A Cascade–Cas3-enabled method called TRIM3 is reported that generates large deletions by targeting a randomly integrated transposon, enabling facile generation of a genome-reduced mutant library and represents a new avenue for large-scale genome modifications and the development of improved bioprocessing hosts.
Abstract
Abstract Genome reduction is widely used to improve microbial bioprocessing hosts by reducing the burden of inessential physiology. Rationally identifying genomic regions that are dispensable or even detrimental to bioprocessing is challenged by our inability to map genome sequence to function across complex regulation and physiology. Thus, there is a need for tools that rapidly generate reduced genome strains with improved performance in process-relevant conditions. Here, we report a Cascade–Cas3-enabled method called TRIM3 that generates large deletions by targeting a randomly integrated transposon, enabling facile generation of a genome-reduced mutant library. Mutants with improved performance were isolated following growth-coupled selection and analyzed by long-read DNA sequencing to identify deletions in their genomes. We deploy this system iteratively in the industrial host Cupriavidus necator H16 on fructose and on formate. After two rounds of TRIM3, we isolate a strain containing a total reduction of 1.4 Mb (18.4% of the genome) that grows 25% faster in a bioreactor on fructose and a strain with a total reduction of 0.5 Mb (7.3% of the genome) that grows 14% faster on formate. This work demonstrates a method for random, iterative, growth-selectable genome reduction that represents a new avenue for large-scale genome modifications and the development of improved bioprocessing hosts.
Cupriavidus necator is a metabolically versatile β-proteobacterium of growing interest for auto- and heterotrophic bioprocesses, yet the genetic determinants governing its biofilm formation remain largely uncharacterized, particularly under process-relevant heterotrophic conditions. Here, we applied a forward-genetics transposon-enrichment approach to identify loci which promote surface-associated growth. A high-density mini-Tn5 mutant library (26,185 insertion clones, exceeding the >17,000 required for genome-wide coverage) was cultivated as a biofilm in a microfluidic flow-cell system on fructose for 168 h, and the surface-associated community was characterized by deep sequencing. Twelve genes showed significantly elevated insertion frequencies, several with documented links to biofilm formation in other bacteria, including the ferrous-iron uptake system (feoA/feoB), galU, and a GSDEF/EAL dual-domain protein. The gene B2043 (E6A55_RS29530), encoding this c-di-GMP-metabolizing protein, was selected for validation by markerless deletion. Under static conditions, the ΔB2043 mutant showed a 1.69 ± 0.06-fold increase in biofilm-associated biomass (p = 5.16 × 10−15). Under flow-through conditions, the mutant attached faster, entered exponential growth ∼10 h earlier, reached its biovolume plateau ∼16 h earlier than the wild-type, and formed distinct tower-like structures. These results identify B2043 as a negative regulator of biofilm formation acting predominantly during attachment, provide the first experimental evidence for c-di-GMP-dependent biofilm regulation in C. necator H16, and establish a functional-genomics framework — together with eleven further candidate loci — for engineering productive biofilms in this organism.
Janek R. Weiler, C. J. Lapp, Johannes Gescher et al.· Biofilm· 0 citations
Cpf1-based genome editing tools were developed for N. punctiforme, and a single-step cloning strategy was devised, allowing for rapid assembly of editing plasmids, and improved conjugation protocols for genetic transfer from E. coli to N. punctiforme were implemented.
J. Ryder, Soohan Woo, Ailea A. Blahm et al.· bioRxiv· 0 citations
Bacterial symbionts of insects undergo dramatic genome reduction during their evolutionary transition from free-living to host-dependent lifestyles, but the dynamics of genome degradation remain poorly understood due to the difficulty of observing these processes in real-time. Sodalis glossinidius, a facultative bacterial endosymbiont of tsetse flies, provides an exceptional opportunity to study this transition experimentally: Unlike highly specialised obligate symbionts, S. glossinidius can be cultured in vitro and retains a large genome (4 Mbp) with extensive pseudogene content (49%, vs. ~ 1% in free-living bacteria), suggesting a recent evolutionary transition. Here, we present a comparative genomic analysis of S. glossinidius strains isolated from laboratory colony-derived Glossina morsitans morsitans, comparing one strain after ten years of serial passaging in laboratory culture (SgGmmC1*) to a counterpart isolated at the same time from the same colony (SgGmmB4). Hybrid genome assembly using Oxford Nanopore and Illumina technologies produced a high-quality 4.29 Mbp genome comprising one circular chromosome and four plasmids. Comparative analysis revealed a significant deletion (16,493 bp) containing 31 genes, including thiM (involved in thiamine biosynthesis) and genes encoding sulfur transporters. Additionally, we identified multiple small-scale chromosomal mutations (8 deletions, 39 insertions, 11 SNPs) resulting in frameshifts in genes including a hemolysin precursor (shlA). Our findings demonstrate that, under stable laboratory conditions without the selective pressures of the host environment, S. glossinidius continues to undergo genome degradation. The loss of thiM supports previous hypotheses of complementary metabolic pathways between S. glossinidius and the primary symbiont Wigglesworthia glossinidia for thiamine biosynthesis. This study provides insights into the evolutionary trajectory of facultative symbionts and has implications for studying the patterns of genome evolution in bacterial symbionts adapting to novel ecological niches, as well as paratransgenic approaches using S. glossinidius for trypanosome control.
