexpensive culture costs. The E. coli BL21(DE3) strain in combination with T7 promoterbased plasmids have been extensively employed to overexpress proteins. In this system, the T7 RNA polymerase gene is inserted in the DE3 prophage of the chromosome under the control of the IPTGinducible L8-UV5 lac promoter, which is a more powerful variant of the wild-type lac promoter. The BL21(DE3) strain is also deficient in Lon and OmpT proteases, and the T7 RNA polymerase transcribes ~ 8 times faster than native E. coli RNA polymerases [4] to generate high level of mRNAs available for protein synthesis. However, this strategy is not the most appropriate for many proteins, especially those that are toxic. The overexpression of membrane proteins may have detrimental effects due to their intrinsic function, improper folding or by exceeding the capabilities of the machineries involved in membrane protein biogenesis and protein secretion [5][START_REF] Klepsch | Consequences of the overexpression of a eukaryotic membrane protein, the human KDEL receptor, in Escherichia coli[END_REF][7]. More than twenty years ago, Miroux and Walker isolated BL21(DE3) mutant strains that displayed improved membrane protein overexpression abilities [8]. These strains were named C41(DE3) and C43(DE3) and are now widely used to overexpress membrane proteins, although they do not improve yields for all tested proteins [START_REF] Dilworth | Microbial expression systems for membrane proteins[END_REF][START_REF] Hattab | Escherichia coli as host for membrane protein structure determination: a global analysis[END_REF]. Much later, it was discovered that the C41(DE3) and C43(DE3) strains contained mutations in the lacUV5 promoter [START_REF] Wagner | Tuning Escherichia coli for membrane protein overexpression[END_REF]. A reduced transcription rate in these derivative strains likely explains why overexpression of many membrane proteins is less toxic for their growth and results in substantially improved membrane protein overexpression yields. Wagner and colleagues hypothesized that high transcription levels of membrane proteins is counterproductive because it leads to the saturation of the Sec translocon [START_REF] Wagner | Tuning Escherichia coli for membrane protein overexpression[END_REF].

As a result, most overexpressed and endogenous membrane proteins fail to insert into the membrane and aggregate, resulting in deleterious effects for the cell. Based on these observations, this team successfully engineered a BL21(DE3) variant strain named Lemo21(DE3), in which the activity of the T7 RNA polymerase can be finely tuned by its natural inhibitor T7 lysozyme, whose gene is under the control of the rhamnose promoter [12,[START_REF] Wagner | Tuning Escherichia coli for membrane protein overexpression[END_REF]. The optimization of membrane protein synthesis and their insertion into the membrane could thus be tuned to strongly minimize the toxic effects associated with membrane protein overexpression, resulting in higher bacterial biomass and protein yield. While misfolded proteins typically aggregate and form inclusions bodies, overexpressing conditions are typically optimized based on the amount of proteins associated with the membrane fraction. A popular strategy to screen experimental conditions and assess the quality of membrane protein expression is to fuse GFP to the C-terminal end of membrane proteins, based on the premise that only properly folded fusion proteins would display GFP fluorescence [START_REF] Drew | Optimization of membrane protein overexpression and purification using GFP fusions[END_REF][START_REF] Drew | Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli[END_REF]. According to our recent work [START_REF] Mathieu | Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain[END_REF], we advocate that functionality of membrane proteins drastically depends on the E. coli expressing strain and cautions against choosing an expression strain based solely on expression levels in membranes. In particular, we showed that several ABC transporters can be expressed and addressed to the membrane equally well in different E. coli strains, but they are substantially more functional and easier to solubilize by mild detergents when overexpressed in the appropriate strain [START_REF] Mathieu | Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain[END_REF]. Hence, we observed a general trend in which mild detergents mainly extract the population of active transporters, whereas a harsher detergent like Fos-choline 12 could solubilize transporters irrespective of their functionality. Similar detergent observations were recently described for membrane proteins expressed in eukaryotic systems [START_REF] Thomas | Quality control in eukaryotic membrane protein overproduction[END_REF]. Hereafter we provide methodological guidelines and advices to optimize the quality of overexpressed membrane proteins in E. coli.

Expression vectors

1. The gene encoding the protein of interest must be cloned into an expression vector compatible with the bacterial host. In this chapter, we will focus on E. coli, which remains the most popular host [3], although L. lactis [START_REF] Linares | Evolved Lactococcus lactis strains for enhanced expression of recombinant membrane proteins[END_REF] and B. subtilis [START_REF] Zweers | Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes[END_REF] can be alternative choices.

2. In E. coli expression vectors, different promoters with various strength and properties are available, such as the commonly used lacUV5, tac, trc, pBAD and T7 promoters [2,[START_REF] Tegel | Enhancing the protein production levels in Escherichia coli with a strong promoter[END_REF]. Due to its strength [START_REF] De Boer | The tac promoter: a functional hybrid derived from the trp and lac promoters[END_REF], the pTac system (hybrid of Trp and Lac promoters) is for instance more appropriate for the production of nontoxic proteins. The pBAD systems, which rely on catabolite repression and positive induction, are tightly regulated by arabinose. The T7 systems have the ability to produce higher levels of expression than pBAD systems, but they are prone to leaky expression [START_REF] Giacalone | Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system[END_REF].

3. Finally, the presence of an encoded affinity tag in the vector should also be considered [2]. The choice of the tag and its position (N-or C-terminal of the produced protein) can be important for downhill applications.

E. coli strains and induction conditions

Finding the appropriate bacterial strain and growth conditions are essential steps to achieve the optimal production of membrane proteins. For that purpose, we recommend optimizing the production by analyzing multiple expression strains and induction conditions.

Selection of E. coli strains

We present here below a selection of useful strains for protein overexpression in E. coli. Recent advances in the development and characterization of a number of T7-based strains offer a variety of opportunities to facilitate the functional overexpression of membrane proteins.

1. BL21(DE3): BL21(DE3) is an early-developed E. coli strain suitable for the overexpression of proteins [START_REF] Studier | Use of bacteriophage T7 RNA polymerase to direct selective highlevel expression of cloned genes[END_REF]. It contains the λDE3 lysogen prophage that carries the gene encoding T7 RNA polymerase, under the control of a lacUV5 promoter for specific IPTG induction. Thanks to 3 mutations located in the -10 promoter sequence and inside the CAP/cAMP binding site, the lacUV5 promoter is a strong variant of the lac promoter and is insensitive to catabolic repression. Moreover, this strain is deficient in Lon protease located in the cytoplasm and OmpT protease located in the outer membrane, reducing degradation of heterologously expressed proteins.

