This study aimed to evaluate the hydrolysates from orange peel waste (OPW) as the low-cost carbon source for lycopene production. Initially, the dilute acid pretreatment combined with enzymatic hydrolysis of OPW resulted in a total sugar concentration of 62.18 g/L. Meanwhile, a four-month adaptive laboratory evolution (ALE) experiment using a d-galacturonic acid minimal medium resulted in an improvement in the growth rate of our previously engineered Escherichia coli strain for lycopene production. After evolutionary adaptation, response surface methodology (RSM) was adapted to optimize the medium composition in fermentation. The results obtained from RSM analysis revealed that the 5.53 % carbon source of orange peel hydrolysate (OPH), 6.57 g/L nitrogen source, and 30 °C temperature boosted lycopene production in the final strain. Subsequently, the optimized treatment for lycopene fermentation was then conducted in a 5 L batch fermenter under the surveillance of a kinetic model that uses the Logistic equation for strain growth (μm = 0.441 h-1), and Luedeking-Piret equations for lycopene production (Pm = 1043 mgL-1) with growth rate constant (α = 0.1491). At last, lycopene biosynthesized from OPH was extracted and analyzed for qualitative validation. Likewise, its data on phytic acid (between 1.01 % and 0.86 %) and DPPH radical scavenging (between 38.06 % and 29.08 %) highlighted the better antioxidant capacity of lycopene. In conclusion, the OPH can be used as a fermentation feedstock which opens new possibilities of exploiting fruit crop residues for food and pharmaceutical applications.

Rhodosporidium toruloides has emerged as an excellent option for microbial lipid production due to its ability to accumulate up to 70 % of lipids per cell dry weight, consume multiple substrates such as glucose and xylose, and tolerate toxic compounds. Despite the potential of Rhodosporidium toruloides for high lipid yields, achieving these remains is a significant hurdle. A comprehensive review is essential to thoroughly evaluate the advancements in processes and technologies to enhance lipid production in R. toruloides. The review covers various strategies for enhancing lipid production like co-culture, adaptive evolution, carbon flux analysis, as well as different modes of fermentation. This review will help researchers to better understand the recent developments in technologies for sustainable and scalable lipid production from R. toruloides and simultaneously emphasize the need for developing an efficient and sustainable bioprocess.

With its versatile metabolism including aerobic and anaerobic respiration, photosynthesis, photo-fermentation and nitrogen fixation, Rhodobacter sphaeroides can adapt to diverse environmental and nutritional conditions, including the presence of various stressors such as heavy metals. Thus, it is an important microorganism to study the molecular mechanisms of bacterial stress response and resistance, and to be used as a microbial cell factory for biotechnological applications or bioremediation. In this study, a highly cobalt-resistant and genetically stable R. sphaeroides strain was obtained by evolutionary engineering, also known as adaptive laboratory evolution (ALE), a powerful strategy to improve and characterize genetically complex, desired microbial phenotypes, such as stress resistance. For this purpose, successive batch selection was performed in the presence of gradually increased cobalt stress levels between 0.1-15 mM CoCl2 for 64 passages and without any mutagenesis of the initial population prior to selection. The mutant individuals were randomly chosen from the last population and analyzed in detail. Among these, a highly cobalt-resistant and genetically stable evolved strain called G7 showed significant cross-resistance against various stressors such as iron, magnesium, nickel, aluminum, and NaCl. Growth profiles and flame atomic absorption spectrometry analysis results revealed that in the presence of 4 mM CoCl2 that significantly inhibited growth of the reference strain, the growth of the evolved strain was unaffected, and higher levels of cobalt ions were associated with G7 cells than the reference strain. This may imply that cobalt ions accumulated in or on G7 cells, indicating the potential of G7 for cobalt bioremediation. Whole genome sequencing of the evolved strain identified 23 single nucleotide polymorphisms in various genes that are associated with transcriptional regulators, NifB family-FeMo cofactor biosynthesis, putative virulence factors, TRAP-T family transporter, sodium/proton antiporter, and also in genes with unknown functions, which may have a potential role in the cobalt resistance of R. sphaeroides.

