Characterization of the microbiota in long- and short-term natural indigo fermentation Abstract The duration for which the indigo-reducing state maintenance in indigo natural fermentation in batch dependent. The microbiota was analyzed in two batches of sukumo fermentation fluids that lasted for different durations (Batch 1: less than 2 months; Batch 2: nearly 1 year) to understand the mechanisms underlying the sustainability and deterioration of this natural fermentation process. The transformation of the microbiota suggested that the deterioration of the fermentation fluid is associated with the relative abundance of Alcaligenaceae. Principal coordinates analysis (PCoA) showed that the microbial community maintained a very stable state in only the long-term Batch 2. Therefore, entry of the microbiota into a stable state under alkaline anaerobic condition is an important factor for maintenance of indigo fermentation for long duration. This is the first report on the total transformation of the microbiota for investigation of long-term maintenance mechanisms and to address the problem of deterioration in indigo fermentation.

Improving ionic liquid tolerance in Saccharomyces cerevisiae through heterologous expression and directed evolution of an ILT1 homolog from Yarrowia lipolytica Abstract Ionic liquids show promise for deconstruction of lignocellulosic biomass prior to fermentation. Yet, imidazolium ionic liquids (IILs) can be toxic to microbes even at concentrations present after recovery. Here, we show that dominant overexpression of an Ilt1p homolog (encoded by YlILT1/YALI0C04884) from the IIL-tolerant yeast Yarrowia lipolytica confers an improvement in 1-ethyl-3-methylimidazolium acetate tolerance in Saccharomyces cerevisiae compared to the endogenous Ilt1p (ScILT1/YDR090C). We subsequently enhance tolerance in S. cerevisiae through directed evolution of YlILT1 using growth-based selection, leading to identification of mutants that grow in up to 3.5% v/v ionic liquid. Lastly, we demonstrate that strains expressing YlILT1 variants demonstrate improved growth rate and ethanol production in the presence of residual IIL. This shows that dominant overexpression of a heterologous protein (wild type or evolved) from an IIL-tolerant yeast can increase tolerance in S. cerevisiae at concentrations relevant to bioethanol production from IIL-treated biomass.

Adaptive mechanism of Acidithiobacillus thiooxidans CCTCC M 2012104 under stress during bioleaching of low-grade chalcopyrite based on physiological and comparative transcriptomic analysis Abstract Acidithiobacillus thiooxidans (A. thiooxidans) is often used for sulfur-bearing ores bioleaching, but its adaptive mechanism to harsh environments remains unclear. Here, we explored the adaptive mechanism of A. thiooxidans in the process of low-grade chalcopyrite bioleaching based on the physiology and comparative transcriptome analysis. It was indicated that A. thiooxidans maintains intracellular pH homeostasis by regulating unsaturated fatty acids, especially cyclopropane fatty acids, intracellular ATP, amino acid metabolism, and antioxidant factors. Comparative transcriptome analysis indicated that the key genes involved in sulfur oxidation, sor and soxABXYZ, were significantly up-regulated, generating more energy to resist extreme environmental stress by more active sulfur metabolism. Confocal laser scanning microscope analysis found that down-regulation of flagellar-related genes was likely to promote the biofilm formation. System-level understanding of leaching microorganisms under extreme stress can contribute to the evolution of these extremophiles via genetic engineering modification work, which further improves bioleaching in future.

The expression, secretion and activity of the aspartic protease MpAPr1 in Metschnikowia pulcherrima IWBT Y1123 Abstract Protease-secreting yeasts have broad biotechnological potential for application to various industrial processes, including winemaking. However, this activity is influenced by the yeast response to environmental factors such as nitrogen and protein sources, as are found in grape juice. In this study, the wine-relevant yeast Metschnikowia pulcherrima IWBT Y1123, with known protease-secreting ability, was subjected to different nitrogen-containing compounds to monitor their impact on protease secretion and activity. Protease activity increased above basal levels for haemoglobin-containing treatments, indicating an inductive influence of proteins. On the other hand, treatments containing both haemoglobin and assimilable nitrogen sources led to a delayed increase in protease activity and protein degradation, suggesting a nitrogen catabolite repression mechanism at work. Protease activity and expression were furthermore evaluated in grape juice, which revealed increased expression and activity levels over time as promising results for further investigations into the impact of this yeast on wine properties.