C41(DE3):C41(DE3

) is a famous strain derived from BL21(DE3) [8]. In 1996, Miroux and Walker selected this mutant strain for improved expression of toxic membrane proteins. This strain was isolated on plates containing IPTG and showed mutations in the lacUV5 promoter: two in the -10 promoter region of T7 RNA polymerase and one in the lac operator [START_REF] Wagner | Tuning Escherichia coli for membrane protein overexpression[END_REF]. These mutations reduced the promoter strength. As a result, smaller amounts of membrane proteins are produced, thereby preventing saturation of the Sec translocon and improving their insertion and proper folding in the membranes.

C43(DE3): C43(DE3

) is derived from C41(DE3) [8]. This strain has an additional mutation in the lacI gene of the DE3 region that may weaken the IPTG inducer affinity for the LacI repressor and therefore reduces the transcription initiation of the lacUV5 promoter [START_REF] Kwon | Comparative genomics and experimental evolution of Escherichia coli BL21(DE3) strains reveal the landscape of toxicity escape from membrane protein overproduction[END_REF]. C43(DE3) can better express some membrane proteins that remain too toxic for C41(DE3), such as the E. coli

F-type ATPase subunit b [8].
4. C44(DE3) et C45(DE3): C44(DE3) and C45(DE3) are recent strains also derived from the BL21(DE3) [START_REF] Angius | A novel regulation mechanism of the T7 RNA polymerase based expression system improves overproduction and folding of membrane proteins[END_REF]. They have stop codons in the T7 RNA polymerase gene (either located before the B catalytic domains of the polymerase for C45(DE3) or right after the B catalytic domain for C44(DE3)). However, as the BL21(DE3) strain exhibits a weak suppressive stop codon activity, the strains are still able to express sufficient but lower amounts of T7 RNA polymerase.

As a result, less proteins will be produced but they may be better inserted and folded into the membranes.

C41(DE3)

ΔompF ΔacrAB: C41(DE3) ΔompF ΔacrAB is derived from C41(DE3) [START_REF] Kanonenberg | Shaping the lipid composition of bacterial membranes for membrane protein production[END_REF]. This strain is deleted of the genes encoding OmpF and AcrB proteins, which are major membrane protein contaminants during purification [START_REF] Wiseman | Stubborn contaminants: influence of detergents on the purity of the multidrug ABC transporter BmrA[END_REF]. Surprisingly, these gene deletions altered the phospholipid composition of the membrane and its lipid to protein ratio. Consequently, this strain showed improved expression for a number of membrane proteins.

6. Lemo21(DE3): Lemo21(DE3) is derived from BL21(DE3) [START_REF] Wagner | Tuning Escherichia coli for membrane protein overexpression[END_REF]. This strain contains an additional plasmid called pLemo that expresses, under the control of a rhamnose-dependent promoter, the T7 Lysozyme. Because the T7 lysozyme is a natural inhibitor of T7 RNA polymerase, one can finely tune the expression of the protein of interest by varying the rhamnose concentration. Lemo21(DE3) is thus tunable for membrane protein overexpression and may allow optimizing expression by using a single strain rather than a multitude of different strains.

7. Other BL21 derivatives : Other BL21 derivatives have been developed with specific advantages. For instance, BL21-AI [START_REF] Bhawsinghka | Complete Genome Sequence of Escherichia coli BL21-AI[END_REF] and BL21-SI [START_REF] Donahue | BL21-SI competent cells for protein expression in E. coli[END_REF] are strains in which the expression of the T7 RNA polymerase is induced and tuned by arabinose or salts, respectively.

Combination with useful plasmids

Most of the aforementioned T7-based strains can be combined with useful plasmids. For instance, the Rosetta strain is a BL21(DE3) strain that contains a plasmid encoding rare tRNA codons to improve the expression of eukaryotic proteins. The pLysS or pLysE plasmids can reduce leaky expression, which is advantageous for toxic proteins. Another example is the CyDisCo system, which encoded a sulfhydryl oxidase and a disulfide bond isomerase that facilitate the formation of disulfide bonds in recombinant proteins [START_REF] Sohail | Production of Extracellular Matrix Proteins in the Cytoplasm of E. coli: Making Giants in Tiny Factories[END_REF].

Induction conditions

In order to optimize the expression of the target membrane protein, multiple conditions can be tested experimentally. The most important ones are presented below.

1. Culture broth: A wide range of media can be tested. Here we suggest the use of three typical media used for membrane protein production: LB, 2xYT and TB.

2. Temperatures: Before induction, bacteria are commonly grown at 37 °C. After induction, temperature has been shown to play a significant role in expression level and protein folding.

For better membrane protein folding, low post-induction temperatures between 18 °C and 25 °C are typically used.

IPTG Induction:

The concentration used for induction does not fine-tune levels of expression, and it is common to use IPTG around 1 mM. However, if low expression levels are observed, different concentrations might be tried. In some cases, omitting the IPTG gave the best expression results [START_REF] Zhang | High-level production of membrane proteins in E. coli BL21(DE3) by omitting the inducer IPTG[END_REF].

4. Cell Density: Exponential growth phase (between 0.4 to 0.8 OD600nm) is commonly used to start induction. OD600nm at 0.6 is often chosen. It is also possible to induce at late OD (>1) or even stationary phase (>2) to improve the yield of toxic proteins.

Induction Time: It should be determined by analyzing expression of the proteins by SDS-PAGE.

Induction time can vary between few hours to overnight. 6. When using Lemo21(DE3) cells, a range of rhamnose concentrations must be tested to find the best expression conditions (e.g. 0, 100, 250, 500, 750, 1000, 2000 µM)

Overexpression of membrane proteins: general guidelines

It is essential to work in sterile conditions during the overexpression.

1. Transformation. We strongly recommend to perform fresh transformations for each overexpression. In our experience, we often lost expression when starting from glycerol stocks saved at -80 °C. Transformation of chemically-competent cells (calcium chloride) using heat shock protocol is a convenient method, but other standard methodologies can be used (electroporation etc.). Cells need to be plated on LB-agar supplemented with the antibiotic used for selection. Importantly, if the Lemo21(DE3) cells are used, further supplement the LBagar plates with 25 µg/mL chloramphenicol for pLemo selection and 2 mM rhamnose for promoter repression. Incubate the plates overnight at 37 °C.

Preculture and culture.

(i) The safest way to proceed is to avoid a preculture step. Thus, inoculate a single or several colonies into fresh medium supplemented with antibiotic(s) (see Note 2). The culture can be incubated at 37 °C with shaking at 180 rpm.

