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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.

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.

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.

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.

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.

Corynebacterium glutamicum ATCC 31831 grew on l -arabinose as the sole carbon source at a specific growth rate that was twice that on d -glucose. The gene cluster responsible for l -arabinose utilization comprised a six-cistron transcriptional unit with a total length of 7.8 kb. Three l -arabinose-catabolizing genes, araA (encoding l -arabinose isomerase), araB ( l -ribulokinase), and araD ( l -ribulose-5-phosphate 4-epimerase), comprised the araBDA operon, upstream of which three other genes, araR (LacI-type transcriptional regulator), araE ( l -arabinose transporter), and galM (putative aldose 1-epimerase), were present in the opposite direction. Inactivation of the araA , araB , or araD gene eliminated growth on l -arabinose, and each of the gene products was functionally homologous to its Escherichia coli counterpart. Moreover, compared to the wild-type strain, an araE disruptant exhibited a >80% decrease in the growth rate at a lower concentration of l -arabinose (3.6 g liter −1 ) but not at a higher concentration of l -arabinose (40 g liter −1 ). The expression of the araBDA operon and the araE gene was l -arabinose inducible and negatively regulated by the transcriptional regulator AraR. Disruption of araR eliminated the repression in the absence of l -arabinose. Expression of the regulon was not repressed by d -glucose, and simultaneous utilization of l -arabinose and d -glucose was observed in aerobically growing wild-type and araR deletion mutant cells. The regulatory mechanism of the l -arabinose regulon is, therefore, distinct from the carbon catabolite repression mechanism in other bacteria.

The current paradigm to produce biotechnological ethanol is to use the yeast Saccharomyces cerevisiae to ferment sugars derived from starch or sugar crops such as maize, sugar cane or sugar beet. Despite its current success, the global impact of this manufacturing model is restricted on the one hand by limits on the availability of these primary raw materials, and on the other hand by the maturity of baker’s yeast fermentation technologies. Revisiting the technical, economic, and value chain aspects of the biotechnological ethanol industry points to the need for radical innovation to complement the current manufacturing model. Implementation of lignocellulosic materials is clearly a key enabler to the billion-ton biofuel vision. However, realization of the full market potential of biofuels will be facilitated by the availability of an array of innovative technological options, as the flexibility generated by these alternative processes will not only enable the exploitation of heretofore untapped local market opportunities, but also it will confer to large biorefinery structures numerous opportunities for increased process integration as well as optimum reactivity to logistic and manufacturing challenges. In turn, all these factors will interplay in synergy to contribute in shifting the economic balance in favor of the global implementation of biotechnological ethanol.

A transcriptional profiling of the metabolism of Corynebacterium glutamicum under oxygen deprivation conditions is reported. It was observed that the glucose consumption rate per cell when C. glutamicum cells were incubated under oxygen deprivation conditions was higher than that achieved by cells incubated under aerobic growth conditions. Furthermore, DNA microarray and quantitative RT-PCR analyses revealed that the genes of several key enzymes of the glycolytic and organic acid production pathways, including gapA, pgk, tpi, ppc, ldhA and mdh, were significantly upregulated under oxygen deprivation conditions. The corresponding enzymic activities consistently correlated with the regulation patterns of the genetic expression observed at the transcriptional level. Studies of lacZ fusions with the gapA, ldhA and mdh genes indicated not only that these genes are strongly induced at the onset of the stationary phase under aerobic growth conditions, but also that high expression levels are maintained under oxygen deprivation conditions. These results indicate that the genetic expression of several key metabolic enzymes in C. glutamicum cells incubated under oxygen deprivation conditions is chiefly regulated at the transcriptional level. The physiological consequence of the observed increased transcription under oxygen deprivation conditions is an increased rate of carbon source consumption, which is accompanied by a concomitant increase in organic acid production.

Corynebacterium glutamicum, a gram-positive soil bacterium, has been regarded as an aerobe because its growth by fermentative catabolism or by anaerobic respiration has, to this date, not been demonstrated. In this study, we report on the anaerobic growth of C. glutamicum in the presence of nitrate as a terminal electron acceptor. C. glutamicum strains R and ATCC13032 consumed nitrate and excreted nitrite during growth under anaerobic, but not aerobic, conditions. This was attributed to the presence of a narKGHJI gene cluster with high similarity to the Escherichia coli narK gene and narGHJI operon. The gene encodes a nitrate/nitrite transporter, whereas the operon encodes a respiratory nitrate reductase. Transposonal inactivation of C. glutamicum narG or narH resulted in mutants with impaired anaerobic growth on nitrate because of their inability to convert nitrate to nitrite. Further analysis revealed that in C. glutamicum, narK and narGHJI are cotranscribed as a single narKGHJI operon, the expression of which is activated under anaerobic conditions in the presence of nitrate. C. glutamicum is therefore a facultative anaerobe.

