Search

Recent advances in technology have brought machine learning and big data to the forefront of next-generation therapies, and notably cell- and gene-based therapies. For the first time, technology has enabled us to amass large cell banks and datasets, from which one can derive novel insights using artificial intelligence. Furthermore, scientific advances have decreased the cost of genomic sequencing by several orders of magnitude, driven by a concomitant increase in the throughput of DNA sequencing, also by orders of magnitude. These have brought parallel advances, not only in segmentation of patient populations and in understanding of disease pathobiology, but also for the tracking of millions of cells in single experiments. This allows both researchers and clinicians to accumulate data at an unprecedented rate and to process datasets to generate novel insights that enable cell biologists to usher in a new kind of regenerative medicine, driven by deep-knowledge. Remarkably, these developments also produce economic benefits by their potential to drastically reduce the cost of drug development and by enabling more accessible personalised medicine treatment strategies. Overall, in silico technologies are deeply changing the nature of the field of cell biology, with process-driven operations slowly becoming automated. This necessarily changes the role of the human and increases the depth and breadth of the analyses that can be performed on and using cellular populations. As machines increasingly simplify routine tasks, and as humans shift their focus to roles that machines cannot perform including making judgment calls, using imagination or relying on “gut instinct”, these technology-derived evolutions will change the nature and cost – both time-wise and economically - of work in the advanced therapy space. The years ahead will bring a game-changing tide-shift, where next generation cell therapies, enabled by advanced technologies, will solve important unknown unknowns and change the nature of healthcare in still unimaginable ways.

With the approval in recent years of several first-in-class gene- and cell-based therapies by the European or American regulatory agencies, including Kymriah, Yescarta, Luxturna, Glybera and Strimvelis, as well as the approval of products under the Sakigake designation by the Japanese regulatory agency, the field of regenerative medicine has at long last moved from the realm of R&D to the reality of clinical practice in the major pharmaceutical market jurisdictions as we are approaching the end of the second decade of the 21st century. The beginning of this journey in the arena of market access has already proven to be a rocky one, from big pharma U-turns to product withdrawals on commercial or strategic grounds. History teaches us that such beginnings for the development and commercialisation of any game-changing innovation are to be expected, a phenomenon that is perhaps best exemplified in the pharmaceutical industry by the slow emergence and adoption of the technology of monoclonal antibodies in the 1990–2000 decade. The first two defining moments of the biotechnology economy occurred with the creation of Cetus in 1971 and Genentech in 1976 following the discovery of restriction enzymes and ligases, and the subsequent development of genetic engineering techniques. A second transformational milestone was reached in 1997 with the approval of MabThera, further reinforced in 2005 with the global market of this life-saving anticancer antibody drug reaching a critical blockbuster commercial milestone with a global market in excess of $4 billion. One of the critical success factors for disruptive innovation products to be truly successful is to unambiguously provide game-changing benefits that conventional products cannot deliver. The S-curve shaped process at play in the development of regenerative medicine products that deliver disease-modifying or curative benefits is detailed here, and its implications in the future development of major innovation chunks is discussed through a predictive practical analysis of the innovation pattern of CAR-T cells.

Checkpoint blockade and adoptive therapy with engineered human immune effector cells represent two paradigm-changing innovations that have already brought to cancer patients clinical benefits that no other therapeutic modalities have delivered to this date. However, to further increase partial and complete remission rates in solid tumour, cancers require the harnessing of multiple mechanisms of actions within a treatment algorithm to offensively destroy the bulk of malignant tumours on the one hand and defensively destroy treatment-resistant cells or prevent their emergence on the other, while ensuring treatment tolerability. The myriad of relevant mechanisms and of possible treatment regimens make it necessary to leverage big data, achieve enhanced patient population stratification, and harness computational power to generate rational hypotheses about what constitutes optimal drug combinations for further testing in adaptive clinical trials. The concept of hallmarks of cancer provide a useful cognitive chunk framework that tightly links immunology and oncology to design multimodal treatments, and recent evidence of checkpoint inhibitors developed in a first step as a monotherapy and completed in a second one with conventional chemotherapy reveal a first glimpse of the clinical benefits that are to come. Such combinations can particularly further enhance the therapeutic effects of living drugs such as chimeric antigen receptor T (CAR-T) cells and CAR-NK (natural killer) cells by directly acting on the immunosuppressive tumour microenvironment, while engineered nonimmune effector adult cells can be leveraged to home to tumours and their metastases for locally releasing a cytotoxic cargo including both by prior ex vivo cell loading or in situ synthesis of an oncolytic virus or a therapeutic cytokine.