Efficient production of glutathione with multi-pathway engineering in Corynebacterium glutamicum Abstract Glutathione is a bioactive tripeptide composed of glycine, l-cysteine, and l-glutamate, and has been widely used in pharmaceutical, food, and healthy products. The current metabolic studies of glutathione were mainly focused on the native producing strains with precursor amino acid supplementation. In the present work, Corynebacterium glutamicum, a workhorse for industrial production of a series of amino acids, was engineered to produce glutathione. First, the introduction of glutathione synthetase gene gshF from Streptococcus agalactiae fulfilled the ability of glutathione production in C. glutamicum and revealed that l-cysteine was the limiting factor. Then, considering the inherent capability of l-glutamate synthesis and the availability of external addition of low-cost glycine, l-cysteine biosynthesis was enhanced using a varieties of pathway engineering methods, such as disrupting the degradation pathways of l-cysteine and l-serine, and removing the repressor responsible for sulfur metabolism. Finally, the simultaneously introduction of gshF and enhancement of cysteine formation enabled C. glutamicum strain to produce glutathione greatly. Without external addition of l-cysteine and l-glutamate, 756 mg/L glutathione was produced. This is first time to demonstrate the potential of the glutathione non-producing strain C. glutamicum for glutathione production and provide a novel strategy to construct glutathione-producing strains.

Systemic understanding of Lactococcus lactis response to acid stress using transcriptomics approaches Abstract During fermentation, acid stress caused by the accumulation of acidic metabolites seriously affects the metabolic activity and production capacity of microbial cells. To elucidate the acid stress-tolerance mechanisms of microbial cells, we performed genome mutagenesis combined with high-throughput technologies to screen acid stress-tolerant strains. Mutant strain Lactococcus lactis WH101 showed a 16,000-fold higher survival rate than that of the parent strain after 5 h of acid shock at pH 4.0 and maintained higher ATP, NH4+, and intracellular pH (pHi) levels during acid stress. Additionally, comparative transcriptomics analysis revealed enhanced regulation of carbohydrate metabolism and sugar transport to provide additional energy, amino acid metabolism and transport to maintain pHi homeostasis and ATP generation, and fatty acid metabolism to enhance cellular acid tolerance. Moreover, overexpression of identified components resulted in 12.6- and 12.9-fold higher survival rates after acid shock for 3 h at pH 4.0 in L. lactis (ArcB) and L. lactis (MalQ) compared to the control strain, respectively. These findings provide valuable insight into the acid stress-response mechanisms of L. lactis and promote the further development of robust industrial strains.

Genomic and physiological analyses reveal that extremely thermophilic Caldicellulosiruptor changbaiensis deploys uncommon cellulose attachment mechanisms Abstract The genus Caldicellulosiruptor is comprised of extremely thermophilic, heterotrophic anaerobes that degrade plant biomass using modular, multifunctional enzymes. Prior pangenome analyses determined that this genus is genetically diverse, with the current pangenome remaining open, meaning that new genes are expected with each additional genome sequence added. Given the high biodiversity observed among the genus Caldicellulosiruptor, we have sequenced and added a 14th species, Caldicellulosiruptor changbaiensis, to the pangenome. The pangenome now includes 3791 ortholog clusters, 120 of which are unique to C. changbaiensis and may be involved in plant biomass degradation. Comparisons between C. changbaiensis and Caldicellulosiruptor bescii on the basis of growth kinetics, cellulose solubilization and cell attachment to polysaccharides highlighted physiological differences between the two species which are supported by their respective gene inventories. Most significantly, these comparisons indicated that C. changbaiensis possesses uncommon cellulose attachment mechanisms not observed among the other strongly cellulolytic members of the genus Caldicellulosiruptor.

