An exploratory in silico comparison of open-source codon harmonization tools.

BACKGROUND: Not changing the native constitution of genes prior to their expression by a heterologous host can affect the amount of proteins synthesized as well as their folding, hampering their activity and even cell viability. Over the past decades, several strategies have been developed to optimize the translation of heterologous genes by accommodating the difference in codon usage between species. While there have been a handful of studies assessing various codon optimization strategies, to the best of our knowledge, no research has been performed towards the evaluation and comparison of codon harmonization algorithms. To highlight their importance and encourage meaningful discussion, we compared different open-source codon harmonization tools pertaining to their in silico performance, and we investigated the influence of different gene-specific factors. RESULTS: In total, 27 genes were harmonized with four tools toward two different heterologous hosts. The difference in %MinMax values between the harmonized and the original sequences was calculated (ΔMinMax), and statistical analysis of the obtained results was carried out. It became clear that not all tools perform similarly, and the choice of tool should depend on the intended application. Almost all biological factors under investigation (GC content, RNA secondary structures and choice of heterologous host) had a significant influence on the harmonization results and thus must be taken into account. These findings were substantiated using a validation dataset consisting of 8 strategically chosen genes. CONCLUSIONS: Due to the size of the dataset, no complex models could be developed. However, this initial study showcases significant differences between the results of various codon harmonization tools. Although more elaborate investigation is needed, it is clear that biological factors such as GC content, RNA secondary structures and heterologous hosts must be taken into account when selecting the codon harmonization tool.

MariClus: Your One-Stop Platform for Information on Marine Natural Products, Their Gene Clusters and Producing Organisms.

BACKGROUND: The marine environment hosts the vast majority of living species and marine microbes that produce natural products with great potential in providing lead compounds for drug development. With over 70% of Earth's surface covered in water and the high interaction rate associated with liquid environments, this has resulted in many marine natural product discoveries. Our improved understanding of the biosynthesis of these molecules, encoded by gene clusters, along with increased genomic information will aid us in uncovering even more novel compounds. RESULTS: We introduce MariClus (https://www.mariclus.com), an online user-friendly platform for mining and visualizing marine gene clusters. The first version contains information on clusters and the predicted molecules for over 500 marine-related prokaryotes. The user-friendly interface allows scientists to easily search by species, cluster type or molecule and visualize the information in table format or graphical representation. CONCLUSIONS: This new online portal simplifies the exploration and comparison of gene clusters in marine species for scientists and assists in characterizing the bioactive molecules they produce. MariClus integrates data from public sources, like GenBank, MIBiG and PubChem, with genome mining results from antiSMASH. This allows users to access and analyze various aspects of marine natural product biosynthesis and diversity.

Comprehensive metabolomics reveals correlation between sophorolipid biosynthesis and autophagy.

Sophorolipids are biobased and biodegradable glycolipid surface-active agents contributing to the shift from petroleum to biobased surfactants, associated with clear environmental benefits. However, their production cost is currently too high to allow commercialisation. Therefore, a continuous sophorolipid production process was evaluated, i.e., a retentostat with an external filtration unit. Despite an initial increase in volumetric productivity, productivity eventually declined to almost 0 g L-1 h-1. Following comprehensive metabolomics on supernatant obtained from a standardised retentostat, we hypothesised exhaustion of the N-starvation-induced autophagy as the main mechanism responsible for the decline in bolaform sophorolipid productivity. Thirty-six metabolites that correlate with RNA/protein autophagy and high sophorolipid productivity were putatively identified. In conclusion, our results unveil a plausible cause of this bola sophorolipid productivity decline in an industrially relevant bioreactor set-up, which may thus impact majorly on future yeast biosurfactant regulation studies and the finetuning of bola sophorolipid production processes.

From bumblebee to bioeconomy: Recent developments and perspectives for sophorolipid biosynthesis.