Medicinal signaling cell (MSC)-based products represent emerging treatments in various therapeutic areas including cardiometabolic, inflammation, autoimmunity, orthopedics, wound healing and oncology. Exploring innovation beyond minimally manipulated plastic-adherent ex vivo expanded allogeneic MSCs enables product delineation. Product delineation is on the critical path to maximize clinical benefits and market access. An innovation framework is presented here along various innovation dimensions comprising composition-of-matter by means of positive cell surface markers, formulation varying for example the cell dose or the preservation mode and medium, manufacturing to adapt the secretome of MSCs to the condition of interest, the mode of delivery and corresponding delivery devices, as well as molecular engineering and biomarkers. The rationale of the innovation space thus described applies generally to all cell-based therapies.

The TERMIS-Europe (EU) Industry committee intended to address the two main critical issues in the clinical/commercial translation of Advanced Therapeutic Medicine Products (ATMP): (1) entrepreneurial exploitation of breakthrough ideas and innovations, and (2) regulatory market approval. Since January 2012, more than 12,000 publications related to regenerative medicine and tissue engineering have been accepted for publications, reflecting the intense academic research activity in this field. The TERMIS-EU 2014 Industry Symposium provided a reflection on the management of innovation and technological breakthroughs in biotechnology first proposed to contextualize the key development milestones and constraints of allocation of financial resources, in the development life-cycle of radical innovation projects. This was illustrated with the biofuels story, sharing similarities with regenerative medicine. The transition was then ensured by an overview of the key identified challenges facing the commercialization of cell therapy products as ATMP examples. Real cases and testimonies were then provided by a palette of medical technologies and regenerative medicine companies from their commercial development of cell and gene therapy products. Although the commercial development of ATMP is still at the proof-of-concept stage due to technology risks, changing policies, changing markets, and management changes, the sector is highly dynamic with a number of explored therapeutic approaches, developed by using a large diversity of business models, both proposed by the experience, pitfalls, and successes of regenerative medicine pioneers, and adapted to the constraint resource allocation and environment in radical innovation projects.

We previously reported on the impacts of the overexpression of individual genes of the glycolytic pathway encoding glucokinase (GLK), glyceraldehyde phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), triosephosphate isomerase (TPI), and bisphosphate aldolase (FBA) on D-lactate productivity in Corynebacterium glutamicum under oxygen-deprived conditions. Searching for synergies, in the current study, we simultaneously overexpressed the five glycolytic genes in a stepwise fashion to evaluate the effect of the cumulative overexpression of glycolytic genes on D-lactate production. Interestingly, the final D-lactate concentration markedly differed depending on whether or not the PFK encoding gene was overexpressed when combined with overexpressing other glycolytic genes. The simultaneous overexpression of the GLK, GAPDH, TPI, and FBA encoding genes led to the highest initial D-lactate concentration at 10 h. However, this particular recombinant strain dramatically slowed producing D-lactate when a concentration of 1300 mM was reached, typically after 32 h. In contrast, the strain overexpressing the PFK encoding gene together with the GLK, GAPDH, TPI, and FBA encoding genes showed 12.7 % lower initial D-lactate concentration at 10 h than that observed with the strain overexpressing the genes coding for GLK, GAPDH, TPI, and FBA. However, this recombinant strain continued to produce D-lactate after 32 h, reaching 2169 mM after a mineral salts medium bioprocess incubation period of 80 h. These results suggest that overexpression of the PFK encoding gene is essential for achieving high production of D-lactate. Our findings provide interesting options to explore for using C. glutamicum for cost-efficient production of D-lactate at the industrial scale.

