Dataset Information

Adaptive laboratory evolution of Bacillus subtilis to overcome toxicity of lignocellulosic hydrolysate derived from Distiller's dried grains with solubles (DDGS).

ABSTRACT: Microbial tolerance to toxic compounds formed during biomass pretreatment is a significant challenge to produce bio-based products from lignocellulose cost effectively. Rational engineering can be problematic due to insufficient prerequisite knowledge of tolerance mechanisms. Therefore, adaptive laboratory evolution was applied to obtain 20 tolerant lineages of Bacillus subtilis strains able to utilize Distiller's Dried Grains with Solubles-derived (DDGS) hydrolysate. Evolved strains showed both improved growth performance and retained heterologous enzyme production using 100% hydrolysate-based medium, whereas growth of the starting strains was essentially absent. Whole-genome resequencing revealed that evolved isolates acquired mutations in the global regulator codY in 15 of the 19 sequenced isolates. Furthermore, mutations in genes related to oxidative stress (katA, perR) and flagella function appeared in both tolerance and control evolution experiments without toxic compounds. Overall, tolerance adaptive laboratory evolution yielded strains able to utilize DDGS-hydrolysate to produce enzymes and hence proved to be a valuable tool for the valorization of lignocellulose.

SUBMITTER: Driessen JLSP

PROVIDER: S-EPMC10206485 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Adaptive laboratory evolution of <i>Bacillus subtilis</i> to overcome toxicity of lignocellulosic hydrolysate derived from Distiller's dried grains with solubles (DDGS).

Driessen Jasper L S P JLSP Johnsen Josefin J Pogrebnyakov Ivan I Mohamed Elsayed T T ETT Mussatto Solange I SI Feist Adam M AM Jensen Sheila I SI Nielsen Alex T AT

Metabolic engineering communications 20230501

Microbial tolerance to toxic compounds formed during biomass pretreatment is a significant challenge to produce bio-based products from lignocellulose cost effectively. Rational engineering can be problematic due to insufficient prerequisite knowledge of tolerance mechanisms. Therefore, adaptive laboratory evolution was applied to obtain 20 tolerant lineages of <i>Bacillus subtilis</i> strains able to utilize Distiller's Dried Grains with Solubles-derived (DDGS) hydrolysate. Evolved strains show ...[more]

PMID: 37234932

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Project description:Background. Steer progeny suckled by cows fed a dried distillers grains and solubles (DDGS) diet the first three months of lactation were heavier during feedlot finishing and had significantly lower marbling and larger longissumus muscles than steer suckled by cows fed a control diet (CON). These differences were profound in that progeny were managed and fed identically from weaning until finishing, and suggested that the developmental program of muscle composition was established during the suckling period. Purpose. Transcriptomes of longissimus muscle were measured using next generation sequencing to investigate whether there were any developmental clues to the differences in marbling scores and muscle content between steers suckled by DDGS (n=5) versus control (CON; n=5) diet fed cows during lactation. Methods. Multiparous Angus × Simmental cows with male progeny were fed one of two diets supplemented with either DDGS or soybean meal (CON), from calving until day 129 postpartum (PP). After conclusion of the treatments at 129 days postpartum, cow–calf pairs were comingled and managed as one group until weaning at 219 days postpartum. Steers were then transitioned to a common diet. Longissimus muscle (LM) biopsies were obtained from steers suckled by cows fed DDGS (n=5) and CON (n=5) 10 d prior to slaughter. RNA was extracted from LM. Total RNA isolated and Library Prep Kit was used to generate library for measuring messenger RNA molecules. Library was sequenced on an Illumina HiSeq2500 platform. FastQC (v 0.10.1) was used to assess sequence quality, and quality trimming was done using FASTX toolkit (v 0.0.13). Bases with less than Phred33 score of 30 were removed and resulted in reads of at least 50 bases. Reads were mapped against the bowtie2-indexed Bos taurus genome using Tophat (v 2.0.9) with default parameters, and the genome annotation file (.gtf) from Ensembl was used to associate the genome with annotated features. Raw read counts of each sample for each gene feature were generated with HTSeq (v 0.5.3p7) using Tophat output and Bos taurus annotations. Counts were merged using custom perl scripts and used for downstream differential gene expression (DE) analysis. DE pairwise analysis between samples CON and DDGS was done with ‘R’ (Version 2.15.2). Counts matrix, library sizes, and experimental design were combined to create an object using edgeR (v 3.0.3) package. Results. Number of reads per sample ranged from 79,530,352 to 58,600,824. After trimming there were 77,569,706 to 57,307,614 per sample. The percent of reads that mapped to bovine genome per sample ranged from 77.3 to 92.3%, and averaged 86.3% across all samples. Number of reads per gene ranged from one to over two-hundred ninety thousand counts per million (CPM) reads. There were 809 genes differentially expressed (P-adj<0.1) between CON and DDGS muscle. Of these 637 were upregulated and 172 downregulated in DDGS relative to CON. Overall the DDGS versus CON muscle transcriptomic signature was pro-myogenic and anti-adipogenic. In particular, myokines/satellite cell maintenance factors were found among upregulated (LIF, CNTF, FGFB1, EPHB1) genes. The anti-adipogenic signature was typified by the upregulation of anti-inflammatory cytokines and receptors (IL1RAP, IL1RL2, IL13RA2, IL1F10,), and downregulation of expression of inflammation/inflammatory cytokines and receptor (TNF, IL6R CXCL9), which suggests a selection of differentiation pathways away from adipogenic line. While the upregulation of TGFB, SPP1 and INHBA supports selection of fibroblast lineage of cells. Conclusion. The lactation phase of production can effect meat quality by affecting transcriptional signatures that favor myogenesis and depress inflammation.

