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Microbial production of low-cost chemicals such as biofuels and bulk chemicals requires high titer, rate and especially yield to be able to compete with petrochemical-based processes. To achieve high yield, the metabolic pathways involved in product formation should be redox-balanced and conserve sufficient metabolic energy to fulfil the cells’ requirements for maintenance and growth. Thanks to advances in omics and genetic engineering tools, redox-balanced product pathways can be created, however, product yield is – in most cases – limited by energy conservation. Capturing additional energy can lead to higher product titer or allow microorganisms to grow under inhibitory conditions (high cell density cultivation and high product titer) depending on the product of interest. In this thesis, we designed a generally applicable strategy to capture additional metabolic energy from sugars in E. coli. We also proposed alternative methods to further increase microbial product formation

Chitin is a natural biopolymer abundantly occurring in nature and is a main source for production of its monomer - N-acetylglucosamine (GlcNAc). GlcNAc is widely used in medical treatment of joints disorders e.g. osteoarthritis, in skin care and dermatology as moisture agent, in food as a food additive. In this thesis we present the development of an enzymatic process for production of GlcNAc from chitin with use of chitinolytic enzymes Chitinase Chi1 and N-acetylglucosaminidase (NAGase) MthNAG from Myceliophthora thermophila C1. We homologously overproduced Chitinase Chi1 and MthNAG in a low protease/(hemi-) cellulase free M. thermophila C1-expression host. Overproduced enzymes were purified and their mode of action, catalytic properties and thermostability were characterised. Both enzymes showed remarkable thermostability that has never been observed for this group of fungal enzymes. Chitinase Chi1 and MthNAG synergistically hydrolysed chitin i.e. the products released from polymeric chitin by Chitinase Chi1 were hydrolysed by MthNAG to a final product, GlcNAc. The synergistic action of Chitinase Chi1 and MthNAG and their thermostability was fundamental for the development of an enzymatic production process of GlcNAc. Furthermore, we investigated the production of GlcNAc from chitin impregnated with H2SO4 and subsequently ball milled (ball-milled-H2SO4-chitin). According to the XRD measurements, the crystallinity of chitin has been drastically decreased upon ball-milling pre-treatment and the accessibility of chitin was considerably improved for the enzymatic action of Chitinase Chi1 and MthNAG. Subsequently, we optimized the production of GlcNAc from ball-milled-H2SO4-chitin based on a catalytic cascade catalysed by Chitinase Chi1 and MthNAG. Using central composite design (CCD) and response surface methodology (RSM), we found conditions that allow production of GlcNAc at high yield. After 6 h incubation at 50 °C, the yield of GlcNAc of 73.2 % from 1.6 % (w/v) ba

Chapter 1 provides the background for the thesis. In it, I provide a short overview of what biotechnology is and how it has been utilized it for thousands of years. I then address how modern biotechnology has evolved over the past few decades. Its progress has been triggered by the discovery of nucleic acids and marked by a focus on genetic understanding of cell and organism function and on the subsequent manipulation to ultimately benefit society in one way or another. Furthermore, computational biology has been increasingly important to determine the success of biotechnological research, in for example an anti-malarial drug- producing yeast. However, for microalgae, which are very promising organisms for biotechnological applications, there are essentially no successful commercialized examples of modern biotechnology. The chapter further discusses the importance of computationally predicting protein functions and its role in bioinformatics and systems biology research, concluding that this is one of the challenges for microalgal biotechnology. This topic is discussed in all chapters of this thesis, as its overarching goal is to develop and deploy tools and methodologies that lead to increase our understanding of microalgae as cell factories.Chapter 2 is an review on the state of microalgal biotechnology in 2014, of which the major discussion points are still valid, and how bioinformatics and systems biology should be used to further microalgal research. It describes the challenges of microalgal genomics, bioinformatics, and systems biology research. The chapter addresses a few challenges for microalgae in particular: a lack of genomic data, a low amount of validated protein functions, and genome-scale metabolic models largely based off of Arabidopsis thaliana. Suggestions are made on how to overcome these challenges, by for example better utilizing bioinformatics methods and databases. Chapter 3 addresses a specific challenge: the need for accurate annotation of t

