Bianca C. Neves

Rhamnolipids in perspective: gene regulatory pathways, metabolic engineering, production and technological forecasting

Rhamnolipids have emerged as a very promising class of biosurfactants in the last decades, exhibiting properties of great interest in several industrial applications, and have represented a suitable alternative to chemically-synthesized surfactants. This class of biosurfactants has been extensively studied in recent years, aiming at their large-scale production based on renewable resources, which still require high financial costs. Development of non-pathogenic, high-producing strains has been the focus of a number of studies involving heterologous microbial hosts as platforms. However, the intricate gene regulation network controlling rhamnolipid biosynthesis represents a challenge to metabolic engineering and remains to be further understood and explored. This article provides an overview of the biosynthetic pathways and the main gene regulatory factors involved in rhamnolipid production within Pseudomonas aeruginosa, the prototypal producing species. In addition, we provide a perspective view into the main strategies applied to metabolic engineering and biotechnological production.

Enhanced xylose fermentation and ethanol production by engineered Saccharomyces cerevisiae strain

We have recently demonstrated that heterologous expression of a bacterial xylose isomerase gene (xylA) of Burkholderia cenocepacia enabled a laboratorial Saccharomyces cerevisiae strain to ferment xylose anaerobically, without xylitol accumulation. However, the recombinant yeast fermented xylose slowly. In this study, an evolutionary engineering strategy was applied to improve xylose fermentation by the xylA-expressing yeast strain, which involved sequential batch cultivation on xylose. The resulting yeast strain co-fermented glucose and xylose rapidly and almost simultaneously, exhibiting improved ethanol production and productivity. It was also observed that when cells were grown in a medium containing higher glucose concentrations before being transferred to fermentation medium, higher rates of xylose consumption and ethanol production were obtained, demonstrating that xylose utilization was not regulated by catabolic repression. Results obtained by qPCR demonstrate that the efficiency in xylose fermentation showed by the evolved strain is associated, to the increase in the expression of genes HXT2 and TAL1, which code for a low-affinity hexose transporter and transaldolase, respectively. The ethanol productivity obtained after the introduction of only one genetic modification and the submission to a one-stage process of evolutionary engineering was equivalent to those of strains submitted to extensive metabolic and evolutionary engineering, providing solid basis for future applications of this strategy in industrial strains.

Functional expression of Burkholderia cenocepacia xylose isomerase in yeast increases ethanol production from a glucose–xylose blend

This study presents results regarding the successful cloning of the bacterial xylose isomerase gene (xylA) of Burkholderia cenocepacia and its functional expression in Saccharomyces cerevisiae. The recombinant yeast showed to be competent to efficiently produce ethanol from both glucose and xylose, which are the main sugars in lignocellulosic hydrolysates. The heterologous expression of the gene xylA enabled a laboratorial yeast strain to ferment xylose anaerobically, improving ethanol production from a fermentation medium containing a glucose-xylose blend similar to that found in sugar cane bagasse hydrolysates. The insertion of xylA caused a 5-fold increase in xylose consumption, and over a 1.5-fold increase in ethanol production and yield, in comparison to that showed by the WT strain, in 24h fermentations, where it was not detected accumulation of xylitol. These findings are encouraging for further studies concerning the expression of B. cenocepacia xylA in an industrial yeast strain.

Short-chain Fatty Acids in Infected Root Canals of Teeth with Apical Periodontitis before and after Treatment

INTRODUCTION Short-chain fatty acids (SCFAs) are bacterial metabolic end products that may function as virulence factors. This study evaluated the occurrence of SCFAs in infected root canals before and after treatment. METHODS Samples were taken from root canals of teeth with apical periodontitis before (S1) and after (S2) chemomechanical preparation with either NaOCl or chlorhexidine as the irrigant and then after interappointment medication with calcium hydroxide (S3). High-performance liquid chromatography was used for detection of SCFAs. Selected bacterial taxa that are recognized producers of the target SCFAs were identified by real-time polymerase chain reaction. RESULTS Butyric acid was the most common fatty acid in S1, followed by propionic acid. Both molecules were also found in S2 and S3 from both NaOCl and chlorhexidine groups. Lactic acid was not present in detectable levels in S1, but it occurred in 1 postinstrumentation sample and in 9 samples taken after calcium hydroxide medication. Of the target taxa, Fusobacterium nucleatum was the most prevalent in S1 (76%), followed by members of the Actinobacteria phylum (71%), Streptococcus species (59%), and Parvimonas micra (53%). Gram-positive taxa, especially streptococci, were the most prevalent bacteria in S2 and S3. SCFA detection was matched with the respective potential producer species in most cases. CONCLUSIONS This first report of SCFAs in infected root canals suggests that these molecules may play a role in the pathogenesis of apical periodontitis. Significance of persistence of SCFAs after treatment and its effects on the long-term outcome await elucidation.

Type III apparatus of Pseudomonas aeruginosa as a tool to diagnose pulmonary infection in cystic fibrosis patients

Pseudomonas aeruginosa is associated with increased mortality in cystic fibrosis (CF) patients, and expresses type III secretion system proteins (TTSP), which is a common mechanism used by gram‐negative pathogens for delivery of anti‐host factors. Our aim was to investigate whether or not these antigens (TTSP) would be recognized by CF sera, by Western blot reaction. We have showed herein that all patients (n = 11) not chronically infected by P. aeruginosa had their first serum positive for TTSP (ExoS, ExoT, PopB, and/or PopD). All chronic patients had a strong positive serology to TTSP, although relatively weak reactions to TTSP were observed for some individuals in the negative control group. Therefore, TTSP that were early produced in P. aeruginosa infected CF patients, induced a detectable antibody response in those patients and were easily detected by Western‐blot reaction.

Enhanced rhamnolipid production by Pseudomonas aeruginosa overexpressing estA in a simple medium

A modified Pseudomonas aeruginosa strain capable of overexpressing the estA gene, an encoding gene for a membrane-bound esterase, was constructed and its rhamnolipid (RML) production was studied. Fermentations using wild-type (WT) and modified P. aeruginosa strains were conducted until exhaustion of glycerol in Medium Salt Production, using two different C/N ratios. At a C/N of 83.2, the modified strain produced up to 3.9 times more RMLs than the WT, yielding a maximum concentration of 14.62 g/L RML when measured by HPLC and 22 g/L by the orcinol assay. Cell-free supernatant from the modified strain reduced surface tension to 29.4 mN/m and had a CMC of 240 mg/L and CMD of 56.05. This is the first report on the construction of an estA-based recombinant strain for RML production.