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The possibility of using agro-industrial residues in bioconversion processes advances with the development of biotechnology and the search for processes in which is possible to add commercial value to previously discarded products. These processes should be low cost, easy to control and chemical-free. The yeast Yarrowia lipolytica (YL) is widely used to produce lipids, enzymes, citric acid, and proteins, among others. This study aimed to evaluate the capacity of this yeast to use agro-industrial residues as a source of carbon without adding extra carbohydrate sources for the development of cells. The study evaluated the production of proteins and lipids from different carbon sources as well as the optimization of the process (agitation, temperature, and nitrogen source). Indeed, YL produced 22.3% of protein and 9.4% of lipids in dry biomass, a 179% of protein and 660% of lipid increase from raw material, respectively, when using cassava residues as a carbon source. However, lipase production was low, indicating that the strain had priority for cell growth. Resumo A possibilidade de utilização de resíduos agroindustriais em processos de bioconversão tem avançado com o desenvolvimento da biotecnologia e a busca por processos nos quais seja possível agregar valor comercial a produtos anteriormente descartados. Esses processos devem ser de baixo custo, fáceis de controlar e livres de produtos químicos. A levedura Yarrowia lipolytica (YL) é amplamente utilizada para a produção de lipídios, enzimas, ácido cítrico, proteínas, entre outros. O objetivo deste estudo foi avaliar a capacidade desta levedura em utilizar resíduos agroindustriais como fonte de carbono sem adição de fontes extras de carboidratos para o desenvolvimento das células. O estudo avaliou a produção de proteínas e lipídios a partir de diferentes fontes de carbono, bem como a otimização do processo (agitação, temperatura e fonte de nitrogênio). A YL produziu 22,3% de proteína e 9,4% de lipídios a partir da biomassa seca, um aumento de 179% de proteína e 660% de lipídio da matéria-prima, respectivamente, ao utilizar resíduos de mandioca como fonte de carbono. No entanto, a produção de lipase foi baixa, indicando que a cepa tem prioridade para o crescimento celular.

Overproduction of recombinant secretory proteins triggers numerous physiological perturbations. Depending on a given heterologous protein characteristics, the producer cell is faced with different challenges which lead to varying responses in terms of its physiology and the target protein production rate. In the present study, we used steady-state-maintained Yarrowia lipolytica cells to investigate the impact of different heterologous proteins on the physiological behavior of the host cells. Such an approach allowed to uncouple the impact of the overproduction of a particular protein from the phenomena that result from growth phase or are caused by the heterogeneity of the analyzed populations. Altogether, eight variants of recombinant strains, individually overproducing heterologous proteins of varying molecular weight (27–65 kDa) and reporting activity (enzymatic and fluorescent) were subjected to chemostat cultivations. The steady-state-maintained cells were analyzed in terms of the substrate utilization, biomass and metabolites production, as well as the reporter protein synthesis. Simplified distribution of carbon and nitrogen between the respective products, as well as expression analysis of the heterologous genes were conducted. The here-obtained data suggest that using a more transcriptionally active promoter results in channeling more C flux towards the target protein, giving significantly higher specific amounts and production rates of the target polypeptide, at the cost of biomass accumulation, and with no significant impact on the polyols production. The extent of the reporter protein’s post-translational modifications, i.e., the number of disulfide bonds and glycosylation pattern, strongly impacts the synthesis process. Specific responses in terms of the protein formation kinetics, the gene expression levels, and transcript-to-protein linearity were observed.Key Points• Eight expression systems, producing different reporter proteins were analyzed.• The cells were maintained in steady-state by continuous chemostat culturing.• Protein- and promoter-specific effects were observed.

Abstract In this research, we were interested in answering a question whether subjecting a Yarrowia lipolytica strain overproducing a recombinant secretory protein (rs-Prot) to pre-optimized stress factors may enhance synthesis of the rs-Prot. Increased osmolarity (3 Osm kg−1) was the primary stress factor implemented alone or in combination with decreased temperature (20 °C), known to promote synthesis of rs-Prots. The treatments were executed in batch bioreactor cultures, and the cellular response was studied in terms of culture progression, gene expression and global proteomics, to get insight into molecular bases underlying an awaken reaction. Primarily, we observed that hyperosmolarity executed by high sorbitol concentration does not enhance synthesis of the rs-Prot but increases its transcription. Expectedly, hyperosmolarity induced synthesis of polyols at the expense of citric acid synthesis and growth, which was severely limited. A number of stress-related proteins were upregulated, including heat-shock proteins (HSPs) and aldo–keto reductases, as observed at transcriptomics and proteomics levels. Concerted downregulation of central carbon metabolism, including glycolysis, tricarboxylic acid cycle and fatty acid synthesis, highlighted redirection of carbon fluxes. Elevated abundance of HSPs and osmolytes did not outbalance the severe limitation of protein synthesis, marked by orchestrated downregulation of translation (elongation factors, several aa-tRNA synthetases), amino acid biosynthesis and ribosome biogenesis in response to the hyperosmolarity. Altogether we settled that increased osmolarity is not beneficial for rs-Prots synthesis in Y. lipolytica, even though some elements of the response could assist this process. Insight into global changes in the yeast proteome under the treatments is provided. Key points • Temp enhances, but Osm decreases rs-Prots synthesis by Y. lipolytica. • Enhanced abundance of HSPs and osmolytes is overweighted by limited translation. • Global proteome under Osm, Temp and Osm Temp treatments was studied.

Background Translational regulation played an important role in the correct folding of heterologous proteins to form bioactive conformations during biogenesis. Translational pausing coordinates protein translation and co-translational folding. Decelerating translation elongation speed has been shown to improve the soluble protein yield when expressing heterologous proteins in industrial expression hosts. However, rational redesign of translational pausing via synonymous mutations may not be feasible in many cases. Our goal was to develop a general and convenient strategy to improve heterologous protein synthesis in Pichia pastoris without mutating the expressed genes. Results Here, a large-scale deletion library of ribosomal protein (RP) genes was constructed for heterologous protein expression in Pichia pastoris, and 59% (16/27) RP deletants have significantly increased heterologous protein yield. This is due to the delay of 60S subunit assembly by deleting non-essential ribosomal protein genes or 60S subunit processing factors, thus globally decreased the translation elongation speed and improved the co-translational folding, without perturbing the relative transcription level and translation initiation. Conclusion Global decrease in the translation elongation speed by RP deletion enhanced co-translational folding efficiency of nascent chains and decreased protein aggregates to improve heterologous protein yield. A potential expression platform for efficient pharmaceutical proteins and industrial enzymes production was provided without synonymous mutation.

International audience; Yarrowia lipolytica has emerged as an attractive solution for screening enzyme activities thanks to the numerous tools available for heterologous protein production and its strong secretory ability. Nowadays, activity screening for improved enzymes mostly relies on the evaluation of independent clones in microtiter plates. However, even with highly robotized screening facilities, the relatively low throughput and high cost of the technology do not enable the screening of large diversities, which significantly reduce the probability of isolating improved variants. Droplet-based microfluidics is an emerging technology that allows the high-throughput and individual picoliter droplets manipulation and sorting based on enzymatic substrate fluorescence. This technology is an attractive alternative to microtiter plate screenings with higher throughputs and drastic reduction of working volume and cost.Here, we present a droplet-based microfluidic platform for the screening of libraries expressed in the yeast Y. lipolytica, from the generation of a random mutagenesis library of a heterologous enzyme and its expression in Y. lipolytica to the droplet-based microfluidic procedures composed of cell encapsulation and growth and activity screening or sorting of improved clones.

Biotechnology advances have allowed bacteria, yeasts, plants, mammalian and insect cells to function as heterologous protein expression systems. Recently, microalgae have gained attention as an innovative platform for recombinant protein production, due to low culture media cost, compared to traditional systems, as well as the fact that microalgae such as Chlamydomonas reinhardtii are considered safe (GRAS) by the Food and Drug Administration (FDA). Previous studies showed that recombinant protein production in traditional platforms by semicontinuous process increased biomass and bio product productivity, when compared to batch process. As there is a lack of studies on semicontinuous process for recombinant protein production in microalgae, the production of recombinant mCherry fluorescent protein was evaluated by semicontinuous cultivation of Chlamydomonas reinhardtii in bubble column photobioreactor. This semicontinuous cultivation process was evaluated in the following conditions: 20%, 40%, and 60% culture portion withdrawal. The highest culture withdrawal percentage (60%) provided the best results, as an up to 161% increase in mCherry productivity (454.5 RFU h-1 - Relative Fluorescence Unit h-1 ), in comparison to batch cultivation (174.0 RFU h-1 ) of the same strain. All cultivations were carried out for 13 days, at pH 7, temperature 25°C and, by semicontinuous process, two culture withdrawals were taken during the cultivations. Throughout the production cycles, it was possible to obtain biomass concentration up to 1.36 g L-1 .