The aerobic microorganism Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar xylose, which is commonly found in agricultural residues and other lignocellulosic biomass. We demonstrated the functionality of the corynebacterial xylB gene encoding xylulokinase and constructed two recombinant C. glutamicum strains capable of utilizing xylose by cloning the Escherichia coli gene xylA encoding xylose isomerase, either alone (strain CRX1) or in combination with the E. coli gene xylB (strain CRX2). These genes were provided on a high-copy-number plasmid and were under the control of the constitutive promoter trc derived from plasmid pTrc99A. Both recombinant strains were able to grow in mineral medium containing xylose as the sole carbon source, but strain CRX2 grew faster on xylose than strain CRX1. We previously reported the use of oxygen deprivation conditions to arrest cell replication in C. glutamicum and divert carbon source utilization towards product production rather than towards vegetative functions (M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A. A. Vertès, and H. Yukawa, J. Mol. Microbiol. Biotechnol. 7 :182-196, 2004). Under these conditions, strain CRX2 efficiently consumed xylose and produced predominantly lactic and succinic acids without growth. Moreover, in mineral medium containing a sugar mixture of 5% glucose and 2.5% xylose, oxygen-deprived strain CRX2 cells simultaneously consumed both sugars, demonstrating the absence of diauxic phenomena relative to the new xylA-xylB construct, albeit glucose-mediated regulation still exerted a measurable influence on xylose consumption kinetics.

The Corynebacterium glutamicum insertion sequence IS31831 was used to construct two artificial transposons: Tn31831 and miniTn31831. The transposition vectors were based on a gram-negative replication origin and do not replicate in coryneform bacteria. Strain Brevibacterium flavum MJ233C was mutagenized by miniTn31831 at an efficiency of 4.3 x 10(4) mutants per microgram DNA. Transposon insertions occurred at different locations on the chromosome and produced a variety of mutants. Auxotrophs could be recovered at a frequency of approximately 0.2%. Transposition of IS31831 derivatives led not only to simple insertion, but also to cointegrate formation (5%). No multiple insertions were observed. Chromosomal loci of B. flavum corresponding to auxotrophic and pigmentation mutants could be rescued in Escherichia coli, demonstrating that these transposable elements are useful genetic tools for studying the biology of coryneform bacteria.

Efficient transformation of Brevibacterium flavum MJ233C and Corynebacterium glutamicum ATCC 31831 (up to 5.0 x 10(7) transformants/microgram DNA) depends on the source of plasmid DNA. The transformation efficiencies of B. flavum MJ233C and C. glutamicum ATCC 31831 increased nearly 10(3)-fold when plasmid DNA was isolated from the recipient strain itself or from a damdcm Escherichia coli mutant, as compared with DNA passed through a modification-proficient E. coli strain. These results suggest the presence of a methyl-specific restriction system in certain strains of coryneform bacteria. In addition, electroporation conditions were optimized.

Taking advantage of highly conserved domains present in the three acetohydroxy acid synthase (AHAS) isozymes from E. coli K-12, we designed degenerate oligonucleotides corresponding to these regions. These synthetic DNA sequences were used as primers in polymerase chain reactions in order to amplify DNA sequences from Brevibacterium flavum MJ233 chromosomal DNA. The polymerase chain reaction product was used as a probe to recover genomic fragments from a lambda library of B. flavum MJ233. A 5.8-kb EcoRI fragment hybridizing to the probe was isolated and amplification of this fragment in a B. flavum strain resulted in increased AHAS-specific activity. Sequence analysis revealed two open reading frames (ilvL and ilvS) highly homologous at the amino acid level to the corresponding domains of the three AHAS isozymes of E. coli K-12. Moreover, disruption of the putative ilvL gene by a kanamycin resistance cassette resulted in L-isoleucine and L-valine auxotrophy. These observations demonstrate that the cloned fragment encodes the AHAS gene of B. flavum MJ233.