Corynebacterium glutamicum exhibits numerous ideal intrinsic attributes as a factory of primary and secondary metabolites. The versatile capabilities of this organism have long been implemented at the industrial scale to produce an array of amino acids at high yields and conversion rates, thereby enabling the development of an entire industry. The postgenomic era provides a new technological platform not only to further optimize the intrinsic attributes of C. glutamicum whole cells as biocatalysts, but also to dramatically expand the product portfolio that can be manufactured by this organism, from amino acids to commodity chemicals. This review addresses the methods and strain optimization strategies enabled by genomic information and associated techniques. Their implementation has provided important additional incremental improvements to the economics of industry-scale manufacturing in which C. glutamicum and its episomal elements are used as a performing host-vector system.

Corynebacterium glutamicum encodes a mannitol catabolic operon, which comprises three genes: the DeoR-type repressor coding gene mtlR (sucR), an MFS transporter gene (mtlT), and a mannitol 2-dehydrogenase gene (mtlD). The mtlR gene is located upstream of the mtlTD genes in the opposite orientation. In spite of this, wild-type C. glutamicum lacks the ability to utilize mannitol. This wild-type phenotype results from the genetic regulation of the genes coding for mannitol transport and catalytic proteins mediated by the autoregulated MtlR protein since mtlR mutants grow on mannitol as the sole carbon source. MtlR binds to sites near the mtlR (two sites) and mtlTD promoters (one site downstream of the promoter), with the consensus sequence 5'-TCTAACA-3' being required for its binding. The newly discovered operon comprises the three basic functional elements required for mannitol utilization: regulation, transport, and metabolism to fructose, further processed to the common intermediate of glycolysis fructose-6-phosphate. When relieved from MtlR repression, C. glutamicum, which lacks a functional fructokinase, excretes the fructose derived from mannitol and imports it by the fructose-specific PTS. In order to use mannitol from seaweed biomass hydrolysates as a carbon source for the production of useful commodity chemicals and materials, an overexpression system using the tac promoter was developed. For congruence with the operon, we propose to rename sucR as the mtlR gene.

Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar L-arabinose, a product of the degradation of lignocellulosic biomass. The resultant CRA1 recombinant strain expressed the Escherichia coli genes araA, araB, and araD encoding L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase, respectively, under the control of a constitutive promoter. Unlike the wild-type strain, CRA1 was able to grow on mineral salts medium containing L-arabinose as the sole carbon and energy source. The three cloned genes were expressed to the same levels whether cells were cultured in the presence of D-glucose or L-arabinose. Under oxygen deprivation and with L-arabinose as the sole carbon and energy source, strain CRA1 carbon flow was redirected to produce up to 40, 37, and 11%, respectively, of the theoretical yields of succinic, lactic, and acetic acids. Using a sugar mixture containing 5% D-glucose and 1% L-arabinose under oxygen deprivation, CRA1 cells metabolized L-arabinose at a constant rate, resulting in combined organic acids yield based on the amount of sugar mixture consumed after D-glucose depletion (83%) that was comparable to that before D-glucose depletion (89%). Strain CRA1 is, therefore, able to utilize L-arabinose as a substrate for organic acid production even in the presence of D-glucose.