U novije vrijeme, veliki interes za heterologne proteine dovodi do potrebe za pronalaskom efikasnih tehnika za njihovu proizvodnju. Tehnika izlaganja heterologih proteina na površini stanične stijenke kvasca je značajan predmet istraživanja u znanosti i industriji. Njezinom primjenom omogućava se istovremena proizvodnja i sekrecija heterolognih proteina te njihova imobilizacija na površini stanice i primijena kvasaca kao staničnih biokatalizatora. Osnovni nedostatak metode je relativno niska koncentracija imobiliziranih proteina na površini stanica. Ta je činjenica glavni razlog sve većeg broja istraživanja u svrhu poboljšanja metode. Nastoje se pronaći optimalni sustavi za izlaganje heterolognih proteina te pogodni domaćini. Uz do sada najviše istraženi kvasac S. cerevisiae, sve veća važnost se pridaje metilotrofnim kvascima. Cilj ovog rada je prikazati nekonvencionalne metilotrofne kvasce, P. pastoris i H. polymorpha, kao pogodne domaćine za proizvodnju heterolognih proteina te njihovo izlaganje na površini stanica. In recent times, the great interest in heterologous proteins has led to the need of finding efficient techniques for their production. Yeast surface display of heterologous proteins has become a significant subject of research in science and industry. Its application enables the simultaneous production and secretion of heterologous proteins, as well as their immobilization on the cell surface and the use of yeast as whole-cell biocatalysts. The main disadvantage of the method is the relatively low concentration of immobilized proteins on the cell surface. This fact is the main reason for the progress of research in order to improve the method. Great effort has been put into finding the optimal systems for surface display of heterologous proteins and finding the suitable host. In addition to the most researched yeast S. cerevisiae so far, increasing importance is given to methylotrophic yeast. The aim of this study is to present non-conventional methylotrophic yeasts, P. pastoris and H. polymorpha, as suitable hosts for the production of heterologous proteins and their display on the cell surface.

While overproduction of recombinant secretory proteins (rs-Prots) triggers multiple changes in the physiology of the producer cell, exposure to suboptimal growth conditions may further increase that biological response. The environmental conditions may modulate the efficiency of both the rs-Prot gene transcription and translation but also the polypeptide folding. Insights into responses elicited by different environmental stresses on the rs-Prots synthesis and host yeast physiology might contribute to a better understanding of fundamental biology processes, thus providing some clues to further optimise bioprocesses. Herein, a series of batch cultivations of Yarrowia lipolytica strains differentially metabolically burdened by the rs-Prots overproduction have been conducted. Combinations of different stress factors, namely pH (3/7) and oxygen availability (kLa 28/110 h−1), have been considered for their impact on cell growth and morphology, substrate consumption, metabolic activity, genes expression, and secretion of the rs-Prots. Amongst others, our data demonstrate that a highly metabolically burdened cell has a higher demand for the carbon source, although presenting a compromised cell growth. Moreover, the observed decrease in rs-Prot production under adverse environmental conditions rather results from the emergence of a less-producing cell subpopulation than from the decrease of the synthetic capacity of the whole cell population.

Chemostat cultivation mode imposes selective pressure on cells, which may result in slow adaptation in the physiological state over time. We applied a two-compartment scale-down chemostat system imposing feast-famine conditions to characterize the long-term (100's of hours) response of S. cerevisiae to fluctuating glucose availability. A wild-type strain and a recombinant strain, expressing an insulin precursor, were cultured in the scale-down system, and analyzed at the physiological and proteomic level. Phenotypes of both strains were compared to those observed in a well-mixed chemostat. Our results show that S. cerevisiae subjected to long term chemostat conditions undergoes a global reproducible shift in its cellular state and that this transition occurs faster and is larger in magnitude for the recombinant strain including a significant decrease in the expression of the insulin product. We find that the transition can be completely avoided in the presence of fluctuations in glucose availability as the strains subjected to feast-famine conditions under otherwise constant culture conditions exhibited constant levels of the measured proteome for over 250 hours. We hypothesize possible mechanisms responsible for the observed phenotypes and suggest experiments that could be used to test these mechanisms. This article is protected by copyright. All rights reserved.

Prokaryotic systems have been considered the most affordable and simplest hosts which are being employed to express recombinant proteins such as allergens; nevertheless, without appropriate signal peptide (SP), these systems cannot be used for secretory proteins. Recently, a lot of effort has been put into assessing the potential of gram-positive strains such as lactic acid bacteria for new applications in the production of heterologous proteins. Ama r 2 is a respiratory allergen from Amaranthus retroflexus, whose recombinant production in the probiotic host could be introduced as a specific and effective way to rapid diagnosis and immunotherapy of this allergy. Consequently, the production of this recombinant protein using the prokaryotic system, requires a suitable SP to protect disulfide bonds and to prevent misfolding. This study was designed to predict the best SPs for the expression of Ama r 2 protein in Lactococcus lactis as the host. In this study, 42 signal sequences were selected from SP databases and the most important features of them were evaluated. First, n, h and c regions of the SPs and their probabilities were investigated by signalP software version 4.1. Then, their physicochemical properties were evaluated by Portparam and SOLpro. Moreover, the secretion sorting and sub-cellular localization sites were evaluated by PRED-TAT and ProtcompB software programs. The results revealed that yjgB, entC2 (Entrotoxine type C-2), ent B (Entrotoxine type), blaZ (Beta lactamase), dex (number 21), blm (Beta lactamase 2), dex (Dextranase; number 20) and number 26 were introduced theatrically as the best SPs to express Ama r 2 in Lactococcus lactis.

Interferon‐beta (IFNβ) is a cytokine involved in the antiviral, anti‐proliferative and immunomodulatory responses of cells, and a potent drug for multiple sclerosis. Human interferon beta (HuIFNβ) gene fused with the glucoamylase signal sequence in the N‐terminus and 6 His Tag in the C‐terminus was cloned into pKlac1 vector and introduced in Kluyveromyces lactis to allow secreted expression and one‐step purification of the protein. Recombinant yeast transformant with the highest level of HuIFNβ production was identified, and this secreted up to 1 mg/L of the cytokine after 72 h of incubation. Glycosylated and non‐glycosylated forms of the cytokine were elaborated by the yeast, the latter in higher quantities. His tag of the protein allowed easy one‐step purification by nickel‐nitriloacetic acid affinity chromatography, yielding close to 100% purity. SDS‐PAGE, western blot and MALDI‐TOF‐TOF confirmed the identity of the protein. The biological activity of the recombinant HuIFNβ was confirmed by its anti cell proliferative activity on HeLa cells. Expression of HuIFNβ in K. lactis is advantageous since it is a very safe organism to produce proteins for therapeutic applications, allows glycosylation and offers a cost effective method for large scale production as it can be grown in cheap carbon sources.

PMID: 30737739 Chapter 6; International audience; Yarrowia lipolytica has emerged as an alternative expression system for heterologous protein production and enzyme evolution. Several different expression systems dedicated for this species have been developed, ranging from the simple cloning of expression vectors to recently developed high-throughput methodologies using efficient cloning and assembly such as Gateway and Golden Gate strategies. The latter strategies, due to their modular character, enable multiple vector construction and the construction of expression cassettes containing different genes or a gene under different promoters of various strengths.Here, we present the Golden Gate cloning strategy for the construction of multiple expression cassettes, the transformation into Y. lipolytica, and the selection of efficient enzyme-producing strains using an insect alpha-amylase as a reporter detected via a thermal cycler-based microassay.

Production of recombinant proteins such as antibody fragments in the periplasm of the bacterium Escherichia coli has a number of advantages, including the ability to form disulphide bonds, aiding correct folding, and the relative ease of release and subsequent capture and purification. In this study, we employed two N-terminal signal peptides, PelB and DsbA, to direct a recombinant scFv antibody (single-chain variable fragment), 13R4, to the periplasm via the Sec and SRP pathways respectively. A design of experiments (DoE) approach was used to optimise process conditions (temperature, inducer concentration and induction point) influencing bacterial physiology and the productivity, solubility and location of scFv. The DoE study indicated that titre and subcellular location of the scFv depend on the temperature and inducer concentration employed, and also revealed the superiority of the PelB signal peptide over the DsbA signal peptide in terms of scFv solubility and cell physiology. Baffled shake flasks were subsequently used to optimise scFv production at higher biomass concentrations. Conditions that minimised stress (low temperature) were shown to be beneficial to production of periplasmic scFv. This study highlights the importance of signal peptide selection and process optimisation for the production of scFv antibodies, and demonstrates the utility of DoE for selection of optimal process parameters. Electronic supplementary material The online version of this article (10.1186/s13568-018-0727-8) contains supplementary material, which is available to authorized users.