(ii) Alternatively, if it works, a preculture step can be employed to facilitate the timescale management of the cultures. A single fresh colony is then taken from the agar plate and inoculated in LB medium supplemented with antibiotic(s) for an overnight preculture at 37 °C with shaking. If using Lemo21(DE3) cells, further supplement the preculture with 25 µg/mL chloramphenicol for pLemo selection and 2 mM rhamnose for promoter repression. The next morning, dilute the preculture into the desired broth (LB, 2YT, TB or another medium) supplemented with antibiotic(s) at a low OD600nm (usually between 0.05-0.1) and incubate at 37 °C with shaking at 180 rpm. Pellets can be frozen at -80 °C for subsequent use.

Preparation of the membrane fraction

Here we present a mechanical cell disruption based on high pressure lysis using a microfluidizer processor, but other methods are possible (French Press, sonication etc.). Then regular differential centrifugation is performed in order to isolate the membrane fraction.

1. Bacterial pellets are resuspended in the desired lysis buffer (e.g. 30 mL per L of culture). Lyse the cells by three successive passages through Microfluidizer TM at 18 000 psi (see Note 4).

2. Remove unbroken cells, cellular debris and insoluble proteins by centrifuging at 15 000 x g for 30 min at 4 °C. Keep the supernatant 3. Harvest membrane vesicles by an ultracentrifugation at 150 000 x g for 1 h (or possibly 200 000

x g for 30 min).

4. Discard the supernatant and resuspend membrane pellets in the desired resuspension buffer (at least 10 mL per L of culture). A potter pestle can be used to resuspend the membranes.

Ultracentrifuge again at 150 000 x g for 1 h.

5.

Resuspend and homogenize the membranes with a potter in the storage buffer (~2 mL per L of culture).

6. Flash-freeze membrane aliquots in liquid nitrogen and store them at -80 °C.

Assess membrane vesicles concentration using a BCA assay and check overexpression by SDS-PAGE gel (load 20 µg of total membrane proteins).

8. If necessary, a western blot analysis can be performed to check the overexpression of the protein of interest. In this case, a lower amount of proteins is usually loaded onto the gel (e.g. 0.5-5 µg).

Quality assessment of membrane protein expression

Although it is tempting to select the experimental conditions (bacterial strain, induction conditions) that produce the best expression levels in the membrane fraction, we strongly advise caution at this step. For instance, while the multidrug ABC transporter PatA/PatB from S. pneumoniae [START_REF] Garvey | Overexpression of patA and patB, which encode ABC transporters, is associated with fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae[END_REF] could be overexpressed equally well in the membrane of different E. coli strains, the transporter was >3 times more active in the C41(DE3) strain as compared to other strains, and this difference was even more drastic in other induction conditions [START_REF] Mathieu | Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain[END_REF]. Similar conclusions were reached with the multidrug ABC transporter BmrA from Bacillus subtilis [START_REF] Steinfels | Characterization of YvcC (BmrA), a multidrug ABC transporter constitutively expressed in Bacillus subtilis[END_REF], since most of the transporters overexpressed in the membrane of some strains were inactive [START_REF] Mathieu | Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain[END_REF]. In other words, a good expression in the membrane can be associated with bad or even ugly functionality of membrane proteins. Here below we present three different methodologies to evaluate the functionality of membrane proteins. If you must choose a strain and induction conditions, choose wisely!

GFP-fusion based systems

Methods employing GFP fusion have been very useful to assess the overexpression and quality of membrane proteins [START_REF] Drew | Optimization of membrane protein overexpression and purification using GFP fusions[END_REF][START_REF] Geertsma | Quality control of overexpressed membrane proteins[END_REF][START_REF] Kawate | Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins[END_REF]. This strategy presumes that proper folding and fluorescence of GFP fused to the C-terminus of a target protein depends on the appropriate folding of the latter. When the fusion protein is present in inclusion bodies, the GFP does not fold properly and thus does not fluoresce. Although we observed a substantial amount of GFP fluorescence associated with poorly functional membrane proteins [START_REF] Mathieu | Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain[END_REF], this methodology remains valuable for high-throughput screening of expression conditions.

Functional characterization of membrane proteins

Some membrane proteins display specific functional activities that are obviously the best way to assess the quality of their overexpression. For example, here we describe a transport assay that is applicable for various bacterial multidrug ABC transporters expressed in E. coli [START_REF] Orelle | A multidrug ABC transporter with a taste for GTP[END_REF][START_REF] Steinfels | Characterization of YvcC (BmrA), a multidrug ABC transporter constitutively expressed in Bacillus subtilis[END_REF]. Membrane vesicles with different conditions of expression may show similar levels of proteins of interest but strong differences in drug transport activities.

1. Use a fluorimeter (e.g. Photon Technology International Quanta Master I fluorimeter) and set the excitation and emission wavelengths according to the fluorescent drug used (e.g. 480 nm and 590 nm for doxorubicin, respectively).

2. In a 2 mL-Quartz cuvette containing a small magnet at the bottom, add 1 mL of transport buffer, and 100 μg of E. coli vesicles (amount to be adapted according to the level of expression). Next, place the cuvette inside the fluorimeter and record for 2 min before adding the fluorescent drug.

3. After 2 more min with the fluorescent drug, induce transport with 2 mM ATP and record the fluorescence for 10 min (until stabilization).

Solubilization of membrane proteins by mild detergents

When comparing the transport activities of multidrug ABC transporters expressed in the membrane of various strains, we observed a positive correlation between the activity of ABC transporters in the membrane and the propensity of mild detergents to solubilize them [START_REF] Mathieu | Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain[END_REF]. Furthermore, mild detergents such as dodecyl-maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG) [START_REF] Breyton | Assemblies of lauryl maltose neopentyl glycol (LMNG) and LMNG-solubilized membrane proteins[END_REF][START_REF] Chae | Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins[END_REF] mainly, if not exclusively, extracted the population of active membrane proteins [START_REF] Mathieu | Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain[END_REF].

On the other hand, harsh detergents like Fos-choline 12 were able to extract all proteins, including the inactive population. Similar detergent trends were described for membrane proteins expressed in eukaryotic systems [START_REF] Thomas | Quality control in eukaryotic membrane protein overproduction[END_REF]. Therefore, in the absence of available functional assays, the efficacy of DDM or LMNG detergents to solubilize the membrane protein of interest is a good indicator of its proper folding and functionality. If the target membrane protein is more solubilized by mild detergents in one expression condition as compared to others, such condition should be favored for subsequent applications.

1. Solubilization of the target membrane protein: Prepare a 2 mL-Eppendorf tube with a small magnet bar and dilute the membranes at 2 to 4 mg/mL in solubilization buffer. Add 0.5 to 1% final concentration of mild detergent (LMNG or DDM) or strong detergent (e.g. FC12). Incubate 2 h at 4 °C with agitation.

