Adaptive laboratory evolution triggers pathogen-dependent broad-spectrum antimicrobial potency in Streptomyces

Background In the present study, adaptive laboratory evolution was used to stimulate antibiotic production in a Streptomyces strain JB140 (wild-type) exhibiting very little antimicrobial activity against bacterial pathogens. The seven different competition experiments utilized three serial passages (3 cycles of adaptation-selection of 15 days each) in which Streptomyces strain (wild-type) was challenged repeatedly to one (bi-culture) or two (tri-culture) or three (quadri-culture) target pathogens. The study demonstrates a simple laboratory model to study the adaptive potential of evolved phenotypes and genotypes in Streptomyces to induce antibiotic production. Results Competition experiments resulted in the evolution of the wild-type Streptomyces strain JB140 into the seven unique mutant phenotypes that acquired the ability to constitutively exhibit increased antimicrobial activity against three bacterial pathogens Salmonella Typhi (NCIM 2051), Staphylococcus aureus (NCIM 2079), and Proteus vulgaris (NCIM 2027). The mutant phenotypes not only effectively inhibited the growth of the tested pathogens but were also observed to exhibit improved antimicrobial responses against one clinical multidrug-resistant (MDR) uropathogenic Escherichia coli (UPEC 1021) isolate. In contrast to the adaptively evolved mutants, only a weak antimicrobial activity was detected in the wild-type parental strain. To get molecular evidence of evolution, RAPD profiles of the wild-type Streptomyces and its evolved mutants were compared which revealed significant polymorphism among them. Conclusion The competition-based adaptive laboratory evolution method can constitute a platform for evolutionary engineering to select improved phenotypes (mutants) with increased antibacterial profiles against targeted pathogens.

Evolutionary engineering of Wickerhamomyces subpelliculosus and Kazachstania gamospora for baking

The conventional baker’s yeast, Saccharomyces cerevisiae , is an indispensable baking workhorse of all times. Its monopoly coupled to its major drawbacks such as streamlined carbon substrate utilisation base and a poor ability to withstand a number of baking associated stresses prompt the need to search for alternative yeasts to leaven bread in the era of increasingly complex consumer lifestyles. Our previous work identified the inefficient baking attributes of Wickerhamomyces subpelliculosus and Kazachstania gamospora as well as preliminarily observations of improving fermentative capacity of potential alternative baker’s yeasts using evolutionary engineering. Here we report the characterisation and improvement in baking traits in five out of six independently evolved lines incubated for longer time and passaged for at least 60 cycles relative to their parental strains as well as the conventional baker’s yeast. In addition, evolved clones produced bread with a higher loaf volume when compared to bread baked with either ancestral strain or the control conventional baker’s yeast. Remarkably, our approach improved the yeasts’ ability to withstand baking associated stresses, a key baking trait exhibited poorly in both the conventional baker’s yeast and their ancestral strains. W. subpelliculosus evolved the best characteristics attractive for alternative baker’s yeasts as compared to the evolved K. gamospora strains. These results demonstrate the robustness of evolutionary engineering in development of alternative baker’s yeasts.

Synthesis of Epitope-Targeting Antibody Based on Native Protein–Protein Interactions for Ftsz Filamentation Suppressor

Phage display and biopanning is a powerful tool for generating binding molecules for a specific target. However, the selection process based on binding affinity provides no assurance for the antibody’s affinity to the target epitope. In this study, we propose a molecular-evolution approach guided by native protein–protein interactions to generate epitope-targeting antibodies. The binding-site sequence in a native protein was grafted into a complementarity-determining region (CDR) in the antibody, and a nonrelated CDR loop (in the grafted antibody) was randomized by phage display techniques. In this construction of antibodies by integrating graft and evolution technology (CAnIGET method), suitable grafting of the functional sequence weakly functionalized the antibody, and the molecular-evolution approach enhanced the binding function to inhibit the native protein–protein interactions. Antibody fragments with an affinity for filamenting temperature-sensitive mutant Z (FtsZ) were constructed and completely inhibited the polymerization of FtsZ. Consequently, the expression of these fragments drastically decreased the cell division rate. We demonstrate the potential of the CAnIGET method with the use of native protein–protein interactions for steady epitope-specific evolutionary engineering.