Sophorolipids are biobased compounds produced by the genera Starmerella and Pseudohyphozyma that gain exponential interest from academic and industrial stakeholders due to their mild and environmental friendly characteristics. Currently, industrially relevant sophorolipid volumetric productivities are reached up to 3.7 g∙L-1∙h-1 and sophorolipids are used in the personal care and cleaning industry at small scale. Moreover, applications in crop protection, food, biohydrometallurgy and medical fields are being extensively researched. The research and development of sophorolipids is at a crucial stage. Therefore, this work presents an overview of the state-of-the-art on sophorolipid research and their applications, while providing a critical assessment of scientific techniques and standardisation in reporting. In this review, the genuine sophorolipid producing organisms and the natural role of sophorolipids are discussed. Subsequently, an evaluation is made of innovations in production processes and the relevance of in-situ product recovery for process performance is discussed. Furthermore, a critical assessment of application research and its future perspectives are portrayed with a focus on the self-assembly of sophorolipid molecules. Following, genetic engineering strategies that affect the sophorolipid physiochemical properties are summarised. Finally, the impact of sophorolipids on the bioeconomy are uncovered, along with relevant future perspectives.

A multi-omics study to boost continuous bolaform sophorolipid production.

Biodegradable and biobased surface active agents are renewable and environmentally friendly alternatives to petroleum derived or oleochemical surfactants. However, they are accompanied by relatively high production costs. In this study, the aim was to reduce the production costs for an innovative type of microbial biosurfactant: bolaform sophorolipids, produced by the yeast Starmerella bombicola ΔsbleΔat. A novel continuous retentostat set-up was performed whereby continuous broth microfiltration retained the biomass in the bioreactor while performing an in situ product separation of bolaform sophorolipids. Although a mean volumetric productivity of 0.56 g L-1 h-1 was achieved, it was not possible to maintain this productivity, which collapsed to almost 0 g L-1 h-1. Therefore, two process adaptations were evaluated, a sequential batch strategy and a phosphate limitation alleviation strategy. The sequential batch set-up restored the mean volumetric productivity to 0.66 g L-1 h-1 for an additional 132 h but was again followed by a productivity decline. A similar result was obtained with the phosphate limitation alleviation strategy where a mean volumetric productivity of 0.54 g L-1 h-1 was reached, but a productivity decline was also observed. Whole genome variant analysis uncovered no evidence for genomic variations for up to 1306 h of retentostat cultivation. Untargeted metabolomics analysis identified 8-hydroxyguanosine, a biomarker for oxidative RNA damage, as a key metabolite correlating with high bolaform sophorolipid productivity. This study showcases the application of a retentostat to increase bolaform sophorolipid productivity and lays the basis of a multi-omics platform for in depth investigation of microbial biosurfactant production with S. bombicola.

Elucidation of the Natural Function of Sophorolipids Produced by Starmerella bombicola.

The yeast Starmerella bombicola distinguishes itself from other yeasts by its potential of producing copious amounts of the secondary metabolites sophorolipids (SLs): these are glycolipid biosurfactants composed out of a(n) (acetylated) sophorose moiety and a lipid tail. Although SLs are the subject of numerous research papers and have been commercialized, e.g., in eco-friendly cleaning solutions, the natural function of SLs still remains elusive. This research article investigates several hypotheses for why S. bombicola invests that much energy in the production of SLs, and we conclude that the main natural function of SLs in S. bombicola is niche protection: (1) the extracellular storage of an energy-rich, yet metabolically less accessible carbon source that can be utilized by S. bombicola upon conditions of starvation with (2) antimicrobial properties. In this way, S. bombicola creates a dual advantage in competition with other microorganisms. Additionally, SLs can expedite growth on rapeseed oil, composed of triacylglycerols which are hydrophobic substrates present in the yeasts' environment, for a non-SL producing strain (Δcyp52M1). It was also found that-at least under lab conditions-SLs do not provide protection against high osmotic pressure prevalent in sugar-rich environments such as honey or nectar present in the natural habitat of S. bombicola.

Novel Alkaloids from Marine Actinobacteria: Discovery and Characterization.

The marine environment is an excellent resource for natural products with therapeutic potential. Its microbial inhabitants, often associated with other marine organisms, are specialized in the synthesis of bioactive secondary metabolites. Similar to their terrestrial counterparts, marine Actinobacteria are a prevalent source of these natural products. Here, we discuss 77 newly discovered alkaloids produced by such marine Actinobacteria between 2017 and mid-2021, as well as the strategies employed in their elucidation. While 12 different classes of alkaloids were unraveled, indoles, diketopiperazines, glutarimides, indolizidines, and pyrroles were most dominant. Discoveries were mainly based on experimental approaches where microbial extracts were analyzed in relation to novel compounds. Although such experimental procedures have proven useful in the past, the methodologies need adaptations to limit the chance of compound rediscovery. On the other hand, genome mining provides a different angle for natural product discovery. While the technology is still relatively young compared to experimental screening, significant improvement has been made in recent years. Together with synthetic biology tools, both genome mining and extract screening provide excellent opportunities for continued drug discovery from marine Actinobacteria.

