Professor Arnold L. (Arny) Demain's historical position in the rise of industrial microbiology and biotechnology.

This perspective text focuses on the pivotal role and historical position that the late Prof. Arnold L. (Arny) Demain has taken since the 1950s in the rise and impact of the field of industrial microbiology and biotechnology. His drive toward academic research with industrial potential-first at Merck & Co. and later at MIT-, his feeling for establishing cordial personal contacts with his students and postdocs (Arny's Army) and his ability for worldwide networking are outlined here, intertwined with the author's personal experiences and impressions. His scientific output is legendary as to research papers, comprehensive reviews, books, and lectures at conferences worldwide. Some of his research experiences in industry and academia are mentioned in a historical context as well as his relentless efforts to advocate the importance and impact of industrial microbiology and biotechnology as an essential green technology for our planet Earth.

Microbial biotechnology for the synthesis of (pro)vitamins, biopigments and antioxidants: challenges and opportunities.

Vitamins and related compounds, such as provitamins, biopigments and antioxidants, belong to those few chemicals that appeal in a positive way to most people. These terms sound synonymous to vitality, good health and mental strenght, even to the layman. Everyone of us needs his/her daily intake of (pro)vitamins and antioxidants, normally provided by a balanced and varied diet. However, current food habits or preferences, food availabilities, as well as food processing, preservation or cooking methodologies and technologies, do not always assure a sufficient balanced natural daily (pro)vitamin supply to a healthy individual, and even more so for a stressed or sick human being. Today, modern society is seldom confronted with the notorious avitaminoses of the past, well known to the Western World, but they do still occur frequently in overpopulated, war-ridden, poverty- or famine-struck regions on our globe, as well as for surprisingly large population groups in developed countries.

Dynamic metabolic flux analysis demonstrated on cultures where the limiting substrate is changed from carbon to nitrogen and vice versa.

The main requirement for metabolic flux analysis (MFA) is that the cells are in a pseudo-steady state, that there is no accumulation or depletion of intracellular metabolites. In the past, the applications of MFA were limited to the analysis of continuous cultures. This contribution introduces the concept of dynamic MFA and extends MFA so that it is applicable to transient cultures. Time series of concentration measurements are transformed into flux values. This transformation involves differentiation, which typically increases the noisiness of the data. Therefore, a noise-reducing step is needed. In this work, polynomial smoothing was used. As a test case, dynamic MFA is applied on Escherichia coli cultivations shifting from carbon limitation to nitrogen limitation and vice versa. After switching the limiting substrate from N to C, a lag phase was observed accompanied with an increase in maintenance energy requirement. This lag phase did not occur in the C- to N-limitation case.

Transient metabolic modeling of Escherichia coli MG1655 and MG1655 DeltaackA-pta, DeltapoxB Deltapppc ppc-p37 for recombinant beta-galactosidase production.

Escherichia coli is one of the most widely used hosts for the production of recombinant proteins, among other reasons because its genetics are far better characterized than those of any other microorganism. To improve the understanding of recombinant protein synthesis in E. coli, the production of a model recombinant protein, beta-galactosidase, was studied in response to the constitutive overexpression of the anaplerotic reaction afforded by PEP carboxylase. To this end, an IPTG wash-in experiment was performed starting from a well-defined steady-state condition for both the wild-type E. coli and a mutant with a defective acetate pathway and a constitutively overexpressed ppc. In order to compare the dynamics of the fluxes over time during the wash-in experiment, a method referred to as transient metabolic flux analysis, which is based on steady-state metabolic flux analysis, was used. This allowed us to track the intracellular changes/fluxes in both strains. It was observed that the flux towards fermentation products was 3.6 times lower in the ppc overexpression mutant compared to the wild-type E. coli. In the former on the other hand, the PPC flux is in general higher. In addition, the flux towards beta-galactosidase was higher (12.4 times), resulting in five times more protein activity. These results indicate that by constitutively overexpressing the anaplerotic ppc gene in E. coli, the TCA cycle intermediates are increasingly replenished. The additional supply of these protein precursors has a positive result on recombinant protein production.

Promoter knock-in: a novel rational method for the fine tuning of genes.

