Dynamic control systems that mimic natural regulation of catabolic pathways enable rapid production of lignocellulose-derived bioproducts

Expanding the catabolic repertoire of engineered microbial bioproduction hosts enables more efficient use of complex feedstocks such as lignocellulosic hydrolysates, but the deleterious effects of existing expression systems limit the maximum carry capacity for heterologous catabolic pathways. Here we demonstrate use of a conditionally beneficial oxidative xylose catabolic pathway to improve performance of a Pseudomonas putida strain that has been engineered for growth-coupled bioconversion of glucose into the valuable bioproduct cis,cis-muconic acid. In the presence of xylose, the pathway enhances growth rate, and therefor productivity, by >60%, but the metabolic burden of constitutive pathway expression reduces the its growth rate by >20% in the absence of xylose. To mitigate this growth defect, we develop a xylose biosensor based on the XylR transcription factor from Caulobacter crescentus NA1000 to autonomously regulate pathway expression. We generate a library of engineered xylose-sensitive promoters that cover a three order-of-magnitude range of expression levels to tune pathway expression. Using structural modeling to guide mutations, we engineer XylR with two and three orders-of-magnitude reduced sensitivity to xylose and L-arabinose, respectively. A previously developed heterologous xylose isomerase pathway is placed under control of the biosensor, which improves the growth rate with xylose as a carbon source by 10% over the original constitutively expressed pathway. Finally, the oxidative xylose catabolic pathway is placed under control of the biosensor, enabling the bioproduction strain to maintain the increased growth rate in the presence of xylose, without the growth defect incurred from constitutive pathway expression in the absence of xylose. Utilizing biosensors to autonomously regulate conditionally beneficial catabolic pathways is generalizable, and will be critical for engineering bioproduction hosts bacteria with the wide range of catabolic pathways required for bioconversion of complex feedstocks.

Microbial protein metabolism in the monogastric gastrointestinal tract: a review

Dietary and endogenous protein that become available for the microbiota in the hindgut can be metabolized via different routes. They can become building blocks for the microbial cells or enter different catabolic pathways. Protein degradation via fermentation pathways is seen as a non-preferred route as it results in the formation and release of metabolites that can interfere with biological systems in the host and can have deleterious outcomes. Reducing protein fermentation and guiding the metabolism towards less toxic end-products might be possible targets for improving host health. To do so, more knowledge on factors manipulating the process of microbial protein metabolism, including on substrate availability, microbial composition and segmental differences in the hindgut, is required.

Novel Insights into Dimethylsulfoniopropionate Catabolism by Cultivable Bacteria in the Arctic Kongsfjorden

Dimethylsulfoniopropionate (DMSP) is one of the most abundant organic sulfur compounds in the oceans, which is mainly degraded by bacteria through two pathways, a cleavage pathway and a demethylation pathway. Its volatile catabolites dimethyl sulfide (DMS) and methanethiol (MT) in these pathways play important roles in the global sulfur cycle and have potential influences on the global climate. Intense DMS/DMSP cycling occurs in the Arctic. However, little is known about the diversity of cultivable DMSP-catabolizing bacteria in the Arctic and how they catabolize DMSP. Here, we screened DMSP-catabolizing bacteria from Arctic samples and found that bacteria of four genera ( Psychrobacter , Pseudoalteromonas , Alteromonas and Vibrio ) could grow with DMSP as the sole carbon source, among which Psychrobacter and Pseudoalteromonas are predominant. Four representative strains ( Psychrobacter sp. K31L, Pseudoalteromonas sp. K222D, Alteromonas sp. K632G and Vibrio sp. G41H) from different genera were selected to probe their DMSP catabolic pathways. All these strains produce DMS and MT simultaneously during their growth on DMSP, indicating that all strains likely possess the two DMSP catabolic pathways. On the basis of genomic and biochemical analyses, the DMSP catabolic pathways in these strains were proposed. Bioinformatic analysis indicated that most bacteria of Psychrobacter and Vibrio have the potential to catabolize DMSP via the demethylation pathway, and that only a small portion of Psychrobacter strains may catabolize DMSP via the cleavage pathway. This study provides novel insights into DMSP catabolism in marine bacteria. IMPORTANCE Dimethylsulfoniopropionate (DMSP) is abundant in the oceans. The catabolism of DMSP is an important step of the global sulfur cycle. Although Gammaproteobacteria are widespread in the oceans, the contribution of Gammaproteobacteria in global DMSP catabolism is not fully understood. Here, we found that bacteria of four genera belonging to Gammaproteobacteria ( Psychrobacter , Pseudoalteromonas , Alteromonas and Vibrio ), which were isolated from Arctic samples, were able to grow on DMSP. The DMSP catabolic pathways of representative strains were proposed. Bioinformatic analysis indicates that most bacteria of Psychrobacter and Vibrio have the potential to catabolize DMSP via the demethylation pathway, and that only a small portion of Psychrobacter strains may catabolize DMSP via the cleavage pathway. Our results suggest that novel DMSP dethiomethylases/demethylases may exist in Pseudoalteromonas , Alteromonas and Vibrio , and that Gammaproteobacteria may be important participants in marine, especially in polar DMSP cycling.