Comprehensive study on Escherichia coli genomic expression: Does position really matter?

As a biorefinery platform host, Escherichia coli has been used extensively to produce metabolites of commercial interest. Integration of foreign DNA onto the bacterial genome allows for stable expression overcoming the need for plasmid expression and its associated instability. Despite the development of numerous tools and genome editing technologies, the question of where to incorporate a synthetic pathway remains unanswered. To address this issue, we studied the genomic expression in E. coli and linked it not only to 26 rationally selected genomic locations, but also to the gene direction in relation to the DNA replication fork, to the carbon and nitrogen source, to DNA folding and supercoiling, and to metabolic burden. To enable these experiments, we have designed a fluorescent expression cassette to eliminate specific local effects on gene expression. Overall it can be concluded that although the expression range obtained by changing the genomic location of a pathway is small compared to the range typically seen in promoter-RBS libraries, the effect of culture medium, environmental stress and metabolic burden can be substantial. The characterization of multiple effects on genomic expression, and the associated libraries of well-characterized strains, will only stimulate and improve the creation of stable production hosts fit for industrial settings.

Alkaloids from Marine Fungi: Promising Antimicrobials.

Resistance of pathogenic microorganisms against antimicrobials is a major threat to contemporary human society. It necessitates a perpetual influx of novel antimicrobial compounds. More specifically, Gram- pathogens emerged as the most exigent danger. In our continuing quest to search for novel antimicrobial molecules, alkaloids from marine fungi show great promise. However, current reports of such newly discovered alkaloids are often limited to cytotoxicity studies and, moreover, neglect to discuss the enigma of their biosynthesis. Yet, the latter is often a prerequisite to make them available through sufficiently efficient processes. This review aims to summarize novel alkaloids with promising antimicrobial properties discovered in the past five years and produced by marine fungi. Several discovery strategies are summarized, and knowledge gaps in biochemical production routes are identified. Finally, links between the structure of the newly discovered molecules and their activity are proposed. Since 2015, a total of 35 new antimicrobial alkaloids from marine fungi were identified, of which 22 showed an antibacterial activity against Gram- microorganisms. Eight of them can be classified as narrow-spectrum Gram- antibiotics. Despite this promising ratio of novel alkaloids active against Gram- microorganisms, the number of newly discovered antimicrobial alkaloids is low, due to the narrow spectrum of discovery protocols that are used and the fact that antimicrobial properties of newly discovered alkaloids are barely characterized. Alternatives are proposed in this review. In conclusion, this review summarizes novel findings on antimicrobial alkaloids from marine fungi, shows their potential as promising therapeutic candidates, and hints on how to further improve this potential.

Unraveling the regulation of sophorolipid biosynthesis in Starmerella bombicola.

Starmerella bombicola very efficiently produces the secondary metabolites sophorolipids (SLs). Their biosynthesis is not-growth associated and highly upregulated in the stationary phase. Despite high industrial and academic interest, the underlying regulation of SL biosynthesis remains unknown. In this paper, potential regulation of SL biosynthesis through the telomere positioning effect (TPE) was investigated, as the SL gene cluster is located adjacent to a telomere. An additional copy of this gene cluster was introduced elsewhere in the genome to investigate if this results in a decoy of regulation. Indeed, for the new strain, the onset of SL production was shifted to the exponential phase. This result was confirmed by RT-qPCR analysis. The TPE effect was further investigated by developing and applying a suitable reporter system for this non-conventional yeast, enabling non-biased comparison of gene expression between the subtelomeric CYP52M1- and the URA3 locus. This was done with a constitutive endogenous promotor (pGAPD) and one of the endogenous promotors of the SL biosynthetic gene cluster (pCYP52M1). A clear positioning effect was observed for both promotors with significantly higher GFP expression levels at the URA3 locus. No clear GFP upregulation was observed in the stationary phase for any of the new strains.

5' untranslated regions: the next regulatory sequence in yeast synthetic biology.

