A split ribozyme that links detection of a native RNA to orthogonal protein outputs

Individual RNA remains a challenging signal to synthetically transduce into different types of cellular information. Here, we describe Ribozyme-ENabled Detection of RNA (RENDR), a plug-and-play strategy that uses cellular transcripts to template the assembly of split ribozymes, triggering splicing reactions that generate orthogonal protein outputs. To identify split ribozymes that require templating for splicing, we used laboratory evolution to evaluate the activities of different split variants of the Tetrahymena thermophila ribozyme. The best design delivered a 93-fold dynamic range of splicing with RENDR controlling fluorescent protein production in response to an RNA input. We resolved a thermodynamic model to guide RENDR design, showed how input signals can be transduced into diverse visual, chemical, and regulatory outputs, and used RENDR to detect an antibiotic resistance phenotype in bacteria. This work shows how transcriptional signals can be monitored in situ using RNA synthetic biology and converted into different types of biochemical information.

Meiosis-Based Laboratory Evolution of the Thermal Tolerance in Kluyveromyces marxianus

Kluyveromyces marxianus is the fastest-growing eukaryote and a promising host for producing bioethanol and heterologous proteins. To perform a laboratory evolution of thermal tolerance in K. marxianus, diploid, triploid and tetraploid strains were constructed, respectively. Considering the genetic diversity caused by genetic recombination in meiosis, we established an iterative cycle of “diploid/polyploid - meiosis - selection of spores at high temperature” to screen thermotolerant strains. Results showed that the evolution of thermal tolerance in diploid strain was more efficient than that in triploid and tetraploid strains. The thermal tolerance of the progenies of diploid and triploid strains after a two-round screen was significantly improved than that after a one-round screen, while the thermal tolerance of the progenies after the one-round screen was better than that of the initial strain. After a two-round screen, the maximum tolerable temperature of Dip2-8, a progeny of diploid strain, was 3°C higher than that of the original strain. Whole-genome sequencing revealed nonsense mutations of PSR1 and PDE2 in the thermotolerant progenies. Deletion of either PSR1 or PDE2 in the original strain improved thermotolerance and two deletions displayed additive effects, suggesting PSR1 and PDE2 negatively regulated the thermotolerance of K. marxianus in parallel pathways. Therefore, the iterative cycle of “meiosis - spore screening” developed in this study provides an efficient way to perform the laboratory evolution of heat resistance in yeast.

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.

A Two-Stage Adaptive Laboratory Evolution Strategy to Enhance Docosahexaenoic Acid Synthesis in Oleaginous Thraustochytrid

Thraustochytrid is a promising algal oil resource with the potential to meet the demand for docosahexaenoic acid (DHA). However, oils with high DHA content produced by genetic modified thraustochytrids are not accepted by the food and pharmaceutical industries in many countries. Therefore, in order to obtain non-transgenic strains with high DHA content, a two-stage adaptive laboratory evolution (ALE) strategy was applied to the thraustochytrid Aurantiochytrium sp. Heavy-ion irradiation technique was first used before the ALE to increase the genetic diversity of strains, and then two-step ALE: low temperature based ALE and ACCase inhibitor quizalofop-p-ethyl based ALE were employed in enhancing the DHA production. Using this strategy, the end-point strain E-81 with a DHA content 51% higher than that of the parental strain was obtained. The performance of E-81 strain was further analyzed by component analysis and quantitative real-time PCR. The results showed that the enhanced in lipid content was due to the up-regulated expression of key enzymes in lipid accumulation, while the increase in DHA content was due to the increased transcriptional levels of polyunsaturated fatty acid synthase. This study demonstrated a non-genetic approach to enhance lipid and DHA content in non-model industrial oleaginous strains.

Application of Microalgal Stress Responses in Industrial Microalgal Production Systems

Adaptive laboratory evolution (ALE) has been widely utilized as a tool for developing new biological and phenotypic functions to explore strain improvement for microalgal production. Specifically, ALE has been utilized to evolve strains to better adapt to defined conditions. It has become a new solution to improve the performance of strains in microalgae biotechnology. This review mainly summarizes the key results from recent microalgal ALE studies in industrial production. ALE designed for improving cell growth rate, product yield, environmental tolerance and wastewater treatment is discussed to exploit microalgae in various applications. Further development of ALE is proposed, to provide theoretical support for producing the high value-added products from microalgal production.