A thermostable Chitinase Chi1 from Myceliophthora thermophila C1 was homologously produced and characterized. Chitinase Chi1 shows high thermostability at 40 °C (>140 h 90% activity), 50 °C (>168 h 90% activity), and 55 °C (half-life 48 h). Chitinase Chi1 has broad substrate specificity and converts chitin, chitosan, modified chitosan, and chitin oligosaccharides. The activity of Chitinase Chi1 is strongly affected by the degree of deacetylation (DDA), molecular weight (Mw), and side chain modification of chitosan. Chitinase Chi1 releases mainly (GlcNAc)2 from insoluble chitin and chitooligosaccharides with a polymerization degree (DP) ranging from 2 to 12 from chitosan, in a processive way. Chitinase Chi1 shows higher activity toward chitin oligosaccharides (GlcNAc)4−6 than toward (GlcNAc)3 and is inactive for (GlcNAc)2. During hydrolysis, oligosaccharides bind at subsites −2 to +2 in the enzyme’s active site. Chitinase Chi1 can be used for chitin valorisation and for production of chitin- and chito-oligosaccharides at industrial scale.

A Monascus ruber strain was isolated that was able to grow on mineral medium at high sugar concentrations and 175 g/l lactic acid at pH 2.8.

From a chemistry perspective, proteins can be thought of as polymers of amino acids, linked by amide bonds. They feature unsurpassed control over monomer sequence and molecular size. The amino acid sequence of proteins determines their three-dimensional folded structure, resulting in unique properties. Proteins such as collagen, elastin, and silk play a crucial structure-forming role in various tissues and animal architecture such as spider webs. These proteins are characterized by highly repetitive amino acid sequences, and can reversibly self-assemble into supramolecular structures through the formation of noncovalent bonds. These unique properties have sparked the interest of material scientists, and mimics of these proteins have been designed and produced as heterologous proteins in suitable expression systems. The most commonly employed host for these so-called protein-based polymers, or protein polymers for short, is the bacterium Escherichia coli. In this thesis, we explored the use of an alternative platform, namely the methylotrophic yeast Pichia pastoris (Komagataella phafii). This organism is well-known for its often relatively high yields, and offers a choice between intracellular and secretory production. Secretion of the polymer into the medium provides a highly effective first purification step, and precludes the need for cell disruption procedures that are cost-prohibitive at an industrial scale. We evaluated the secretory production in P. pastoris of various protein polymers: murine collagen fragments (gelatins), a de novo-designed highly hydrophilic gelatin, silk-like proteins, hydrogel-forming triblock copolymers with collagen-inspired end blocks, block copolymers with heterodimer-forming modules, and silk-inspired triblock copolymers that feature integrin-binding or proteoglycan-binding cell-adhesive motifs. All of these protein polymers were produced at g/L levels, and various bioprocessing and strain engineering strategies were employed to addr

The microalgae Tetradesmus obliquus is able to maintain a high photosynthetic efficiency under nitrogen limitation and is considered a promising green microalgae for sustainable production of diverse compounds, including biofuels. Here, we report the first draft whole-genome shotgun sequencing of T. obliquus. The final assembly comprises 108,715,903 bp with over 1,368 scaffolds.

The first draft genome of Scenedesmus obliquus (UTEX 393). The assembly yielded 9191 contigs (min length 200bp) for a total length of 96197736 bp. The scaffolding yielded 1680 sequences for a total of 126848291 bp. We were not able to recover four chromosomes and the two organelle genomes. Few scaffolds were found to cover 99.97% of the reference chloroplast genome. The mitochondrial genome was too fragmented and the scaffolds covered only 46% of UTEX78 and KS3-2., The first draft genome of Scenedesmus obliquus (UTEX 393). The assembly yielded 9191 contigs (min length 200bp) for a total length of 96197736 bp. The scaffolding yielded 1680 sequences for a total of 126848291 bp. We were not able to recover four chromosomes and the two organelle genomes. Few scaffolds were found to cover 99.97% of the reference chloroplast genome. The mitochondrial genome was too fragmented and the scaffolds covered only 46% of UTEX78 and KS3-2.

