Production of biodegradable plastics using sugarcane bagasse hemicellulosic hydrolysate.

O objetivo deste trabalho foi produzir poli-3-hidroxibutirato (P3HB) e poli-3-hidroxibutirato-co-3-hidroxivalerato (PHB-co-3HV), polímeros biodegradáveis, utilizando hidrolisado hemicelulósico, rico em xilose, de bagaço de cana-de-açúcar. O estudo dos fluxos metabólicos de xilose in silico indicou que, através do redirecionamento do metabolismo, é possível aumentar o rendimento P3HB a partir de xilose de 0.25 g g-1 para 0.40 g g-1. Obtiveram-se mutantes no sistema repressão catabólica nos quais se verificaram consumo simultâneo de carboidratos e redução do tempo de consumo dos açúcares. Porém, diferenças de fluxos de carbono resultaram em menores valores de crescimento e produção de PH3B em relação às linhagens parentais. Um programa de bioprospecção destacou Burkholderia sp. F24, em experimentos em biorreator obteve-se 25.04 g l-1 de biomassa, 49.31% de acúmulo de P3HB na massa seca celular, alcançando uma produtividade de 0.28 g l-1 h-1. Além disso, foi possível controlar a fração molar de 3HV na síntese PHB-3HV em F24 utilizando xilose e ácido levulínico.The aim of this thesis is to produce poly3-hydroxybutyrate (P3HB) and poli-3-hidroxibutirate-co-3-hydroxyvalerate (PHB-co-3HV), biodegradable polymers, using hemicellulosic hydrolysate, rich in xylose, from sugarcane bagasse. Metabolic flux analysis in silico of xylose metabolism indicated that, though metabolism redirection is possible to increase P3HB yield from 0.25 g g-1 to 0.40 g g-1. It was observed simultaneous consumption of sugars and reduction of time necessary to exhaust of all sugars in the media culture in mutants with catabolite repression partially abolished. However, differences in carbon flux resulted in lower growth and P3HB production in comparison to the parental strain. A bioprospecting program selected Burkholderia sp. F24, in experiments in bioreactor it reached 25.04 g l-1, 49.31% of P3HB accumulation of the dry cell mass and 0.28 g l-1 h-1 of productivity. Moreover, it was possible to modulate to molar fraction of 3HV in PHB-co-3HV biosyntheses with Burkholderia sp. F24 using xylose and levulinic acid

Cloning and overexpression of the xylose isomerase gene from Burkholderia sacchari and production of polyhydroxybutyrate from xylose

A different organization for the xyl operon was found in different genomes of Burkholderia and Pseudomomas species. Degenerated primers were designed based on Burkholderia genomes and used to amplify the xylose isomerase gene (xylA) from Burkholderia sacchari IPT101 The gene encoded a protein of 329 amino acids, which showed the highest similarity (90%) to the homologous gene of Burkholderia dolosa. It was cloned in the broad host range plasmid pBBR1MCS-2, which partially restored growth and polyhydroxybutyrate production capability in xylose to a B. sacchari xyl(-) mutant. When xylA was overexpressed in the wild-type strain, it was not able to increase growth and polyhydroxybutyrate production, suggesting that XylA activity is not limiting for xylose utilization in B. sacchari.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[04/15369-0

PHB Biosynthesis in Catabolite Repression Mutant of Burkholderia sacchari

Due to the effect of catabolite repression, sugar mixtures cannot be metabolized in a rapid and efficient way implicating in lower productivity in bioprocesses using lignocellulosic hydrolysates. In gram-negative bacteria, this mechanism is mediated by the phosphotransferase system (PTS), which concomitantly internalizes and phosphorylates sugars. In this study, we isolated a UV mutant of Burkholderia sacchari, called LFM828, which transports hexoses and pentoses by a non-PTS uptake system. This mutant presented released glucose catabolite repression over the pentoses. In mixtures of glucose, xylose, and arabinose, specific growth rates and the specific sugar consumption rates were, respectively, 10 and 23% higher in LFM828, resulting in a reduced time to exhaust all sugars in the medium. However, in polyhydroxybutyrate (PHB) biosynthesis experiments it was necessary the supplementation of yeast extract to maintain higher values of growth rate and sugar consumption rate. The deficient growth in mineral medium was partially recovered by replacing the ammonium nitrogen source by glutamate. It was demonstrated that the ammonium metabolism is not defective in LFM828, differently from ammonium, glutamate can also be used as carbon and energy allowing an improvement on the carbohydrates utilization for PHB production in LFM828. In contrast, higher rates of ammonia consumption and CO(2) production in LFM828 indicate altered fluxes through the central metabolism in LFM828 and the parental. In conclusion, PTS plays an important role in cell physiology and the elimination of its components has a significant impact on catabolite repression, carbon flux distribution, and PHB biosynthesis in B. sacchari.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)International Mobility of Santander BankInternational Mobility of Santander Ban

