Conversion of levoglucosan and cellobiosan by Pseudomonas putida KT2440

Pyrolysis offers a straightforward approach for the deconstruction of plant cell wall polymers into bio-oil. Recently, there has been substantial interest in bio-oil fractionation and subsequent use of biological approaches to selectively upgrade some of the resulting fractions. A fraction of particular interest for biological upgrading consists of polysaccharide-derived substrates including sugars and sugar dehydration products such as levoglucosan and cellobiosan, which are two of the most abundant pyrolysis products of cellulose. Levoglucosan can be converted to glucose-6-phosphate through the use of a levoglucosan kinase (LGK), but to date, the mechanism for cellobiosan utilization has not been demonstrated. Here, we engineer the microbe Pseudomonas putida KT2440 to use levoglucosan as a sole carbon and energy source through LGK integration. Moreover, we demonstrate that cellobiosan can be enzymatically converted to levoglucosan and glucose with β-glucosidase enzymes from both Glycoside Hydrolase Family 1 and Family 3. β-glucosidases are commonly used in both natural and industrial cellulase cocktails to convert cellobiose to glucose to relieve cellulase product inhibition and to facilitate microbial uptake of glucose. Using an exogenous β-glucosidase, we demonstrate that the engineered strain of P. putida can grow on levoglucosan up to 60 g/L and can also utilize cellobiosan. Overall, this study elucidates the biological pathway to co-utilize levoglucosan and cellobiosan, which will be a key transformation for the biological upgrading of pyrolysis-derived substrates. Keywords: Pseudomonas putida KT2440, Levoglucosan kinase, B-glucosidase, Cellobiosan, Pyrolysis, Biofuel

Microbial sensor variation across biogeochemical conditions in the terrestrial deep subsurface.

ImportanceThe ability to detect extracellular environmental conditions is a fundamental property of all life forms. Because microbial two-component sensor systems convert information about extracellular conditions into biochemical information that controls their behaviors, we evaluated how two-component sensor systems evolved within the deep Earth across multiple sites where abiotic and biotic properties vary. We show that these sensor systems remain abundant in microbial consortia at all subterranean sampling sites and observe correlations between sensor system abundances and abiotic (dissolved organic carbon variation) and biotic (consortia diversity) properties. These results suggest that multiple environmental properties may drive sensor protein evolution and highlight the need for further studies of metagenomic and geochemical data in parallel to understand the drivers of microbial sensor evolution

Microbial sensor variation across biogeochemical conditions in the terrestrial deep subsurface

ABSTRACTMicrobes can be found in abundance many kilometers underground. While microbial metabolic capabilities have been examined across different geochemical settings, it remains unclear how changes in subsurface niches affect microbial needs to sense and respond to their environment. To address this question, we examined how microbial extracellular sensor systems vary with environmental conditions across metagenomes at different Deep Mine Microbial Observatory (DeMMO) subsurface sites. Because two-component systems (TCSs) directly sense extracellular conditions and convert this information into intracellular biochemical responses, we expected that this sensor family would vary across isolated oligotrophic subterranean environments that differ in abiotic and biotic conditions. TCSs were found at all six subsurface sites, the service water control, and the surface site, with an average of 0.88 sensor histidine kinases (HKs) per 100 genes across all sites. Abundance was greater in subsurface fracture fluids compared with surface-derived fluids, and candidate phyla radiation (CPR) bacteria presented the lowest HK frequencies. Measures of microbial diversity, such as the Shannon diversity index, revealed that HK abundance is inversely correlated with microbial diversity (r2 = 0.81). Among the geochemical parameters measured, HK frequency correlated most strongly with variance in dissolved organic carbon (r2 = 0.82). Taken together, these results implicate the abiotic and biotic properties of an ecological niche as drivers of sensor needs, and they suggest that microbes in environments with large fluctuations in organic nutrients (e.g., lacustrine, terrestrial, and coastal ecosystems) may require greater TCS diversity than ecosystems with low nutrients (e.g., open ocean).IMPORTANCEThe ability to detect extracellular environmental conditions is a fundamental property of all life forms. Because microbial two-component sensor systems convert information about extracellular conditions into biochemical information that controls their behaviors, we evaluated how two-component sensor systems evolved within the deep Earth across multiple sites where abiotic and biotic properties vary. We show that these sensor systems remain abundant in microbial consortia at all subterranean sampling sites and observe correlations between sensor system abundances and abiotic (dissolved organic carbon variation) and biotic (consortia diversity) properties. These results suggest that multiple environmental properties may drive sensor protein evolution and highlight the need for further studies of metagenomic and geochemical data in parallel to understand the drivers of microbial sensor evolution