Project description:Two sets of in vitro rumen fermentation experiments were conducted to determine effects of diets that included wet distiller's grains plus solubles (WDGS) and tannin-rich peanut skin (PS) on the in vitro digestibility, greenhouse gas (GHG) and other gas emissions, fermentation rate, and microbial changes. The objectives were to assess associative effects of various levels of PS or WDGS on the in vitro digestibility, GHG and other gas emissions, fermentation rate, and microbial changes in the rumen. All gases were collected using an ANKOM Gas Production system for methane (CH4), carbon dioxide (CO2), nitrous oxide (N2O), and hydrogen sulfide (H2S) analyses. Cumulative ruminal gas production was determined using 250 mL ANKOM sampling bottles containing 50 mL of ruminal fluid (pH 5.8), 40 mL of artificial saliva (pH 6.8), and 6 g of mixed diets after a maximum of 24 h of incubation. Fermenters were flushed with CO2 gas and held at 39 °C in a shaking incubator for 24 h. Triplicate quantitative real-time polymerase chain reaction (qPCR) analyses were conducted to determine microbial diversity. When WDGS was supplied in the diet, in the absence of PS, cumulative CH4 production increased (P < 0.05) with 40% WDGS. In the presence of PS, production of CH4 was reduced but the reduction was less at 40% WDGS. In the presence of PS, ruminal lactate, succinate, and acetate/propionate (A/P) ratio tended to be less with a WDGS interaction (P < 0.01). In the presence of PS and with 40% WDGS, average populations of Bacteroidetes, total methanogens, Methanobrevibacter sp. AbM4, and total protozoa were less. The population of total methanogens (R2 = 0.57; P < 0.01), Firmicutes (R2 = 0.46: P < 0.05), and Firmicutes/Bacteroidetes (F/B) ratio (R2 = 0.46; P < 0.03) were strongly correlated with ruminal CH4 production. Therefore, there was an associative effect of tannin-rich PS and WDGS, which suppressed methanogenesis both directly and indirectly by modifying populations of ruminal methanogens.

Project description:Dried distillers' grains with solubles (DDGS) are rich in nutrients, and partially alternative feeding of DDGS effectively reduces cost of feed and improves animals' growth. We used 16S rDNA gene sequencing and LC/MS-based metabolomics to explore the effect of feeding cattle with a basal diet (BD) and a Jiang-flavor DDGS diet (replaces 25% concentrate of the diet) on microbiome and metabolome of ruminal and cecal contents in Guanling yellow cattle. The results showed that the ruminal and cecal contents shared the same dominance of Bacteroidetes, Firmicutes and Proteobacteria in two groups. The ruminal dominant genera were Prevotella_1, Rikenellaceae_RC9_gut_group, and Ruminococcaceae_UCG-010; and the cecal dominant genera were Ruminococcaceae_UCG-005, Ruminococcaceae_UCG-010, and Rikenellaceae_RC9_gut_group. Linear discriminant analysis effect size analysis (LDA > 2, P < 0.05) revealed the significantly differential bacteria enriched in the DDGS group, including Ruminococcaceae_UCG_012, Prevotellaceae_UCG_004 and Anaerococcus in the ruminal contents, which was associated with degradation of plant polysaccharides. Besides, Anaerosporobacter, Anaerovibrio, and Caproiciproducens in the cecal contents were involved in fatty acid metabolism. Compared with the BD group, 20 significantly different metabolites obtained in the ruminal contents of DDGS group were down-regulated (P < 0.05), and based on them, 4 significantly different metabolic pathways (P < 0.05) were enriched including "Linoleic acid metabolism," "Biosynthesis of unsaturated fatty acids," "Taste transduction," and "Carbohydrate digestion and absorption." There were 65 significantly different metabolites (47 were upregulated, 18 were downregulated) in the cecal contents of DDGS group when compared with the BD group, and 4 significantly different metabolic pathways (P < 0.05) were enriched including "Longevity regulating pathway," "Bile secretion," "Choline metabolism in cancer," and "HIF-1 signaling pathway." Spearman analysis revealed close negative relationships between the top 20 significantly differential metabolites and Anaerococcus in the ruminal contents. Bacteria with high relevance to cecal differential metabolites were Erysipelotrichaceae_UCG-003, Dielma, and Solobacterium that affect specific metabolic pathways in cattle. Collectively, our results suggest that feeding cattle with a DDGS diet improves the microbial structure and the metabolic patterns of lipids and carbohydrates, thus contributing to the utilization efficiency of nutrients and physical health to some extent. Our findings will provide scientific reference for the utilization of DDGS as feed in cattle industry.

Dataset Information

Adaptive laboratory evolution of Bacillus subtilis to overcome toxicity of lignocellulosic hydrolysate derived from Distiller's dried grains with solubles (DDGS).

Publications

Adaptive laboratory evolution of <i>Bacillus subtilis</i> to overcome toxicity of lignocellulosic hydrolysate derived from Distiller's dried grains with solubles (DDGS).

Similar Datasets

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