Improvement of thermotolerance in Lachancea thermotolerans using a bacterial selection pressure
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The use of thermotolerant yeast strains is an important attribute for a cost-effective high temperature biofermentation processes. However, the availability of thermotolerant yeast strains remains a major challenge. Isolation of temperature resistant strains from extreme environments or the improvements of current strains are two major strategies known to date. We hypothesised that bacteria are potential “hurdles” in the life cycle of yeasts, which could influence the evolution of extreme phenotypes, such as thermotolerance. We subjected a wild-type yeast, Lachancea thermotolerans to six species of bacteria sequentially for several generations. After coevolution, we observed that three replicate lines of yeasts grown in the presence of bacteria grew up to 37 °C whereas the controls run in parallel without bacteria could only grow poorly at 35 °C retaining the ancestral mesophilic trait. In addition to improvement of thermotolerance, our results show that the fermentative ability was also elevated, making the strains more ideal for the alcoholic fermentation process because the overall productivity and ethanol titers per unit volume of substrate consumed during the fermentation process was increased. Our unique method is attractive for the development of thermotolerant strains or to augment the available strain development approaches for high temperature industrial biofermentation.
KeywordsThermotolerance Experimental evolution Yeast-bacteria coevolution Alcoholic fermentation
Microbial alcoholic fermentation processes remain at the heart of humankind since the dawn of civilization. The efficiency of this indispensable process is often hampered by the paucity of microbes to withstand harsh environmental stresses . One of the most important environmental factors that affect microbial growth and survival is temperature. Thermotolerant industrial production strains are important for the reduction of cooling costs and protection of fermentation processes from failure due to accidental thermal management faults or increased ambient temperatures . The need for thermotolerant strains, exacerbated by the current global warming effects, which require a more stringent temperature control in large-scale industrial fermentors, is a major concern in modern bioprocesses. Approaches to obtain thermotolerant strains are strongly required. One possible approach is the isolation of naturally thermotolerant yeast species from their native habitats. However, the availability of thermotolerant yeast strains from nature is a major challenge because such characteristics are less commonly found in natural yeasts isolates. This is so, because in nature microorganisms tend to optimize their traits exclusively for survival and reproduction. Such characteristics are mostly irrelevant for modern technical applications, for example, in stressful large-scale industrial conditions . Only a handful of thermotolerant species isolated from tropical environments as well extreme environments around wine production plants and environments with increased levels of solar radiation have been reviewed . Yeasts isolated around the production plants are adaptively evolved and in most cases possess robust abilities to survive other extreme stresses . Another approach to obtain thermotolerant strains is generation of artificial diversity, for example, directed evolutionary engineering. This approach involves the pre-adaptation of yeasts to stressors common in specific industrial conditions. This includes, the pre-exposure of yeasts to mild forms and sub-lethal stressors such as high temperatures, high concentrations of toxic or growth impairing substances, for example, organic compounds, osmolytes, ethanol and weak acids, among others [5, 6, 7]. However, such a method is often associated with reduction in productivity and loss of phenotypes or trade-offs in phenotypes important for a specific production process [8, 9]. The reduction in growth rates and inability to utilize the intended source of carbon are an example of major drawbacks associated with this approach reported elsewhere [8, 9]. Another possible approach is genetic engineering. Knowledge-based genetic modification remains less effective as thermotolerance is encoded by multiple quantitative traits [10, 11, 12]. Other methods hinged on generation of artificial diversity such as mutagenesis and classical breeding exist albeit with their limitations similar to evolutionary engineering. New methods to develop thermotolerant yeasts retaining fermentative traits are, therefore, of interest.
In this work, we extended the development of thermotolerant yeast strains not only beyond the conventional strategies but also beyond the industrial “workhorse”, Sacharomyces cerevisiae, using non-conventional yeasts. Lachancea thermotolerans, a yeast that diverged from the S. cerevisiae lineage prior to its whole genome duplication , was sequentially cocultured with six bacteria of increasing tolerance to ethanol for several generations as reported in our previous studies [14, 15, 16]. Our evolution strategy ascertains that microorganisms’ adaptive phenotypes in nature are hinged on the interactions with others . We hypothesised that mimicking the natural habitat where yeasts are sympatric to bacteria may yield isolates with tolerance to extreme stress [14, 16], while keeping their fermentative capabilities as ethanol is key to their survival strategy with respect to bacteria .