International audience; Microbial production of secretory proteins constitutes one of the key branches of current industrial biotechnology, earning billion dollar (USD) revenues each year. That industrial branch strongly relies on fluent operation of the secretory machinery within a microbial cell. The secretory machinery, directing the nascent polypeptide to its final destination, constitutes a highly complex system located across the eukaryotic cell. Numerous molecular identities of diverse structure and function not only build the advanced network assisting folding, maturation and secretion of polypeptides but also serve as sensors and effectors of quality control points. All these events must be harmoniously orchestrated to enable fluent processing of the protein traffic. Availability of these elements is considered to be the limiting factor determining capacity of protein traffic, which is of crucial importance upon biotechnological production of secretory proteins. The main purpose of this work is to review and discuss findings concerning secretory machinery operating in a non-conventional yeast species, Yarrowia lipolytica, and to highlight peculiarities of this system prompting its use as the production host. The reviewed literature supports the thesis that secretory machinery in Y. lipolytica is characterized by significantly higher complexity than a canonical yeast protein secretion pathway, making it more similar to filamentous fungi-like systems in this regard.

Background Translational regulation played an important role in the correct folding of heterologous proteins to form bioactive conformations during biogenesis. Translational pausing coordinates protein translation and co-translational folding. Decelerating translation elongation speed has been shown to improve the soluble protein yield when expressing heterologous proteins in industrial expression hosts. However, rational redesign of translational pausing via synonymous mutations may not be feasible in many cases. Our goal was to develop a general and convenient strategy to improve heterologous protein synthesis in Pichia pastoris without mutating the expressed genes. Results Here, a large-scale deletion library of ribosomal protein (RP) genes was constructed for heterologous protein expression in Pichia pastoris, and 59% (16/27) RP deletants have significantly increased heterologous protein yield. This is due to the delay of 60S subunit assembly by deleting non-essential ribosomal protein genes or 60S subunit processing factors, thus globally decreased the translation elongation speed and improved the co-translational folding, without perturbing the relative transcription level and translation initiation. Conclusion Global decrease in the translation elongation speed by RP deletion enhanced co-translational folding efficiency of nascent chains and decreased protein aggregates to improve heterologous protein yield. A potential expression platform for efficient pharmaceutical proteins and industrial enzymes production was provided without synonymous mutation. Electronic supplementary material The online version of this article (10.1186/s13068-019-1377-z) contains supplementary material, which is available to authorized users.

In the past decade much effort has been devoted to the development of new expression systems and novel techniques for the surface display of heterologous proteins in yeast in order to improve their applications in biotechnology, food technology, pharmacology and medicine. Heterologous protein-encoding genes are generally fused with genes coding for yeast cell wall proteins or their fragments required for anchoring. The variety of reactions by which a protein can be displayed at the cell surface enables finding the appropriate one for each individual protein. However, it is still challenging how to improve the efficiency of display of protein complexes and increase the quantity of protein displayed on the yeast surface. Recently, synthetic protein chimeras that self-assemble into the scaffolds on the yeast surface displaying different proteins have been constructed. This review focuses on systems and techniques for display of recombinant proteins on the yeast cell surfaces and applications afforded by this technology.

El present document està centrat en un punt de vista d’enginyeria de bioprocés, de cara a la producció heteròloga de la lipasa de Rhizopus oryzae, expressada en el llevat metilotròfic Pichia pastoris sota el control del promotor de l’alcohol oxidasa 1, induïble amb metanol. Especialment, l’objectiu es focalitza a la millora de l’eficiència del bioprocés mitjançant l’ús d’estratègies d’operació en discontinu alimentat en bioreactors de diferent escala. Amb això, es pretén establir P. pastoris com una plataforma versàtil, robusta i competent per a la producció de proteïnes recombinants. A la primera part de la tesi, l’èmfasi es troba en l’assoliment de nivells òptims dels principals índexs de rendiment d’interès industrial a escala de laboratori, utilitzant estratègies d’operació amb metanol com a font única de carboni i inductor del promotor. Prèviament a aquest objectiu, però, es van realitzar estudis preliminars i millores operacionals per tal de superar certes limitacions del sistema i per aportar-hi versatilitat i robustesa. Entre ells, es va actualitzar i incorporar millores al programa de supervisió i control del procés en LabVIEW, s’hi instal·là nou hardware d’instrumentació i es va implementar una nova estratègia d’alimentació de la font de nitrogen. Posteriorment, un estudi sistem àtic de l’efecte de la disponibilitat d’oxigen es va dur a terme. Es va obtenir que, en condicions de normòxia, l’oxigen dissolt pot limitar el procés tant per un excés com per nivells escassos. El 25 % de saturació d’aire es va establir com a valor òptim de cara a les velocitats específiques, i els resultats van ser validats per un test de consistència i reconciliació de dades. A partir d’aquest punt, s’assolí l’optimització del bioprocés a escala de laboratori mitjançant el disseny de perfils d’alimentació de metanol amb eines matemàtiques d’optimització. A partir de dades de modelització i amb una eina d’optimització simple i versàtil, es van dur a terme les simulacions de perfils òptims de concentració de metanol per maximitzar la concentració de producte i la productivitat volumètrica, es van comparar amb estratègies anteriors i es van aplicar experimentalment amb èxit. Per altra banda, la següent part del document se centra en estudis de canvi d’escala. El primer que es realitza és a escala de microbioreactor. Es van reproduir uns cultius realitzats a aquella escala al laboratori per verificar aquest sistema com a vàlid per realitzar-hi assajos d’expressió i selecció de soques i per dissenyar-hi estratègies simples d’operació en discontinu alimentat. Finalment, el bioprocés va ser portat a escala pilot de 50 L. Malauradament, els intents realitzats no van assolir reproduir l’eficiència del bioprocés, tot i que va ser possible descartar-ne algunes hipòtesis plantejades i proposarne d’altres que hauran de ser investigades en un futur, com ara limitacions en la transferència d’oxigen., The present work is focused, with a bioprocess engineering point of view, in the production of the heterologous Rhizopus oryzae lipase expressed in the methylotrophic yeast Pichia pastoris under the control of the methanol-induced alcohol oxidase 1 promoter; and, specially, the improvement of the bioprocess efficiency by the use of proper operational strategies in fed-batch mode for bioreactors of different scale. With that, P. pastoris is stablished as a versatile, robust and competent platform for the production of recombinant proteins. In a first part of the dissertation, the emphasis is put on reaching optimal levels of the main performance indexes of industrial interest at lab scale in methanol feeding strategies as the only carbon source and inducer. Prior to this final goal, though, preliminary studies and operational improvements were carried out to outcome system limitations and to give more versatility and robustness to the system. They included upgrades in system control and supervising in LabVIEW, installation of new hardware for instrumentation and a new feeding strategy for the nitrogen source added to the cultures. Later, a systematic study on the effect of the oxygen tension was conducted. It was found that, in normoxic conditions, dissolved oxygen can limit the process in both excess and lack. An optimal value of 25 % air saturation was found for all specific rates and results were validated by a consistency check and further data reconciling. From that point, the optimization of the bioprocess in bench-top bioreactor by the mathematical design of methanol feeding profiles was fulfilled. From modelling data, and by the use of a simple, versatile optimization tool, the simulation of the optimal methanol concentration profiles were carried out to maximize product titer and volumetric productivity, and then compared to previous strategies and then successfully applied experimentally. On the other hand, the next part of this document focuses on studies of scale down and up from the bench-top scale. The first change is downwards to a microbioreactor system of a 1000 μL scale. Cultures from that scale were successfully reproduced succesfully at lab scale to verify the system as suitable for clone screening and design of simple fedbatch strategies. Finally, the bioprocess was brought up to a 50 L scale. Unfortunately, the attempts did not succeed on reproduce the bioprocess efficiency, but it was possible to discard some of the hypothesis that were brought up, and to settle some other ones that can be further investigated in the future, like oxygen mass transfer limitations.

A descoberta das monoxigenases de polissacarídeos líticas dependentes de cobre (LPMOs), que agem em sinergismo com outras enzimas na desconstrução da celulose, gerou um grande interesse da comunidade científica por seu potencial de aplicação na produção de biocombustíveis a partir de resíduos lignocelulósicos. A busca por essas proteínas auxiliares em microrganismos surgiu como uma estratégia promissora, pois há grande diversidade de isoformas e disponibilidade de sequências genômicas dessas proteínas. Diante disso, o presente trabalho teve como objetivo expressar LPMOs recombinantes de origem bacteriana em levedura Komagataella phafii. Foram obtidos clones de K. phafii transformados com 6 genes selecionados a partir de banco de dados. Análises da cinética de expressão proteica por técnica de western blot indicaram secreção apenas da LPMO de 25 kDa codificada pelo gene de Thermobifida fusca YX. Ensaios de produção da LPMO de T. fusca foram realizados em biorreator de 1L e Erlenmeyers de 250 mL e 1000 mL. Nos ensaios em Erlenmeyers houve a detecção da proteína, confirmada por SDS-PAGE e western blot, acompanhado de um alto crescimento microbiano. No ensaio em biorreator não houve detecção da proteína de interesse e o crescimento microbiano foi baixo. Com a confirmação da expressão da LPMO nos ensaios em Erlenmeyers, estes foram parcialmente purificados e avaliados por gel de proteínas e western blot. Os resultados obtidos mostram que o sistema de expressão de K. phafii foi eficiente, expressando a LPMO de T. fusca. The discovery of copper-dependent lytic polysaccharide monooxygenase (LPMOs), auxiliary proteins which act in synergy with other enzymes on the cellulose degradation, generated a great interest from the scientific community due to their potential application in biofuels production from lignocellulosic residues. The search for these auxiliary proteins in microorganisms has emerged as a promising strategy because there is a great diversity of isoforms and availability of genomic sequences. Thus, the present work aimed to express bacterial LPMOs in Komagataella phafii. Clones of K. phafii transformed with 6 genes selected from database were obtained. Analysis of protein expression kinetics by western blot technique showed accumulation only of the LPMO of 25 kDa coded by the gene from Thermobifida fusca YX. Production of LPMO from T. fusca was tested in 1 L bioreactor and 250 mL and 1000 mL Erlenmeyer flasks. In Erlenmeyer flasks experiments, there was protein detection, confirmed by SDS-PAGE and western blot, and a high microbial growth. In the bioreactor assay, there was no target protein detection, and microbial growth was low. After confirmation of LPMO expression in Erlenmeyer flasks assays, protein were partially purified and confirmed by protein gel and western blot. Results obtained showed that the expression system of K. phafii was effective, expressing the LPMO from T. fusca.

The baculovirus expression vector system (BEVS) is a versatile and powerful platform for protein expression in insect cells. With the ability to approach similar post-translational modifications as in mammalian cells, the BEVS offers a number of advantages including high levels of expression as well as an inherent safety during manufacture and of the final product. Many BEVS products include proteins and protein complexes that require expression from more than one gene. This review examines the expression strategies that have been used to this end and focuses on the distinguishing features between those that make use of single polycistronic baculovirus (co-expression) and those that use multiple monocistronic baculoviruses (co-infection). Three major areas in which researchers have been able to take advantage of co-expression/co-infection are addressed, including compound structure-function studies, insect cell functionality augmentation, and VLP production. The core of the review discusses the parameters of interest for co-infection and co-expression with time of infection (TOI) and multiplicity of infection (MOI) highlighted for the former and the choice of promoter for the latter. In addition, an overview of modeling approaches is presented, with a suggested trajectory for future exploration. The review concludes with an examination of the gaps that still remain in co-expression/co-infection knowledge and practice.

To circumvent cell wall retention commonly associated to Saccharomyces cerevisiae when used as a host for heterologous protein production, we have created a translational fusion of human interleukin-1β (IL-1β) to the Pir4 cell wall protein, so as to drive the secretion of the recombinant product to the growth medium. The auxotrophic S. cerevisiae BY4741 was used as host to express the Pir4-IL1β fusion protein. Once it was ascertained that the fusion protein was secreted to the culture medium and behaved as a growth-linked product, S. cerevisiae BY4741 [PIR4-IL1β] was cultured in an aerated fed-batch reactor to achieve high cell density and, consequently, high product concentration in the medium. Two cultivation media were employed, a rich complex and a defined mineral medium, the latter suitably supplemented with bacto-casamino acids as ACA (auxotrophy-complementing amino acid) source. The rich complex medium allowed a good performance of the producer strain only during batch growth, but was revealed to be inadequate for long-term fed-batch operations. The defined mineral medium ensured a better performance, even though not yet satisfactory in spite of a proper ACA supplementation. The behaviour of BY4741 was attributed to an intrinsic sensitivity of the producer strain to long-term aerated fed-batch operations.

Today, sufficient data are available to support the use of lactic acid bacteria (LAB), notably lactococci and lactobacilli, as delivery vehicles for the development of new mucosal vaccines. These non-pathogenic Gram-positive bacteria have been safely consumed by humans for centuries in fermented foods. They thus constitute an attractive alternative to the attenuated pathogens (most popular live vectors actually studied) which could recover their pathogenic potential and are thus not totally safe for use in humans. This chapter reviews the current research and advances in the use of LAB as live delivery vectors of proteins of interest for the development of new safe mucosal vaccines. The use of LAB as DNA vaccine vehicles to deliver DNA directly to antigen-presenting cells of the immune system is also discussed.

Background Numerous strategies have been developed for the display of heterologous proteins in the surface of live bacterial carriers, which can be used as vaccines, immune-modulators, cancer therapy or bioremediation. Bacterial biofilms have emerged as an interesting approach for the expression of proteins of interest. Bacillus subtilis is a well-described, endospore-forming organism that is able to form biofilms and also used as a probiotic, thus making it a suitable candidate for the display of heterologous proteins within the biofilm. Here, we describe the use of TasA, an important structural component of the biofilms formed by B. subtilis, as a genetic tool for the display of heterologous proteins. Results We first engineered the fusion protein TasA-mCherry and showed that was widely deployed within the B. subtilis biofilms. A significant enhancement of the expression of TasA-mCherry within the biofilm was obtained when depleting both tasA and sinR genes. We subsequently engineered fusion proteins of TasA to antigenic peptides of the E. granulosus parasite, paramyosin and tropomyosin. Our results show that the antigens were well expressed within the biofilm as denoted by macrostructure complementation and by the detection of the fusion protein in both immunoblot and immunohistochemistry. In addition, we show that the recombinant endospores of B. subtilis preserve their biophysical and morphological properties. Conclusions In this work we provide strong evidence pointing that TasA is a suitable candidate for the display of heterologous peptides, such as antigens, cytokines, enzymes or antibodies, in the B. subtilis biofilms. Finally, our data portray that the recombinant endospores preserve their morphological and biophysical properties and could be an excellent tool to facilitate the transport and the administration., Microbial Cell Factories, 15, ISSN:1475-2859

Lactococcus lactis is of great importance for the nutrition of hundreds of millions of people worldwide. This paper describes the genome sequence of Lactococcus lactis subsp. cremoris MG1363, the lactococcal strain most intensively studied throughout the world. The 2,529,478-bp genome contains 81 pseudogenes and encodes 2,436 proteins. Of the 530 unique proteins, 47 belong to the COG (clusters of orthologous groups) functional category "carbohydrate metabolism and transport," by far the largest category of novel proteins in comparison with L. lactis subsp. lactis IL1403. Nearly one-fifth of the 71 insertion elements are concentrated in a specific 56-kb region. This integration hot-spot region carries genes that are typically associated with lactococcal plasmids and a repeat sequence specifically found on plasmids and in the "lateral gene transfer hot spot" in the genome of Streptococcus thermophilus. Although the parent of L. lactis MG1363 was used to demonstrate lysogeny in Lactococcus, L. lactis MG1363 carries four remnant/satellite phages and two apparently complete prophages. The availability of the L. lactis MG1363 genome sequence will reinforce its status as the prototype among lactic acid bacteria through facilitation of further applied and fundamental research.

