Influence of Mono- and Oligosaccharides on the cbh1 Gene Transcripion in the Penicillium verruculosum Filamentous Fungus

A method has been developed for the analysis of the induction of the cbh1 gene transcription in the filamentous fungus Penicillium verruculosum after treatment with mono- and oligosaccharides. The method allows obtaining mRNA, which encodes cellobiohydrolase-1 (CBH1) in an amount sufficient for RT-PCR. Citrate, a compound that does not cause carbon catabolic repression, was used as a carbon source. It was shown for the first time that xylose, gentiobiose, and mainly cellobiose induce the expression of the cbh1 gene in P. verruculosum, while sophorose and a mixture of xylooligosaccharides with a degree of polymerization from 3 to 5 are most likely to serve as precursors of inducers. Penicillium verruculosum, qPCR, RT-PCR, monosaccharides, oligosaccharides, inducer The work was supported by the Russian Foundation for Basic Research (RFBR) (grant no.18-29-07070).

Development of a genetic toolset for the highly engineerable and metabolically versatile Acinetobacter baylyi ADP1

One primary objective of synthetic biology is to improve the sustainability of chemical manufacturing. Naturally occurring biological systems can utilize a variety of carbon sources, including waste streams that pose challenges to traditional chemical processing, such as lignin biomass, providing opportunity for remediation and valorization of these materials. Success, however, depends on identifying micro-organisms that are both metabolically versatile and engineerable. Identifying organisms with this combination of traits has been a historic hindrance. Here, we leverage the facile genetics of the metabolically versatile bacterium Acinetobacter baylyi ADP1 to create easy and rapid molecular cloning workflows, including a Cas9-based single-step marker-less and scar-less genomic integration method. In addition, we create a promoter library, ribosomal binding site (RBS) variants and test an unprecedented number of rationally integrated bacterial chromosomal protein expression sites and variants. At last, we demonstrate the utility of these tools by examining ADP1’s catabolic repression regulation, creating a strain with improved potential for lignin bioprocessing. Taken together, this work highlights ADP1 as an ideal host for a variety of sustainability and synthetic biology applications.

Convergence between Regulation of Carbon Utilization and Catabolic Repression in Xanthophyllomyces dendrorhous

ABSTRACT Xanthophyllomyces dendrorhous is a carotenogenic yeast with a singular metabolic capacity to produce astaxanthin, a valuable antioxidant pigment. This yeast can assimilate several carbon sources and sustain fermentation even under aerobic conditions. Since astaxanthin biosynthesis is affected by the carbon source, the study of carotenogenesis regulatory mechanisms is key for improving astaxanthin yield in X. dendrorhous. This study aimed to elucidate the regulation of the metabolism of different carbon sources and the phenomenon of catabolic repression in this yeast. To this end, protein and transcript levels were quantified by iTRAQ (isobaric tags for relative and absolute quantification) and transcriptomic sequencing (RNA-seq) in the wild-type strain under conditions of glucose, maltose, or succinate treatment and in the mutant strains for genes MIG1, CYC8, and TUP1 under conditions of glucose treatment. Alternative carbon sources such as maltose and succinate affected the relative abundances of 14% of the wild-type proteins, which were mainly grouped into the carbohydrate metabolism category, with the glycolysis/gluconeogenesis and citrate cycle pathways being the most highly represented pathways. Each mutant strain showed significant proteomic profile changes, affecting approximately 2% of the total proteins identified, compared to the wild-type strain under glucose treatment conditions. Similarly to the results seen with the alternative carbon sources, the changes in the mutant strains mainly affected carbohydrate metabolism, with glycolysis/gluconeogenesis and the pentose phosphate and citrate cycle pathways being the most highly represented pathways. Our results showed convergence between carbon assimilation and catabolic repression in the strains studied. Interestingly, indications of cooperative, opposing, and overlapping processes during catabolic regulation were found. We also identified target proteins of the regulatory processes, reinforcing the likelihood of catabolic repression at the posttranscriptional level. IMPORTANCE The conditions affecting catabolic regulation in X. dendrorhous are complex and suggest the presence of an alternative mechanism of regulation. The repressors Mig1, Cyc8, and Tup1 are essential elements for the regulation of the use of glucose and other carbon sources. All play different roles but, depending on the growth conditions, can work in convergent, synergistic, and complementary ways to use carbon sources and to regulate other targets for yeast metabolism. Our results reinforced the belief that further studies in X. dendrorhous are needed to clarify a specific regulatory mechanism at the domain level of the repressors as well as its relationship with those of other metabolic repressors, i.e., the stress response, to elucidate carotenogenic regulation at the transcriptomic and proteomic levels in this yeast.