2. After 2 h, ultracentrifuge at 150 000 x g for 1 h at 4 °C or >200 000 x g for 30 min at 4 °C.

3. Collect supernatant and resuspend insoluble pellets in equal volume of the same buffer.

4. Run a SDS-PAGE with 5 to 10 μL of each fraction to check if the target membrane protein is in the supernatant (solubilized) or in the pellet (non-solubilized).

The choice of the best expression conditions has to be tuned up together with the ability of mild detergent to efficiently solubilize membrane proteins in a functional state. Gold standards are activity assays (in detergent and after reconstitution in lipid bilayers) (e.g. [START_REF] Alvarez | Full engagement of liganded maltose-binding protein stabilizes a semi-open ATP-binding cassette dimer in the maltose transporter[END_REF][START_REF] Herget | Purification and reconstitution of the antigen transport complex TAP: a prerequisite for determination of peptide stoichiometry and ATP hydrolysis[END_REF][START_REF] Souabni | Quantitative real-time analysis of the efflux by the MacAB-TolC tripartite efflux pump clarifies the role of ATP hydrolysis within mechanotransmission mechanism[END_REF][START_REF] Wagner | In vitro NTPase activity of highly purified Pdr5, a major yeast ABC multidrug transporter[END_REF] for ABC transporters) and the homogeneous profile in size-exclusion chromatography [START_REF] Kawate | Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins[END_REF][START_REF] Wagner | In vitro NTPase activity of highly purified Pdr5, a major yeast ABC multidrug transporter[END_REF]. In addition, high-throughput thermal stability screening can be useful to find detergents that efficiently stabilize membrane proteins [START_REF] Kotov | High-throughput stability screening for detergent-solubilized membrane proteins[END_REF].

Case studies

BmrA, a multidrug ABC transporter from Bacillus subtilis

The production of the multidrug ABC transporter BmrA [START_REF] Steinfels | Characterization of YvcC (BmrA), a multidrug ABC transporter constitutively expressed in Bacillus subtilis[END_REF] has been historically and successfully performed in C41(DE3) strain [START_REF] Lacabanne | Flexible-to-rigid transition is central for substrate transport in the ABC transporter BmrA from Bacillus subtilis[END_REF][START_REF] Steinfels | Highly efficient overproduction in E. coli of YvcC, a multidrug-like ATP-binding cassette transporter from Bacillus subtilis[END_REF]. Recent work suggested that BmrA could be overexpressed even more efficiently in Lemo21(DE3) strain [START_REF] Mathieu | Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain[END_REF]. In section 3.3, we strongly recommended to prevent T7 promoter leakage by growing the Lemo21(DE3) cells in the presence of a high concentration of rhamnose (such as 2 mM) during transformation and preculture of cells. For membrane proteins or toxic proteins in general, this step is critical. The objective is to completely repress the expression of the protein of interest during these steps and consequently prevent toxicity effects that can severely impair the production at the induction step.

As seen in Fig. 1, the use of 2 mM rhamnose was indeed essential during transformation and preculture for the effective induction of BmrA. The optimal concentration of rhamnose during the induction stage has to be determined for each protein of interest and 0.5 mM was appropriate for BmrA.

Using this procedure, we observed similar expressions in the membrane fraction of C41(DE3) and Lemo21(DE3) strains (Fig. 2A and2B). However, even though these levels of expression were similar, the transport activity of the protein in the membranes of Lemo21(DE3) strain was substantially improved (Fig. 2C) and a higher fraction of BmrA could be solubilized by DDM from these membranes (Fig. 2D).

A BceAB-type transporter from Streptococcus pneumoniae

The C41(DE3) strain proved to be excellent for overexpression of ABC transporters [START_REF] Mathieu | Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain[END_REF] and a large range of other membrane proteins [START_REF] Hattab | Escherichia coli as host for membrane protein structure determination: a global analysis[END_REF]. Due to the strength of induction in BL21(DE3), overexpression of membrane proteins in this strain was often toxic [START_REF] Klepsch | Consequences of the overexpression of a eukaryotic membrane protein, the human KDEL receptor, in Escherichia coli[END_REF]7]. However, this strain can still be helpful in some circumstances. In the case of an ABC transporter that confer resistance to bacitracin [START_REF] Becker | An ABC transporter of Streptococcus pneumoniae involved in susceptibility to vancoresmycin and bacitracin[END_REF], only BL21(DE3) expressed the protein (Fig. 3A and3B) and LMNG successfully extracted a substantial fraction of the membrane protein (Fig. 3C).

Expression of soluble proteins

Although this chapter is focused on membrane proteins, the choice of the strain may also affect the expression and folding of soluble proteins. We compared the expression of a soluble kinase, UbK [START_REF] Nguyen | Expanding the Kinome World: A New Protein Kinase Family Widely Conserved in Bacteria[END_REF], using the BL21(DE3) and the Lemo21(DE3). As shown in Fig. 4, the leaky expression seen in the absence of IPTG in the BL21(DE3) is strongly reduced in the Lemo21(DE3) strain either in the absence or presence of rhamnose. Therefore, if the protein of interest is toxic, the latter strain could prove to be advantageous but this should be further verified by functional assays. Although preliminary work with UbK suggest differences in functionality depending on the strain and induction conditions, additional work on a variety of soluble proteins should be conducted to confirm these observations. 1. The buffers indicated are those used for the multidrug ABC transporter BmrA. However, the appropriate buffers need to be chosen according to the membrane protein of interest.

2. For proper oxygenation, the culture broth should ideally represent 1/5 of the total volume of the glass flask. If baffled flasks are used, this volume can be increased.

3. Depending on the levels of expression, it may be possible to see the protein of interest in the total fraction. If not, assess protein expression in the membrane fraction.

4. The high pressure used here favors the formation of inverted (i.e. inside-out) membrane vesicles [START_REF] Altendorf | Orientation of membrane vesicles from Escherichia coli as detected by freeze-cleave electron microscopy[END_REF][START_REF] Seckler | Sidedness of native membrane vesicles of Escherichia coli and orientation of the reconstituted lactose: H+ carrier[END_REF]. It is advantageous to use for transport assays. In the case of the multidrug exporter BmrA, the nucleotide-binding domains are exposed outside of the inverted membrane vesicles and transport of fluorescent drugs occurs toward the lumen of vesicles.

3 . 4 . 5 .