BACKGROUND: Metabolic engineering aims at channeling the metabolic fluxes towards a desired compound. An important strategy to achieve this is the modification of the expression level of specific genes. Several methods for the modification or the replacement of promoters have been proposed, but most of them involve time-consuming screening steps. We describe here a novel optimized method for the insertion of constitutive promoters (referred to as "promoter knock-in") whose strength can be compared with the native promoter by applying a promoter strength predictive (PSP) model. RESULTS: Our method was successfully applied to fine tune the ppc gene of Escherichia coli. While developing the promoter knock-in methodology, we showed the importance of conserving the natural leader region containing the ribosome binding site (RBS) of the gene of interest and of eliminating upstream regulatory elements (transcription factor binding sites). The gene expression was down regulated instead of up regulated when the natural RBS was not conserved and when the upstream regulatory elements were eliminated. Next, three different promoter knock-ins were created for the ppc gene selecting three different artificial promoters. The measured constitutive expression of the ppc gene in these knock-ins reflected the relative strength of the different promoters as predicted by the PSP model. The applicability of our PSP model and promoter knock-in methodology was further demonstrated by showing that the constitutivity and the relative levels of expression were independent of the genetic background (comparing wild-type and mutant E. coli strains). No differences were observed during scaling up from shake flask to bioreactor-scale, confirming that the obtained expression was independent of environmental conditions. CONCLUSION: We are proposing a novel methodology for obtaining appropriate levels of expression of genes of interest, based on the prediction of the relative strength of selected synthetic promoters combined with an optimized promoter knock-in strategy. The obtained expression levels are independent of the genetic background and scale conditions. The method constitutes therefore a valuable addition to the genetic toolbox for the metabolic engineering of E. coli.

Creating lactose phosphorylase enzymes by directed evolution of cellobiose phosphorylase.

Disaccharide phosphorylases are interesting enzymes for the production of sugar phosphates from cheap starting materials and for the synthesis of novel glycosides. Cellobiose phosphorylase (CP) from Cellulomonas uda was subjected to directed evolution in order to create enzyme variants with significantly increased lactose phosphorylase (LP) activity, useful for the production of alpha-D-galactose 1-phosphate. In a first round, random mutagenesis was performed on part of the CP gene and the resultant library was selected on minimal lactose medium. One clone containing six amino acid mutations was found with increased LP activity compared with the wild-type CP enzyme. The negative and neutral mutations were eliminated by site-directed mutagenesis and the resultant enzyme variant containing two amino acid substitutions (T508A/N667T) showed more LP activity than the parent mutant. Saturation mutagenesis of the beneficial sites and screening for improved mutants allowed us to identify the T508I/N667A mutant which has 7.5 times higher specific activity on lactose than the wild-type. The kinetic parameters of the mutants were determined and showed that the increased LP activity was caused by a higher k(cat) value. This is the first report of an engineered CP with modified substrate specificity.

Knocking out the MFE-2 gene of Candida bombicola leads to improved medium-chain sophorolipid production.

The nonpathogenic yeast Candida bombicola synthesizes sophorolipids. These biosurfactants are composed of the disaccharide sophorose linked to a long-chain hydroxy fatty acid and have potential applications in the food, pharmaceutical, cosmetic and cleaning industries. In order to expand the range of application, a shift of the fatty acid moiety towards medium-chain lengths would be recommendable. However, the synthesis of medium-chain sophorolipids by C. bombicola is a challenging objective. First of all, these sophorolipids can only be obtained by fermentations on unconventional carbon sources, which often have a toxic effect on the cells. Furthermore, medium-chain substrates are partially metabolized in the beta-oxidation pathway. In order to redirect unconventional substrates towards sophorolipid synthesis, the beta-oxidation pathway was blocked on the genome level by knocking out the multifunctional enzyme type 2 (MFE-2) gene. The total gene sequence of the C. bombicola MFE-2 (6033 bp) was cloned (GenBank accession number EU371724), and the obtained nucleotide sequence was used to construct a knock-out cassette. Several knock-out mutants with the correct geno- and phenotype were evaluated in a fermentation on 1-dodecanol. All mutants showed a 1.7-2.9 times higher production of sophorolipids, indicating that in those strains the substrate is redirected towards the sophorolipid synthesis.