Development of a genetic toolset for the highly engineerable and metabolically versatile Acinetobacter baylyi ADP1

One primary objective of synthetic biology is to improve the sustainability of chemical manufacturing. Biological systems can utilize a variety of carbon sources, including waste streams that pose challenges to traditional chemical processing such as lignin biomass, providing opportunity for remediation and valorization of these materials. Success, however, depends on identifying microorganisms that are both metabolically versatile and engineerable. This has been a historic hindrance. Here, we leverage the facile genetics of the metabolically versatile bacterium Acinetobacter baylyi ADP1 to create easy and rapid molecular cloning workflows, a promoter library, ribosomal binding site (RBS) variants, and an unprecedented number of bacterial chromosomal protein expression sites and variants. Moreover, we demonstrate the utility of these tools by examining ADP1’s catabolic repression regulation, creating a strain with improved potential for lignin bioprocessing. Taken together, this work establishes ADP1 as an ideal host for a variety of sustainability and synthetic biology applications.

ThefruRBAOperon Is Necessary for Group A Streptococcal Growth in Fructose and for Resistance to Neutrophil Killing during Growth in Whole Human Blood

Bacterial pathogens rely on the availability of nutrients for survival in the host environment. The phosphoenolpyruvate-phosphotransferase system (PTS) is a global regulatory network connecting sugar uptake with signal transduction. Since the fructose PTS has been shown to impact virulence in several streptococci, including the human pathogenStreptococcus pyogenes(the group AStreptococcus[GAS]), we characterized its role in carbon metabolism and pathogenesis in the M1T1 strain 5448. Growth in fructose as a sole carbon source resulted in 103 genes affected transcriptionally, where thefrulocus (fruRBA) was the most induced. Reverse transcriptase PCR showed thatfruRBAformed an operon which was repressed by FruR in the absence of fructose, in addition to being under carbon catabolic repression. Growth assays and carbon utilization profiles revealed that although the entirefruoperon was required for growth in fructose, FruA was the main transporter for fructose and also was involved in the utilization of three additional PTS sugars: cellobiose, mannitol, andN-acetyl-d-galactosamine. The inactivation ofsloR, afruAhomolog that also was upregulated in the presence of fructose, failed to reveal a role as a secondary fructose transporter. Whereas the ability of both ΔfruRand ΔfruBmutants to survive in the presence of whole human blood or neutrophils was impaired, the phenotype was not reproduced in murine whole blood, and those mutants were not attenuated in a mouse intraperitoneal infection. Since the ΔfruAmutant exhibited no phenotype in the human or mouse assays, we propose that FruR and FruB are important for GAS survival in a human-specific environment.

Polysaccharides and Proteins Added to Flowing Drinking Water at Microgram-per-Liter Levels Promote the Formation of Biofilms Predominated by Bacteroidetes and Proteobacteria

ABSTRACTBiopolymers are important substrates for heterotrophic bacteria in (ultra)oligotrophic freshwater environments, but information about their utilization at microgram-per-liter levels by attached freshwater bacteria is lacking. This study aimed at characterizing biopolymer utilization in drinking-water-related biofilms by exposing such biofilms to added carbohydrates or proteins at 10 μg C liter−1in flowing tap water for up to 3 months. Individually added amylopectin was not utilized by the biofilms, whereas laminarin, gelatin, and caseinate were. Amylopectin was utilized during steady-state biofilm growth with simultaneously added maltose but not with simultaneously added acetate. Biofilm formation rates (BFR) at 10 μg C liter−1per substrate were ranked as follows, from lowest to highest: blank or amylopectin (≤6 pg ATP cm−2day−1), gelatin or caseinate, laminarin, maltose, acetate alone or acetate plus amylopectin, and maltose plus amylopectin (980 pg ATP cm−2day−1). Terminal restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene sequence analyses revealed that the predominant maltose-utilizing bacteria also dominated subsequent amylopectin utilization, indicating catabolic repression and (extracellular) enzyme induction. The accelerated BFR with amylopectin in the presence of maltose probably resulted from efficient amylopectin binding to and hydrolysis by inductive enzymes attached to the bacterial cells.Cytophagia,Flavobacteriia,Gammaproteobacteria, andSphingobacteriiagrew during polysaccharide addition, andAlpha-,Beta-, andGammaproteobacteria,Cytophagia,Flavobacteriia, andSphingobacteriiagrew during protein addition. The succession of bacterial populations in the biofilms coincided with the decrease in the specific growth rate during biofilm formation. Biopolymers can clearly promote biofilm formation at microgram-per-liter levels in drinking water distribution systems and, depending on their concentrations, might impair the biological stability of distributed drinking water.