Background The DHFR negative CHO DXB11 cell line (also known as DUX-B11 and DUKX) was historically the first CHO cell line to be used for large scale production of heterologous proteins and is still used for production of a number of complex proteins. Results Here we present the genomic sequence of the CHO DXB11 genome sequenced to a depth of 33x. Overall a significant genomic drift was seen favoring GC → AT point mutations in line with the chemical mutagenesis strategy used for generation of the cell line. The sequencing depth for each gene in the genome revealed distinct peaks at sequencing depths of 0x, 16x, 33x and 49x coverage corresponding to a copy number in the genome of 0, 1, 2 and 3 copies. This indicate that 17% of the genes are haploid revealing a large number of genes which can be knocked out with relative ease. This tendency of haploidy was furthermore shown to be present in eight additional analyzed CHO genomes (15-20% haploidy) but not in the genome of the Chinese hamster. The dhfr gene is confirmed to be haploid in CHO DXB11; transcriptionally active and the remaining allele contains a G410C point mutation causing a Thr137Arg missense mutation. We find ~2.5 million single nucleotide polymorphisms (SNP’s), 44 gene deletions in the CHO DXB11 genome and 9357 SNP's, which interfere with the coding regions of 3458 genes. Copy number variations for nine CHO genomes were mapped to the chromosomes of the Chinese hamster showing unique signatures for each chromosome. The data indicate that chromosome one and four appear to be more stable over the course of the CHO evolution compared to the other chromosomes thus might presenting the most attractive landing platforms for knock-ins of heterologous genes. Conclusions Our studies reveal an unexpected degree of haploidy in CHO DXB11 and CHO cells in general and highlight the chromosomal changes that have occurred among the CHO cell lines sequenced to date. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1391-x) contains supplementary material, which is available to authorized users.

The effect of induction of protein production was studied in bioreactor cultures of T. reesei strain Rut-C30 and its transformant expressing endoglucanase I core domain (EGI, Cel7B) fused with a hydrophobic peptide tag. The tag was previously designed for efficient purification of the fusion protein in aqueous two-phase separation. The fungi were first grown on glucose-containing minimal medium after which rich medium with lactose as a carbon source was added to induce cellulase production. Production of extracellular protein and cellulase activity and the transcript levels of the major cellulase genes were analyzed during the cultivations. Induction of the cellulase genes followed a similar temporal pattern in both strains. The first phase of induction took place after addition of lactose as soon as glucose was depleted, and the second phase after lactose was consumed. Western analysis showed that a decreased amount of fusion protein was produced in the culture medium compared with the endogenous EGI, although the strain harbors several copies of the recombinant gene under the strong cbh1 promoter. The fusion protein appeared to accumulate within the cells, indicating impaired secretion of the protein. The mRNA levels of the UPR (unfolded protein response) target genes, bip1 and pdi1, and the level of the active form of hac1 transcript encoding the UPR transcription factor increased concurrently with induction of the cellulase genes in both strains, indicating increased requirement of the folding machinery under these conditions. However, only a minor increase in bip1 and pdi1 transcript level was observed in the transformant compared with the parental strain.

Bacillus subtilis is a rod-shaped, Gram-positive soil bacterium that secretes numerous enzymes to degrade a variety of substrates. enabling the bacterium to survive in a continuously changing environment. These enzymes are produced commercially and this production represents about 60% of the industrial-enzyme market. Unfortunately, the secretion of heterologous proteins. originating from Gram-negative bacteria or from eukaryotes, is often severely hampered. Several bottlenecks in the B. subtilis secretion pathway, such as poor targeting to the translocase, degradation of the secretory protein, and incorrect folding, have been revealed. Nevertheless. research into the mechanisms and control of the secretion pathways will lead to improved Bacillus protein secretion systems and broaden the applications as industrial production host. This review focuses on studies that aimed at optimizing B. subtilis as cell factory for commercially interesting heterologous proteins. (C) 2004 Elsevier B.V. All rights reserved.

Bacillus subtilis is a rod-shaped, Gram-positive soil bacterium that secretes numerous enzymes to degrade a variety of substrates. enabling the bacterium to survive in a continuously changing environment. These enzymes are produced commercially and this production represents about 60% of the industrial-enzyme market. Unfortunately, the secretion of heterologous proteins. originating from Gram-negative bacteria or from eukaryotes, is often severely hampered. Several bottlenecks in the B. subtilis secretion pathway, such as poor targeting to the translocase, degradation of the secretory protein, and incorrect folding, have been revealed. Nevertheless. research into the mechanisms and control of the secretion pathways will lead to improved Bacillus protein secretion systems and broaden the applications as industrial production host. This review focuses on studies that aimed at optimizing B. subtilis as cell factory for commercially interesting heterologous proteins. (C) 2004 Elsevier B.V. All rights reserved.

International audience; The study of most membrane proteins remains challenging due to their hydrophobicity and their low natural abundance in cells. Lactococcus lactis, a Gram-positive lactic bacterium, has been traditionally used in food fermentations and is nowadays widely used in biotechnology for large-scale production of heterologous proteins. This system has been successfully used for the production of prokaryotic and eukaryotic membrane proteins. The purpose of this chapter is to provide detailed protocols for (1) the expression of plant peripheral or intrinsic membrane proteins and then for (2) their solubilization, from Lactococcus membranes, for further purification steps and biochemical characterization.

This study focused on the growth of Saccha-romyces cerevisiae MM01 recombinant strains and the respective production of three extracellular heterologous cutinases: a wild-type cutinase and two cutinases in which the primary structure was fused with the peptides (WP)2 and (WP)4, respectively. Different cultivation and strategies were tested in a 2-L shake flask and a 5-L bioreactor, and the respective cell growth and cutinase production were analyzed and compared for the three yeast strains. The highest cutinase productions and productivities were obtained in the fed-batch culture, where wild-type cutinase was secreted up to a level of cutinase activity per dry cell weight (specific cell activity) of 4.1 Umg−1 with activity per protein broth (specific activity) of 266 Umg−1, whereas cutinase-(WP)2 was secreted with a specific cell activity of 2.1 Umg−1 with a specific activity of 200 Umg−1, and cutinase-(WP)4 with a specific cell activity of 0.7 Umg−1 with a specific activity of 15 Umg−1. The results indicate that the fusion of hydrophobic peptides to cutinase that changes the physical properties of the fused protein limits cutinase secretion and subsequently leads to a lower plasmid stability and lower yeast cell growth. These effects were observed under different cultivation conditions (shake flask and bioreactor) and cultivation strategies (batch culture versus fed-batch culture). © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 692–698, 2002.

"In this thesis, methanol biofiltration coupled with heterologous protein production was studied. A P. pastoris strain transformed with the gen Ech42 responsible for producing Endochitinase 42 (Ech42) was used (Pérez-Martínez et al., 2007). Three different biofilters were designed and tested under different methanol loads, nitrogen concentrations, pH and EBRT (Empty Bed Residence Time) conditions. The maximum elimination capacity observed was 643.5 g/m3*h under a methanol load of 800 g/m3*h, 5 g/L (NH4)2SO4, pH 5 and 60 seconds of EBRT. Also, the Ech42 activity ranged from 578 to 1172 U/L and was mainly affected by pH changes. Additionally, it was observed that EC was very sensitive to changes under the mentioned operational conditions. However, the heterologous protein activity was quite stable. On the other hand, divergence in the system was approached. It was noted that two or more systems operating under identical conditions can behave differently over time. Thus, repeatability in biofiltration shouldn’t be taken for granted. Some probable explanations for such divergence are the random colonization of foreign species as well as the unpredictable evolution of microbial communities. In any case, this is the first report of operational divergence in biofilters. Finally, a dynamic mathematical model was developed based on CSTR equations. A Monod kinetic was used in order to describe methanol consumption behavior. Also, microbial growth was considered for two different kinds of microorganisms: P. pastoris and others. The model fit well with experimental elimination capacities and protein production. However, total biomass volume was not well described. The source of the model discrepancy could be the failure to consider inert biomass. Therefore, the mathematical model must be further explored." "En la presente tesis se estudió la biofiltración de metanol acoplada con la producción de proteínas heterólogas. Para ello, se utilizó un cultivo de P. pastoris transformado con el gen Ech42 responsable de producir la Endoquitinasa 42 de Trichoderma Atroviride (Pérez-Martínez et al., 2007). Se diseñaron tres biofiltros idénticos y se operaron bajo diferentes condiciones de carga de metanol, concentración de nitrógeno, pH y tiempo de residencia. La máxima capacidad de eliminación de metanol observada fue de 643.5 g/m3*h a una carga de metanol de 800 g/m3*h, 5 g/L (NH4)2SO4, pH 5 y 60 segundos de tiempo de residencia. La actividad de Ech42 varió entre 578 y 1172 U/L siendo el pH el parámetro más importante para el proceso. Además, se encontró que la capacidad de eliminación del sistema fue muy sensible a los cambios de condiciones manejadas. Sin embargo, la actividad de la proteína heteróloga fue notablemente estable. Por otro lado, también se estudió la divergencia del sistema a través de un análisis estadístico simple, microscopía electrónica de barrido y análisis cinético, durante las primeras tres etapas de operación. Se observó que dos o más sistemas de biofiltración pueden comportarse de distinta manera con el tiempo, aún y cuando son operados bajo las mismas condiciones. Algunas explicaciones probables son los procesos aleatorios de colonización de otras especias así como la impredecible evolución de comunidades microbianas. En cualquier caso, los resultados mostrados en esta tesis son el primer reporte de divergencia en biofiltros. Finalmente, se desarrolló un modelo matemático dinámico para describir el desempeño de los sistemas de biofiltración acoplados a la producción de proteínas. Éste se basó en ecuaciones para reactores CSTR (Completely Stirred Tank Reactor) pero en una estructura discretizada del sistema. Para la degradación de metanol se utilizó el modelo de Monod. Se consideró la presencia de dos grupos de microorganismos creciendo en los sistemas: P. pastoris y otros microorganismos. El modelo mostró un buen ajuste en relación con las capacidades de eliminación experimentales y la producción proteica. Sin embargo, fue difícil ajustar los valores experimentales de biomasa total presente (volumen). Tal discrepancia puede deberse a que el modelo no considera biomasa inerte. Por lo anterior, es recomendable que el modelo matemático sea modificado o replanteado."