Production of glucolipids and specialty fatty acids from sophorolipids by Penicillium decumbens naringinase: Optimization and kinetics.

In view of global environmental concerns and the awakening to the exhaustibility of our natural resources, an increasing importance of biologically derived surfactants can be expected in the near future. Enzymatic modification of these biosurfactants allows to improve their characteristics and so extend their application field. In view of this, glucolipids are interesting substrates e.g., for the synthesis of new glycolipids with increased biological activity. Here, we describe the optimization of glucolipid production from Candida bombicola sophorolipids by Penicillium decumbens naringinase and show that the enzyme might be useful for production of specialty fatty acids as well. Optimum conditions for production of glucolipids were found to be pH 7.0 and 50 degrees C with a yield of 80% (w/w) glucolipids after 3 h of incubation. The K(m) for sophorolipids was 1.67 mM, while V(max) was 0.035 mM sophorolipids/min. At pH 3.0, glucolipids were immediately further hydrolyzed and completely converted to fatty acids after 24 h of incubation, offering a biological route to the synthesis of unique specialty fatty acids. The K(m) for glucolipids was 11 mM while V(max) was 0.21 mM glucolipids/min. Glucose inhibited the enzyme in a competitive way with K(I) around 10-15 mM glucose. Surfactant properties of the produced glucolipids were comparable to those of the acidic sophorolipids.

Importance of the cytochrome P450 monooxygenase CYP52 family for the sophorolipid-producing yeast Candida bombicola.

Three cytochrome P450 monooxygenases belonging to the CYP52 family were isolated from the genome of the sophorolipid-producing yeast Candida bombicola using degenerate PCR and genomic walking. One gene displayed high identity with the CYP52E members and was classified into this group (CYP52E3), whereas the other genes belonged to new groups: CYP52M and CYP52N. CYP52E3 and CYP52N1 turned out to be of no relevance for sophorolipid production, but show clear upregulation when the yeast cells are grown on alkanes as the sole carbon source. On the other hand, CYP52M1 is clearly upregulated during sophorolipid synthesis and very likely takes part in sophorolipid formation.

Cloning and characterisation of the glyceraldehyde 3-phosphate dehydrogenase gene of Candida bombicola and use of its promoter.

The glyceraldehyde-3-phosphate dehydrogenase gene (GPD) of the sophorolipid producing yeast Candida bombicola was isolated using degenerated PCR and genome walking. The obtained 3,740 bp contain the 1,008 bases of the coding sequence and 1,613 and 783 bp of the upstream and downstream regions, respectively. The corresponding protein shows high homology to the other known GPD genes and is 74% identical to the gyceraldehyde-3-phosphate dehydrogenase of Yarrowia lipolytica. The particular interest in the C. bombicola GPD gene sequence originates from the potential use of its promoter for high and constitutive expression of homologous and heterologous genes. Southern blot analysis did not give any indication for the presence of multiple GPD genes and it can therefore be expected that the promoter can be used for efficient and high expression. This hypothesis was further confirmed by the biased codon usage in the GPD gene. GDP promoter fragments of different lengths were used to construct hygromycin resistance cassettes. The constructs were used for the transformation of C. bombicola and all of them, even the ones with only 190 bp of the GPD promoter, were able to render the cells resistant to hygromycin. The efficacy of a short GPD promoter can be a convenient characteristic for the construction of compact expression cassettes or vectors for C. bombicola. The GenBank accession number of the sequence described in this article is EU315245.

Development of a transformation and selection system for the glycolipid-producing yeast Candida bombicola.

Sophorolipids are surface-active compounds synthesized by the non-pathogenic yeast Candida bombicola. Over recent decades much effort has been spent to optimize culture conditions in order to improve the yield and production process. As far as we know, however, hardly any attention has been given to the genetics of the producing yeast strain itself and there are no published results available on the genetic engineering of C. bombicola. Nevertheless, this can be a useful tool for the study of the sophorolipid synthesis pathway and open up perspectives for improved production. A first step is the development of a suitable transformation and selection method. This article describes the creation and selection of an uracil auxotrophic C. bombicola mutant, which can be transformed back to prototrophy with the species' own orotidine 5'-phosphate decarboxylase or URA3 gene. Successful transformation was confirmed by a PCR-based method discriminating between the wild-type and mutated URA3 gene.