In this study, we sought to develop strains with elevated thermotolerance. To screen for the emergence of elevated thermotolerant populations of L. thermotolerans after evolution in the presence of bacteria, we investigated their thermal sensitivity and growth between mesophilic, i.e., 30 °C to growth inhibitory temperatures, i.e. ~ 40 °C . Other than thermotolerance, the strain’s ability to produce ethanol is dependent on its tolerance to other stressors that are associated with biofermentation. High ethanol stress, chemical surfactants, inhibitors and ROS exerted by substances found in raw materials as well as others are common examples [19, 20, 21]. Thus, we investigated the evolution of cross-protection from other extreme environmental stressors such as high ethanol titers, reactive oxygen species as well as ability to withstand surfactants. Further comparative analyses of molecular changes of the evolved strains were carried out using pulse field gel electrophoresis (PFGE). The fermentative capacity, before and after evolution, was then investigated to ascertain the suitability of the method in developing strains for highly productive high temperature fermentations.
Materials and methods
Strains used in this study
A wild type non-conventional yeast, L. thermotolerans, type strain CBS 6340, was the parental strain used in this study. Bacterial species, Pantoea agglomerans Eh318 (CUCPB 2140), Serattia plymuthica AS9 (CCUG 61396), Bacillus subtilis PS216 (BGSC 3A36), Streptomyces venezuelae (ATCC 10712), Lactococcus lactis (NCDO 2118) and Pseudomonas fluorescens (NCIMB 10462) previously reported [14, 15, 16] were sequentially used as a selection pressure to evolve L. thermotolerans. S. cerevisiae (S288c) and Schizosaccharomyces pombe (SJA148) strains were used as standards for karyotyping.
Adaptive laboratory evolution experiments
A unique experimental evolution strategy involving the sequential introduction of bacteria to compete with yeasts and the subsequent elimination of bacteria through the addition of antibiotics before transferring yeasts into fresh media, previously reported [14, 15, 16] was used. In brief, six flasks containing rich medium (YPD: 2% glucose, 0.5% yeast extract and 1% peptone, at a pH of 6.2 in 250 mL baffled-bottom flasks at 25 °C) were inoculated with an isogenic colony of L. thermotolerans (CBS6340 strain). Three flasks with and three without bacteria (controls) were then incubated and refreshed for several passages as reported [14, 15, 16]. More specifically, we grew 25 mL of yeast culture (5 ± 0.05 log10 CFUs/mL) in YPD in baffled-bottom flasks in an incubating shaker for 4 h (end of lag phase) at 200 revolutions per minute (r.p.m) at 25 °C before a predetermined amount of bacteria (4 ± 0.05 log10 CFUs/mL) was inoculated into an already adapted yeast population. The co-culture was incubated for 40 h, which was a predetermined time point before diauxic shift, and then bacteria were killed by addition of streptomycin (100 μg/mL). After sufficient time, at least 4 h after addition of the antibiotics, we transferred 50 μL of exponentially growing yeasts (7.7 ± 0.1 log10 CFUs/mL) into 25 mL fresh YPD. We carried out 20 such transfer passages before exchanging the bacterium with another species. Each transfer passage was approximately 8 generations amounting to a total of at least 180 generations, per bacterial species used. We froze 500 μL of the cell culture suspension in 25% glycerol at − 80 °C for analyses before each transfer cycle. This procedure was repeated for several generations allowing yeasts to compete with each bacterial species sequentially in order of increasing tolerance to ethanol (P. agglomerans, S. plymuthica, B. subtilis, S. venezuelae, L. lactis, and P. flourescens) as reported in . The order with which yeast encounter bacteria is significant for selection towards better ethanol producers [14, 16], thus in this study it was intended to select for elevated ethanol producers in addition to thermotolerant strains. We sequenced rDNA D1/D2 region, amplified by universal primers NL1 (forward primer)(5′-GCATATCAATAAGCGGAGGA AAAG-3′) and NL4 (reverse primer) (5′-GGTCCGTGTTTCAAGACGG-3′) of the yeast before proceeding with the next bacterium to examine contamination despite periodic plating and microscopy as reported by . Transfer cycles were carried out before the stationary phase of the yeasts to exclude other possible selection pressures and outcomes that could arise when a substrate (glucose) becomes limiting or is exhausted in the medium as reported elsewhere [22, 23], and thus could mask our bacterial competition selection pressure hypothesis set in the experiment. A detailed description of the method was fully reported elsewhere .