 345 Induction. When the OD600nm reaches the desired phase (e.g. ~0.6), proceed with the induction step by adding IPTG (e.g. 1 mM) and by switching to the chosen temperature for the desired incubation time. Induction analysis. Harvest 1 mL aliquot of the cultures before and after IPTG addition for protein expression analysis by SDS-PAGE and/or Western Blot (see Note 3) by centrifugation at 10,000 x g for 1 min at 4 °C. Discard supernatants. Bacteria pellets are either frozen at -80 °C or used directly. Resuspend the pellets in Laemmli buffer 1X (~10 µL per 0.1 OD600nm). Run a SDS-PAGE with the equivalent of 0.1 OD600nm of resuspended bacteria. Collecting the cells. Pellet the cells at 5 000 g for 20 min at 4 °C. Discard the supernatant.

Figures.Figure 1 .

 1 Figures.

Figure 2 .

 2 Figure 2. Overexpression of BmrA in membranes of C41(DE3) or Lemo21(DE3) strains. A, Expression

Figure 3 .

 3 Figure 3. Western blot analysis of SpABC expression in several E. coli strains and solubilization from

Figure 4 .

 4 Figure 4. Expression of UbK kinase from Bacillus subtilis in BL21(DE3) and Lemo21(DE3) strains. The

Acknowledgments

This work was supported by the Agence Nationale de la Recherche (grants ANR-17-CE11-0045 and ANR-19-CE11-0023).


F Bourahima
email: fazati.bourahima@chpolansky.fr

T Baudin

M Rege

V Ji

F Brisset

A Zavdoveev

A L Helbert

Formation of a joint between deposited and base metals during laser cladding of a nickelbased powder onto a copper-based alloy

Keywords: Laser cladding, Power attenuation, Powder distribution, Dilution zone

Laser metal deposition (LMD) is an alternative method to other cladding techniques such as Plasma Transfer Arc (PTA) or blowtorch for surface treatment in the glass industry. It aims to produce dense, high-quality coatings on a non-planar surface without affecting its thermal and mechanical properties. In this study, Ni-based coatings were coated onto Cu-Ni-Al substrate using a 3 JET nozzle technique. During laser cladding, good metallurgical bonding is necessary to ensure the further surfacing process technique. A microstructural analysis was conducted, and the mechanical properties were then evaluated with microhardness analysis to link process parameters to coating bonding quality. A calculation of the power attenuation attempts to explain the impact of the powder distribution on the bonding. This work revealed that a chemical dilution zone exists between coating and substrate and is necessary for perfect metallurgical bonding. The heterogeneous bonding, observed through the section, along the curved interface coating/substrate, has been linked to the Gaussian distribution of the powder that attenuates the input power. The attenuated power was measured all along the interface.

Introduction

During glass bottle production, viscous glass is poured into Cupro-Nickel-Aluminum (Cu-Ni-Al) molds at temperatures ranging from 700 to 1200°C. Cu-Ni-Al or cast-iron glass molds must absorb the high glass temperature to cool it homogeneously, playing the role of thermal exchangers [START_REF] Rege | Stage de formation aux techniques de rechargement dans l'industrie verrière[END_REF]. However, during this process, corrosion or abrasion can appear on the molds' sensitive parts (neck ring and match). Also, thermal fatigue can be observed during the molding cycle. Therefore, it is important to modify the mold surface properties before the production to extend its lifespan. In order to do that, Ni-based or Co-based powder is cladded on those parts. The most famous cladding methods for glass industry are Plasma Transferred Arc (PTA) [START_REF] Oberländer | Comparison of properties of coatings produced by laser cladding and conventional methods[END_REF] and blowtorch [START_REF] Rege | Stage de formation aux techniques de rechargement dans l'industrie verrière[END_REF]. But with these techniques, a high Heat Affected Zone (HAZ) is present. It corresponds to a zone belonging to the substrate that has not reached its fusion temperature but underwent a change in its structure due to the process. To avoid that, a pre-heating is operated with those techniques. In mold industry, the aim is to limit this Affected Zone to prevent changes in the substrate thermal properties. To be able to do that, laser cladding is operated instead of former techniques without mold pre-heating.

Laser cladding is an innovative surfacing technique, developed in the 90's [START_REF] Steen | Laser Material Processing[END_REF][START_REF] Ryabtsev | Properties of alloys based on Fe, Ni, Co, deposited by laserpowder method[END_REF]. It is used to obtain well-bonded high-quality materials, free of pores and cracks without affecting the substrate thermal properties (so, a low HAZ). Some researchers have investigated the impact of laser cladding on the substrate microstructural change. In most of the studies, the HAZ is observed during laser cladding on cast iron substrates with the appearance of metastable phases like martensite or ledeburite [START_REF] Jeshvaghani | Microstructural study and wear behavior of ductile iron surface alloyed by Inconel 617[END_REF][START_REF] Liu | Microstructural evolution and bonding characteristic in multi-layer laser cladding of NiCoCr alloy on compacted graphite cast iron[END_REF].

Balu et al. [START_REF] Balu | Laser cladding of nickel-based alloy coatings on copper substrates[END_REF] have operated laser cladding with two kinds of Ni-based powder on Cu substrate with and without pre-heating. They observed that the grain size in the HAZ is high distanced from the interface. It has also been seen that this size increases at low laser scanning speed. Hardness is lower in the HAZ than distanced from the interface. They found that a substrate pre-heating leads to a better bonding due to a larger melting pool and HAZ.

Despite the limited HAZ, laser cladding often induces an area of local chemical dilution between the powder and the substrate. Authors explained that dilution is obtained at the interface when a fusion between the substrate and the coating occur [START_REF] Balu | Laser cladding of nickel-based alloy coatings on copper substrates[END_REF][START_REF] Saqib | Analysis of laser cladding bead morphology for developing additive manufacturing travel paths[END_REF][START_REF] Tao | Effect of heat treatment on residual stress and wear behaviors of the TiNi/Ti2Ni based laser cladding composite coatings[END_REF]. This dilution, dependent on the input laser power [START_REF] Kim | Characterization of surface modification by laser cladding using low melting point metal[END_REF], has to be restricted in order to avoid a large HAZ but seems necessary to ensure good metallurgical bonding. Many researches have observed that according to the powder nature, a high power attenuation can appear [START_REF] Kumar | The effect of Marangoni-Rayleigh-Benard convection on the process parameters in blown-powder laser cladding process-A numerical investigation[END_REF][START_REF] Rienstra | Analytical approximations to the viscous glass-flow problem in the mould-plunger pressing process, including an investigation of boundary conditions[END_REF]. There are two ways of describing the dilution (rate) [START_REF] Toyserkani | Laser Cladding[END_REF]: in geometrical terms, it in calculating the ratio between the melted area (including the HAZ) and the coating area. The second is in metallurgical terms, which consists in measuring the depth of the chemical mixture between the coating and the substrate. Liu et al. [START_REF] Liu | Research and development status of laser cladding on magnesium alloys: A review[END_REF] present a dilution rate by using the geometrical method during laser cladding of magnesium alloys.

