Genome-Scale Transcription-Translation Mapping Reveals Features of Zymomonas mobilis Transcription Units and Promoters.

Zymomonas mobilis is an ethanologenic alphaproteobacterium with promise for the industrial conversion of renewable plant biomass into fuels and chemical bioproducts. Limited functional annotation of the Z. mobilis genome is a current barrier to both fundamental studies of Z. mobilis and its development as a synthetic biology chassis. To gain insight, we collected sample-matched multiomics data, including RNA sequencing (RNA-seq), transcription start site (TSS) sequencing (TSS-seq), termination sequencing (term-seq), ribosome profiling, and label-free shotgun proteomic mass spectrometry, across different growth conditions and used these data to improve annotation and assign functional sites in the Z. mobilis genome. Proteomics and ribosome profiling informed revisions of protein-coding genes, which included 44 start codon changes and 42 added proteins. We developed statistical methods for annotating transcript 5' and 3' ends, enabling the identification of 3,940 TSSs and their corresponding promoters and 2,091 transcription termination sites, which were distinguished from RNA processing sites by the lack of an adjacent RNA 5' end. Our results revealed that Z. mobilis σA -35 and -10 promoter elements closely resemble canonical Escherichia coli -35 and -10 elements, with one notable exception: the Z. mobilis -10 element lacks the highly conserved -7 thymine observed in E. coli and other previously characterized σA promoters. The σA promoters of another alphaproteobacterium, Caulobacter crescentus, similarly lack the conservation of -7 thymine in their -10 elements. Our results anchor the development of Z. mobilis as a platform for synthetic biology and establish strategies for empirical genome annotation that can complement purely computational methods.IMPORTANCE Efforts to rationally engineer synthetic pathways in Zymomonas mobilis are impeded by a lack of knowledge and tools for predictable and quantitative programming of gene regulation at the transcriptional, posttranscriptional, and posttranslational levels. With the detailed functional characterization of the Z. mobilis genome presented in this work, we provide crucial knowledge for the development of synthetic genetic parts tailored to Z. mobilis This information is vital as researchers continue to develop Z. mobilis for synthetic biology applications. Our methods and statistical analyses also provide ways to rapidly advance the understanding of poorly characterized bacteria via empirical data that enable the experimental validation of sequence-based prediction for genome characterization and annotation.

Regulated redirection of central carbon flux enhances anaerobic production of bioproducts in Zymomonas mobilis.

The microbial production of chemicals and fuels from plant biomass offers a sustainable alternative to fossilized carbon but requires high rates and yields of bioproduct synthesis. Z. mobilis is a promising chassis microbe due to its high glycolytic rate in anaerobic conditions that are favorable for large-scale production. However, diverting flux from its robust ethanol fermentation pathway to nonnative pathways remains a major engineering hurdle. To enable controlled, high-yield synthesis of bioproducts, we implemented a central-carbon metabolism control-valve strategy using regulated, ectopic expression of pyruvate decarboxylase (Pdc) and deletion of chromosomal pdc. Metabolomic and genetic analyses revealed that glycolytic intermediates and NADH accumulate when Pdc is depleted and that Pdc is essential for anaerobic growth of Z. mobilis. Aerobically, all flux can be redirected to a 2,3-butanediol pathway for which respiration maintains redox balance. Anaerobically, flux can be redirected to redox-balanced lactate or isobutanol pathways with ≥65% overall yield from glucose. This strategy provides a promising path for future metabolic engineering of Z. mobilis.

OptSSeq: High-Throughput Sequencing Readout of Growth Enrichment Defines Optimal Gene Expression Elements for Homoethanologenesis.

The optimization of synthetic pathways is a central challenge in metabolic engineering. OptSSeq (Optimization by Selection and Sequencing) is one approach to this challenge. OptSSeq couples selection of optimal enzyme expression levels linked to cell growth rate with high-throughput sequencing to track enrichment of gene expression elements (promoters and ribosome-binding sites) from a combinatorial library. OptSSeq yields information on both optimal and suboptimal enzyme levels, and helps identify constraints that limit maximal product formation. Here we report a proof-of-concept implementation of OptSSeq using homoethanologenesis, a two-step pathway consisting of pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) that converts pyruvate to ethanol and is naturally optimized in the bacterium Zymomonas mobilis. We used OptSSeq to determine optimal gene expression elements and enzyme levels for Z. mobilis Pdc, AdhA, and AdhB expressed in Escherichia coli. By varying both expression signals and gene order, we identified an optimal solution using only Pdc and AdhB. We resolved current uncertainty about the functions of the Fe2+-dependent AdhB and Zn2+-dependent AdhA by showing that AdhB is preferred over AdhA for rapid growth in both E. coli and Z. mobilis. Finally, by comparing predictions of growth-linked metabolic flux to enzyme synthesis costs, we established that optimal E. coli homoethanologenesis was achieved by our best pdc-adhB expression cassette and that the remaining constraints lie in the E. coli metabolic network or inefficient Pdc or AdhB function in E. coli. OptSSeq is a general tool for synthetic biology to tune enzyme levels in any pathway whose optimal function can be linked to cell growth or survival.

Synergistic action of cinnamaldehyde with silver nanoparticles against spore-forming bacteria: a case for judicious use of silver nanoparticles for antibacterial applications.

Silver has long been advocated as an effective antimicrobial. However, toxicity issues with silver have led to limited use of silver in nanoform, especially for food preservation. With the aim of exploring combinatorial options that could increase the antibacterial potency of silver nanoparticles and reduce the effective dosage of silver, we evaluated the extent of synergy that a combination of silver nanoparticles and an essential oil representative (cinnamaldehyde) could offer. A battery of gram-positive and gram-negative bacterial strains was utilized for antibacterial assays, and extents of synergism were calculated from fractional inhibitory concentration indices. The activity of nanoparticles was greatly enhanced when utilized in the presence of cinnamaldehyde. We observed combinatorial effects that were strongly additive against all the bacterial strains tested, and genuine synergy was found against spore forming Bacillus cereus and Clostridium perfringens - bacterial strains associated with release of cytotoxins in contaminated food and known for their persistence. Bacterial kill curve analysis revealed a very fast bactericidal action when a combination of two agents was used. The electron and atomic force microscopy also revealed extensive damage to the bacterial cell envelop in the presence of both agents. We also performed hemolysis assays to investigate and approximate the extent of toxicity exhibited by the two agents, and observed no adverse effect at the concentrations required for synergy. This study shows that safe levels of silver in nanoform in combination with essential oil component cinnamaldehyde can be effectively used for controlling the spore-forming bacterial species.