Development of scalable high throughput fermentation approaches for physiological characterisation of yeast and filamentous fungi

I denne afhandling præsenteres tre validerede ”high throughput” metoder til fysiologisk karakterisering af mikroorganismer under bioreaktor sammenlignelige forhold. Rent eksperimentelt blev dette gennemført via anvendelse af bl.a. genmodificerede Saccharomyces cerevisiae (gær) og Aspergillus nidulans (skimmelsvamp) stammer, designet til at producere det heterologe model polyketid 6-metylsalicyl syre (6-MSA). I studiet blev der etableret en automatiseret ”high throughput” screeningsmetode til gær med primær fokus på vækst hastigheder. Videreudvikling af denne metode resulterede i en fuld automatiseret metode til kvantitativ fysiologisk karakterisering i mikrotiter bakker. Fuld skalerbarhed blev demonstreret gennem komparativ fysiologisk karakterisering af gær dyrket i både mikrotiter bakker og bioreaktorer, hvor både væksthastigheder og udbytte koefficienter blev vist sammenlignelige. Disse resultater blev yderligere understøttet ved hjælp af metabolsk flux analyse resulterende i til alle formål identiske flux distributionerne. I afhandlingen præsenteres endvidere en nyskabende automatiseret ”high throughput” metode til kultivering af skimmelsvampe i mikrotiter bakker. Dette blev muliggjort ved hjælp af den anioniske polymer carboxypolymetylen, der sikrede en fordelagtig morfologi, hvilket muliggjorde estimering af væksthastigheder via optisk densitets målinger. I lighed med det tidligere studie på gær, blev der også her demonstreret fuld skalerbarhed, med ens væksthastigheder og 6-MSA udbytte koefficienter for A. nidulans dyrket i både mikrotiter bakker og i bioreaktorer. Denne alsidige og robuste metode viste sig yderligere, anvendelig for en bred vifte af forskellige skimmelsvampe, resulterende i litteratur sammenlignelige væksthastigheder. Afslutningsvis udførtes et komparativt studie til afklaring af cellefabrik potentialet af de, i afhandlingen, anvendte 6-MSA producerende gær og skimmelsvampe. Højest produktivitet og udbytte blev identificeret for en A. nidulans stamme, hvori 2 kopier af 6-MSA genet var blevet kromosomalt integreret.I dette studie endvidere udviklet en A. nidulans kemostat kultiveringsmetode til anvendelse i komparative studier. Tilsvarende S. cerevisae kultiveringer blev kompromitteret af metaboliske oscilleringer under bestemte forhold, hvilket besværliggjorde sammenligning. Den oscillerende fænotype blev stabiliseret ved tilsætning af en mindre mængde ethanol til fødemediet, hvilket muliggjorde sammenligning af de to forskellige microorganismer. På baggrund af disse forsøg kunne det konkluderes, at A. nidulans er væsentlig overlegen, som celle fabrik til produktion af 6-MSA i forhold til S. cerevisiae. Samlet set har de i afhandlingen udviklede metoder bidraget til en progression imod højere evalueringskapacitet med et væsentlig højere detaljeringsniveau i relation til fysiologisk karakterisering af mikro-skala fermenteringer. Med disse studier er der banet vej for yderligere fremskridt i detaljeret kvantitativ fysiologi baseret på omics analyser, hvilket yderligere vil øge mulighederne for anvendelse af avanceret kvantitativ fysiologi i en bredere vifte af fermenteringssystemer.The pursuit of identifying efficient cell factory candidates for production of pharmaceutically relevant products and commodity chemicals relies heavily on the successful selection of process suitable microorganisms. Hence the selection of efficient cell factories is paramount for successful scale-up to economically viable industrial processes. Accurate quantitative assessment of cellular performance is required for the evaluation of the overall suitability of a microorganism as an industrial cell factory, ensuring that not only product, but also process parameters are optimised. With the increasing number of strains generated through genetic engineering programmes, the traditionally applied methods for strain characterisation, which are typically labour intensive and time consuming, have become somewhat limited due to throughput capacity. Unfortunately, most high throughput methods only provide low levels of information compared to larger scale cultivations, explaining why these systems have not been broadly implemented. The overall aim of the thesis was, therefore, to shift this paradigm towards higher throughput systems for assessment of cellular performance with a higher level of information. This was pursued through development and validation of small, scalable microtiter based systems, for cultivating yeast and filamentous fungi, validated by comparable results from bioreactors. The experimental work was performed using Saccharomyces cerevisiae (yeast) and Aspergillus nidulans (filamentous fungus) strains producing the heterologous model polyketide, 6-methylsalicylic acid (6-MSA). An automated methodology for high throughput screening focusing on growth rates, together with a fully automated method for quantitative physiological characterisation in microtiter plates, was established for yeast. Full scalability was demonstrated through comparative physiological characterisation of yeasts, cultivated in both microtiter plates and bioreactors, revealing that the growth rate and yield coefficients of all non-volatile products including biomass could be correlated. The highly correlated results were taken asan indication of comparable growth physiology in both microtiter plates and bioreactors, which was substantiated by metabolic flux analysis resulting in identical flux distributions over micro-scale and lab scale cultivations. The thesis further presents a novel automated high throughput method for cultivating filamentous fungi in microtiter plates, without compromise to morphology and with product yields and growth rates identical to bioreactors. This was made possible by the dispersive effect on morphology of the anionic polymer carboxypolymethylene, enabling the application of optical density measurements as a means to evaluate growth rates. Again full scalability was demonstrated for a heterologous 6-MSA producing A. nidulans strain, cultivated in both microtiter plates and in bioreactors, displaying identical growth rates and 6-MSA yields on glucose. This versatile and robust method was shown to be applicable for a wide range of different filamentous fungi and conditions, with growth rates comparable with those reported in the literature. A final comparative study of cell factory potential, involving the heterologous 6-MSA producing S. cerevisiae and A. nidulans strains, applied throughout the thesis, demonstrated superior yields and productivities for an A. nidulans strain where two copies of the 6-MSA gene had been chromosomally integrated. Implementation of a chemostat cultivation strategy for A. nidulans proved successful for conditional comparison, while metabolic oscillations and low productivities in continuous cultivations of S. cerevisiae rendered it less favorable for production of 6-MSA. Overall the methods developed and validated during the course of this study, have contributed to a progression towards higher throughput, together with an improved level of detail in physiological characterisation of cultivations at micro-scale. This paves the way for further advances in detailed quantitative physiology based on omics analyses, which would further increase the possibilities for advanced quantitative physiology at different scales.