Isolation of thermotolerant strains
From each frozen sample stored after every 80 generations (10 passages) we plated out cells (7.7 ± 0.1 log10 CFUs/mL). Single independent colonies were picked and used to initiate cultures for thermal stability investigation. Cultures were grown in 2 mL YPD (2% glucose, 0.5% yeast extract and 1% peptone, at a pH of 6.2) in test tubes and incubated overnight at 25 °C in an orbital shaker (Infors HT). Cells were then pelleted using a microfuge and washed twice with phosphate buffered saline (PBS) and adjusted to OD600nm 0.06, which corresponds to 5.75 ± 0.2 log10 CFUs/mL. 100 μL of each of the independent cultures cells was then transferred to 96-well plates. Cells were then spotted on solid YPD plates using a 6 × 8 array spot replicator (Sigma Aldrich) before incubation at 30 °C, 35 °C, 37 °C, 39 °C, and 40 °C for 5 days. All assays were repeated at least twice, each time starting with an independent colony.
Investigation of tolerance to other stressors
Thermotolerant colonies from each of the six evolution lines (three from control-evolved and three from bacterial competition) were further used to determine their sensitivity to different environmental stresses. Single colonies were used to initiate overnight cultures in 2 mL YPD (2% glucose, 0.5% yeast extract and 1% peptone, at a pH of 6.2) or YNB (Yeast Nitrogen Base: 1.7 g/L; 5 g/L ammonium sulphate supplemented with 2% glucose, pH 6.2), in test tubes and incubated overnight at 25 °C in an orbital shaker (Infors HT). The cultures were then pelleted using a microfuge and washed twice in 1000 μL of PBS and then resuspend in 500 μL. The cultures were then adjusted to an OD600nm of 0.06 before being spotted on plates supplemented with reagents responsible for respective stresses. Effects of stressors were established by scoring growth of the evolved strains in comparisons to the ancestral strain. For record keeping and analyses, spots on petri dishes were scanned after 2 days unless otherwise specified.
Ethanol tolerance assay
Ethanol tolerance was assayed using the same procedures as above except that yeasts were spotted on solid YPD containing different concentrations of ethanol (6–10%) and incubated at 25 °C. Growth was scored for 10 days.
Oxidative stress (menadione and H2O2)
Sensitivity to H2O2 induced oxidative stress was determined using cells at stationary phase. Cells were grown overnight in YNB (Yeast Nitrogen Base: 1.7 g/L; 5 g/L ammonium sulphate supplemented with 2% glucose, pH 6.2), washed and diluted to OD600nm 0.06, which corresponds to 5.75 ± 0.2 log10 CFUs/mL. Cells were then spotted on solid YNB containing different concentrations of H2O2 (0–4 mM) using a spot test replicator. Plates were incubated up to 5 days at 25 °C. The same procedures were repeated with menadione of concentration ranges between 0.14 and 0.18 mM. Experiments were carried out in triplicate and repeated twice.
Sensitivity to sodium dodecyl-sulfate (SDS)
The same procedure was carried out as above except that SDS was used as a stressor at concentration ranges of 0.01–0.02%. Experiments were carried out in triplicate and repeated twice.
Karyotyping of evolved strains
From each frozen sample stored after every 80 generations (10 passages) we plated out cells (7.7 ± 0.1 log10 CFUs/mL) and randomly selected three independent colonies and determined their karyotypes using a CHEF Mapper XA PFGE apparatus (Bio-Rad) as described . To determine the genetic stability of the colonies, 20 sequential transfers of the colonies under non-selective conditions (YPD at 25 °C) were carried out and followed by karyotype analyses.
Fermentative capacity of thermotolerant strains under aerobic conditions in flasks was investigated. We used the most thermotolerant and ethanol tolerant strains, i.e., passage 50 from evolution lines B and C. Cells from evolution line A were not included due to their ethanol sensitivity. In addition, those evolved in the absence of bacteria (lines D, E, and F) were not included due to their thermal sensitivity. We revived frozen cultures from evolution lines B as well as C by plating them out (7.7 ± 0.1 log10 CFUs/mL) on solidified YPD agar. The plates were incubated at 25 °C. Three independent colonies were randomly selected for inoculation in YPD as overnight seed cultures. After incubation, the cells were pelleted and washed with sterile water before inoculating 25 mL YPD (2% glucose, 0.5% yeast extract and 1% peptone), at a pH of 6.2 in 250 mL baffled-bottom Erlenmeyer shake flasks. The flasks were incubated at 37 °C at 200 r.p.m in an Infors HT Ecotron shaker unit (Infors HT). The starting OD600nm was set at 1. The ancestral strain was used as a control but was grown at 30 °C as it could not grow at 37 °C. We performed these batch cultures in triplicate. Samples withdrawn at appropriate intervals were used to monitor cell growth and analyze metabolites using an HPLC with specifications as reported . Product yields; maximum specific rates of glucose consumption and ethanol production rates were calculated as reported [16, 25]. The experiments were carried out in triplicate and repeated twice.