In Vitro Gut Modeling as a Tool for Adaptive Evolutionary Engineering of Lactiplantibacillus plantarum

ABSTRACT Research and marketing of probiotics demand holistic strain improvement considering both the biotic and abiotic gut environment. Here, we aim to establish the continuous in vitro colonic fermentation model PolyFermS as a tool for adaptive evolutionary engineering. Immobilized fecal microbiota from adult donors were steadily cultivated up to 72 days in PolyFermS reactors, providing a long-term compositional and functional stable ecosystem akin to the donor’s gut. Inoculation of the gut microbiota with immobilized or planktonic Lactiplantibacillus plantarum NZ3400, a derivative of the probiotic model strain WCFS1, led to successful colonization. Whole-genome sequencing of 45 recovered strains revealed mutations in 16 genes involved in signaling, metabolism, transport, and cell surface. Remarkably, mutations in LP_RS14990, LP_RS15205, and intergenic region LP_RS05100<LP_RS05095 were found in recovered strains from different adaptation experiments. Combined addition of the reference strain NZ3400 and each of those mutants to the gut microbiota resulted in increased abundance of the corresponding mutant in PolyFermS microbiota after 10 days, showing the beneficial nature of these mutations. Our data show that the PolyFermS system is a suitable technology to generate adapted mutants for colonization under colonic conditions. Analysis thereof will provide knowledge about factors involved in gut microbiota colonization and persistence. IMPORTANCE Improvement of bacterial strains in regard to specific abiotic environmental factors is broadly used to enhance strain characteristics for processing and product quality. However, there is currently no multidimensional probiotic strain improvement approach for both abiotic and biotic factors of a colon microbiota. The continuous PolyFermS fermentation model allows stable and reproducible continuous cultivation of colonic microbiota and provides conditions akin to the host gut with high control and easy sampling. This study investigated the suitability of PolyFermS for adaptive evolutionary engineering of a probiotic model organism for lactobacilli, Lactiplantibacillus plantarum, to an adult human colonic microbiota. The application of PolyFermS controlled gut microbiota environment led to adaptive evolution of L. plantarum strains for enhanced gut colonization characteristics. This novel tool for strain improvement can be used to reveal relevant factors involved in gut microbiota colonization and develop adapted probiotic strains with improved functionality in the gut.

Adaptive laboratory evolution triggers pathogen-dependent broad-spectrum antimicrobial potency in Streptomyces

In the present study, adaptive laboratory evolution was used to stimulate antibiotic production in a weak antibiotic-producing Streptomyces strain JB140. The seven different competition experiments utilized three serial passages (three cycles of adaptation-selection of 15 days each) of a weak antibiotic-producing Streptomyces strain (wild-type) against one (biculture) or two (triculture) or three (quadriculture) target pathogens. This resulted in the evolution of a weak antibiotic-producing strain into the seven unique mutant phenotypes that acquired the ability to constitutively exhibit increased antimicrobial activity against bacterial pathogens. The mutant not only effectively inhibited the growth of the tested pathogens but also observed to produce antimicrobial against multidrug-resistant (MDR) E. coli. Intriguingly, the highest antimicrobial activity was registered with the Streptomyces mutants that were adaptively evolved against the three pathogens (quadriculture competition). In contrast to the adaptively evolved mutants, a weak antimicrobial activity was detected in the un-evolved, wild-type Streptomyces. To get molecular evidence of evolution, RAPD profiles of the wild-type Streptomyces and its evolved mutants were compared that revealed significant polymorphism among them. These results demonstrated that competition-based adaptive laboratory evolution method can constitute a platform for evolutionary engineering to select improved phenotypes (mutants) with increased production of antibiotics against targeted pathogens.

Does co-expression of Yarrowia lipolytica genes encoding Yas1p, Yas2p and Yas3p make a potential alkane-responsive biosensor in Saccharomyces cerevisiae?

