Sofie De Maeseneire

Towards the industrialization of new biosurfactants: Biotechnological opportunities for the lactone esterase gene from Starmerella bombicola

Although sophorolipids (SLs) produced by S. bombicola are a real showcase for the industrialization of microbial biosurfactants, some important drawbacks are associated with this efficient biological process, e.g., the simultaneous production of acidic and lactonic SLs. Depending on the application, there is a requirement for the naturally produced mixture to be manipulated to give defined ratios of the components. Recently, the enzyme responsible for the lactonization of SLs was discovered. The discovery of the gene encoding this lactone esterase (sble) enabled the development of promising S. bombicola strains producing either solely lactonic (using a sble overexpression strain described in this paper: oe sble) or solely acidic SLs (using a sble deletion strain, which was recently described, but not characterized yet: Δsble). The new S. bombicola strains were used to investigate the production processes (fermentation and purification) of either lactonic or acidic SLs. The strains maintain the high inherent productivities of the wild‐type or even perform slightly better and thus represent a realistic industrial opportunity. 100% acidic SLs with a mixed acetylation pattern were obtained for the Δsble strain, while the inherent capacity to selectively produce lactonic SLs was significantly increased (+42%) for the oe sble strain (99% lactonic SLs). Moreover, the regulatory effect of citrate on lactone SL formation for the wild‐type was absent in this new strain, which indicates that it is more robust and better suited for the industrial production of lactonic SLs. Basic parameters were determined for the purified SLs, which confirm that the two new strains produce molecules with distinctive properties of which the application potential can now easily be investigated independently. Biotechnol. Bioeng. 2016;113: 550–559.

Candida bombicola as a platform organism for the production of tailor-made biomolecules

The yeast Candida bombicola is capable of producing high amounts (400 g/L) of the biosurfactant sophorolipids. The genetic makeup of this industrially important yeast has recently been uncovered and molecular manipulation techniques have been developed. Hence, all tools for the development of new bioprocesses with C. bombicola are now available. As a proof of concept, the production of two totally different molecules was aimed for: the bioplastic polyhydroxyalkanoate (PHA) and a new‐to‐nature cellobioselipid‐biosurfactant. Integration of the new functionalities at genomic loci necessary for sophorolipid production safeguards the new biomolecules from sophorolipid contamination, while taking advantage of the regulation of the sophorolipid gene cluster. A maximum yield of 2.0% wt/dwt PHA was obtained; furthermore, this is the first time cellobioselipid synthesis by a non‐natural producer is reported. We here provided proof of concept that C. bombicola can be transformed into a platform organism for the production of tailor‐made biomolecules. Biotechnol. Bioeng. 2013; 110:2494–2503.

Cloning, characterization and functionality of the orotidine-5 '-phosphate decarboxylase gene (URA3) of the glycolipid-producing yeast Candida bombicola

Candida bombicola is a yeast species known to synthesize glycolipids. Although these glycolipids find several industrial, cosmetic and pharmaceutical applications, very little is known about the genetics of C. bombicola. A basic tool for genetic study and modification is the availability of an efficient transformation and selection system. In order to develop such a system, the URA3 gene of Candida bombicola was isolated using degenerate PCR and genomic walking. The gene encodes for an enzyme of 262 amino acids and shows high homology with the known orotidine‐5′‐phosphate decarboxylases of several other yeast species. The functionality of the gene was proved by complementation of a URA3‐negative Saccharomyces cerevisiae strain. The GenBank Accession No. of the sequence described in this article is DQ916828. Copyright

Biosurfactant gene clusters in eukaryotes: regulation and biotechnological potential

Biosurfactants (BSs) are a class of secondary metabolites representing a wide variety of structures that can be produced from renewable feedstock by a wide variety of micro-organisms. They have (potential) applications in the medical world, personal care sector, mining processes, food industry, cosmetics, crop protection, pharmaceuticals, bio-remediation, household detergents, paper and pulp industry, textiles, paint industries, etc. Especially glycolipid BSs like sophorolipids (SLs), rhamnolipids (RLs), mannosylerythritol lipids (MELs) and cellobioselipids (CBLs) have been described to provide significant opportunities to (partially) replace chemical surfactants. The major two factors currently limiting the penetration of BSs into the market are firstly the limited structural variety and secondly the rather high production price linked with the productivity. One of the keys to resolve the abovementioned bottlenecks can be found in the genetic engineering of natural producers. This could not only result in more efficient (economical) recombinant producers, but also in a diversification of the spectrum of available BSs as such resolving both limiting factors at once. Unraveling the genetics behind the biosynthesis of these interesting biological compounds is indispensable for the tinkering, fine tuning and rearrangement of these biological pathways with the aim of obtaining higher yields and a more extensive structural variety. Therefore, this review focuses on recent developments in the investigation of the biosynthesis, genetics and regulation of some important members of the family of the eukaryotic glycolipid BSs (MELs, CBLs and SLs). Moreover, recent biotechnological achievements and the industrial potential of engineered strains are discussed.