Acquisition of thermotolerance
Evolution of resistance to other stresses
Resistance to surfactants
Large-scale chromosomal rearrangements
In nature, yeasts like any other organisms compete for resources amongst either themselves or other yeasts of different species as well as against co-habitants from across the kingdom, such as bacteria. Competition is a crucial driver of evolution . Interspecific competition is one such ecological force that reduces population density [37, 38], which subsequently reduces the accumulation of adaptive mutations increasing the risks of extinction [38, 39]. On the other hand, such an increased strength of selection pressure increases the rate of evolution [40, 41, 42].
Based on the fact that organisms adapt to environments they live in and may as well cope with extreme environments they may encounter in their evolutionary life history , we created an ecological battlefield by subjecting yeasts to the competition against bacteria with a goal to develop thermotolerant yeasts without compromising their fermentative capabilities. We hypothesized that probing yeasts would force them to define mechanisms to adapt as well as to out-grow competitors; such as efficient resource utilization, ability to tolerate a variety of stresses associated with such a habitat, and the ability to mount a refined niche defense strategy in quest to dominate habitats. A comprehensive review of how microorganisms aggressively deploy niche-dominating strategies is available elsewhere .
Competition is known to increase rates of evolution by increasing a selective pressure that pushes organisms to explore multiple paths to survive [40, 41, 45]. In our study, we forced yeasts to evolve survival responses against the presence and activities of bacteria. A basic principle of this selection pressures is based on the relatively faster growth rates of bacteria when compared to that of yeasts coupled to their faster consumption of the available carbon sources, endowing them with a competitive advantage. Among other studied selection pressures exerted by bacteria is the alteration of environmental pH, secretion of antifungals and chitinases and other growth inhibitory molecules [46, 47].
We show that in response to an increasing bacterial selection pressure, yeasts evolved elevated thermotolerance as well as ethanol tolerance and resistance to surfactants. There is an increase in ethanol production accompanied by an increased ethanol tolerance, however, based on our data available this is an observation is not necessarily a causative factor. We also show that adaptive evolution experiments using poorly studied and less exploited non-conventional yeasts may yield insights in the study of stress resistance and development of stress resistant strains of other species.
Conventional industrial yeast production strains are exposed to several stressful environments such as low and high temperatures, exposure to ethanol, osmotic stress, and oxidative stress during the preparation as starter cultures as well as during fermentation reviewed in . Strain robustness in such stressful environments is essential for increasing productivity of fermentation, which often culminates in an economically viable bioprocess. There has been extensive research going on to ameliorate the negative effects of sensitivity to stress in conventional yeasts [49, 50]. This research could benefit from the knowledge derived from the studies of non-conventional yeasts, which could point towards novelties in stress resistance strategies when compared to conventional yeasts.
The evolution of a phenotype protecting against thermal stress when yeasts were co-cultured with bacteria suggests that our method is an attractive, inexpensive and a simple alternative to develop yeast strains with robust resistance to a number of stressful conditions. The emergence of stress tolerance phenotypes in all three independent evolution lines, which was not evident in the control strains, is suggestive of an adaptive and not transient phenotype. This is a key factor for reproducibility and adoption of the method. A more pronounced thermotolerance with increasing selection pressure as evident in the terminally evolved strains, suggestive of a stepwise evolution of the mechanism involved, makes this method more attractive because there is a probable room for further improvement thermotolerance improvement.
The exposure to a bacterial selection pressure cross-protected the evolved strains from ethanol and surfactants. These results are in agreement with reported cases, where stressors are known to induce cross-protection against an impending stressor [51, 52, 53]. A functional overlap of responses behind thermotolerance and ethanol tolerance observed, is not surprising as they are in agreement with the use of similar stress response mechanisms under these two scenarios in yeast [29, 54]. Both stressors cause similar plasma membrane protein changes. In addition, heat-shock proteins involved in mounting stress responses against these two stressors also overlap [29, 54]. In addition, we noted that in addition co-culture evolved strains were protected against surfactant, SDS, whose response mechanism is based on membrane structures [30, 31].
One of the drawbacks of experimental evolution is the emergence of trade-offs, which negatively impact the robustness and applicability of the developed strains . There was a loss of resistance to oxidative stress, which may be considered as an undesirable trade-off when the method is transferred for the development of S. cerevisiae strains for biofuels production. This is because cells are in constant exposure to oxygen during the propagation stages that induce oxidative stress . Current strategies to reduce the toxicity of the lignocellulosic feedstocks reviewed in  may also mitigate the possible effects.