Anticancer and antifungal compounds from <em>Aspergillus</em>, <em>Penicillium</em> and other filamentous fungi

This review covers important anticancer and antifungal compounds reported from filamentous fungi and in particular from Aspergillus, Penicillium and Talaromyces. The taxonomy of these fungi is not trivial, so a focus of this review has been to report the correct identity of the producing organisms based on substantial previous in-house chemotaxonomic studies

The expression of glycerol facilitators from various yeast species improves growth on glycerol of <i>Saccharomyces cerevisiae</i>

Glycerol is an abundant by-product during biodiesel production and additionally has several assets compared to sugars when used as a carbon source for growing microorganisms in the context of biotechnological applications. However, most strains of the platform production organism Saccharomyces cerevisiae grow poorly in synthetic glycerol medium. It has been hypothesized that the uptake of glycerol could be a major bottleneck for the utilization of glycerol in S. cerevisiae. This species exclusively relies on an active transport system for glycerol uptake. This work demonstrates that the expression of predicted glycerol facilitators (Fps1 homologues) from superior glycerol-utilizing yeast species such as Pachysolen tannophilus, Komagataella pastoris, Yarrowia lipolytica and Cyberlindnera jadinii significantly improves the growth performance on glycerol of the previously selected glycerol-consuming S. cerevisiae wild-type strain (CBS 6412-13A). The maximum specific growth rate increased from 0.13 up to 0.18 h−1 and a biomass yield coefficient of 0.56 gDW/gglycerol was observed. These results pave the way for exploiting the assets of glycerol in the production of fuels, chemicals and pharmaceuticals based on baker's yeast. Keywords: Yeast, Saccharomyces cerevisiae, Glycerol, Transport, Glycerol facilitator, Fps1, Stl

Black perithecial pigmentation in <i>Fusarium </i>species is due to the accumulation of 5-deoxybostrycoidin-based melanin

Biosynthesis of the black perithecial pigment in the filamentous fungus Fusarium graminearum is dependent on the polyketide synthase PGL1 (oPKS3). A seven-membered PGL1 gene cluster was identified by over-expression of the cluster specific transcription factor pglR. Targeted gene replacement showed that PGL1, pglJ, pglM and pglV were essential for the production of the perithecial pigment. Over-expression of PGL1 resulted in the production of 6-O-demethyl-5-deoxybostrycoidin (1), 5-deoxybostrycoidin (2), and three novel compounds 5-deoxybostrycoidin anthrone (3), 6-O-demethyl-5-deoxybostrycoidin anthrone (4) and purpurfusarin (5). The novel dimeric bostrycoidin purpurfusarin (5) was found to inhibit the growth of Candida albicans with an IC(50) of 8.0 +/− 1.9 μM. The results show that Fusarium species with black perithecia have a previously undescribed form of 5-deoxybostrycoidin based melanin in their fruiting bodies