The challenges of implementing biorefineries on a global scale include socioeconomic, financial, and technological constraints. In particular, the development of biorefineries is tightly linked to the continued availability of fermentation raw materials. These constraints can be relaxed by the use of diverse raw materials, while advances that confer higher flexibility would enable biotechnological plant managers to swiftly react to volatile markets. In conventional processes, Saccharomyces cerevisiae grows on a relatively limited range of substrates, and produces only a single product—ethanol. Given the observed maturity of the S. cerevisiae fermentation technology, alternatives to baker's yeast may be needed to tip the economic balance in favour of biotechnological ethanol. These alternative fermentation technologies may allow a greater diversity of substrates to be used to produce an individually tailored mix of ethanol and other chemicals. Copyright © 2007 Society of Chemical Industry

Hematopoietic stem cell transplantation (HSCT) is a life-saving treatment for inherited anemias, immunodeficiencies or hematologic malignancies. A major complication of allo-HSCT associated with high transplant-related mortality rates is graft-versus-host disease (GvHD). Current and future clinical benefits in HSCT enabled by advances in hematopoietic stem cells, mesenchymal stem cells, Tregs and natural killer cells technologies are reviewed here and discussed. Among these evolutions, based on the need for mesenchymal stem cells to be recruited by an inflammatory environment, the development and use of novel GvHD biomarkers could be explored further to deliver the right pharmaceutical to the right patient at the right time. The successful commercialization of cytotherapeutics to efficiently manage GvHD will create a virtuous ‘halo’ effect for regenerative medicine.

Hematopoietic stem cell transplantation is a life-saving treatment for inherited anemias, immunodeficiencies or hematologic malignancies. When using partially HLA-matched allografts, a major complication is graft-versus-host disease (GvHD). The ideal attributes of a transformational new GvHD treatment include improved survival, decreased corticosteroids, decreased antifungals, improved quality of life through decreased infections, reduced number of hospital stay days, reduced risks of developing chronic GvHD impact on lower GI tract and liver, prophylactic benefits and decreased GvHD relapses, but, in the case of cancer, without negatively impacting beneficial graft-versus-tumor effects. The current practice of hematopoietic stem cell transplantation, its incidence and its unmet medical needs are reviewed here and discussed.

Is the introduction of renewable biofuels a simple problem of technology development and diffusion or does it require an industrial revolution?

A native composite transposon was isolated from Corynebacterium glutamicum ATCC 14751. This transposon comprises two functional copies of a corynebacterial IS 31831 -like insertion sequence organized as converging terminal inverted repeats. This novel 20.3-kb element, Tn 14751 , carries 17.4 kb of C. glutamicum chromosomal DNA containing various genes, including genes involved in purine biosynthesis but not genes related to bacterial warfare, such as genes encoding mediators of antibiotic resistance or extracellular toxins. A derivative of this element carrying a kanamycin resistance cassette, minicomposite Tn 14751 , transposed into the genome of C. glutamicum at an efficiency of 1.8 × 10 2 transformants per μg of DNA. Random insertion of the Tn 14751 derivative carrying the kanamycin resistance cassette into the chromosome was verified by Southern hybridization. This work paves the way for realization of the concept of minimum genome factories in the search for metabolic engineering via genome-scale directed evolution through a combination of random and directed approaches.

Lactate and succinate were produced from glucose by Corynebacterium glutamicum under oxygen deprivation conditions without growth. Addition of bicarbonate to the reaction mixture led not only to a 3.6-fold increase in succinate production rate, but also to a 2.3- and 2.5-fold increase, respectively, of the rates of lactate production and glucose consumption, compared to the control. Furthermore, when small amounts of pyruvate were added to the reaction mixture, acid production rates and the glucose consumption rate were multiplied by a factor ranging from 2 to 3. These phenomena were paralleled by an increase in the NAD+/NADH ratio, thus corroborating the view that the efficient regeneration of NAD+ could be triggered by the addition of either bicarbonate or pyruvate. To investigate the global metabolism of corynebacteria under oxygen deprivation conditions, we engineered several strains where the genes coding for key metabolic enzymes had been inactivated by gene disruption and replacement. A lactate dehydrogenase (LDH)-deficient mutant was not able to produce lactate, suggesting this enzyme has no other isozyme. Although a pyruvate carboxylase (pyc) mutant exhibited similar behavior to that of the wild type, phosphoenolpyruvate carboxylase (ppc) mutants were characterized by a dramatic decrease in succinate production, which was concomitant to decreased lactate production and glucose consumption rates. This set of observations corroborates the view that in coryneform bacteria under oxygen deprivation conditions the major anaplerotic reaction is driven by the ppc gene product rather than by the pyc gene product. Moreover, intracellular NADH concentrations in C. glutamicum were observed to correlate to oxygen-deprived metabolic flows.

