Genome shuffling: Progress and applications for phenotype improvement

Although rational method and global technique have been successfully applied in strain improvement respectively, the demand for engineering complex phenotypes required combinatorial approach. The technology of genome shuffling has been presented as a novel whole genome engineering approach for the rapid improvement of cellular phenotypes. This approach using recursive protoplast fusion with multi-parental strains offers the advantage of recombination throughout the entire genome without the necessity for genome sequence data or network information. Genome shuffling has been demonstrated as an effective method, which is not only for producing improved strain but also for providing information on complex phenotype. In this review we attempt to present the advantage of genome shuffling, introduce the procedure of this technology, summarize the applications of this approach for phenotype improvement and then give perspective on the development of this method in the future.     

A genome-wide set of congenic mouse strains derived from DBA/2J on a C57BL/6J background

In the analysis of complex traits, congenic strains are powerful tools because they allow characterization of a single locus in the absence of genetic variation throughout the remainder of the genome. Here, we report the construction and initial characterization of a genome-wide panel of congenic strains derived from the donor strain DBA/2J on the background strain C57BL/6J. For many strains, we have carried out high-density SNP genotyping to precisely map the congenic interval and to identify any contaminating regions. Certain strains exhibit striking variation in litter size and in the ratio of females to males. We illustrate the utility of the set by "Mendelizing" the complex trait of myocardial calcification. These 65 strains cover more than 95% of the autosomal genome and should facilitate the analysis of the many genetic trait differences that have been reported between these parental strains.     

A novel strategy to construct yeast Saccharomyces cerevisiae strains for very high gravity fermentation

Very high gravity (VHG) fermentation is aimed to considerably increase both the fermentation rate and the ethanol concentration, thereby reducing capital costs and the risk of bacterial contamination. This process results in critical issues, such as adverse stress factors (ie., osmotic pressure and ethanol inhibition) and high concentrations of metabolic byproducts which are difficult to overcome by a single breeding method. In the present paper, a novel strategy that combines metabolic engineering and genome shuffling to circumvent these limitations and improve the bioethanol production performance of Saccharomyces cerevisiae strains under VHG conditions was developed. First, in strain Z5, which performed better than other widely used industrial strains, the gene GPD2 encoding glycerol 3-phosphate dehydrogenase was deleted, resulting in a mutant (Z5ΔGPD2) with a lower glycerol yield and poor ethanol productivity. Second, strain Z5ΔGPD2 was subjected to three rounds of genome shuffling to improve its VHG fermentation performance, and the best performing strain SZ3-1 was obtained. Results showed that strain SZ3-1 not only produced less glycerol, but also increased the ethanol yield by up to 8% compared with the parent strain Z5. Further analysis suggested that the improved ethanol yield in strain SZ3-1 was mainly contributed by the enhanced ethanol tolerance of the strain. The differences in ethanol tolerance between strains Z5 and SZ3-1 were closely associated with the cell membrane fatty acid compositions and intracellular trehalose concentrations. Finally, genome rearrangements in the optimized strain were confirmed by karyotype analysis. Hence, a combination of genome shuffling and metabolic engineering is an efficient approach for the rapid improvement of yeast strains for desirable industrial phenotypes.     

Evolutionary engineering by genome shuffling

An upsurge in the bioeconomy drives the need for engineering microorganisms with increasingly complex phenotypes. Gains in productivity of industrial microbes depend on the development of improved strains. Classical strain improvement programmes for the generation, screening and isolation of such mutant strains have existed for several decades. An alternative to traditional strain improvement methods, genome shuffling, allows the directed evolution of whole organisms via recursive recombination at the genome level. This review deals chiefly with the technical aspects of genome shuffling. It first presents the diversity of organisms and phenotypes typically evolved using this technology and then reviews available sources of genetic diversity and recombination methodologies. Analysis of the literature reveals that genome shuffling has so far been restricted to microorganisms, both prokaryotes and eukaryotes, with an overepresentation of antibiotics- and biofuel-producing microbes. Mutagenesis is the main source of genetic diversity, with few studies adopting alternative strategies. Recombination is usually done by protoplast fusion or sexual recombination, again with few exceptions. For both diversity and recombination, prospective methods that have not yet been used are also presented. Finally, the potential of genome shuffling for gaining insight into the genetic basis of complex phenotypes is also discussed.     