According to them, a very low dilution rate prevents from good metallurgical bonding. Using the same definition of the dilution rate, Luo et al. [START_REF] Luo | Effect of NiCrBSi content on microstructural evolution, cracking susceptibility and wear behaviors of laser cladding WC/Ni-NiCrBSi composite coatings[END_REF] present the same observation during laser cladding of NiCrBSi. Balu et al. [START_REF] Balu | Laser cladding of nickel-based alloy coatings on copper substrates[END_REF] used the metallurgical method to measure the dilution rate during laser cladding of Ni-based powder on a Cu substrate. They found that a high dilution can lead to a high HAZ. Pereira et al. [START_REF] Pereira | Mechanical and microstructural characterization of MCrAlY coatings produced by laser cladding: The influence of the Ni, Co and Al content[END_REF] observed that during laser cladding of NiCoCrBSi on stainless steel, a very low dilution zone (metallurgical method) at about 2 μm is enough to obtain a perfect bonding without affecting the substrate mechanical properties (no cracking behavior observed). Many authors [START_REF] Adesina | Influence of phase composition and microstructure on corrosion behavior of laser based Ti-Co-Ni ternary coatings on Ti-6Al-4V alloy[END_REF][START_REF] Wang | Real-time monitoring of chemical composition in nickel-based laser cladding layer by emission spectroscopy analysis[END_REF] presented an EDS analysis to measure the dilution between two materials and its components' chemical evolution.

The main challenge in laser cladding on a copper-based substrate is to obtain good bonding despite the low absorptivity of the substrate [START_REF] Babinets | Problems and prospects of surfacing of copper and copper parts by wear-resistant layers (Review)[END_REF]. Zhang et al. [START_REF] Zhang | Characterization on laser clad nickel based alloy coating on pure copper[END_REF] explained that it was impossible to obtain bonding without preheating, so they pre-heated the substrate to 300°C. It appears that a compromise must be found to respect both the restricted HAZ and the dilution zone needed to guarantee the cohesion between the coating and the substrate. We should bear in mind that this cohesion is crucial to allow further machining of the repaired mold. Moreover, the coating has to be free of cracks and pores to retain a perfect glass surface.

The aim of this study is to obtain a perfect bonding with a limited or non-existent HAZ after cladding of nickel-based material on a Cu-Ni-Al substrate. The novelty in this process is the laser cladding performed without preheating on a curved surface. Also, the high thermal conductivity of the copper-based material is challenging because it can prevent from obtaining the perfect bonding.

This can be achieved by identifying the relationship between the dilution zone (existence and thickness) and the bonding quality with regards to the laser process parameters. A microstructural and chemical analysis of the coating/substrate interface will be conducted. Also, microhardness measurements will be presented. The influence of the Gaussian distribution of Ni powder on the laser power attenuation, and thus on the bonding, will be discussed.

Materials and experimental techniques

Laser cladding consists in melting an injected powder with a very thin surface layer on the substrate by a laser beam to produce a metallurgical bonding. During this study, a 4KW Nd: YAG laser with a wavelength of 1030 nm was used. An optical fiber with a diameter of 600 µm was used for guiding the beam. Ni powder was injected coaxially into the laser beam. A cross-section of the Cu-Ni-Al mold of the bottle ring is shown in Figure 1a after cladding. Figure 1b process, the interaction distance, fs, between the laser and the powder is considered (Figure 2): At the focal plane (position B), the powder flow follows a Gaussian distribution. This has been proved by many research studies [START_REF] Ferreira | Experimental and numerical analysis of gas/powder flow for different LMD nozzles[END_REF][START_REF] Cheikh | Analysis and prediction of single laser tracks geometrical characteristics in coaxial laser cladding process[END_REF][START_REF] Pouzet | Additive layer manufacturing of titanium matrix composites using the direct metal deposition laser process[END_REF]. In the present study, the Gaussian distribution has been centered at the midpoint of the mold curved surfaces of the samples.

X=0

Laser input

Powder

The chemical compositions of the powder and the substrate are listed in Table 1. The process parameters used during this study are presented in Table 2. These samples are extracted using Taguchi design of experiment. Only samples with a partial or perfect bonding have been selected. To be characterized, samples were mechanically polished using SiC papers and then diamond polished to 1 µm for Optical Microscopy (OM), Scanning Electron Microscopy (SEM)

and Energy Dispersive Spectroscopy (EDS) analyses. To operate Electron Backscatter Diffraction (EBSD), an extra OPS polishing was done. Vickers hardness tests were performed on the crosssection, along a line perpendicular to the coating surface with a 10-gf load for 15 seconds. The measurements were taken from either side of the interface for all the 6 samples.

3 Results and discussion

Microstructure features and associated chemical and mechanical properties

Figure 3 describes the three different kinds of bonding behavior that can be observed after laser cladding of Ni-based powder on a Cu-Ni-Al substrate: perfect bonding (Figure 3a), partial bonding (Figure 3b) where the bonding is partially present at the interface and no bonding (Figure 3c). A bonding is considered partial when 15% bonding default is observed in the coating/substrate interface. samples (see Table 2). Figure 4a shows that CNC 5 presents perfect bonding (case of Figure 3c)

whereas Figure 4b shows a sample (CNC 6) with a discontinuous bonding (Figure 3b). Indeed, in the curved center, there is a lack of bonding. No analyses have been performed on samples with no bonding (Figure 3a) since the clad is easily separated from the substrate.

For both samples, the substrate is composed of a dendritic matrix. No heat affected zone It can be observed that microstructure is very similar in both cases (Figure 5a and5b).The solidification features are in accordance with the literature [START_REF] Song | The relationship between convection mechanism and solidification structure of the iron-based molten pool in metal laser direct deposition[END_REF][START_REF] Pereira | High temperature oxidation behavior of laser cladding MCrAlY coatings on austenitic stainless steel[END_REF][START_REF] Pan | Research on microstructural and property evolution in laser cladded HAZ[END_REF][START_REF] Hemmati | Effects of the alloy composition on phase constitution and properties of laser deposited Ni-Cr-B-Si coatings[END_REF]. A nucleation phase, characterized by small equiaxed grains, is evidenced at the interface, then a columnar growth directed mainly parallel to the <001> direction of the dendrites is observed (Figure 5a). So, the bonding quality does not change the solidification behavior during laser cladding.