We speculate that the presence and activity of bacteria could have induced adaptive changes either in the plasma membrane protein structures and permeability or an altered expression of either plasma membrane associated genes and heat shock protein family of genes. An investigation of the molecular karyotypes of evolved strains revealed a significant polymorphism. Although changes involving large-scale genomic rearrangements allow a swift mechanism of evolutionary adaptation , by regulating the expression of several genes in a metabolic pathway as well as gene dosage [57, 58, 59], the link between rearrangements and the observed phenotypes remain to be proven. In our previous studies, we noted the emergence of new metabolic traits due to a large-scale genomic rearrangement following a segmental duplication and translocation event. Whole genome sequencing revealed the duplication of vital genes involved in osmotolerance, DNA replication stress, and thermotolerance . In addition, several other yeasts with large-scale rearrangements were reported [14, 60]. Our method does not impair growth and fermentation of the yeasts, and thus it is suitable for industrial strain development. In conclusion, our study highlights the importance of ecological battlefields for the induction of adaptive phenotypes. Interspecific competitions in nature are often aggressive , so microorganisms reshape molecular mechanisms to avoid being annihilated or develop new strategies to dominate the niches. The deployment of such versatile strategies may also entail the protection against other types of stress not tested or contemplated in this work.
We propose the use of this method as a valuable approach to develop robust stress resistant industrial strains. Our tool mimicks the natural environment characteristic of cross-kingdom competition for sugars in nature when fruits ripen, every autumn, availing excess sugars for a short period of time. Although complex interactions among a multitudes of microorganism exist in nature, here we created a less complex competitive environment in which yeasts were allowed to sequentially encounter each of the six bacterial species used in this study, one at a time, for a time long enough to effect an evolutionary change. The order with which yeasts encountered bacteria was assumed to be a gradient of strength of selection, from weak (ethanol sensitive bacteria) to strong (ethanol tolerant bacteria). A weak selection pressure during the early stages of evolution was ascertained to allow the emergence of more genetically diverse variants, an attribute important for organisms to explore a multiple paths to adaptation as well as avoiding populations being driven to extinction when the solution becomes inaccessible; associated with strong selection pressure as reviewed elsewhere . Nutrient limitation, exhaustion and end product toxicity characteristic of the stationary phase of growth have been shown to force yeasts to adaptively evolve mechanisms to either utilise alternative or limiting nutrients reviewed in . To exclude such possible selection pressures and outcomes that could mask our bacterial competition selection pressure hypothesis set in our development tool, transfer of cultures was carried out before reaching the stationary phases. This allowed us to explore the evolvability of yeasts only during the exponential phases, which is a competitive growth against bacteria in the presence of a carbon source depreciating over the course of time. The use of antibiotic drugs to kill the surviving bacteria before transfer into fresh media was an important step to avoid emergence of antibiotic resistant bacteria. Antibiotics were added even in the absence of a bacterial challenge (controls) so that the consequence of a bacterial selection pressure on evolving yeast populations instead of unknown selection pressures could be investigated. This is due to the fact that it is characteristic of yeasts to undergo metabolic shifts even in a simple laboratory environment, as they tend to maximize their survival and reproduction.
Until now, thermal stress tolerant yeasts have been developed through the exposure to a mild form and gradually increasing thermal stress, which unfortunately, negatively affects the productivity of the production strains due to major trade-offs. Our tool suggests that improvement of the production capacity as well as cross-protection from other environmental stresses encountered during production is possible. Both of these attributes add up to the most desirable phenotypes of industrial yeasts. It should be noted that for a specific application of thermotolerance strains, such as bioethanol production from inexpensive lignocellulosic feedstocks, further studies are need to investigate if the acquisition of thermotolerance affects the utilisation of alternative carbon sources (xylose, arabinose, galactose and mannitol), abundant in lignocellulosic hydrolysates.
Our novel two-species cross-kingdom competition strategy is an attractive tool to develop stress tolerant industrial strains.
We would like to dedicate this work to our late friend, teacher and mentor, Professor Jure Piskur. Our continued respect and admiration of him is the reason why this study was continued.
Conceptualization: JP CC. Funding acquisition: JP CC WK. Investigation: NZ OPI. Methodology: NZ OPI. Project administration: WK. Resources: JP WK. Supervision: JP CC WK. Writing—original draft: NZ. Writing—review and editing: NZ CC OPI WK.
This work was funded by the Marie Curie, Cornucopia Project (264717), Fysiografen and Lindström Foundations.
Compliance with ethical standards
Conflict of interest
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