The Bacillus pumilus 3-19 Glutamyl peptidase (EC 3.4.21.19) was isolated from the culture medium of the B. subtilis recombinant strain at the following stages of the bacillus growth: a decelerating growth phase and a stationary growth phase. The action of the purified preparations of the enzyme on different phases of its growth was studied on the insulin B-chain and various protein and peptide substrates. Physicochemical properties of the enzyme were compared for different phases of its growth. The glutamyl endopeptidase preparations differed in their catalytic characteristics and their sensitivity to the metal cations. © 2013 Pleiades Publishing, Ltd.

The gammaproteobacterium Pseudomonas syringae pv. tomato DC3000 is the causal agent of bacterial speck, a common disease of tomato. The mode of infection of this pathogen is not well understood, but according to molecular biological, genomic and proteomic data it produces a number of proteins that may promote infection and draw nutrients from the plant. Glyceraldehyde-3- phosphate dehydrogenase (GAPDH) is a major enzyme of carbon metabolism that was reported to be a surface antigen and virulence factor in other pathogenic microorganisms, but its possible role in the infection process of P. syringae has so far not been studied. Whole-genome sequence analyses revealed the occurrence in this phytopathogenic bacterium of three paralogous gap genes encoding distinct GAPDHs, namely two class I enzymes having different molecular mass subunits and one class III bifunctional D-erythrose-4-phosphate dehydrogenase/GAPDH enzyme. By using genome bioinformatics data, as well as alignments of both DNA and deduced protein sequences, the three gap genes of P. syringae were one-step cloned with a His-Tag in pET21a vector using a PCR-based strategy, and its expression optimized in E. coli BL21 to achieve high yield of the heterologous proteins. In accordance with their distinct molecular phylogenies, these bacterial gap genes encode functional GAPDHs of diverse molecular masses and nicotinamide-coenzyme specificities, suggesting specific metabolic and/or cellular roles.

Conselho Nacional de Desenvolvimento Científico e Tecnológico Selection of the most suitable yeast host for heterologous protein expression is important for the success of a biotechnological process. However despite the diversity of works with protein expression in yeasts the secretion level in these organisms are still low when compared with filamentous fungi. The yeast Kluyveromyces marxianus has a great potential for fermentation and protein production. Although, there are few studies reporting heterologous expression in this yeast. It shows the need for more studies to develop an efficient system for the recombinant protein production in this yeast. The aim of this work was the construction of linear cassettes for expression heterologous proteins in K. marxianus. The cassettes were constructed by fusion PCR. For this purpose we performed a first reaction cycle in which each fragment was amplified individually (the promoter, the gene of interest and the selection mark). The promoters (ScHXT1p, ScPGKp, ScTDH3p, KmINU1p and KmPCPL3p) were amplified from the genome of S. cerevisiae and K. marxianus. The endoglucanase and celobiohidrolase genes from A. niger (eng1 and cbhA) were cloned in pKLAC1 vector that was then used as template to amplify the sequence of genes flanked by the sequences of the Kluyveromyces lactis signal peptide α-mating factor and LAC4 gene terminator. The gene KanMX, which confers resistance to geneticin (G418) in eukaryotes, was amplified from genome of a S. cerevisiae mutant. The second step was a fusion PCR reaction of the fragments: the promoter desired, α-MF-gene of interest-TT and the KanMX. This construction was efficient, reproducible and capable of being held in a shorter time than tradicional cloning methods. The constructions were used in K. marxianus UFV-3 transformation by two different methods, the lithium acetate and electroporation. The lithium acetate protocol was more efficient and the transformants were selected by resistance to increased G418 concentrations. A escolha da levedura hospedeira para produção de uma proteína heteróloga é fundamental para o sucesso de um processo biotecnológico, porém, apesar da diversidade de trabalhos com expressão de proteínas em leveduras, os níveis de secreção nesses organismos ainda são considerados baixos quando comparadas aos fungos filamentosos. A levedura K. marxianus possui grande potencial biotecnológico para fermentação e produção de proteínas. Apesar desse potencial existem poucos trabalhos relatando a expressão heteróloga de proteínas nessa levedura. Este quadro demonstra a necessidade de mais estudos para padronização de um sistema fácil e eficiente para obtenção de K. marxianus recombinantes para produção de proteínas de interesse. O objetivo deste trabalho foi padronizar a construção de cassetes lineares para expressão de proteínas heterólogas em K. marxianus. Os cassetes de expressão foram construídos pela técnica de PCR de fusão. Para tanto foi realizado um primeiro ciclo de reação na qual foi amplificado individualmente cada fragmento integrante do cassete (o promotor, o gene de interesse e a marca de seleção). Os promotores escolhidos, ScHXT1p, ScPGKp, ScTDH3p, KmINU1p e KmPCPL3p, foram amplificados do genoma de S. cerevisiae ou K. marxianus. Os genes das enzimas endoglucanase e celobiohidrolase de Aspergillus níger (eng1e cbhA) foram clonados no vetor KLAC1 e este foi utilizado como molde para amplificação da sequência dos genes flanqueados pelas sequências do peptídeo sinal α-mating factor de Kluyveromyces lactis e do terminador do gene lac4 de Kluyveromyces lactis. A sequência codificadora do gene KanMX, marca de seleção que confere resistência em eucariotos para o antibiótico geneticina (G418), foi amplificado do DNA genômico de uma linhagem mutante de S. cerevisiae. O segundo passo consistiu da reação de fusão dos fragmentos de cada promotor com os fragmentos αMF-gene-TT e KanMX por PCR. Esse tipo de construção se mostrou eficiente, reprodutível e passível de ser realizada em tempo muito inferior àquele de métodos de clonagem tradicionais. Os cassetes construídos foram utilizados na transformação de leveduras Kluyveromyces marxianus UFV-3 por dois métodos, acetato de lítio e eletroporação. O protocolo de acetato de lítio foi mais eficiente e as colônias obtidas foram selecionadas por resistência às concentrações crescentes de G418.

International audience; Most large-scale microbial production of recombinant proteins are based on Escherichia coli, yeasts, or filamentous fungi systems. Using eukaryotic hosts, antibody fragments are generally expressed by targeting to the secretory pathway. This enables not only efficient disulfide bond formation but also secretion of soluble and correctly folded product. For this goal, a recombinant vector was constructed to produce a single-domain antibody (NbAahI'22) directed against AahI' scorpion toxin using the methylotrophic yeast Pichia pastoris. The corresponding complementary DNA was cloned under control of the alcohol oxidase promoter in frame with the Saccharomyces α-factor secretion signal and then transferred to P. pastoris cell strain X-33. Using Western blot, we detected the expression of the recombinant NbAahI'22 exclusively in the culture medium. Targeting to the histidine label, the secreted nanobody was easily purified on nickel-nitrilotriacetic acid resin and then tested in enzyme-linked immunosorbent assay. Interestingly, the production level of the NbAahI'22 in its new glycosylated form reached more than sixfold that obtained in E. coli. These findings give more evidence for the utilization of P. pastoris as a heterologous expression system.