Nutritional and Volatile Characterisation of Milk Inoculated with Thermo-Tolerant Lactobacillus bulgaricus through Adaptive Laboratory Evolution

In this study, thermo-tolerant strain of Lactobacillus bulgaricus (L. bulgaricus) was developed using gradual increase in temperature to induce Adaptive Laboratory Evolution (ALE). Viable colony count of 1.87 ± 0.98 log cfu/mL was achieved at 52 °C, using MRS agar supplemented with 2% lactose. Changes in bacteria morphology were discovered, from rod (control) to filament (52 °C) to cocci after frozen storage (−80 °C). When milk was inoculated with thermo-tolerant L. bulgaricus, lactic acid production was absent, leaving pH at 6.84 ± 0.13. This has caused weakening of the protein network, resulting in high whey separation and lower water-holding capacity (37.1 ± 0.35%) compared to the control (98.10 ± 0.60%). Significantly higher proteolytic activity was observed through free amino acids analysis by LC-MS. Arginine and methionine (237.24 ± 5.94 and 98.83 ± 1.78 µg/100 g, respectively) were found to be 115- and 275-fold higher than the control, contributing to changing the aroma similar to cheese. Further volatile analysis through SPME-GC-MS has confirmed significant increase in cheese-aroma volatiles compared to the control, with increase in diacetyl formation. Further work on DNA profiling, metabolomics and peptidomics will help to answer mechanisms behind the observed changes made in the study.

Activating silent glycolysis bypasses in Escherichia coli

All living organisms share similar reactions within their central metabolism to provide precursors for all essential building blocks and reducing power. To identify whether alternative metabolic routes of glycolysis can operate in E. coli, we complementarily employed in silico design, rational engineering, and adaptive laboratory evolution. First, we used a genome-scale model and identified two potential pathways within the metabolic network of this organism replacing canonical Embden-Meyerhof-Parnas (EMP) glycolysis to convert phosphosugars into organic acids. One of these glycolytic routes proceeds via methylglyoxal, the other via serine biosynthesis and degradation. Then, we implemented both pathways in E. coli strains harboring defective EMP glycolysis. Surprisingly, the pathway via methylglyoxal immediately operated in a triosephosphate isomerase deletion strain cultivated on glycerol. By contrast, in a phosphoglycerate kinase deletion strain, the overexpression of methylglyoxal synthase was necessary for implementing a functional methylglyoxal pathway. Furthermore, we engineered the serine shunt which converts 3-phosphoglycerate via serine biosynthesis and degradation to pyruvate, bypassing an enolase deletion. Finally, to explore which of these alternatives would emerge by natural selection we performed an adaptive laboratory evolution study using an enolase deletion strain. The evolved mutants were shown to use the serine shunt. Our study reveals the flexible redesignation of metabolic pathways to create new metabolite links and rewire central metabolism.

Characterization of highly ferulate-tolerant Acinetobacter baylyi ADP1 isolates by a rapid reverse-engineering method

Adaptive laboratory evolution (ALE) is a powerful approach for improving phenotypes of microbial hosts. Evolved strains typically contain numerous mutations that can be revealed by whole-genome sequencing. However, determining the contribution of specific mutations to new phenotypes is typically challenging and laborious. This task is complicated by factors such as the mutation type, the genomic context, and the interplay between different mutations. Here, a novel approach was developed to identify the significance of mutations in strains evolved from Acinetobacter baylyi ADP1. This method, termed Rapid Advantageous Mutation ScrEening and Selection (RAMSES), was used to analyze mutants that emerged from stepwise adaptation to, and consumption of, high levels of ferulate, a common lignin-derived aromatic compound. After whole-genome sequence analysis, RAMSES allowed rapid determination of effective mutations and seamless introduction of the beneficial mutations into the chromosomes of new strains with different genetic backgrounds. This simple approach to reverse-engineering exploits the natural competence and high recombination efficiency of ADP1. Mutated DNA, added directly to growing cells, replaces homologous chromosomal regions to generate transformants that will become enriched if there is selective benefit. Thus, advantageous mutations can be rapidly identified. Here, the growth advantage of transformants under selective pressure revealed key mutations in genes related to aromatic transport, including hcaE , hcaK , and vanK , and a gene, ACIAD0482 , which is associated with lipopolysaccharide synthesis. This study provides insights into enhanced utilization of industrially relevant aromatic substrates and demonstrates the use of A. baylyi ADP1 as a convenient platform for strain development and evolution studies. Importance Microbial conversion of lignin-enriched streams is a promising approach for lignin valorization. However, the lignin-derived aromatic compounds are toxic to cells at relevant concentrations. Although adaptive laboratory evolution (ALE) is a powerful approach to develop more tolerant strains, it is typically laborious to identify the mechanisms underlying phenotypic improvement. We employed Acinetobacter baylyi ADP1, an aromatic compound degrading strain that may be useful for biotechnology. The natural competence and high recombination efficiency of this strain can be exploited for critical applications such as the breakdown of lignin and plastics, abundant polymers composed of aromatic subunits. The natural transformability of this bacterium enabled us to develop a novel approach for rapid screening of advantageous mutations from ALE-derived aromatic-tolerant ADP1-derived strains. We clarified the mechanisms and genetic targets for improved tolerance towards common lignin-derived aromatic compounds. This study facilitates metabolic engineering for lignin valorization.