GapMind for Carbon Sources: Automated annotations of catabolic pathways

GapMind for carbon sources is an automated tool for annotating catabolic pathways in bacterial and archaeal genomes. GapMind includes 62 compounds and identifies potential transporters and enzymes by their similarity to experimentally-characterized proteins. To improve GapMind's coverage, we used high-throughput genetic data from 29 bacteria and systematically examined the gaps. We identified novel pathways or enzymes for the utilization of glucosamine, citrulline, myo-inositol, lactose, and phenylacetate, and we annotated 299 diverged enzymes and transporters. We also curated 125 proteins from published reports. For the 29 bacteria with genetic data, GapMind finds high-confidence paths for 85% of utilized carbon sources. In diverse bacteria and archaea, 38% of utilized carbon sources have high-confidence paths, which was improved from 27% by incorporating the fitness-based annotations and our curation. GapMind for carbon sources is available as a web server (http://papers.genomics.lbl.gov/carbon) and takes just 30 seconds for the typical genome.

A Recently Assembled Degradation Pathway for 2,3-Dichloronitrobenzene in Diaphorobacter sp. Strain JS3051

Because anthropogenic nitroaromatic compounds have entered the biosphere relatively recently, exploration of the recently evolved catabolic pathways can provide clues for adaptive evolutionary mechanisms in bacteria. The concept that nitroarene dioxygenases shared a common ancestor with naphthalene dioxygenase is well established.

Separate Upper Pathway Ring Cleavage Dioxygenases Are Required for Growth of Sphingomonas wittichii RW1 on Dibenzofuran and Dibenzo-p-dioxin

Sphingomonas wittichii RW1 is one of a few strains known to grow on the related compounds dibenzofuran (DBF) and dibenzo-p-dioxin (DXN) as the sole source of carbon. Previous work by others (B. Happe, L. D. Eltis, H. Poth, R. Hedderich, and K. N. Timmis, J Bacteriol 175:7313-20, 1993, doi: 10.1128/jb.175.22.7313-7320.1993) showed that purified DbfB had significant ring cleavage activity against the DBF metabolite trihydroxybiphenyl but little activity against the DXN metabolite trihydroxybiphenylether. We took a physiological approach to positively identify ring cleavage enzymes involved in the DBF and DXN pathways. Knockout of dbfB on the RW1 megaplasmid pSWIT02 results in a strain that grows slowly on DBF but normally on DXN confirming that DbfB is not involved in DXN degradation. Knockout of SWIT3046 on the RW1 chromosome results in a strain that grows normally on DBF but that does not grow on DXN demonstrating that SWIT3046 is required for DXN degradation. A double knockout strain does not grow on either DBF or DXN demonstrating that these are the only ring cleavage enzymes involved in RW1 DBF and DXN degradation. Substitution of dbfB by SWIT3046 results in a strain that grows normally (equal to wild type) on both DBF and DXN showing that promoter strength is important for SWIT3046 to take the place of DbfB in DBF degradation. Thus both dbfB and SWIT3046 encoded enzymes are involved in DBF degradation but only the SWIT3046 encoded enzyme is involved in DXN degradation. Importance S. wittichii RW1 has been the subject of numerous investigations due to the fact that it is one of only a few strains known to grow on DXN as the sole carbon and energy source. However, while the genome has been sequenced and several DBF pathway enzymes have been purified, there has been very little research using physiological techniques to precisely identify the genes and enzymes involved in the RW1 DBF and DXN catabolic pathways. Using knockout and gene replacement mutagenesis our work identifies separate upper pathway ring cleavage enzymes involved in the related catabolic pathways for DBF and DXN degradation. The identification of a new enzyme involved in DXN biodegradation explains why the pathway of DBF degradation on the RW1 megaplasmid pSWIT02 is inefficient for DXN degradation. In addition, our work demonstrates that both plasmid and chromosomally encoded enzymes are necessary for DXN degradation suggesting that the DXN pathway has only recently evolved.