The central metabolic pathway of Corynebacterium glutamicum was engineered to produce ethanol. A recombinant strain which expressed the Zymomonas mobilis genes coding for pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) was constructed. Both genes placed under the control of the C. glutamicum ldhA promoter were expressed at high levels in C. glutamicum, resulting, under oxygen-deprivation conditions, in a significant yield ofethanol from glucose in a process characterized by the absence of cellular growth. Addition of pyruvate in trace amounts to the reaction mixture induced a 2-fold increase in the ethanol production rate. A similar effect was observed when acetaldehyde was added. Disruption of the lactate dehydrogenase (ldhA) gene led to a 3-fold higher ethanol yield than wild type, with no lactate production. Moreover, inactivation of the phosphoenolpyruvate carboxylase (ppc) and ldhA genes revealed a significant amount of ethanol production and a dramatic decrease in succinate without any lactate production, when pyruvate was added. Since the reaction occurred in the absence of cell growth, the ethanol volumetric productivity increased in proportion to cell density of ethanologenic C. glutamicum in a process under oxygen-deprivation conditions. These observations corroborate the view that intracellular NADH concentrations in C. glutamicum are correlated to oxygen-deprived metabolic flows.

We produced bovine trypsinogen in the yeast Pichia pastoris . Little or no trypsinogen was detected when the gene with its native leader sequence was expressed under the control of the strong aox1 promoter, suggesting that expression of the wild-type bovine trypsinogen was toxic to the cells. We altered the trypsinogen native propeptide sequence by replacing the lysine at position 6 with an aspartic acid, thus destroying the site in the propeptide cleaved by enterokinase and by trypsin. This mutant accumulated up to 10 mg of trypsinogen per liter in shake flask cultures and about 40 mg/liter in 6-liter fermentors. Trypsinogen could be activated in vitro with a dipeptidyl-aminopeptidase, which selectively removed the modified trypsinogen propeptide; the resulting trypsin was fully active and showed evidence of glycosylation. Thus, we have developed a novel protein production scheme that can be used for the expression of proteins, such as proteases, that are deleterious to the producing organism. This system relies on the expression of a zymogen that cannot be activated in vivo coupled with its in vitro purification and activation.

A 3.4-kb cryptic plasmid was obtained from a new isolate of Rhodobacter blasticus . This plasmid, designated pMG160, was mobilizable by the conjugative strain Escherichia coli S17.1 into Rhodobacter sphaeroides, Rhodobacter capsulatus , and Rhodopseudomonas palustris . It replicated in the latter strains but not in Rhodospirillum rubrum, Rhodocyclus gelatinosus , or Bradyrhizobium species. Plasmid pMG160 was stably maintained in R. sphaeroides for more than 100 generations in the absence of selection but showed segregational instability in R. palustris . Instability in R. palustris correlated with a decrease in plasmid copy number compared to the copy number in R. sphaeroides . The complete nucleotide sequence of plasmid pMG160 contained three open reading frames (ORFs). The deduced amino acid sequences encoded by ORF1 and ORF2 showed high degrees of homology to the MobS and MobL proteins that are involved in plasmid mobilization of certain plasmids. Based on homology with the Rep protein of several other plasmids, ORF3 encodes a putative rep gene initiator of plasmid replication. The functions of these sequences were demonstrated by deletion mapping, frameshift analysis, and analysis of point mutations. Two 6.1-kb pMG160-based E. coli-R. sphaeroides shuttle cloning vectors were constructed and designated pMG170 and pMG171. These two novel shuttle vectors were segregationally stable in R. sphaeroides growing under nonselective conditions.

The slow adoption of cytotherapeutics remains a vexing hurdle given clinical progress achieved to date with a variety of stem cell lineages. Big and midsize pharmaceutical companies as an asset class still delay large-scale investments in this arena until technological and market risks will have been further reduced. Nonetheless, a handful of stem cell strategic alliance and licensing transactions have already been implemented, indicating that progress is actively monitored, although most of these involve midsize firms. The greatest difficulty is, perhaps, that the regenerative medicine industry is currently only approaching the point of inflexion of the technology development S-curve, as many more clinical trials read out. A path to accelerating technology adoption is to focus on innovation outliers among healthcare actors. These can be identified by analyzing systemic factors (e.g., national science policies and industry fragmentation) and intrinsic factors (corporate culture, e.g., nimble decision-making structures; corporate finance, e.g., opportunity costs and ownership structure; and operations, e.g., portfolio management strategies, threats on existing businesses and patent expirations). Another path is to accelerate the full clinical translation and commercialization of an allogeneic cytotherapeutic product in any indication to demonstrate the disease-modifying potential of the new products for treatment and prophylaxis, ideally for a large unmet medical need such as dry age-related macular degeneration, or for an orphan disease such as biologics-refractory acute graft-versus-host disease. In times of decreased industry average research productivities, regenerative medicine products provide important prospects for creating new franchises with a market potential that could very well mirror that achieved with the technology of monoclonal antibodies.

The implementation of the biorefinery vision requires that several major goals be met: (1) the supply of a subset of existing fuels and chemical building blocks, (2) the supply of novel chemical building blocks to create novel materials with novel attributes, (3) the management of some risks associated with the breakdown of the petrochemical supply chain, (4) the creation of jobs including positions in rural areas that offer suitable logistics, (5) the increased management of global-warming risks, and (6) the valorization of waste including agricultural and certain types of urban or industrial waste. The roadmap for achieving a sustainable chemical industry is determined by the management of several risk factors, including (1) technological risk, (2) feedstock price volatility and supply risk, (3) market risk, (4) financing risk, and (5) policy risk. Integrated processing should manufacture an array of commodity chemicals and high-value-added chemicals from various feedstocks comprising lignocellulosics, and it should make use of the carbohydrate fractions of the hydrolyzates derived from these raw materials. Integrated processing can then lead to efficient risk management, moving governments, companies, and individuals closer to achieving the broad objectives and far-reaching impacts of this new industrial revolution related to sustainable chemical value chains.

In conventional fermentation processes used in industrial microbiology, product formation and biomass formation often occur in parallel. The yield and cost of goods might be optimized by separating these two phenomena into two distinct phases, however. This so-called growth-arrested bioprocess can be operated particularly efficiently using cells of Corynebacterium glutamicum as biocatalysts for the production of biofuels and other commodity chemicals. Compared to traditional fermentation processes, growth-arrested bioprocesses exhibit properties that make them particularly suited to a variety of applications in lignocellulosic biorefineries. In this chapter, we describe the fundamental attributes of growth-arrested bioprocesses and review recent advances in the genetic engineering of high-performing strains tailor-designed for the production of biofuels, such as cellulosic ethanol, and commodity chemicals.

Biorefineries outlines the processes and steps to successfully scale up production of two types of biofuels, butanol and ethanol, from cellulosic residues for commercial purposes. It covers practical topics, including biomass availability, pretreatment, fermentation, and water recycling, as well as policy and economic factors. This reflects the unique expertise of the editor team, whose backgrounds range from wood and herbaceous feedstocks to process economics and industrial expertise. The strategies presented in this book help readers to design integrated and efficient processes to reduce the cost of production and achieve an economically viable end product * Outlines the economic benefits of designing a single operational process.* Includes all currently available processes on pretreatment, fermentation and recovery* Covers all pretreatment, fermentation, and product recovery options* Focuses on biofuels but acts as a stepping stone to develop cost-efficient processes for an array of commodity chemicals

We have detected cyclic AMP in the culture medium of amino acid-producing coryneform bacteria. Extracellular concentrations of cyclic AMP vary depending on the nutritional medium, the growth phase and the carbon source. It is also shown that the activity of Brevibacterium flavum adenylate cyclase in intact cells is stimulated in the presence of glucose. Furthermore, addition of cyclic AMP to B. flavum cultures decreased amino acid production similarly to the effect produced by the addition of inorganic phosphate.

