The Xylose Metabolic Engineering Of Kluyveromyces Marxianus And Changing The Substrate Specificity Of Rhll In Bacterial Quorum Sensing Using Directed Evolution

This study contains two parts.1. Study of xylose metabolic in Kluyveromyces marxianusMicrobial conversion of hemicellulose to produce the clean renewable energy ethanol has been one of the hot research fields of bioenergy. However, there are no microbial strains that directly efficient conversion of hemicellulose to ethanol in nature. In recent years, there are many studies on fermenting xylose using genetic engineering yeast, such as Saccharomyces cerevisiae. The difficulty lies in the using of five-carbon sugars especially xylose with yeast. In yeast cell, xylose was first reduced by xylose reductase and produced xylitol, following by xylitol dehydrogenase converted xylitol to xylulose and further metabolized to produce ethanol under oxygen limiting conditions. The studies of xylose metabolism in different strains will promoter the renewable energy produce from biomass.The ability of Kluyveromyces marxianus using glucose fermentation is comparable to Saccharomyces cerevisiae. However, K. marxianus could ferment xylose which S. cerevisiae could not. As a result, engineered K. marxianus has a great potential for xylose fenmentation. Exploring the K. marxianus xylose fermentation limiting factor is an important part for its industrial applications. In this study, we cloning the xylose reductase gene Kmxyll in K. marxianus and determined the characters of xylose reductase which indicated the reason of its low ability during xylose fermentation. According to the conservative part of the amino acid sequence, the xylose reductase in K. marxianus was cloned by TAIL-PCR for the first time and sequenced. Xylose reductase was then recombinant expressed in E. coli and analysized. Xylose reductase from K. marxianus prefers NADPH to NADH as coenzyme and the xylose reductase coenzyme preference may cause the low xylose fermentation in K. marxianus. To further verify the cloned xylose reductase, we constructed a xylose reductase gene disruption strain K. marxianus YZB001and it can not growth with xylose. In order to improve the xylose fermentation ability of K. marxianus, xylose reductase (PsXR) and its mutants PsXRN272D and PsXRK21A/N272D from Pichia stipitis (Scheffersomyces stipitis NBRC10063) were transformed into YZB001. The recombination strains significantly improved the K. marxianus xylose fermentation ability and the PsXRN272D mutants showed the best result compared to the wild-type K. marxiamis. K. marxiamis recombinant strains YZB013(PsXR) and YZB014(PsXRN272D) fermented20g/L xylose at42could increased3.63times and11.83times of ethanol production to1.10g/L and3.55g/L compared to the control strain YHJ010. When the temperature rises to45℃, YZB014could still fermented20g/L xylose and produced2.27g/L ethanol. When fermented with corncob acid hydrolyzate feedstock at42℃, YZB014produced4.09g/L ethanol. Recombinant K. marxianus strains showed its great potential in the transformation of biomass to bioethanol.2. Exploring the substrate specificity of Rhll in bacterial quorum sensing using directed evolutionIt is well established that quorum sensing is importing in regulation virulence factor generation during bacterial infections. There are large amounts of evidence that using quorum sensing inhibitors along with traditional antibiotics can effectively eliminate bacteria with multidrug resistance being increasingly observed in hospital bacterial infections, indicating quorum sensing inhibitors represent a novel class of antibacterial agents. There is also evidence that inhibiting quorum sensing is less likely to cause drug resistance development in bacteria when used strategically, making quorum sensing inhibitors interesting candidates as novel antibacterial drugs. In gram-negative bacteria, chemicals targeting LuxI and LuxR homologues can effectively inhibit quorum sensing, and drugs specifically inhibiting LuxI and LuxR homologues have been increasingly reported. In this research, we focus on enhancing the understanding of the substrate specificity of LuxI homologues to provide insightful information for developing drugs effectively targeting Luxl homologues. To achieve the goal, we use directed evolution, a highly successfully protein engineering strategy, to alter the substrate specificity of Rhll. RhlI catalyzes the synthesis of butanoyl homoserine lactone (C4HSL), with minor production of hexanoyl homosserine (C6HSL). Using directed evolution, we engineering RhlI for production of3-oxo-hexanoyl homoserine lactone (OHHL). An output and semi-quantitative screening system was building based on a green fluorescent protein. In the first round of screening, three mutants M8, S7and2M26were obtained and the output signal of fluorescence were increased80.02%,167.45%and170.59%compared to the parent protein. In the second round of screening, signals producted by mutant4M1activation the green fluorescent20.27%higher than2M26 did. With such changes of substrate specificity, it is expected that an enhanced understanding of the substrate specificity of RhlI will be rebealed, and such information will be valuable for developing novel drugs effectively targeting RhlI, in particular the RhlI enzyme in the RhlI-RhlR quorum sensing system of the opportunistic bacterium P. aeruginosa.