A SEM-EDS analysis is presented (Figure 6) to observe the chemical impact of the laser cladding at the interface nucleation/substrate. In the Figures 6a andb, deconstruct dendrites from the substrate are observed in a mixed zone at the coating/substrate interface delimited by white lines. Also, the Figure 6c clearly shows a local chemical mixture where copper seems to be replaced by nickel over a thin layer of the substrate. This can be the consequence of a local fusion of the substrate and correspond to the dilution zone that has been reported in the literature [START_REF] Adesina | Influence of phase composition and microstructure on corrosion behavior of laser based Ti-Co-Ni ternary coatings on Ti-6Al-4V alloy[END_REF][START_REF] Pereira | High temperature oxidation behavior of laser cladding MCrAlY coatings on austenitic stainless steel[END_REF]. Apart from this area, there are no discernible chemical changes in the substrate or the Ni coating. So, no HAZ can be chemically observed in this study.

A measurement of the dilution zone thickness was made by chemical analysis from the coating to the substrate (Figure 7). A diminution of the Ni element from the coating to the substrate can be observed. At the same time, the Cu composition progressively increases. Overall, Ni is at around 96% in the coating, then decreases down to 40% close to the interface and stays below 10% in the substrate matrix except inside the dendrites where the Ni increases to 39%. The dendrites also present a higher level of Al (the high concentration of Ni and Al inside the dendrites is in accordance with Figure 6). The dilution zone exhibits a chemical mixture over about 35µm for this sample.

Figure 8 shows the hardness measurements through the interface for four typical features of the microstructure: the columnar zone of the coating, the nucleation zone, the dilution zone, and the substrate.

Coating Substrate 3) in this area as described by Hemmatie et al. [START_REF] Hemmati | Effects of the alloy composition on phase constitution and properties of laser deposited Ni-Cr-B-Si coatings[END_REF]. Indeed, they mention that this compound is of high hardness.

Dilution zone

In the coating and the substrate, the values are not higher than 317 HV. This hardness does not indicate the presence of the HAZ in the substrate. No increase of the hardness is noticed in the nucleation area despite the presence of small grains. In fact, the Hall and Petch law which describes that the hardness is higher when the grain size is lower [START_REF] Gourges-Lorenzon | Durcissement et renforcement des materiaux[END_REF]. But this couldn't be verified is the present work.

Since the dilution zone can be quantified using the EDS analysis it could be interesting to observe its evolution along the curved interface in order to corroborate its existence/thickness with the metallurgical bonding quality.

Dilution zone thickness along the curved interface

To state if the discontinuous bonding along the interface in the studied section is linked to the chemical dilution presence or thickness, a protocol of systematic DZ quantification has been implemented across the section of all samples and is presented in Figure 9. Five lines separated from each other by an angle of 22.5° along which the chemical composition have been measured from the coating to the substrate within 400 μm, and where the DZ depth has been determined. This method allows to plot the DZ evolution along the curved interface (Figure 9). Then at the center of the curve (c zone) this DZ is minimum (zero in the case of no bonding).

Moreover, for all the studied samples, the bonding is lower at the center of the curved section.

Therefore, it is possible to draw a direct connection between DZ depth and bonding quality. Indeed, the deeper the DZ, the better the bonding will be.

Power attenuation

It is well known in the laser cladding process that powder particles contribute to attenuate the incident laser power. Obviously for full consideration it is necessary to calculate the total heat balance of laser surfacing using filler powder, the total amount of heat released by the laser is spent on heating and melting the filler powder, heating the base metal before melting and maintaining the weld pool in a liquid state (the useful part of the heat), as well as radiation into the surrounding space (including as a result of reflection from powder particles, depending on its reflectivity) and heat removal to the base metal (heat loss). The thermal efficiency of the laser surfacing process and the parameters of its regime depend on these indicators: laser power, the amount of powder supplied per unit time, its granulometric composition, etc. [29,[START_REF] Yu | Electroslag technologies for surfacing and recycling of metal and metal-containing wastes[END_REF]. This is complex calculation and for simplification of the description in current research we have considered only the effect of the powder particles (i.e. filler powder). Tabernero et al. [START_REF] Tabernero | Modelling of energy attenuation due to powder flow-laser beam interaction during laser cladding process[END_REF] have described the impact of a shadow of particles during the interaction of the laser and the powder while cladding is in progress. They have assessed the attenuation undergone by the beam and characterized the density of energy that reaches the surface of the substrate. They were able to perform this calculation with a computational fluid dynamics (CFD) model, which has been experimentally validated. Moreover, the present authors used the calculation of the attenuated power to determine the minimum power necessary to ensure an accurate clad/substrate bonding [START_REF] Pereira | High temperature oxidation behavior of laser cladding MCrAlY coatings on austenitic stainless steel[END_REF]. Nevertheless, the local attenuation due to the Gaussian powder distribution has not been studied along the curve section.

El Cheikh et al. [START_REF] Cheikh | Analysis and prediction of single laser tracks geometrical characteristics in coaxial laser cladding process[END_REF] have discovered that at the focal position, fs, presented in Figure 2, a Gaussian distribution of powder can be observed. Qi et al. [START_REF] Qi | Numerical simulation of heat transfer and fluid flow in coaxial laser cladding process for direct metal deposition[END_REF] have proposed an equation of the powder distribution depending on the radial distance x and the axial distance z in the case of a coaxial nozzle as shown:

(

With the peak concentration at the center of powder flow (x=0), x[mm] the radial distance and R[mm] the powder stream.

To understand how the power is impacted, the Beer Lambert Law can be applied [START_REF] Qi | Numerical simulation of heat transfer and fluid flow in coaxial laser cladding process for direct metal deposition[END_REF][START_REF] Peyre | Analytical and numerical modelling of the direct metal deposition laser process[END_REF]:

(

With the power attenuated by the powder flow, the initial input power during laser cladding, S [ the particle section and α a factor which considers phenomena like the scattering effect, plasma generation... α was obtained from an experiment of the attenuated power during laser cladding. A power meter is placed under an optically neutral glass to protect it from the powder injected. During the test, an air flow was injected horizontally in order to deflect the projected powder. As an example, for an incident measured power of 1916 W, the measured attenuated power was 1498 W for a PFR of 28.5 g/min. This led to adjusting the α coefficient at a value of 6 (this result is in accordance with literature [START_REF] Tabernero | Modelling of energy attenuation due to powder flow-laser beam interaction during laser cladding process[END_REF]). If α is equal to 1, the attenuation is only due to particle shadow. The present value of 6 indicates that other phenomena (scattering effect, plasma generation...) also contribute to the power attenuation.

