| Sažetak | U proizvodnji bioetanola kvasac Dekkera bruxellensis vrlo je zanimljiva alternativa kvascu Saccharomyces cerevisiae. Obje vrste brzo fermentiraju šećere i proizvode visoke koncentracije etanola, ali kvasac D. bruxellensis fermentira i disaharid celobiozu te kao izvor dušika koristi i nitrat. Međutim, biotehnološki potencijal ove vrste danas je neiskorišten jer do sada nije bilo moguće ciljano mijenjati njegov genom, tj. metodama genetičkog inženjerstva nije bilo moguće konstruirati sojeve željenih karakteristika. Stoga je cilj ovog rada bio razviti metodologiju za preciznu modifikaciju genoma ovog kvasca, sličnu onoj koja se svakodnevno provodi u modelnom kvascu S. cerevisiae. Razvijena je metoda genetičke transformacije, temeljena na elektroporaciji, kojom se u stanice kvasca D. bruxellensis unosi transformirajuća DNA. Nadalje, konstruirani su integrativni i replikativni plazmidi koji omogućavaju ekspresiju gena iz drugih organizama u ovom kvascu. U ovoj vrsti uspješnost uvođenja preciznih modifikacija u genom relativno je niska (5 – 10%) te ovisi o duljini homologne DNA na krajevima transformirajućeg fragmenta i mehanizmu njegove ugradnje. Međutim, ekspresija gena RAD54 iz kvasca S. cerevisiae, gena koji sudjeluje u procesu homologne genetičke rekombinacije, podigla je uspješnost genskog ciljanja u kvascu D. bruxellensis za oko 3 puta. S druge strane, ekspresija gena RAD52 iz kvasca S. cerevisiae, također uključenog u proces homologne rekombinacije, smanjila je već prethodno nisku uspješnost preciznih modifikacija genoma. Zahvaljujući razvijenoj metodologiji, u ovom radu konstruiran je i soj kvasca D. bruxellensis koji eksprimira transporter ksiloze, ksiloza reduktazu, ksilitol dehidrogenazu i ksilulokinazu pa stoga može rasti na podlozi na kojoj je ksiloza jedini izvor ugljika. Ovaj soj biotehnološki je značajan jer se u proizvodnji bioetanola druge generacije kao supstrat koriste lignocelulozni hidrolizati bogati ovim teško iskoristivim šećerom. |
| Sažetak (engleski) | BACKGROUND: In biotechnology, yeast Dekkera (Brettanomyces) bruxellensis plays a few opposing roles. It irreparably spoils wine by imparting on it the taste and smell of horse-sweat, urine, and burnt plastic. On the other hand, it is irreplaceable in the secondary fermentation of Lambic and English beers to which it bestows fruity aromas and aged character. Furthermore, its robust physiology, lifestyle, and capacity for vigorous fermentation advocate its use in the production of bioethanol. However, despite its versatility and importance, the biology of D. bruxellensis remains mostly unexplored. One of the obstacles to a better understanding of D. bruxellensis lies in the fact that until now was impossible to genetically modify this yeast, i.e., it was impossible to introduce and integrate foreign DNA into its genome, preferably at specific loci. This constraint also hindered construction of strains with improved properties, such as strains that produce higher alcohol content or that ferment xylose, a sugar that is abundant in raw materials used for the production of second-generation bioethanol. To overcome such limitations, this work aimed to develop a protocol for genetic transformation of D. bruxellensis and to determine how often the transforming fragment integrates at a homologous site in the genome. METHODS AND RESULTS: In this work, strain CBS 2499 of yeast D. bruxellensis was microbiologically characterized, genetically transformed, and used to investigate the efficiency of gene targeting. To ease and expedite the cultivation of D. bruxellensis and its transformants, different microbiological media were tested and, when required, new media were formulated. Bioinformatic analysis with newly developed software LIDD showed that almost all genes in strain CBS 2499 are present in two copies. This fact hindered but did not prevent isolation of auxotrophic mutants. Protocols used for routine transformation of model yeast Saccharomyces cerevisiae did not produce transformants of D. bruxellensis until they were reinforced with additional steps. Once fine-tuned, the protocols produced over 5000 transformants per microgram of circular plasmid DNA when they were combined with appropriate selective markers and newly developed set of centromeric plasmids. Analyses of transformants by Southern blot showed that in D. bruxellensis gene targeting is inefficient. The transforming fragment integrated at a specific locus only when it carried few kilobases of homologous DNA at its ends. Even then, gene targeting occurred in only 5-10% of the transformants. However, when D. bruxellensis expressed Rad54 protein from yeast S. cerevisiae, the frequency of ends-in targeting increased threefold. At the same time, the frequency of ends-out targeting remained unaffected. When D. bruxellensis expressed Rad52 protein from yeast S. cerevisiae, the efficiency of both ends-in and ends-out targeting decreased unexpectedly. Low efficiency of gene targeting suggests that illegitimate recombination is very efficient in D. bruxellensis. This high efficiency is also evident when multiple transforming fragments enter the cell as they are first ligated together and then illegitimately integrated into the genome. As such, they form tandem repeats which undergo frequent crossing-overs and are therefore unstable. By employing developed transformation protocol, a new strain of D. bruxellensis was constructed which encodes for xylose transporter, xylose reductase, xylitol dehydrogenase, and xylulokinase. Following UV mutagenesis and adaptive evolution, derivatives of this strain grew on a medium in which xylose was the only carbon source. SIGNIFICANCE AND IMPACT OF STUDY: Protocols described here for the first time allow the introduction of heterologous DNA into yeast D. bruxellensis. Subsequent measurements of gene targeting efficiency defined the length of the homologies at the end of the transforming fragment needed to introduce specific modifications into the genome. With this information, it is possible to construct biotechnologically interesting strains of D. bruxellensis which express new or lack existing genes. As such, these results represent a turning point for both fundamental and practical research of D. bruxellensis. As a proof-of-concept, a strain of D. bruxellensis was developed which can utilize xylose as a carbon source and as such is very interesting for the production of second-generation bioethanol from lignocellulose hydrolysates. |