When developing industrial biotechnology processes, Saccharomyces cerevisiae (baker's yeast or brewer's yeast) is a popular choice as a microbial host. Many tools have been developed in the fields of synthetic biology and metabolic engineering to introduce heterologous pathways and tune their expression in yeast. Such tools mainly focus on controlling transcription, whereas post-transcriptional regulation is often overlooked. Herein we discuss regulatory elements found in the 5' untranslated region (UTR) and their influence on protein synthesis. We provide not only an overall picture, but also a set of design rules on how to engineer a 5' UTR. The reader is also referred to currently available models that allow gene expression to be tuned predictably using different 5' UTRs.

Unraveling and resolving inefficient glucolipid biosurfactants production through quantitative multiomics analyses of Starmerella bombicola strains.

Glucolipids (GLs) are glycolipid biosurfactants with promising properties. These GLs are composed of glucose attached to a hydroxy fatty acid through a ω and/or ω-1 glycosidic linkage. Up until today these interesting molecules could only be produced using an engineered Starmerella bombicola strain (∆ugtB1::URA3 G9) producing GLs instead of sophorolipids, albeit with a very low average productivity (0.01 g·L-1 ·h-1 ). In this study, we investigated the reason(s) for this via reverse-transcription quantitative polymerase chain reaction and Liquid chromatography-multireaction monitoring-mass spectrometry. We found that all glycolipid biosynthetic genes and enzymes were downregulated in the ∆ugtB1 G9 strain in comparison to the wild type. The underlying reason for this downregulation was further investigated by performing quantitative metabolome comparison of the ∆ugtB1 G9 strain with the wild type and two other engineered strains also tinkered in their glycolipid biosynthetic gene cluster. This analysis revealed a clear distortion of the entire metabolism of the ∆ugtB1 G9 strain compared to all the other strains. Because the parental strain of the former was a spontaneous ∆ura3 mutant potentially containing other "hidden" mutations, a new GL production strain was generated based on a rationally engineered ∆ura3 mutant (PT36). Indeed, a 50-fold GL productivity increase (0.51 g·L-1 ·h-1 ) was obtained with the new ∆ugtB1::URA3 PT36 strain compared with the G9-based strain (0.01 g·L-1 ·h-1 ) in a 10 L bioreactor experiment, yielding 118 g/L GLs instead of 8.39 g/L. Purification was investigated and basic properties of the purified GLs were determined. This study forms the base for further development and optimization of S. bombicola as a production platform strain for (new) biochemicals.

Modulating transcription through development of semi-synthetic yeast core promoters.

Altering gene expression regulation by promoter engineering is a very effective way to fine-tune heterologous pathways in eukaryotic hosts. Typically, pathway building approaches in yeast still use a limited set of long, native promoters. With the today's introduction of longer and more complex pathways, an expansion of this synthetic biology toolbox is necessary. In this study we elucidated the core promoter structure of the well-characterized yeast TEF1 promoter and determined the minimal length needed for sufficient protein expression. Furthermore, this minimal core promoter sequence was used for the creation of a promoter library covering different expression strengths. This resulted in a group of short, 69 bp promoters with an 8.0-fold expression range. One exemplar had a two and four times higher expression compared to the native CYC1 and ADH1 promoter, respectively. Additionally, as it was described that the protein expression range could be broadened by upstream activating sequences (UASs), we integrated earlier described single and multiple short, synthetic UASs in front of the strongest yeast core promoter. This approach resulted to further variation in protein expression and an overall promoter library spanning a 20-fold activity range and covering a length from 69 bp to maximally 129 bp. Furthermore, the robustness of this library was assessed on three alternative carbon sources besides glucose. As such, the suitability of short yeast core promoters for metabolic engineering applications on different media, either in an individual context or combined with UAS elements, was demonstrated.

Production of long-chain hydroxy fatty acids by Starmerella bombicola.

To decrease our dependency for the diminishing source of fossils resources, bio-based alternatives are being explored for the synthesis of commodity and high-value molecules. One example in this ecological initiative is the microbial production of the biosurfactant sophorolipids by the yeast Starmerella bombicola. Sophorolipids are surface-active molecules mainly used as household and laundry detergents. Because S. bombicola is able to produce high titers of sophorolipids, the yeast is also used to increase the portfolio of lipophilic compounds through strain engineering. Here, the one-step microbial production of hydroxy fatty acids by S. bombicola was accomplished by the selective blockage of three catabolic pathways through metabolic engineering. Successful production of 17.39 g/l (ω-1) linked hydroxy fatty acids was obtained by the successive blockage of the sophorolipid biosynthesis, the ß-oxidation and the ω-oxidation pathways. Minor contamination of dicarboxylic acids and fatty aldehydes were successfully removed using flash chromatography. This way, S. bombicola was further expanded into a flexible production platform of economical relevant compounds in the chemical, food and cosmetic industries.