Genetic diversity of toxigenic and nontoxigenic Vibrio cholerae serogroups O1 and O139 revealed by array-based comparative genomic hybridization

Toxigenic serogroups O1 and O139 of Vibrio cholerae may cause cholera epidemics or pandemics. Nontoxigenic strains within these serogroups also exist in the environment, and also some may cause sporadic cases of disease. Herein, we investigate the genomic diversity among toxigenic and nontoxigenic O1 and O139 strains by comparative genomic microarray hybridization with the genome of El Tor strain N16961 as a base. Conservation of the toxigenic O1 El Tor and O139 strains is found as previously reported, whereas accumulation of genome changes was documented in toxigenic El Tor strains isolated within the 40 years of the seventh pandemic. High phylogenetic diversity in nontoxigenic O1 and O139 strains is observed, and most of the genes absent from nontoxigenic strains are clustered together in the N16961 genome. By comparing these toxigenic and nontoxigenic strains, we observed that the small chromosome of V. cholerae is quite conservative and stable, outside of the superintegron region. In contrast to the general stability of the genome, the superintegron demonstrates pronounced divergence among toxigenic and nontoxigenic strains. Additionally, sequence variation in virulence-related genes is found in nontoxigenic El Tor strains, and we speculate that these intermediate strains may have pathogenic potential should they acquire CTX prophage alleles and other gene clusters. This genome-wide comparison of toxigenic and nontoxigenic V. cholerae strains may promote understanding of clonal differentiation of V. cholerae and contribute to an understanding of the origins and clonal selection of epidemic strains.     

Pure and mixed genetic lines of Saccharomyces bayanus and Saccharomyces pastorianus and their contribution to the lager brewing strain genome

The yeast species Saccharomyces bayanus and Saccharomyces pastorianus are of industrial importance since they are involved in the production process of common beverages such as wine and lager beer; however, they contain strains whose variability has been neither fully investigated nor exploited in genetic improvement programs. We evaluated this variability by using PCR-restriction fragment length polymorphism analysis of 48 genes and partial sequences of 16. Within these two species, we identified "pure" strains containing a single type of genome and "hybrid" strains that contained portions of the genomes from the "pure" lines, as well as alleles termed "Lager" that represent a third genome commonly associated with lager brewing strains. The two pure lines represent S. uvarum and S. bayanus, the latter a novel group of strains that may be of use in strain improvement programs. Hybrid lines identified include (i) S. cerevisiae/S. bayanus/Lager, (ii) S. bayanus/S. uvarum/Lager, and (iii) S. cerevisiae/S. bayanus/S. uvarum/Lager. The genome of the lager strains may have resulted from chromosomal loss, replacement, or rearrangement within the hybrid genetic lines. This study identifies brewing strains that could be used as novel genetic sources in strain improvement programs and provides data that can be used to generate a model of how naturally occurring and industrial hybrid strains may have evolved.     

Imputation of single-nucleotide polymorphisms in inbred mice using local phylogeny

We present full-genome genotype imputations for 100 classical laboratory mouse strains, using a novel method. Using genotypes at 549,683 SNP loci obtained with the Mouse Diversity Array, we partitioned the genome of 100 mouse strains into 40,647 intervals that exhibit no evidence of historical recombination. For each of these intervals we inferred a local phylogenetic tree. We combined these data with 12 million loci with sequence variations recently discovered by whole-genome sequencing in a common subset of 12 classical laboratory strains. For each phylogenetic tree we identified strains sharing a leaf node with one or more of the sequenced strains. We then imputed high- and medium-confidence genotypes for each of 88 nonsequenced genomes. Among inbred strains, we imputed 92% of SNPs genome-wide, with 71% in high-confidence regions. Our method produced 977 million new genotypes with an estimated per-SNP error rate of 0.083% in high-confidence regions and 0.37% genome-wide. Our analysis identified which of the 88 nonsequenced strains would be the most informative for improving full-genome imputation, as well as which additional strain sequences will reveal more new genetic variants. Imputed sequences and quality scores can be downloaded and visualized online.     

Extensive Recombination of a Yeast Diploid Hybrid through Meiotic Reversion

In somatic cells, recombination between the homologous chromosomes followed by equational segregation leads to loss of heterozygosity events (LOH), allowing the expression of recessive alleles and the production of novel allele combinations that are potentially beneficial upon Darwinian selection. However, inter-homolog recombination in somatic cells is rare, thus reducing potential genetic variation. Here, we explored the property of S. cerevisiae to enter the meiotic developmental program, induce meiotic Spo11-dependent double-strand breaks genome-wide and return to mitotic growth, a process known as Return To Growth (RTG). Whole genome sequencing of 36 RTG strains derived from the hybrid S288c/SK1 diploid strain demonstrates that the RTGs are bona fide diploids with mosaic recombined genome, derived from either parental origin. Individual RTG genome-wide genotypes are comprised of 5 to 87 homozygous regions due to the loss of heterozygous (LOH) events of various lengths, varying between a few nucleotides up to several hundred kilobases. Furthermore, we show that reiteration of the RTG process shows incremental increases of homozygosity. Phenotype/genotype analysis of the RTG strains for the auxotrophic and arsenate resistance traits validates the potential of this procedure of genome diversification to rapidly map complex traits loci (QTLs) in diploid strains without undergoing sexual reproduction.     