Screening of bacteria to produce polyhydroxyalkanoates from xylose

Although xylose is a major constituent of lignocellulosic feedstock and the second most abundant sugar in nature, only 22% of 3,152 screened bacterial isolates showed significant growth in xylose in 24 h. Of those 684, only 24% accumulated polyhydroxyalkanoates after 72 h. A mangrove isolate, identified as Bacillus sp. MA3.3, yielded the best results in literature thus far for Gram-positive strains in experiments with glucose and xylose as the sole carbon source. When glucose or xylose were supplied, poly-3-hydroxybutyrate (PHB) contents of cell dry weight were, respectively, 62 and 64%, PHB yield 0.25 and 0.24 g g(-1) and PHB productivity (P(PHB)) 0.10 and 0.06 g l(-1) h(-1). This 40% P(PHB) difference may be related to the theoretical ATP production per 3-hydroxybutyrate (3HB) monomer calculated as 3 mol mol(-1) for xylose, less than half of the ATP/3HB produced from glucose (7 mol mol(-1)). In PHB production using sugar mixtures, all parameters were strongly reduced due to carbon catabolite repression. PHB production using Gram-positive strains is particularly interesting for medical applications because these bacteria do not produce lipopolysaccharide endotoxins which can induce immunogenic reactions. Moreover, the combination of inexpensive substrates and products of more value may lead to the economical sustainability of industrial PHB production.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

The diversity of polyketide synthase genes from sugarcane-derived fungi

The chemical ecology and biotechnological potential of metabolites from endophytic and rhizosphere fungi are receiving much attention. A collection of 17 sugarcane-derived fungi were identified and assessed by PCR for the presence of polyketide synthase (PKS) genes. The fungi were all various genera of ascomycetes, the genomes of which encoded 36 putative PKS sequences, 26 shared sequence homology with beta-ketoacyl synthase domains, while 10 sequences showed homology to known fungal C-methyltransferase domains. A neighbour-joining phylogenetic analysis of the translated sequences could group the domains into previously established chemistry-based clades that represented non-reducing, partially reducing and highly reducing fungal PKSs. We observed that, in many cases, the membership of each clade also reflected the taxonomy of the fungal isolates. The functional assignment of the domains was further confirmed by in silico secondary and tertiary protein structure predictions. This genome mining study reveals, for the first time, the genetic potential of specific taxonomic groups of sugarcane-derived fungi to produce specific types of polyketides. Future work will focus on isolating these compounds with a view to understanding their chemical ecology and likely biotechnological potential633565577CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPThe Academy of Science for the Developing World (TWAS

The Diversity of Polyketide Synthase Genes from Sugarcane-Derived Fungi

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The chemical ecology and biotechnological potential of metabolites from endophytic and rhizosphere fungi are receiving much attention. A collection of 17 sugarcane-derived fungi were identified and assessed by PCR for the presence of polyketide synthase (PKS) genes. The fungi were all various genera of ascomycetes, the genomes of which encoded 36 putative PKS sequences, 26 shared sequence homology with beta-ketoacyl synthase domains, while 10 sequences showed homology to known fungal C-methyltransferase domains. A neighbour-joining phylogenetic analysis of the translated sequences could group the domains into previously established chemistry-based clades that represented non-reducing, partially reducing and highly reducing fungal PKSs. We observed that, in many cases, the membership of each clade also reflected the taxonomy of the fungal isolates. The functional assignment of the domains was further confirmed by in silico secondary and tertiary protein structure predictions. This genome mining study reveals, for the first time, the genetic potential of specific taxonomic groups of sugarcane-derived fungi to produce specific types of polyketides. Future work will focus on isolating these compounds with a view to understanding their chemical ecology and likely biotechnological potential.633565577The Academy of Science for the Developing World (TWAS)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq