High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration.

Efficient genome engineering is critical to understand and use microbial functions. Despite recent development of tools such as CRISPR-Cas gene editing, efficient integration of exogenous DNA with well-characterized functions remains limited to model bacteria. Here, we describe serine recombinase-assisted genome engineering, or SAGE, an easy-to-use, highly efficient, and extensible technology that enables selection marker-free, site-specific genome integration of up to 10 DNA constructs, often with efficiency on par with or superior to replicating plasmids. SAGE uses no replicating plasmids and thus lacks the host range limitations of other genome engineering technologies. We demonstrate the value of SAGE by characterizing genome integration efficiency in five bacteria that span multiple taxonomy groups and biotechnology applications and by identifying more than 95 heterologous promoters in each host with consistent transcription across environmental and genetic contexts. We anticipate that SAGE will rapidly expand the number of industrial and environmental bacteria compatible with high-throughput genetics and synthetic biology.

Simple and Rapid Site-Specific Integration of Multiple Heterologous DNAs into the Escherichia coli Chromosome.

Escherichia coli is the most studied and well understood microorganism, but research in this system can still be limited by available genetic tools, including the ability to rapidly integrate multiple DNA constructs efficiently into the chromosome. Site-specific, large serine-recombinases can be useful tools, catalyzing a single, unidirectional recombination event between 2 specific DNA sequences, attB and attP, without requiring host proteins for functionality. Using these recombinases, we have developed a system to integrate up to 12 genetic constructs sequentially and stably into in the E. coli chromosome. A cassette of attB sites was inserted into the chromosome and the corresponding recombinases were cloned onto temperature sensitive plasmids to mediate recombination between a non-replicating, attP-containing "cargo" plasmid and the corresponding attB site on the chromosome. The efficiency of DNA insertion into the E. coli chromosome was approximately 107 CFU/µg DNA for six of the recombinases when the competent cells already contained the recombinase-expressing plasmid and approximately 105 CFU/µg DNA or higher when the recombinase-expressing plasmid and "cargo" plasmid were co-transformed. The "cargo" plasmid contains ΦC31 recombination sites flanking the antibiotic gene, allowing for resistance markers to be removed and reused following transient expression of the ΦC31 recombinase. As an example of the utility of this system, eight DNA methyltransferases from Clostridium clariflavum 4-2a were inserted into the E. coli chromosome to methylate plasmid DNA for evasion of the C. clariflavum restriction systems, enabling the first demonstration of transformation of this cellulose-degrading species. IMPORTANCE More rapid genetic tools can help accelerate strain engineering, even in advanced hosts like Escherichia coli. Here, we adapt a suite of site-specific recombinases to enable simple, rapid, and highly efficient site-specific integration of heterologous DNA into the chromosome. This utility of this system was demonstrated by sequential insertion of eight DNA methyltransferases into the E. coli chromosome, allowing plasmid DNA to be protected from restriction in Clostridium clariflavum and enabling genetic transformation of this organism. This integration system should also be highly portable into non-model organisms.

Approaches to genetic tool development for rapid domestication of non-model microorganisms.

Non-model microorganisms often possess complex phenotypes that could be important for the future of biofuel and chemical production. They have received significant interest the last several years, but advancement is still slow due to the lack of a robust genetic toolbox in most organisms. Typically, "domestication" of a new non-model microorganism has been done on an ad hoc basis, and historically, it can take years to develop transformation and basic genetic tools. Here, we review the barriers and solutions to rapid development of genetic transformation tools in new hosts, with a major focus on Restriction-Modification systems, which are a well-known and significant barrier to efficient transformation. We further explore the tools and approaches used for efficient gene deletion, DNA insertion, and heterologous gene expression. Finally, more advanced and high-throughput tools are now being developed in diverse non-model microbes, paving the way for rapid and multiplexed genome engineering for biotechnology.

Methylome and Complete Genome Sequence of Parageobacillus toebii DSM 14590T, a Thermophilic Bacterium.

Here, we present the first complete genome assembly of the thermophilic bacterium Parageobacillus toebii DSM 14590T The P. toebii DSM 14590T genome consists of a 3,270,071-bp circular chromosome and a 52,989-bp native plasmid.

Rational development of transformation in Clostridium thermocellum ATCC 27405 via complete methylome analysis and evasion of native restriction-modification systems.