We have detected cyclic AMP in the culture medium of amino acid-producing coryneform bacteria. Extracellular concentrations of cyclic AMP vary depending on the nutritional medium, the growth phase and the carbon source. It is also shown that the activity of Brevibacterium flavum adenylate cyclase in intact cells is stimulated in the presence of glucose. Furthermore, addition of cyclic AMP to B. flavum cultures decreased amino acid production similarly to the effect produced by the addition of inorganic phosphate.

The corynebacterial insertion sequence IS31831 was demonstrated to promote in vivo formation of an excised transposon fragment (ETF). The results presented in this report support the view that ETF formation is a general feature of conservative transposition.

Corynebacterium glutamicum exhibits numerous ideal intrinsic attributes as a protein factory, including particularly the secretion of a limited number and quantities of endogenous proteins, a very low level of extracellular protease activity, and the presence of two different native protein secretion mechanisms that have been demonstrated to drive the excretion of homologous and heterologous proteins (the general secretory pathway and the twin-arginine pathway). Moreover, it is capable of glycosylation, a property that opens the possibility to manufacture humanized proteins in addition to industrial enzymes. What is more, efficient signal peptides and prodomain regions have already been identified in this bacterium together with zymogen activation protocols. Similarly, surface expression was demonstrated by successfully decorating the surface of C. glutamicum with α-amylase. However, the technology of corynebacterial host vector systems for protein production is still emerging since only a limited number of proteins have been produced to this date with this organism. The construction in optimized strains of optimized secretion vectors that are stable and in high copy numbers and that combine a strong and controllable promoter with efficient translation stabilization region, secretion and maturation signals remains to be achieved. Likewise, the fundamental biology underlying protein secretion in Corynebacteria, and the definition of the capabilities of this novel protein secretion system and its limitations need to be defined further in order to bring to the biotechnological practitioner an additional technological option for industrial enzymes and pharmaceutical biologics manufacturing. Results attained to date, however, demonstrate the strong potential of Corynebacteria for protein manufacturing.

Corynebacterium glutamicum exhibits numerous ideal intrinsic attributes as a microbial factory to produce not only amino acids but also chemicals. The large range of products that can now be biomanufactured mediates a transformational change in the deployment of this microorganism, from umami applications, referring to food enhancers, to chemurgy applications, referring to chemical commodity products. The deep fundamental knowledge of the corynebacterial physiology, the experience curve gained by manufacturing for decades at the industrial scale numerous amino acids, and postgenomic tools to model processes or design synthetic pathways in combinatorial approaches constitute a foundational basis to design efficient and versatile corynebacterial biorefineries. Moreover, the detailed knowledge in amino acid biosynthetic routes can be leveraged to harness the chemical space around these molecules, as exemplified by producing isobutanol from an engineered valine pathway; biosynthesis and chemical synthesis can also be coupled to produce sustainable intermediates. A wide range of applications can nowadays be addressed using C. glutamicum owing to its intrinsic safety and process performance, including also industrial enzymes, therapeutic proteins, antibody fragments, and secondary metabolites comprising small molecule natural products. The economics of the corynebacterial biorefinery could further be enhanced by valorizing its effluents in innovative ways, such as the use of the spent biomass in bioremediation or mining. A critical success factor is to compute an economically optimized process/product mix to create an interconnected product line for serving various markets offering attractive profit margins, covering not only the food and chemical industry but also the cosmetics and pharmaceutical ones.