Cloning and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene and the use of its promoter for expression in Myrothecium gramineum, a novel expression host.

At our laboratory, research has focused on the development of Myrothecium gramineum as a novel expression host. The glyceraldehyde-3-phosphate dehydrogenase (gpd)-promoter of M. gramineum was isolated and characterized (Genbank accession number EF486690). In order to prove its functionality and to explore the potential of M. gramineum as a novel fungal expression host, use of this gpd-promoter for the expression of a fungal alpha-amylase was investigated. Myrothecium gramineum was transformed with pGPDlpAmyAO, containing the gpd-promoter followed by the amy3 encoding sequence of Aspergillus oryzae. Study of the amylase production indicated that the promoter can be successfully used for the expression of heterologous proteins in M. gramineum. To the best of our knowledge, this is the first time a homologous expression system has been described for M. gramineum.

Comparison of protein quantification and extraction methods suitable for E. coli cultures.

Many different extraction and analysis methods exist to determine the protein fraction of microbial cells. For metabolic engineering purposes it is important to have precise and accurate measurements. Therefore six different protein extraction protocols and seven protein quantification methods were tested and compared. Comparison was based on the reliability of the methods and boxplots of the normalized residuals. Some extraction techniques (SDS/chloroform and toluene) should never be used: the measurements are neither precise nor accurate. Bugbuster extraction combined with UV280 quantification gives the best results, followed by the combinations Sonication-UV280 and EasyLyse-UV280. However, if one does not want to use the quantification method UV280, one can opt to use Bugbuster, EasyLyse or sonication extraction combined with any quantification method with exception of the EasyLyse-BCA_P and Sonication-BCA_P combinations.

The genus Gluconobacter oxydans: comprehensive overview of biochemistry and biotechnological applications.

The genus Gluconobacter comprises some of the most frequently used microorganisms when it comes to biotechnological applications. Not only has it been involved in "historical" production processes, such as vinegar production, but in the last decades many bioconversion routes for special and rare sugars involving Gluconobacter have been developed. Among the most recent are the biotransformations involved in the production of L-ribose and miglitol, both very promising pharmaceutical lead molecules. Most of these processes make use of Gluconobacter's membrane-bound polyol dehydrogenases. However, recently other enzymes have also caught the eye of industrial biotechnology. Among them are dextran dextrinase, capable of transglucosylating substrate molecules, and intracellular NAD-dependent polyol dehydrogenases, of interest for co-enzyme regeneration. As such, Gluconobacter is an important industrial microbial strain, but it also finds use in other fields of biotechnology, such as biosensor-technology. This review aims to give an overview of the myriad of applications for Gluconobacter, with a special focus on some recent developments.

Comparison of different strategies to reduce acetate formation in Escherichia coli.

E. coli cells produce acetate as an extracellular coproduct of aerobic cultures. Acetate is undesirable because it retards growth and inhibits protein formation. Most process designs or genetic modifications to minimize acetate formation aim at balancing growth rate and oxygen consumption. In this research, three genetic approaches to reduce acetate formation were investigated: (1) direct reduction of the carbon flow to acetate (ackA-pta, poxB knock-out); (2) anticipation on the underlying metabolic and regulatory mechanisms that lead to acetate (constitutive ppc expression mutant); and (3) both (1) and (2). Initially, these mutants were compared to the wild-type E. coli via batch cultures under aerobic conditions. Subsequently, these mutants were further characterized using metabolic flux analysis on continuous cultures. It is concluded that a combination of directly reducing the carbon flow to acetate and anticipating on the underlying metabolic and regulatory mechanism that lead to acetate, is the most promising approach to overcome acetate formation and improve recombinant protein production. These genetic modifications have no significant influence on the metabolism when growing the micro-organisms under steady state at relatively low dilution rates (less than 0.4 h(-1)).