A major barrier to both metabolic engineering and fundamental biological studies is the lack of genetic tools in most microorganisms. One example is Clostridium thermocellum ATCC 27405T, where genetic tools are not available to help validate decades of hypotheses. A significant barrier to DNA transformation is restriction-modification systems, which defend against foreign DNA methylated differently than the host. To determine the active restriction-modification systems in this strain, we performed complete methylome analysis via single-molecule, real-time sequencing to detect 6-methyladenine and 4-methylcytosine and the rarely used whole-genome bisulfite sequencing to detect 5-methylcytosine. Multiple active systems were identified, and corresponding DNA methyltransferases were expressed from the Escherichia coli chromosome to mimic the C. thermocellum methylome. Plasmid methylation was experimentally validated and successfully electroporated into C. thermocellum ATCC 27405. This combined approach enabled genetic modification of the C. thermocellum-type strain and acts as a blueprint for transformation of other non-model microorganisms.

Complete Genome Sequences of Two Megasphaera elsdenii Strains, NCIMB 702410 and ATCC 25940.

Here, we report the complete genome sequences of two Megasphaera elsdenii strains, ATCC 25940 and NCIMB 702410. M. elsdenii is an anaerobic bacterium capable of producing butanoate and hexanoate and is a member of the Negativicutes.

Five Alternative Myosin Converter Domains Influence Muscle Power, Stretch Activation, and Kinetics.

Muscles have evolved to power a wide variety of movements. A protein component critical to varying power generation is the myosin isoform present in the muscle. However, how functional variation in muscle arises from myosin structure is not well understood. We studied the influence of the converter, a myosin structural region at the junction of the lever arm and catalytic domain, using Drosophila because its single myosin heavy chain gene expresses five alternative converter versions (11a-e). We created five transgenic fly lines, each forced to express one of the converter versions in their indirect flight muscle (IFM) fibers. Electron microscopy showed that the converter exchanges did not alter muscle ultrastructure. The four lines expressing converter versions (11b-e) other than the native IFM 11a converter displayed decreased flight ability. IFM fibers expressing converters normally found in the adult stage muscles generated up to 2.8-fold more power and displayed up to 2.2-fold faster muscle kinetics than fibers with converters found in the embryonic and larval stage muscles. Small changes to stretch-activated force generation only played a minor role in altering power output of IFM. Muscle apparent rate constants, derived from sinusoidal analysis of the chimeric converter fibers, showed a strong positive correlation between optimal muscle oscillation frequency and myosin attachment kinetics to actin, and an inverse correlation with detachment related cross-bridge kinetics. This suggests the myosin converter alters at least two rate constants of the cross-bridge cycle with changes to attachment and power stroke related kinetics having the most influence on setting muscle oscillatory power kinetics.

Complete Genome Sequence of Industrial Dairy Strain Streptococcus thermophilus DGCC 7710.

We report here the complete genome sequence of Streptococcus thermophilus DGCC 7710. S. thermophilus is widely used in industrial dairy production.

Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum.

Consolidated bioprocessing (CBP) has the potential to reduce biofuel or biochemical production costs by processing cellulose hydrolysis and fermentation simultaneously without the addition of pre-manufactured cellulases. In particular, Clostridium thermocellum is a promising thermophilic CBP host because of its high cellulose decomposition rate. Here we report the engineering of C. thermocellum to produce isobutanol. Metabolic engineering for isobutanol production in C. thermocellum is hampered by enzyme toxicity during cloning, time-consuming pathway engineering procedures, and slow turnaround in production tests. In this work, we first cloned essential isobutanol pathway genes under different promoters to create various plasmid constructs in Escherichia coli. Then, these constructs were transformed and tested in C. thermocellum. Among these engineered strains, the best isobutanol producer was selected and the production conditions were optimized. We confirmed the expression of the overexpressed genes by their mRNA quantities. We also determined that both the native ketoisovalerate oxidoreductase (KOR) and the heterologous ketoisovalerate decarboxylase (KIVD) expressed were responsible for isobutanol production. We further found that the plasmid was integrated into the chromosome by single crossover. The resulting strain was stable without antibiotic selection pressure. This strain produced 5.4 g/L of isobutanol from cellulose in minimal medium at 50(o)C within 75 h, corresponding to 41% of theoretical yield.