Serine integrase recombinational engineering (SIRE): A versatile toolbox for genome editing.

Chromosomal integration of biosynthetic pathways for the biotechnological production of high-value chemicals is a necessity to develop industrial strains with a high long-term stability and a low production variability. However, the introduction of multiple transcription units into the microbial genome remains a difficult task. Despite recent advances, current methodologies are either laborious or efficiencies highly fluctuate depending on the length and the type of the construct. Here we present serine integrase recombinational engineering (SIRE), a novel methodology which combines the ease of recombinase-mediated cassette exchange (RMCE) with the selectivity of orthogonal att sites of the PhiC31 integrase. As a proof of concept, this toolbox is developed for Escherichia coli. Using SIRE we were able to introduce a 10.3 kb biosynthetic gene cluster on different locations throughout the genome with an efficiency of 100% for the integrating step and without the need for selection markers on the knock-in cassette. Next to integrating large fragments, the option for multitargeting, for deleting operons, as well as for performing in vivo assemblies further expand and proof the versatility of the SIRE toolbox for E. coli. Finally, the serine integrase PhiC31 was also applied in the yeast Saccharomyces cerevisiae as a marker recovery tool, indicating the potential and portability of this toolbox.

Hidden antibiotics: Where to uncover?

The struggle of humans versus pathogens is a never ending battle. Since the discovery of antibiotics humans have tipped the scales in their favour, but today bacteria are nullifying this advantage by developing resistance mechanisms against these molecules. The plethora of different antibiotics active against pathogens is shrinking while the discovery of new molecules is arduous. Especially the development of drugs active against Gram- pathogens continues slowly. New strategies to discover novel, potent antibiotics are hence needed. Adopting the optimistic view of technological singularity, innovative and disruptive approaches are required and hence proposed to lift the current conundrum. In this review, questions are answered on where and how to look for new natural product hit molecules with antibacterial activity, on how the field of synthetic biology can aid the contemporary pharmaceutical challenge and whether we are ready to make the transition towards other approaches, such as narrow-spectrum antibiotics and phage therapy.

Starmerella bombicola, an industrially relevant, yet fundamentally underexplored yeast.

In this review, we focus on one of the most important microbial producers of biosurfactants, Starmerella bombicola. Emphasis is laid on the discovery, taxonomy, habitat, cellular characteristics, biochemistry and genetics of this non-pathogenic yeast. Biosurfactants are natural surface-active compounds produced by several types of microorganisms and have been considered an interesting alternative to synthetic surfactants. The sophorolipids produced by S. bombicola are promising biosurfactants, with application potential in food, pharmaceutical, cosmetic and cleaning industries. The fundamental knowledge described in this review is of crucial interest to optimize production of these promising compounds. Furthermore, it can be translated to produce novel non-native bioactive molecules with S. bombicola, and to deepen fundamental knowledge on other non-conventional yeast species and in the end to broaden their application potential as well.

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.

Toward Predictable 5'UTRs in Saccharomyces cerevisiae: Development of a yUTR Calculator.

Fine-tuning biosynthetic pathways is crucial for the development of economic feasible microbial cell factories. Therefore, the use of computational models able to predictably design regulatory sequences for pathway engineering proves to be a valuable tool, especially for modifying genes at the translational level. In this study we developed a computational approach for the de novo design of 5'-untranslated regions (5'UTRs) in Saccharomyces cerevisiae with a predictive outcome on translation initiation rate. On the basis of existing data, a partial least-squares (PLS) regression model was trained and showed good performance on predicting protein abundances of an independent test set. This model was further used for the construction of a "yUTR calculator" that can design 5'UTR sequences with a diverse range of desired translation efficiencies. The predictive power of our yUTR calculator was confirmed in vivo by different representative case studies. As such, these results show the great potential of data driven approaches for reliable pathway engineering in S. cerevisiae.

Standardization in synthetic biology: an engineering discipline coming of age.

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.