Antibiotic resistance is a pressing health issue, with the emergence of resistance in bacteria outcompeting the discovery of novel drug candidates. While many studies have used Adaptive Laboratory Evolution (ALE) to understand the determinants of resistance, the influence of the drug dosing profile on the evolutionary trajectory remains understudied. In this study, we employed ALE on Mycobacterium smegmatis exposed to various concentrations of Norfloxacin using both cyclic constant and stepwise increasing drug dosages to examine their impact on the resistance mechanisms selected. Mutations in an efflux pump regulator, LfrR, were found in all of the evolved populations irrespective of the drug profile and population bottleneck, indicating a conserved efflux-based resistance mechanism. This mutation appeared early in the evolutionary trajectory, providing low-level resistance when present alone, with a further increase in resistance resulting from successive accumulation of other mutations. Notably, drug target mutations, similar to those observed in clinical isolates, were only seen above a threshold of greater than 4× the minimum inhibitory concentration (MIC). A combination of three mutations in the genes, lfrR, MSMEG_1959, and MSMEG_5045, was conserved across multiple lineages, leading to high-level resistance and preceding the appearance of drug target mutations. Interestingly, in populations evolved from parental strains lacking the lfrA efflux pump, the primary target of the lfrR regulator, no lfrR gene mutations are selected. Furthermore, evolutional trajectories originating from the ΔlfrA strain displayed early arrest in some lineages and the absence of target gene mutations in those that evolved, albeit delayed. Thus, blocking or inhibiting the expression of efflux pumps can arrest or delay the fixation of drug target mutations, potentially limiting the maximum attainable resistance levels.

Levilactobacillus brevis is crucial in food fermentation, particularly in sourdough production. However, the cultivation of L. brevis faces a challenge with accumulation of lactic acid, a major inhibitor. We aimed to increase the acid tolerance of L. brevis, an industrial strain for sourdough fermentation. We used the adaptive laboratory evolution (ALE) to obtain lactic acid tolerant strains. The evolved strain\'s fermentation and metabolite profiles, alongside sensory evaluation, were compared with the parental strain by using various analytical techniques. The ALE approach increased lactic acid tolerance in the evolved strain showing an increased growth rate by 1.1 and 1.9 times higher than the parental strain at pH 4.1 and 6.5, respectively. Comprehensive analyses demonstrated its potential application in sourdough fermentation, promising reduced downstream costs. The evolved strain, free from genetically modified organisms concerns, has great potential for industrial use by exhibiting enhanced growth in acidic conditions without affecting consumers\' bread preferences.

Biological conversion of lignin from biomass offers a promising strategy for sustainable production of fuels and chemicals. However, aromatic compounds derived from lignin commonly contain methoxy groups, and O-demethylation of these substrates is often a rate-limiting reaction that influences catabolic efficiency. Several enzyme families catalyze aromatic O-demethylation, but they are rarely compared in vivo to determine an optimal biocatalytic strategy. Here, two pathways for aromatic O-demethylation were compared in Pseudomonas putida KT2440. The native Rieske non-heme iron monooxygenase (VanAB) and, separately, a heterologous tetrahydrofolate-dependent demethylase (LigM) were constitutively expressed in P. putida, and the strains were optimized via adaptive laboratory evolution (ALE) with vanillate as a model substrate. All evolved strains displayed improved growth phenotypes, with the evolved strains harboring the native VanAB pathway exhibiting growth rates ∼1.8x faster than those harboring the heterologous LigM pathway. Enzyme kinetics and transcriptomics studies investigated the contribution of selected mutations toward enhanced utilization of vanillate. The VanAB-overexpressing strains contained the most impactful mutations, including those in VanB, the reductase for vanillate O-demethylase, PP_3494, a global regulator of vanillate catabolism, and fghA, involved in formaldehyde detoxification. These three mutations were combined into a single strain, which exhibited approximately 5x faster vanillate consumption than the wild-type strain in the first 8 h of cultivation. Overall, this study illuminates the details of vanillate catabolism in the context of two distinct enzymatic mechanisms, yielding a platform strain for efficient O-demethylation of lignin-related aromatic compounds to value-added products.

Adaptive laboratory evolution (ALE) is a widely used microbial strain development and optimization method. ALE experiments, to select for faster-growing strains, are commonly performed as serial batch cultivations in shake flasks, serum bottles, or microtiter plates or as continuous cultivations in bioreactors on a laboratory scale. To combine the advantages of higher throughput in parallel shaken cultures with continuous fermentations for conducting ALE experiments, a new Continuous parallel shaken pH-auxostat (CPA) was developed. The CPA consists of six autonomous parallel shaken cylindrical reactors, equipped with real-time pH control of the culture medium. The noninvasive pH measurement and control are realized by biocompatible pH sensor spots and a programmable pump module, to adjust the dilution rate of fresh medium for each reactor separately. Two different strains of the methylotrophic yeast Ogataea polymorpha were used as microbial model systems for parallel chemostat and pH-auxostat cultivations. During cultivation, the medium is acidified by the microbial activity of the yeast. For pH-auxostat cultivations, the growth-dependent acidification triggers the addition of fresh feed medium into the reactors, leading to a pH increase and thereby to the control of the pH to a predetermined set value. By controlling the pH to a predetermined set value, the dilution rate of the continuous cultivation is adjusted to values close to the washout point, in the range of the maximum specific growth rate of the yeast. The pH control was optimized by conducting a step-response experiment and obtaining tuned PI controller parameters by the Chien-Hrones-Reswick (CHR) PID tuning method. Two pH-auxostat cultivations were performed with two different O. polymorpha strains at high dilution rates for up to 18 days. As a result, up to 4.8-fold faster-growing strains were selected. The increased specific maximum growth rates of the selected strains were confirmed in subsequent batch cultivations.