Towards a metabolic engineering strain “commons”: An Escherichia coli platform strain for ethanol production

In the genome‐engineering era, it is increasingly important that researchers have access to a common set of platform strains that can serve as debugged production chassis and the basis for applying new metabolic engineering strategies for modeling and characterizing flux, engineering complex traits, and optimizing overall performance. Here, we describe such a platform strain of E. coli engineered for ethanol production. Starting with a fully characterized host strain (BW25113), we site‐specifically integrated the genes required for homoethanol production under the control of a strong inducible promoter into the genome and deleted the genes encoding four enzymes from competing pathways. This strain is capable of producing >30 g/L of ethanol in minimal media with <2 g/L produced of any fermentative byproduct. Using this platform strain, we tested previously identified ethanol tolerance genes and found that while tolerance was improved under certain conditions, any effect on ethanol production or tolerance was lost when grown under production conditions. Thus, our findings reinforce the need for a metabolic engineering “commons” that could provide a set of platform strains for use in more sophisticated genome‐engineering strategies. Towards this end, we have made this production strain available to the scientific community. Biotechnol. Bioeng. 2013; 110: 1520–1526. © 2013 Wiley Periodicals, Inc.     

Engineering genomes in multiplex

Efficiently engineering robust complex traits is a key challenge facing metabolic engineering efforts to synthesize valuable products in vivo. Recent advances in genome engineering confront this barrier and significantly enhance the ability to map functional changes targeted throughout the genome and combinatorially optimize complex (multigenic) traits using multiplex recombineering. We describe a framework for efficiently searching genome-wide combinatorial space to optimize complex traits and highlight recent advances in genome engineering that enable this approach.     

Genomic Confirmation of Hybridisation and Recent Inbreeding in a Vector-Isolated Leishmania Population

Although asexual reproduction via clonal propagation has been proposed as the principal reproductive mechanism across parasitic protozoa of the Leishmania genus, sexual recombination has long been suspected, based on hybrid marker profiles detected in field isolates from different geographical locations. The recent experimental demonstration of a sexual cycle in Leishmania within sand flies has confirmed the occurrence of hybridisation, but knowledge of the parasite life cycle in the wild still remains limited. Here, we use whole genome sequencing to investigate the frequency of sexual reproduction in Leishmania, by sequencing the genomes of 11 Leishmania infantum isolates from sand flies and 1 patient isolate in a focus of cutaneous leishmaniasis in the Çukurova province of southeast Turkey. This is the first genome-wide examination of a vector-isolated population of Leishmania parasites. A genome-wide pattern of patchy heterozygosity and SNP density was observed both within individual strains and across the whole group. Comparisons with other Leishmania donovani complex genome sequences suggest that these isolates are derived from a single cross of two diverse strains with subsequent recombination within the population. This interpretation is supported by a statistical model of the genomic variability for each strain compared to the L. infantum reference genome strain as well as genome-wide scans for recombination within the population. Further analysis of these heterozygous blocks indicates that the two parents were phylogenetically distinct. Patterns of linkage disequilibrium indicate that this population reproduced primarily clonally following the original hybridisation event, but that some recombination also occurred. This observation allowed us to estimate the relative rates of sexual and asexual reproduction within this population, to our knowledge the first quantitative estimate of these events during the Leishmania life cycle.     

The Corynebacterium glutamicum genome: features and impacts on biotechnological processes

Corynebacterium glutamicum has played a principal role in the progress of the amino acid fermentation industry. The complete genome sequence of the representative wild-type strain of C. glutamicum, ATCC 13032, has been determined and analyzed to improve our understanding of the molecular biology and physiology of this organism, and to advance the development of more efficient production strains. Genome annotation has helped in elucidation of the gene repertoire defining a desired pathway, which is accelerating pathway engineering. Post genome technologies such as DNA arrays and proteomics are currently undergoing rapid development in C. glutamicum. Such progress has already exposed new regulatory networks and functions that had so far been unidentified in this microbe. The next goal of these studies is to integrate the fruits of genomics into strain development technology. A novel methodology that merges genomics with classical strain improvement has been developed and applied for the reconstruction of classically derived production strains. How can traditional fermentation benefit from the C. glutamicum genomic data? The path from genomics to biotechnological processes is presented.     

Engineering and adaptive evolution of Escherichia coli for d-lactate fermentation reveals GatC as a xylose transporter

Despite the abundance of xylose in nature, the production of chemicals from C5 sugars remains challenging in metabolic engineering. By deleting xylFGH genes and using adaptive evolution, an efficient E. coli strain capable of producing d-lactate from xylose was engineered. Quantitative proteomics and genome sequencing were used to understand the new phenotype and the metabolic limitations of xylose conversion to d-lactate. Proteomics identified major changes in enzyme concentration in the glycolytic and tricarboxylic acid pathways. Whole genome sequencing of the evolved strain identified a point mutation in the gatC gene, which resulted in a change from serine to leucine at position 184 of the GatC protein. The knockout of gatC in a number of strains and the insertion of the mutation in the non-evolved strain confirmed its activity as a xylose transporter and demonstrated that the mutation is responsible for the high xylose consumption phenotype in the evolved strain. The newly found xylose transporter is a candidate for future strain engineering for converting C5-C6 syrups into valuable chemicals.