Endoplasmic reticulum associated degradation (ERAD) is a conserved mechanism to remove misfolded proteins from the ER by targeting them to the proteasome for degradation. To assess the role of ERAD in filamentous fungi, we have examined the consequences of disrupting putative ERAD components in the filamentous fungus Aspergillus niger. Deletion of derA, doaA, hrdC, mifA, or mnsA in A. niger yields viable strains, and with the exception of doaA, no significant growth phenotype is observed when compared to the parental strain. The gene deletion mutants were also made in A. niger strains containing single- or multicopies of a glucoamylase–glucuronidase (GlaGus) gene fusion. The induction of the unfolded protein response (UPR) target genes (bipA and pdiA) was dependent on the copy number of the heterologous gene and the ERAD gene deleted. The highest induction of UPR target genes was observed in ERAD mutants containing multiple copies of the GlaGus gene. Western blot analysis revealed that deletion of the derA gene in the multicopy GlaGus overexpressing strain resulted in a 6-fold increase in the intracellular amount of GlaGus protein detected. Our results suggest that impairing some components of the ERAD pathway in combination with high expression levels of the heterologous protein results in higher intracellular protein levels, indicating a delay in protein degradation.

Recombinant protein expression in Escherichia coli ( E. coli ) is simple, fast, inexpensive, and robust, with the expressed protein comprising up to 50 percent of the total cellular protein. However, it also has disadvantages. For example, the rapidity of bacterial protein expression often results in unfolded/misfolded proteins, especially for heterologous proteins that require longer times and/or molecular chaperones to fold correctly. In addition, the highly reductive environment of the bacterial cytosol and the inability of E. coli to perform several eukaryotic post‐translational modifications results in the insoluble expression of proteins that require these modifications for folding and activity. Fortunately, multiple, novel reagents and techniques have been developed that allow for the efficient, soluble production of a diverse range of heterologous proteins in E. coli . This overview describes variables at each stage of a protein expression experiment that can influence solubility and offers a summary of strategies used to optimize soluble expression in E. coli . Curr. Protoc. Protein Sci. 61:5.24.1‐5.24.29. © 2010 by John Wiley & Sons, Inc.

The synthesis of heterologous proteins in lactobacilli is strongly influenced by the promoter selected for the expression. In addition, the activity of the promoters themselves may vary among different bacterial hosts. Three different promoters were investigated for their capability to drive enhanced green fluorescent protein (EGFP) expression in Lactococcus lactis spp. cremoris MG1363, in Lactobacillus reuteri DSM 20016(T) and in five L. reuteri strains isolated from chicken crops. The promoters of the Lactobacillus acidophilus surface layer protein gene (slp), L. acidophilus lactate dehydrogenase gene (ldhL) and enterococcal rRNA adenine N-6-methyltransferase gene (ermB) were fused to the coding sequence of EGFP and inserted into the backbone of the pTRKH3 shuttle vector (pTRKH3-slpGFP, pTRKH3-ldhGFP, pTRKH3-ermGFP). Besides conventional analytical methods, a new quick fluorimetric approach was set up to quantify the EGFP fluorescence in transformed clones using the Qubit() fluorometer. ermB proved to be the most effective promoter in L. reuteri isolates, producing 3.90 x 10(-7) g of fluorescent EGFP (mL OD(stationary culture))(-1). Under the same conditions, the ldhL promoter produced 2.66 x 10(-7) g of fluorescent EGFP (mL OD(stationary culture))(-1). Even though the slp promoter was efficient in L. lactis spp. cremoris MG1363, it was nearly inactive both in L. reuteri DSM 20016(T) and in L. reuteri isolates.

A cultural system, aimed at the production of human interleukin-1β (IL-1β) with cells of a non-conventional yeast transformed for interleukin expression, Zygosaccharomyces bailii [pZ3KlIL-1β], was realized. Interleukin production was accomplished in a reactor operating in fed-batch mode to avoid sugar overflow metabolism, limitations with respect to oxygen transfer, and achieve high cell density. Batch operation mode was employed only to characterise the producer strain and experimentally estimate kinetic parameters. In parallel with strain characterisation, a mathematical model was developed. The comparison between simulations and experimental data allowed to evidence the importance of physiological state of inoculum, being only a fermentative one suitable to sustain a given exponential growth. The respiratory capacity of Z. bailii [pZ3KlIL-1β], resulted to be affected by stirring. The theoretical and experimental approach allowed the bioprocess optimisation. © 2009 Elsevier Ltd. All rights reserved.

Laccases have numerous biotechnological applications, among them food processing. The widespread use of laccases has increased the demand for an inexpensive and safe source of recombinant enzyme. We explored the use of a rice-based system for the production of two fungal laccases derived from the ascomycete Melanocarpus albomyces and the basidiomycete Pycnoporus cinnabarinus. High-expression levels of active recombinant laccases were achieved by targeting expression to the endosperm of rice seeds. The laccase cDNAs were fused to a plant-derived signal sequence for targeting to the secretory pathway, and placed under the control of a constitutive seed-specific promoter fused to an intron for enhanced expression. This construct enabled the recovery of on average 0.1–1% of soluble laccase in total soluble proteins (TSP). The highest yields of recombinant laccases obtained in rice seeds were 13 and 39 ppm for riceMaL and ricePycL, respectively. The rice-produced laccases were purified and characterized. The wild-type and the recombinant proteins showed similar biochemical features in terms of molecular mass, pI, temperature and optimal pH and the N-terminus was correctly processed. Although presenting lower kinetic parameters, the rice-produced laccases were also suitable for the oxidative cross-linking of a food model substrate [maize-bran feruloylated arabinoxylans (AX)].

Bottlenecks in protein expression and secretion often limit the development of industrial processes. By manipulating chaperone and foldase levels, improvements in yeast secretion were found for a number of proteins. Recently, sustained endoplasmic reticulum stress, occurring due to recombinant protein production, was reported to cause oxidative stress in yeast. Saccharomyces cerevisiae cells are able to trigger an adaptive response to oxidative-stress conditions, resulting in the upregulation of both primary and secondary antioxidant defenses. SOD1 encodes for a superoxide dismutase that catalyzes the dismutation of superoxide anions (O 2 − ) into oxygen and hydrogen peroxide. It is a Cu 2+ /Zn 2+ metalloenzyme and represents an important antioxidant defense in nearly all aerobic and aerotolerant organisms. We found that overexpression of the Kluyveromyces lactis SOD1 (Kl SOD1 ) gene was able to increase the production of two different heterologous proteins, human serum albumin (HSA) and glucoamylase from Arxula adeninivorans . In addition, Kl SOD1 overexpression led to a significant decrease in the amount of reactive oxygen species (ROS) that originated during protein production. The yield of HSA also increased when K. lactis cells were grown in the presence of the antioxidant agent ascorbic acid and decreased when cells were challenged with menadione, a ROS generator compound. Moreover, we observed that, in high-osmolarity medium, cells overexpressing Kl SOD1 showed higher growth rates than control cells. Our results thus further support the notion that the production of some heterologous proteins may be improved by manipulating genes involved in general stress responses.

Lactococcus lactis is of great importance for the nutrition of hundreds of millions of people worldwide. This paper describes the genome sequence of Lactococcus lactis subsp. cremoris MG1363, the lactococcal strain most intensively studied throughout the world. The 2,529,478-bp genome contains 81 pseudogenes and encodes 2,436 proteins. Of the 530 unique proteins, 47 belong to the COG (clusters of orthologous groups) functional category “carbohydrate metabolism and transport,” by far the largest category of novel proteins in comparison with L. lactis subsp. lactis IL1403. Nearly one-fifth of the 71 insertion elements are concentrated in a specific 56-kb region. This integration hot-spot region carries genes that are typically associated with lactococcal plasmids and a repeat sequence specifically found on plasmids and in the “lateral gene transfer hot spot” in the genome of Streptococcus thermophilus . Although the parent of L. lactis MG1363 was used to demonstrate lysogeny in Lactococcus , L. lactis MG1363 carries four remnant/satellite phages and two apparently complete prophages. The availability of the L. lactis MG1363 genome sequence will reinforce its status as the prototype among lactic acid bacteria through facilitation of further applied and fundamental research.