Synthetic biology is contributing to the advancement of the global net-negative carbon economy, with emphasis on formate as a member of the one-carbon substrate garnering substantial attention. In this study, we employed base editing tools to facilitate adaptive evolution, achieving a formate tolerance of Yarrowia lipolytica to 1 M within 2 months. This effort resulted in two mutant strains, designated as M25-70 and M25-14, both exhibiting significantly enhanced formate utilization capabilities. Transcriptomic analysis revealed the upregulation of nine endogenous genes encoding formate dehydrogenases when cultivated utilizing formate as the sole carbon source. Furthermore, we uncovered the pivotal role of the glyoxylate and threonine-based serine pathway in enhancing glycine supply to promote formate assimilation. The full potential of Y. lipolytica to tolerate and utilize formate establishing the foundation for pyruvate carboxylase-based carbon sequestration pathways. Importantly, this study highlights the existence of a natural formate metabolic pathway in Y. lipolytica.

Microbes able to convert gaseous one-carbon (C1) waste feedstocks are increasingly important to transition to the sustainable production of renewable chemicals and fuels. Acetogens are interesting biocatalysts since gas fermentation using Clostridium autoethanogenum has been commercialised. However, most acetogen strains need complex nutrients, display slow growth, and are not robust for bioreactor fermentations. In this work, we used three different and independent adaptive laboratory evolution (ALE) strategies to evolve the wild-type C. autoethanogenum to grow faster, without yeast extract and to be robust in operating continuous bioreactor cultures. Multiple evolved strains with improved phenotypes were isolated on minimal media with one strain, named \"LAbrini\", exhibiting superior performance regarding the maximum specific growth rate, product profile, and robustness in continuous cultures. Whole-genome sequencing of the evolved strains identified 25 mutations. Of particular interest are two genes that acquired seven different mutations across the three ALE strategies, potentially as a result of convergent evolution. Reverse genetic engineering of mutations in potentially sporulation-related genes CLAU_3129 (spo0A) and CLAU_1957 recovered all three superior features of our ALE strains through triggering significant proteomic rearrangements. This work provides a robust C. autoethanogenum strain \"LAbrini\" to accelerate phenotyping and genetic engineering and to better understand acetogen metabolism.

BACKGROUND: The red oleaginous yeast Rhodotorula toruloides is a promising cell factory to produce microbial oils and carotenoids from lignocellulosic hydrolysates (LCH). A multi-stress tolerant strain towards four major inhibitory compounds present in LCH and methanol, was derived in our laboratory from strain IST536 (PYCC 5615) through adaptive laboratory evolution (ALE) under methanol and high glycerol selective pressure.
RESULTS: Comparative genomic analysis suggested the reduction of the original strain ploidy from triploid to diploid, the occurrence of 21,489 mutations, and 242 genes displaying copy number variants in the evolved strain. Transcriptomic analysis identified 634 genes with altered transcript levels (465 up, 178 down) in the multi-stress tolerant strain. Genes associated with cell surface biogenesis, integrity, and remodelling and involved in stress-responsive pathways exhibit the most substantial alterations at the genome and transcriptome levels. Guided by the suggested stress responses, the multi-stress tolerance phenotype was extended to osmotic, salt, ethanol, oxidative, genotoxic, and medium-chain fatty acid-induced stresses.
CONCLUSIONS: The comprehensive analysis of this evolved strain provided the opportunity to get mechanistic insights into the acquisition of multi-stress tolerance and a list of promising genes, pathways, and regulatory networks, as targets for synthetic biology approaches applied to promising cell factories, toward more robust and superior industrial strains. This study lays the foundations for understanding the mechanisms underlying tolerance to multiple stresses in R. toruloides, underscoring the potential of ALE for enhancing the robustness of industrial yeast strains.