Ultra-high-throughput picoliter-droplet microfluidics screening of the industrial cellulase-producing filamentous fungus Trichoderma reesei Abstract The selection of improved producers among the huge number of variants in mutant libraries is a key issue in filamentous fungi of industrial biotechnology. Here, we developed a droplet-based microfluidic high-throughput screening platform for selection of high-cellulase producers from filamentous fungus Trichoderma reesei. The screening system used a fluorogenic assay to measure amount of cellulase and its activity. The key effectors such as cellulase-inducing medium, spore germination, droplet cultivation time, droplet fluorescence signal detection, and droplet cell sorting were studied. An artificial pre-mixed library of high- and low-cellulase-producing T. reesei strains was screened successfully to verify the feasibility of our method. Finally, two cellulase hyperproducers exhibiting improvements in cellulase activity of 27% and 46% were isolated from an atmospheric and room-temperature plasma (ARTP)-mutated library. This high-throughput screening system could be applied to the engineering of T. reesei strains and other industrially valuable protein-producing filamentous fungi.

Improved production of clavulanic acid by reverse engineering and overexpression of the regulatory genes in an industrial Streptomyces clavuligerus strain Abstract Genomic analysis of the clavulanic acid (CA)-high-producing Streptomyces clavuligerus strains, OL13 and OR, developed through random mutagenesis revealed a frameshift mutation in the cas1 gene-encoding clavaminate synthase 1. Overexpression of the intact cas1 in S. clavuligerus OR enhanced the CA titer by approximately 25%, producing ~ 4.95 g/L of CA, over the OR strain in the flask culture. Moreover, overexpression of the pathway-specific positive regulatory genes, ccaR and claR, in the OR strain improved CA yield by approximately 43% (~ 5.66 g/L) in the flask. However, co-expression of the intact cas1 with ccaR-claR did not further improve CA production. In the 7 L fermenter culture, maximum CA production by the OR strain expressing the wild-type cas1 and ccaR-claR reached approximately 5.52 g/L and 6.01 g/L, respectively, demonstrating that reverse engineering or simple rational metabolic engineering is an efficient method for further improvement of industrial strains.

Redox self-sufficient biocatalyst system for conversion of 3,4-Dihydroxyphenyl- l -alanine into ( R )- or ( S )-3,4-Dihydroxyphenyllactic acid Abstract We developed an efficient multi-enzyme cascade reaction to produce (R)- or (S)-3,4-Dihydroxyphenyllactic acid [(R)- or (S)-Danshensu, (R)- or (S)-DSS] from 3,4-Dihydroxyphenyl-l-alanine (l-DOPA) in Escherichia coli by introducing tyrosine aminotransferase (tyrB), glutamate dehydrogenase (cdgdh) and d-aromatic lactate dehydrogenase (csldhD) or l-aromatic lactate dehydrogenase (tcldhL). First, the genes in the pathway were overexpressed and fine-tuned for (R)- or (S)-DSS production. The resulting strain, E. coli TGL 2.1 and E. coli TGL 2.2, which overexpressed tyrB with the stronger T7 promoter and cdgdh, csldhD or tcldhL with the weaker Trc promoter, E. coli TGL 2.1 yielded 57% increase in (R)-DSS production: 59.8 ± 2.9 mM. Meanwhile, E. coli TGL 2.2 yielded 54% increase in (S)-DSS production: 52.2 ± 2.4 mM. The optimal concentration of L-glutamate was found to be 20 mM for production of (R)- or (S)-DSS. Finally, l-DOPA were transformed into (R)- or (S)-DSS with an excellent enantiopure form (enantiomeric excess > 99.99%) and productivity of 6.61 mM/h and 4.48 mM/h, respectively.