Itaconic acid, a C5-dicarboxylic acid, is a potential biobased building block for the polymer industry. It is obtained from the citric acid cycle by decarboxylation of cis-aconitic acid. This reaction is catalyzed by CadA in the native itaconic acid producer Aspergillus terreus. Recently, another enzyme encoded by the mammalian immunoresponsive gene 1 (irg1), was found to decarboxylate cis-aconitate to itaconate in vitro. We show that heterologous expression of irg1 enabled itaconate production in Escherichia coli with production titres up to 560 mg/L.

Background: Microalgae are a promising platform for producing neutral lipids, to be used in the application for biofuels or commodities in the feed and food industry. A very promising candidate is the oleaginous green microalga Scenedesmus obliquus, because it accumulates up to 45% w/w triacylglycerol (TAG) under nitrogen starvation. Under these conditions, starch is accumulated as well. Starch can amount up to 38% w/w under nitrogen starvation, which is a substantial part of the total carbon captured. When aiming for optimized TAG production, blocking the formation of starch could potentially increase carbon allocation towards TAG. In an attempt to increase TAG content, productivity and yield, starchless mutants of this high potential strain were generated using UV mutagenesis. Previous studies in Chlamydomonas reinhardtii have shown that blocking the starch synthesis yields higher TAG contents, although these TAG contents do not surpass those of oleaginous microalgae yet. So far no starchless mutants in oleaginous green microalgae have been isolated that result in higher TAG productivities. Results: Five starchless mutants have been isolated successfully from over 3,500 mutants. The effect of the mutation on biomass and total fatty acid (TFA) and TAG productivity under nitrogen-replete and nitrogen-depleted conditions was studied. All five starchless mutants showed a decreased or completely absent starch content. In parallel, an increased TAG accumulation rate was observed for the starchless mutants and no substantial decrease in biomass productivity was perceived. The most promising mutant showed an increase in TFA productivity of 41% at 4 days after nitrogen depletion, reached a TAG content of 49.4% (% of dry weight) and had no substantial change in biomass productivity compared to the wild type. Conclusions: The improved S. obliquus TAG production strains are the first starchless mutants in an oleaginous green microalga that show enhanced TAG content under phot

Background Many microalgae accumulate carbohydrates simultaneously with triacylglycerol (TAG) upon nitrogen starvation, and these products compete for photosynthetic products and metabolites from the central carbon metabolism. As shown for starchless mutants of the non-oleaginous model alga Chlamydomonas reinhardtii, reduced carbohydrate synthesis can enhance TAG production. However, these mutants still have a lower TAG productivity than wild-type oleaginous microalgae. Recently, several starchless mutants of the oleaginous microalga Scenedesmus obliquus were obtained which showed improved TAG content and productivity. Results The most promising mutant, slm1, is compared in detail to wild-type S. obliquus in controlled photobioreactors. In the slm1 mutant, the maximum TAG content increased to 57¿±¿0.2% of dry weight versus 45¿±¿1% in the wild type. In the wild type, TAG and starch were accumulated simultaneously during initial nitrogen starvation, and starch was subsequently degraded and likely converted into TAG. The starchless mutant did not produce starch and the liberated photosynthetic capacity was directed towards TAG synthesis. This increased the maximum yield of TAG on light by 51%, from 0.144¿±¿0.004 in the wild type to 0.217¿±¿0.011 g TAG/mol photon in the slm1 mutant. No differences in photosynthetic efficiency between the slm1 mutant and the wild type were observed, indicating that the mutation specifically altered carbon partitioning while leaving the photosynthetic capacity unaffected. Conclusions The yield of TAG on light can be improved by 51% by using the slm1 starchless mutant of S. obliquus, and a similar improvement seems realistic for the areal productivity in outdoor cultivation. The photosynthetic performance is not negatively affected in the slm1 and the main difference with the wild type is an improved carbon partitioning towards TAG.