Recombinant DNA (rDNA) technologies (genetic, protein, and metabolic engineering) allow the production of a wide range of peptides, proteins, and biochemicals from naturally nonproducing cells. These technologies, now approx 25 yr old, have become one of the most important technologies developed in the twentieth century. Pharmaceutical products and industrial enzymes were the first biotech products on the world market made by means of rDNA. Despite important advances in rDNA applications in mammalian cells, yeasts still represent attractive hosts for the production of heterologous proteins. In this review we summarize advantages and limitations of the main and most promising yeast hosts.

The effect of induction of protein production was studied in bioreactor cultures of T. reesei strain Rut-C30 and its transformant expressing endoglucanase I core domain (EGI, Cel7B) fused with a hydrophobic peptide tag. The tag was previously designed for efficient purification of the fusion protein in aqueous two-phase separation. The fungi were first grown on glucose-containing minimal medium after which rich medium with lactose as a carbon source was added to induce cellulase production. Production of extracellular protein and cellulase activity and the transcript levels of the major cellulase genes were analyzed during the cultivations. Induction of the cellulase genes followed a similar temporal pattern in both strains. The first phase of induction took place after addition of lactose as soon as glucose was depleted, and the second phase after lactose was consumed. Western analysis showed that a decreased amount of fusion protein was produced in the culture medium compared with the endogenous EGI, although the strain harbors several copies of the recombinant gene under the strong cbh1 promoter. The fusion protein appeared to accumulate within the cells, indicating impaired secretion of the protein. The mRNA levels of the UPR (unfolded protein response) target genes, bip1 and pdi1, and the level of the active form of hac1 transcript encoding the UPR transcription factor increased concurrently with induction of the cellulase genes in both strains, indicating increased requirement of the folding machinery under these conditions. However, only a minor increase in bip1 and pdi1 transcript level was observed in the transformant compared with the parental strain.

Over the past few years considerable research attention has been assigned to extremophiles as sources of extremozymes due to their applicability in industrial processes, and the development of eco-friendly technologies. The establishment of efficient production strategies for heterologous proteins is an empirical process requiring broad background knowledge on available expression systems together with their major advantages and shortcomings. The studies conducted during the course of this thesis has included four enzymes originating from thermophiles namely, thermostable glycoside hydrolases, xylanase and cellulase from Rhodothermus marinus, cyclomaltodextrinase from Anoxybacillus flavithermus and a phospholipase from alkaliphilic Bacillus halodurans. Batch cultivation of R. marinus in the presence of xylan allowed low production of the native xylanase in sufficient amounts to probe cell-attachment studies by enzymatic and immunological techniques. Higher levels of the target proteins were achieved by intracellular and extracellular heterologous production using an Escherichia coli and Pichia pastoris based expression system respectively. The production of a functional enzyme is intimately related to the host's cellular machinery furthermore, as a prerequisite, the establishment of efficient bioprocess strategies is crucial for attaining optimum enzyme production yields. The results presented include bench-scale production strategies employing high cell density fed-batch cultivations with E. coli as a host. Also efficient extracellular production of thermostable xylanase and alkaliphilic phospholipase production using the methylotrophic yeast P. pastoris as a host is reported.

The optimisation and scale-up of a specific protein production process have to take into account cultivation conditions as well as cell physiology of growth and the influence of foreign protein expression on host cell metabolism. The ability of Zygosaccharomyces bailii to tolerate high sugar concentrations as well as high temperatures and acidic environments renders this "non-conventional" yeast suitable for the development of biotechnological processes like heterologous protein production. This work addresses the production of human interleukin-1β by a recombinant Z. bailii strain. We found that the heterologous protein production causes some modifications of the Z. bailii carbon metabolism, leading to a reduced biomass yield. The other important factor is the dependence of the recombinant IL-1β production/secretion on the growth rate. Among the cultivation strategies studied, the most appropriate in terms of production and productivity was the fed-batch mode. © 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

We have set up methods for intracellular and extracellular proteome analysis in Trichoderma. These include analysis of steady state total protein pattern using 2D gel electrophoresis as well as detection of actively synthesised proteins using metabolic labelling. In addition, phosphoprotein staining and Western analysis with phosphoprotein antibodies have been used to detect differences in the cellular levels of phosphorylated proteins. Proteome analysis has been applied to study the cellular responses activated in Trichoderma reesei upon production of a heterologous protein, tPA (tissue plasminogen activator). Protein samples from chemostat cultures of the tPA-producing transformant and the parental strain Rut-C30 were subjected to 2D gel electrophoresis and protein spots with altered intensity were identified using LC-MS/MS analysis and comparison of the obtained peptide masses and sequence tags with public protein sequence data bases. The analysis revealed up-regulation of a number of proteins involved in protein glycosylation and folding, and members of heat shock protein families. Many of these proteins have not been previously reported from T. reesei. The responses detected in the strain producing the heterologous protein were also compared to those observed in cultures treated with chemical agents to inhibit protein folding and transport, dithiothreitol (DTT) and Brefeldin A, respectively.

International audience; Characterization of host factors affecting heterologous protein secretion in Lactococcus lactis.Lactococcus lactis is the model organism for Lactic Acid Bacteria (LAB) and is a GRAS bacterium. It is therefore a good candidate for the secretion of heterologous proteins of therapeutic interest. In order to investigate new potential uses for LAB, we study host factors involved in the production, the stability and/or the secretion of heterologous proteins in L. lactis. Our strategy uses a random insertional mutagenesis performed on a L. lactis MG1363 derivative strain carrying a uspnuc cassette in its chromosome. The uspnuc cassette encodes a hybrid precursor composed of the signal peptide of the Usp45 secreted protein and the staphylococcal nuclease (Nuc) which is used here as a reporter protein for the secretion process. Nuc activity of bacterial colonies is readily detectable in vivo and this provides an efficient screening for the selection of secretion mutants. We thus selected and analyzed six L. lactis mutants affected in Nuc secretion. Five out of the 6 corresponding genes were identified. Three of them seem to be involved in the production and/or secretion of proteins: 2 genes are homologous to unknown peptidases and one is homologous to a gene located downstream of a transcription regulator. Combination of different strategies should provide new insight of the secretion process in L. lactis and could result in the construction of secretion mutants with enhanced capacities.; Lactococcus lactis, bactérie lactique modèle, présente une parfaite innocuité hygiénique. Elle constitue, de ce fait, un bon candidat pour la sécrétion de protéines d'intérêt thérapeutique. Une partie de nos travaux dans ce domaine concerne la caractérisation des facteurs d'hôte impliqués dans la production, la stabilité et/ou la sécrétion de protéines hétérologues chez L. lactis. La stratégie employée consiste en une mutagenèse par transposition aléatoire sur le chromosome. Cette mutagenèse est menée sur une souche de L. lactis portant une cassette uspnuc intégrée sur le chromosome. La cassette uspnuc code pour un précurseur hybride composé du peptide signal d'Usp45, protéine sécrétée par L. lactis, et de la partie mature de la nucléase de Staphylococcus aureus (Nuc) utilisée ici comme protéine rapporteur de la sécrétion. L'activité de Nuc, facilement détectable in vivo sur les colonies bactériennes, constitue un crible efficace pour la sélection de mutants affectés dans la sécrétion. Six mutants de L. lactis affectés dans la sécrétion de Nuc ont ainsi été sélectionnés et analysés. Cinq des 6 gènes correspondants ont été identifiés. Trois semblent particulièrement impliqués dans la production et la sécrétion de protéines : 2 sont homologues à des gènes de peptidases inconnues et un est homologue à un gène situé dans une région potentiellement régulatrice d'un facteur de transcription. Différentes approches par mutagenèse devraient déboucher sur une meilleure connaissance de la machinerie de sécrétion de L. lactis et la construction de mutants hyperproducteurs ou hypersécréteurs.

Praxis XXI (BD/11306/97)., European Science Foundation (ESF)., Center for International Mobility (CIMO).

Yeasts are an attractive group of lower eukaryotic microorganisms, some of which are used in several industrial processes that include brewing, baking and the production of a variety of biochemical compounds. More recently, yeasts have been developed as host organisms for the production of foreign (heterologous) proteins. Saccharomyces cerevisiae has usually been the yeast of choice, but an increasing number of alternative non-Saccharomyces yeasts has now become accessible for modern molecular genetics techniques. Some of them exhibit certain favourable traits such as high-level secretion or very strong and tightly regulated promoters, offering significant advantages over traditional bakers' yeast. In the present work, the current status of Kluyveromyces lactis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris (the best-known alternative yeast systems) is reviewed. The advantages and limitations of these systems are discussed in relation to S. cerevisiae. Spanish Society for Microbiology