Desulfitobacterium hafniense Y51 is a dechlorinating bacterium that encodes an unusually large set of O-demethylase paralogs and specialized respiratory systems including specialized electron donors and acceptors. To use this organism in bioremediation of tetrachloroethene (PCE) or trichloroethene (TCE) pollution, expression patterns of its 5,060 genes were determined under different conditions using 60-mer probes in DNA microarrays. PCE, TCE, fumarate, nitrate, and dimethyl sulfoxide (DMSO) respiration all sustain the growth of strain Y51. Global transcriptome analyses were thus performed using various electron donor and acceptor couples (respectively, pyruvate and either fumarate, TCE, nitrate, or DMSO, and vanillate/fumarate). When TCE is used as terminal electron acceptor, resulting in its detoxification, a series of electron carriers comprising a cytochrome bd-type quinol oxidase (DSY4055-4056), a ferredoxin (DSY1451), and four Fe–S proteins (DSY1626, DSY1629, DSY0733, DSY3309) are upregulated, suggesting that the products of these genes are involved in PCE oxidoreduction. Interestingly, the PCE dehalogenase cluster (pceABCT) is constitutively expressed in the media tested, with pceT being upregulated and pceC downregulated in pyruvate/TCE-containing medium. In addition, another dehalogenation enzyme (DSY1155 coding for a putative chlorophenol reductive dehalogenase), is induced 225-fold in that medium, despite not being involved in PCE respiration. Remarkably since the reducing equivalents formed during pyruvate conversion to acetyl-CoA are channeled to electron acceptors including halogenated compounds, pyruvate induces expression of a pyruvate:ferredoxin oxidoreductase. This study paves the way to understanding the physiology of D. hafniense, optimizing this microbe as a bioremediation agent, and designing bioarray sensors to monitor the presence of dechlorinating organisms in the environment.

Three coryneform bacteria, Brevibacterium flavum, Brevibacterium lactofermentum and Corynebacterium glutamicum have been shown to be able to convert 7-keto-8-aminopelargonic acid to biotin through a biotin synthetic pathway identical to that from Escherichia coli (Hatakeyama et al., DNA Sequence, in press, 1993). We report in this paper the cloning and sequencing of the biotin biosynthetic genes encoding the 7,8-diaminopelargonic acid aminotransferase (bioA) and the dethiobiotin synthetase (bioD) of B. flavum MJ233, by complementation of E. coli bioA and bioD mutants. Both bioA and bioD genes from B. flavum were located on a 4.0-kb SalI DNA fragment. Nucleotide sequence analysis of this fragment revealed that these genes consist of a 1272 bp and a 675 bp open reading frame, respectively. The deduced amino acid sequence of the 7,8-diaminopelargonic acid aminotransferase (BioA) is 51.3% and 31.9% identical to that of the E. coli and Bacillus spaericus bioA gene products, respectively. The deduced amino acid sequence of the dethiobiotin synthetase (BioD) is 25.9% and 32.7% identical to that of the E. coli and B. sphaericus bioD gene products, respectively. In addition, the genomic organization of the bioA, bioB and bioD genes in B. flavum has been shown to be different from that in E. coli and B. sphaericus.

The biotin biosynthetic pathway of three coryneform bacteria, Brevibacterium flavum, Brevibacterium lactofermentum, and Corynebacterium glutamicum were analysed by cross-feeding experiments using several Escherichia coli biotin-requiring mutants. The three strains of coryneform bacteria tested were able to convert 7-keto-8-aminopelargonic acid to biotin, through a biotin synthetic pathway identical to that from E. coli. The biotin biosynthetic gene, bioB, of B. flavum was cloned by phenotypic complementation of E. coli bioB mutants. The bioB gene was located on a 1.7 kb HindIII-SacI DNA fragment. Nucleotide sequence analysis of this fragment revealed that the bioB gene of B. flavum consists of a 1005 bp open reading frame. Its deduced amino acid sequence is 35.7% and 31.5% identical to that of the E. coli and Bacillus sphaericus bioB gene products, respectively. B. flavum mutants obtained by in vivo disruption of the bioB gene lost their ability to grow on minimal medium containing dethiobiotin, indicating that the bioB gene product is necessary for the conversion of dethiobiotin to biotin.