Poppy Pescod, Lee R. Haines, Alistair C. Darby et al.· PLoS Neglected Tropical Dise...· 0 citations
Second-generation (2G) bioethanol from lignocellulosic feedstocks is a sustainable alternative to fossil fuels. However, its production is constrained by the poor performance of industrial microbes in hydrolysates that are generated during biomass pretreatment. Scheffersomyces stipitis is a native xylose fermenting yeast and a promising platform for 2G bioethanol production, and adaptive evolution under hydrolysate stress has yielded strains with enhanced performance. However, the chromosomal basis of this adaptation is unknown. Here, we demonstrate that chromosome scale structural variation, rather than point mutations, underlies the improved phenotype of the evolved strains. By integrating long- and short-read genome sequencing, we identify two major chromosomal rearrangements in the top performing isolate: a reciprocal translocation between chromosomes 1 and 2 that disrupts the NUDIX hydrolase gene YSA1, and the formation of a mitotically stable 175 kb minichromosome derived from chromosome 5. Functional analyses show that disruption of YSA1 enhances xylose utilisation and ethanol yield, while the minichromosome contributes to improved performance in hydrolysate conditions. These findings provide direct evidence that balanced rearrangements and minichromosome formation can be selected during prolonged stress and can generate adaptive phenotypes. Taken together, our study establishes genome reorganisation as a key driver of adaptation in S. stipitis.
Samuel Vega-Estévez, A. Armitage, B. Dien et al.· Chromosome Research· 0 citations
It is demonstrated that CRISPR can be employed to edit Acanthamoeba genes using a knock-in approach and serves as a foundation to further develop A. castellanii as a model system with which to study diverse questions in cell and molecular biology, biochemistry and evolution.
Dudley Chung, Sari Matar, John M. Archibald· bioRxiv· 0 citations
The global emergence of vancomycin-resistant Gram-positive pathogens underscores the urgent need for efficient production of novel lipoglycopeptide antibiotics. Dalbavancin, a last-resort therapeutic agent, relies on its key biosynthetic precursor A40926B0, whose industrial manufacture is severely limited by the low yield of wild-type Nonomuraea gerenzanensis and inefficient genetic tools for this rare actinomycete. Here, we developed a high-efficiency CRISPR/AsCas12f1 genome editing system and applied systematic metabolic engineering to boost A40926B0 biosynthesis. First, conjugation conditions were optimized to elevate the transfer efficiency in N. gerenzanensis D11. The hypercompact AsCas12f1 nuclease showed markedly lower cytotoxicity than SpCas9 and enabled 100% gene deletion efficiency with preferred PAMs (TTTG, CTTG, GTTG). Second, we strengthened the shikimate pathway via multiple genetic strategies: overexpressing feedback-resistant DAHP synthase (aroGfbr) and chorismate mutase/prephenate dehydrogenase (tyrAfbr), as well as knocking out pheA. This manipulation blocks the phenylalanine synthetic branch and redirects metabolic flux toward the l-tyrosine branch. Third, we engineered the branched-chain fatty acid (BCFA) pathway via promoter replacement of bkdA2B2C2, LipAB, fabF and deletion of acdH to enhance isododecanoyl side-chain supply. The combinatorial engineering yielded strain B-13, which produced 1740 mg/L A40926B0 in shake flasks. Finally, 50-L fed-batch fermentation with continuous maltodextrin feeding further increased the titer to 1817 mg/L, the highest reported titer to date. This work establishes a robust CRISPR editing tool for N. gerenzanensis and provides valuable engineering references for precursor-oriented strain improvement targeting lipoglycopeptide antibiotics, offering insights for the industrial scale production of A40926B0.
Xiao-Ru Wang, Hong-Mei Zhai, Dan-Dan Gu et al.· Synthetic and Systems Biotec...· 0 citations
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