By inputting the powder distribution on each position of x it is possible to observe the power attenuation on the radial distance for an incident power of 2800W as an example (Figure 10). Figure 10 indicates that, at the center of the powder flow (the focal point fs, Figure 2), the power is attenuated from 2800 to 1200W. At the center of the powder flow (x=0), 57% of the power is lost due to the powder flow (absorbed or reflected). So, only 43% is available for the fusion.

Moreover, given that the absorptivity of Cu-based materials is about 0.06, only 72W remains to reach the substrate (and probably melt it). Since the focal plane of the powder flow corresponds to the center of the mold curved surface, it can be assumed that this shadow effect explains the lack of dilution DZ observed at this position. Therefore, this phenomenon may be at the origin of the absence of bonding for some cladding conditions with too weak incident laser power.

A link between power attenuation, dilution and HAZ

No HAZ was observed here when laser cladding on a Cu-based substrate. But the heterogeneous dilution in the mold's curved section can lead to a lack of bonding at the curved center and induce the coating fall out during machining [START_REF] Rege | Stage de formation aux techniques de rechargement dans l'industrie verrière[END_REF]. In the meantime, on the edges (according to the input laser power), less powder generates more power available for the interface fusion. According to the substrate composition, a high HAZ can be generated and induces a cracking behavior due to the high hardness as it was observed in previous researches conducted by the authors during laser cladding on cast iron [START_REF] Bourahima | Multi-Scale Characterization by Neutronography and Electron Diffraction of Ni Coating on Cu-Ni-Al or Cast-Iron Glass Molds after Laser Cladding[END_REF].

Conclusion

Ni-based powder was deposited by laser cladding on a Cu-based substrate with a curved surface. The cladding process parameters were widely explored in order to verify the bonding quality of the clad to the substrate. Three kinds of metallurgical bonding have been noted: lack of bonding, partial bonding or perfect bonding. In addition, a constant lack of bonding is observed in the center of the substrate curved surface. Moreover, the chemical dilution depth between Ni and Cu was measured at the interface clad/substrate depending on process parameters. It appears that the dilution depth is also lower at the same location of the curved surface. This could be explained by the Gaussian distribution of the powder flow around this point. Indeed, at the curved center the powder density is at maximum, so the input laser power is significantly attenuated. Less energy is then available for the substrate to melt and to ensure a perfect metallurgical bonding. The attenuated power evolution moving away from this central point would explain the discontinuous bonding along the interface in the cross-section. As it was mentioned above, a compromise was found in the present work to ensure a minimal dilution (DZ) in the curved center without affecting the based material properties (limit the HAZ). This optimization can correspond for example to a laser power of 3200 W with a PFR of 28.5 g/min and a scanning speed of 8.5 mm/s.

Tables caption

Table 1 : Chemical compositions of the substrate and powder 5 Table 2 : Process parameters 6

Figures caption

  Figure 1: a) Half of the mold ring after Ni-laser cladding and b) studied cross section

Figure 2 :

 2 Figure 2: Powder distribution according to the focal position fs[START_REF] Babinets | Problems and prospects of surfacing of copper and copper parts by wear-resistant layers (Review)[END_REF]

Figure 3 :

 3 Figure 3: Three kinds of bonding behavior a) no bonding b) partial bonding and c) Perfect bonding

(Figure 4 :

 4 Figure 4: Typical microstructure of the cross-section of the samples a) CNC 5 with good bonding, b) CNC 6 with partial bonding obtained by SEM-BSE Figure 5 shows an EBSD analysis of the microstructure and texture around the interface

Figure 5 :

 5 Figure 5: Crystallographic axes parallel to the solidification direction (black arrows). a) EBSD analysis of the black square area in Figure 4a and b) in Figure 4b.

Figure 6 :

 6 Figure 6: EDS maps of the principal chemical elements composing the deposit and the substrate of sample CNC6 (a) Al, (b) Cu, (c) Ni

Figure 7 :

 7 Figure 7: Chemical composition of the dilution zone from the coating to the substrate (sample CNC 6)

Figure 8 :

 8 Figure 8: Hardness measurements corresponding to the characteristic features encountered from the coating to the substrate (red points)It can be observed from Figure8that the micro-hardness of the dilution zone increased to

Figure 9 :

 9 Figure 9: Measurement the DZ depth over the entire cross-section and DZ depth along the five red lines for all the samples

Figure 9

 9 Figure 9 reveals that the DZ evolution is the same across the section for all the samples

  The discontinuous bonding is well highlighted by the DZ evolution along the curve of the cross-section. It is important to understand what 175 µm to a weaker chemical dilution at the section curve center during laser cladding, as a function of process parameters.

Figure 10 :

 10 Figure 10: Distribution of the power attenuation on the substrate (x=0 is the focal point) for an input laser power of 2800 W

4 Figure 2 : 5 Figure 3 : 7 Figure 4 : 7 Figure 5 : 8 Figure 6 : 9 Figure 7 :) 9 Figure 8 : 10 Figure 9 : 11 Figure 10 :

 425374758697981091110 Figure 1: a) Half of the mold ring after Ni-laser cladding and b) studied cross-section 4 Figure 2: Powder distribution according to the focal position fs [17] 5 Figure 3: Three kinds of bonding behavior a) perfect bonding b) partial bonding and c) no bonding 7

Table 1 :

 1 Chemical compositions of the substrate and powder

	Elements (wt%)	Fe	Mn	Al	Ni	Zn	Pb	Sn	Si Cu	B Cr C
	Cu-Ni-Al	<1	0.5 8.5	15	8	<0.1 0.15 1	Bal	-	-	-
	Ni powder	1	0.1	-	Bal	-	-	-	2.5	-	1.7 0.3 0.5

Table 2 :

 2 Process parameters

	Sample	Power (W)	Speed	Powder Feeding Rate	Spot diameter (mm)
			(mm/s)	(g/min)
	CNC 1	2400	6.5	26.5 g/mm	4 mm
	CNC 2	2600	10	24.5 g/mm	3 mm
	CNC 3	2800	6.5	32.5 g/mm	3 mm
	CNC 4	2800	8.5	24.5 g/mm	4 mm
	CNC 5	3200	8.5	28.5 g/mm	3 mm
	CNC 6	3200	10	30.5 g/mm	4 mm

Acknowledgments

The authors wish to thank C. Aboud, N. Barbosa and C. Lafarge from the ETS CHPOLANSKY for their help in the sample elaboration by laser cladding and their great skill in the field of this technology applied to non-planar surfaces. This research was supported by the ANRT (French National Agency for Research and Technology) and ETS Chpolansky.

For the