Alkane-based biofuels are desirable to produce at a commercial scale as these have properties similar to current petroleum-derived transportation fuels. Rationally engineering microorganisms to produce a desirable compound, such as alkanes, is, however, challenging. Metabolic engineers are therefore increasingly implementing evolutionary engineering approaches combined with high-throughput screening tools, including metabolite biosensors, to identify productive cells. Engineering Saccharomyces cerevisiae to produce alkanes could be facilitated by using an alkane-responsive biosensor, which can potentially be developed from the native alkane-sensing system in Yarrowia lipolytica, a well-known alkane-assimilating yeast. This putative alkane-sensing system is, at least, based on three different transcription factors (TFs) named Yas1p, Yas2p and Yas3p. Although this system is not fully elucidated in Y. lipolytica, we were interested in evaluating the possibility of translating this system into an alkane-responsive biosensor in S. cerevisiae. We evaluated the alkane-sensing system in S. cerevisiae by developing one sensor based on the native Y. lipolytica ALK1 promoter and one sensor based on the native S. cerevisiae CYC1 promoter. In both systems, we found that the TFs Yas1p, Yas2p and Yas3p do not seem to act in the same way as these have been reported to do in their native host. Additional analysis of the TFs suggests that more knowledge regarding their mechanism is needed before a potential alkane-responsive sensor based on the Y. lipolytica system can be established in S. cerevisiae.

D-glucose overflow metabolism in an evolutionary engineered high-performance D-xylose consuming Saccharomyces cerevisiae strain

Co-consumption of D-xylose and D-glucose by Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. There is a need for improved sugar conversion rates to minimize fermentation times. Previously, we have employed evolutionary engineering to enhance D-xylose transport and metabolism in the presence of D-glucose in a xylose-fermenting S. cerevisiae strain devoid of hexokinases. Re-introduction of Hxk2 in the high performance xylose-consuming strains restored D-glucose utilization during D-xylose/D-glucose co-metabolism, but at rates lower than the non-evolved strain. In the absence of D-xylose, D-glucose consumption was similar to the parental strain. The evolved strains accumulated trehalose-6-phosphate during sugar co-metabolism, and showed an increased expression of trehalose pathway genes. Upon the deletion of TSL1, trehalose-6-phosphate levels were decreased and D-glucose consumption and growth on mixed sugars was improved. The data suggest that D-glucose/D-xylose co-consumption in high-performance D-xylose consuming strains causes the glycolytic flux to saturate. Excess D-glucose is phosphorylated enters the trehalose pathway resulting in glucose recycling and energy dissipation, accumulation of trehalose-6-phosphate which inhibits the hexokinase activity, and release of trehalose into the medium.

Evolutionary engineering of E. coli MG1655 for tolerance against isoprenol

Background Isoprenol is the basis for industrial flavor and vitamin synthesis and also a promising biofuel. Biotechnological production of isoprenol with E. coli is currently limited by the high toxicity of the final product. Adaptive laboratory evolution (ALE) is a promising method to address complex biological problems such as toxicity. Results Here we applied this method successfully to evolve E. coli towards higher tolerance against isoprenol, increasing growth at the half-maximal inhibitory concentration by 47%. Whole-genome re-sequencing of strains isolated from three replicate evolutions at seven time-points identified four major target genes for isoprenol tolerance: fabF, marC, yghB, and rob. We could show that knock-out of marC and expression of mutated Rob H(48) → frameshift increased tolerance against isoprenol and butanol. RNA-sequencing showed that the deletion identified upstream of yghB correlated with a strong overexpression of the gene. The knock-out of yghB demonstrated that it was essential for isoprenol tolerance. The mutated Rob protein and yghB deletion also lead to increased vanillin tolerance. Conclusion Through ALE, novel targets for strain optimization in isoprenol production and also the production of other fuels, such as butanol, could be obtained. Their effectiveness could be shown through re-engineering. This paves the way for further optimization of E. coli for biofuel production.