Citrobacter werkmanii, a new candidate for the production of 1,3-propanediol: strain selection and carbon source optimization

In the past decade 1,3-propanediol (PDO) has been identified as one of the top added value bio-based chemical building blocks in many reports, leading to Klebsiella sp., Clostridium sp., and Escherichia coli based production platforms. However, Citrobacter sp. are also known to naturally produce PDO. In this work a range of Citrobacter sp. has been screened for their PDO production capacity and their natural resistance against high PDO titers, leading to the selection of a new candidate for the production of PDO from glycerol, namely Citrobacter werkmanii DSM17579. In batch fermentation, a production rate of 2.84 g L−1 h−1 and a yield of 0.62 mol mol−1 glycerol were obtained starting from 60 g L−1 and 20 g L−1 of glycerol, respectively. The metabolism of this organism was further studied by perturbing it with 18 carbon sources as co-substrates. These results pointed to the potential use of cheap waste streams such as ligno/hemicellulosic hydrolysates for the production of PDO. Furthermore, the sugar alcohol D-mannitol and D-galactose enhance the production yield significantly (0.83 mol mol−1 and 0.81 mol mol−1, respectively, an enhancement of about 30% compared to glycerol as the sole carbon source). The latter indicates the potential of whey based waste streams for the production of PDO. These are, to date, the highest yields reported for natural producing Enterobacteriaceae using co-substrates for the production of PDO from glycerol.

Development of a selection system for the detection of L-ribose isomerase expressing mutants of Escherichia coli

L-Arabinose isomerase (E.C. 5.3.1.14) catalyzes the reversible isomerization between L-arabinose and L-ribulose and is highly selective towards L-arabinose. By using a directed evolution approach, enzyme variants with altered substrate specificity were created and screened in this research. More specifically, the screening was directed towards the identification of isomerase mutants with L-ribose isomerizing activity. Random mutagenesis was performed on the Escherichia coli L-arabinose isomerase gene (araA) by error-prone polymerase chain reaction to construct a mutant library. To enable screening of this library, a selection host was first constructed in which the mutant genes were transformed. In this selection host, the genes encoding for L-ribulokinase and L-ribulose-5-phosphate-4-epimerase were brought to constitutive expression and the gene encoding for the native L-arabinose isomerase was knocked out. L-Ribulokinase and L-ribulose-5-phosphate-4-epimerase are necessary to ensure the channeling of the formed product, L-ribulose, to the pentose phosphate pathway. Hence, the mutant clones could be screened on a minimal medium with L-ribose as the sole carbon source. Through the screening, two first-generation mutants were isolated, which expressed a small amount of L-ribose isomerase activity.

1,3-propanediol production with Citrobacter werkmanii DSM17579: effect of a dhaD knock-out

Background1,3-propanediol (PDO) is a substantially industrial metabolite used in the polymer industry. Although several natural PDO production hosts exist, e.g. Klebsiella sp., Citrobacter sp. and Clostridium sp., the PDO yield on glycerol is insufficient for an economically viable bio-process. Enhancing this yield via strain improvement can be achieved by disconnecting the production and growth pathways. In the case of PDO formation, this approach results in a microorganism metabolizing glycerol strictly for PDO production, while catabolizing a co-substrate for growth and maintenance. We applied this strategy to improve the PDO production with Citrobacter werkmanii DSM17579.ResultsGenetic tools were developed and used to create Citrobacter werkmanii DSM17579 ∆dhaD in which dhaD, encoding for glycerol dehydrogenase, was deleted. Since this strain was unable to grow on glycerol anaerobically, both pathways were disconnected. The knock-out strain was perturbed with 13 different co-substrates for growth and maintenance. Glucose was the most promising, although a competition between NADH-consuming enzymes and 1,3-propanediol dehydrogenase emerged.ConclusionDue to the deletion of dhaD in Citrobacter werkmanii DSM17579, the PDO production and growth pathway were split. As a consequence, the PDO yield on glycerol was improved 1,5 times, strengthening the idea that Citrobacter werkmanii DSM17579 could become an industrially interesting host for PDO production.

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.

Standardization in synthetic biology: an engineering discipline coming of age

Abstract Background: Leaping DNA read-and-write technologies, and extensive automation and miniaturization are radically transforming the field of biological experimentation by providing the tools that enable the cost-effective high-throughput required to address the enormous complexity of biological systems. However, standardization of the synthetic biology workflow has not kept abreast with dwindling technical and resource constraints, leading, for example, to the collection of multi-level and multi-omics large data sets that end up disconnected or remain under- or even unexploited. Purpose: In this contribution, we critically evaluate the various efforts, and the (limited) success thereof, in order to introduce standards for defining, designing, assembling, characterizing, and sharing synthetic biology parts. The causes for this success or the lack thereof, as well as possible solutions to overcome these, are discussed. Conclusion: Akin to other engineering disciplines, extensive standardization will undoubtedly speed-up and reduce the cost of bioprocess development. In this respect, further implementation of synthetic biology standards will be crucial for the field in order to redeem its promise, i.e. to enable predictable forward engineering.

Transformation of an Exotic Yeast Species into a Platform Organism: A Case Study for Engineering Glycolipid Production in the Yeast Starmerella bombicola

In this chapter, a step-by-step approach on how to transform non-conventional yeasts or fungi into platform organisms is described. The non-conventional glycolipid producing yeast Starmerella bombicola (and in some cases also Pseudohyphozyma bogoriensis) is used as a case study. And more specifically how to engineer it toward production of new-to-nature glycolipids like bola sophorolipids. When starting genetic engineering efforts for non-lab strains, one should start at the very basis: identifying selection markers and possibly developing auxotrophic strains. Once this is done, the quest for the development of an optimal transformation method can be started. After optimization thereof, knock-out and knock-in strains can be generated based upon the specific strategy/aim. Sometimes this can lead to unexpected, but yet very interesting findings. To fully and efficiently expand the potential as a production platform of these yeast strains, a range of additional molecular tools are required. A well-equipped molecular toolbox should contain a set of characterized promotors, terminators, and defined genomic landing paths. The availability of an episomal system greatly facilitates engineering and screening efforts, but also offers the possibility of developing more advanced engineering techniques such as Crispr-Cas. InBio.be is a world leading pioneer to do this for the yeast S. bombicola and combined, these efforts will result in the commercialization of new types of glycolipids in the next few years.