An Extracellular Cell-Attached Pullulanase Confers Branched α-Glucan Utilization in Human Gut Lactobacillus acidophilus.

Of the few predicted extracellular glycan-active enzymes, glycoside hydrolase family 13 subfamily 14 (GH13_14) pullulanases are the most common in human gut lactobacilli. These enzymes share a unique modular organization, not observed in other bacteria, featuring a catalytic module, two starch binding modules, a domain of unknown function, and a C-terminal surface layer association protein (SLAP) domain. Here, we explore the specificity of a representative of this group of pullulanases, Lactobacillus acidophilus Pul13_14 (LaPul13_14), and its role in branched α-glucan metabolism in the well-characterized Lactobacillus acidophilus NCFM, which is widely used as a probiotic. Growth experiments with L. acidophilus NCFM on starch-derived branched substrates revealed a preference for α-glucans with short branches of about two to three glucosyl moieties over amylopectin with longer branches. Cell-attached debranching activity was measurable in the presence of α-glucans but was repressed by glucose. The debranching activity is conferred exclusively by LaPul13_14 and is abolished in a mutant strain lacking a functional LaPul13_14 gene. Hydrolysis kinetics of recombinant LaPul13_14 confirmed the preference for short-branched α-glucan oligomers consistent with the growth data. Curiously, this enzyme displayed the highest catalytic efficiency and the lowest Km reported for a pullulanase. Inhibition kinetics revealed mixed inhibition by ß-cyclodextrin, suggesting the presence of additional glucan binding sites besides the active site of the enzyme, which may contribute to the unprecedented substrate affinity. The enzyme also displays high thermostability and higher activity in the acidic pH range, reflecting adaptation to the physiologically challenging conditions in the human gut.IMPORTANCE Starch is one of the most abundant glycans in the human diet. Branched α-1,6-glucans in dietary starch and glycogen are nondegradable by human enzymes and constitute a metabolic resource for the gut microbiota. The role of health-beneficial lactobacilli prevalent in the human small intestine in starch metabolism remains unexplored in contrast to colonic bacterial residents. This study highlights the pivotal role of debranching enzymes in the breakdown of starchy branched α-glucan oligomers (α-limit dextrins) by human gut lactobacilli exemplified by Lactobacillus acidophilus NCFM, which is one of the best-characterized strains used as probiotics. Our data bring novel insight into the metabolic preference of L. acidophilus for α-glucans with short α-1,6-branches. The unprecedented affinity of the debranching enzyme that confers growth on these substrates reflects its adaptation to the nutrient-competitive gut ecological niche and constitutes a potential advantage in cross-feeding from human and bacterial dietary starch metabolism.

Genome Sequence of Talaromyces atroroseus, Which Produces Red Colorants for the Food Industry.

Talaromyces atroroseus is a known producer of Monascus colorants suitable for the food industry. Furthermore, genetic tools have been established that facilitate elucidation and engineering of its biosynthetic pathways. Here, we report the draft genome of a potential fungal cell factory, T. atroroseus IBT 11181 (CBS 123796).

Genes Linked to Production of Secondary Metabolites in Talaromyces atroroseus Revealed Using CRISPR-Cas9.

The full potential of fungal secondary metabolism has until recently been impeded by the lack of universal genetic tools for most species. However, the emergence of several CRISPR-Cas9-based genome editing systems adapted for several genera of filamentous fungi have now opened the doors for future efforts in discovery of novel natural products and elucidation and engineering of their biosynthetic pathways in fungi where no genetic tools are in place. So far, most studies have focused on demonstrating the performance of CRISPR-Cas9 in various fungal model species, and recently we presented a versatile CRISPR-Cas9 system that can be successfully applied in several diverse Aspergillus species. Here we take it one step further and show that our system can be used also in a phylogenetically distinct and largely unexplored species from the genus of Talaromyces. Specifically, we exploit CRISPR-Cas9-based genome editing to identify a new gene in T. atroroseus responsible for production of polyketide-nonribosomal peptide hybrid products, hence, linking fungal secondary metabolites to their genetic origin in a species where no genetic engineering has previously been performed.

Talaromyces atroroseus, a new species efficiently producing industrially relevant red pigments.

Some species of Talaromyces secrete large amounts of red pigments. Literature has linked this character to species such as Talaromyces purpurogenus, T. albobiverticillius, T. marneffei, and T. minioluteus often under earlier Penicillium names. Isolates identified as T. purpurogenus have been reported to be interesting industrially and they can produce extracellular enzymes and red pigments, but they can also produce mycotoxins such as rubratoxin A and B and luteoskyrin. Production of mycotoxins limits the use of isolates of a particular species in biotechnology. Talaromyces atroroseus sp. nov., described in this study, produces the azaphilone biosynthetic families mitorubrins and Monascus pigments without any production of mycotoxins. Within the red pigment producing clade, T. atroroseus resolved in a distinct clade separate from all the other species in multigene phylogenies (ITS, ß-tubulin and RPB1), which confirm its unique nature. Talaromyces atroroseus resembles T. purpurogenus and T. albobiverticillius in producing red diffusible pigments, but differs from the latter two species by the production of glauconic acid, purpuride and ZG-1494α and by the dull to dark green, thick walled ellipsoidal conidia produced. The type strain of Talaromyces atroroseus is CBS 133442.