Enhanced production of ectoine from methane using metabolically engineered Methylomicrobium alcaliphilum 20Z

Background Ectoine (1,3,4,5-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is an attractive compatible solute because of its wide industrial applications. Previous studies on the microbial production of ectoine have focused on sugar fermentation. Alternatively, methane can be used as an inexpensive and abundant resource for ectoine production by using the halophilic methanotroph, Methylomicrobium alcaliphilum 20Z. However, there are some limitations, including the low production of ectoine from methane and the limited tools for the genetic manipulation of methanotrophs to facilitate their use as industrial strains. Results We constructed M. alcaliphilum 20ZDP with a high conjugation efficiency and stability of the episomal plasmid by the removal of its native plasmid. To improve the ectoine production in M. alcaliphilum 20Z from methane, the ectD (encoding ectoine hydroxylase) and ectR (transcription repressor of the ectABC-ask operon) were deleted to reduce the formation of by-products (such as hydroxyectoine) and induce ectoine production. When the double mutant was batch cultured with methane, ectoine production was enhanced 1.6-fold compared to that obtained with M. alcaliphilum 20ZDP (45.58 mg/L vs. 27.26 mg/L) without growth inhibition. Notably, a maximum titer of 142.32 mg/L was reached by the use of an optimized medium for ectoine production containing 6% NaCl and 0.05 μM of tungsten without hydroxyectoine production. This result demonstrates the highest ectoine production from methane to date. Conclusions Ectoine production was significantly enhanced by the disruption of the ectD and ectR genes in M. alcaliphilum 20Z under optimized conditions favoring ectoine accumulation. We demonstrated effective genetic engineering in a methanotrophic bacterium, with enhanced production of ectoine from methane as the sole carbon source. This study suggests a potentially transformational path to commercial sugar-based ectoine production. Graphical Abstract

Mechanism of differential expression of β-glucosidase genes in functional microbial communities in response to carbon catabolite repression

Background β-Glucosidase is the rate-limiting enzyme of cellulose degradation. It has been stipulated and established that β-glucosidase-producing microbial communities differentially regulate the expression of glucose/non-glucose tolerant β-glucosidase genes. However, it is still unknown if this differential expression of functional microbial community happens accidentally or as a general regulatory mechanism, and of what biological significance it has. To investigate the composition and function of microbial communities and how they respond to different carbon metabolism pressures and the transcriptional regulation of functional genes, the different carbon metabolism pressure was constructed by setting up the static chamber during composting. Results The composition and function of functional microbial communities demonstrated different behaviors under the carbon metabolism pressure. Functional microbial community up-regulated glucose tolerant β-glucosidase genes expression to maintain the carbon metabolism rate by enhancing the transglycosylation activity of β-glucosidase to compensate for the decrease of hydrolysis activity under carbon catabolite repression (CCR). Micrococcales play a vital role in the resistance of functional microbial community under CCR. The transcription regulation of GH1 family β-glucosidase genes from Proteobacteria showed more obvious inhibition than other phyla under CCR. Conclusion Microbial functional communities differentially regulate the expression of glucose/non-glucose tolerant β-glucosidase genes under CCR, which is a general regulatory mechanism, not accidental. Furthermore, the differentially expressed β-glucosidase gene exhibited species characteristics at the phylogenetic level.

Integration of Aspergillus niger transcriptomic profile with metabolic model identifies potential targets to optimise citric acid production from lignocellulosic hydrolysate

Background Citric acid is typically produced industrially by Aspergillus niger-mediated fermentation of a sucrose-based feedstock, such as molasses. The fungus Aspergillus niger has the potential to utilise lignocellulosic biomass, such as bagasse, for industrial-scale citric acid production, but realising this potential requires strain optimisation. Systems biology can accelerate strain engineering by systematic target identification, facilitated by methods for the integration of omics data into a high-quality metabolic model. In this work, we perform transcriptomic analysis to determine the temporal expression changes during fermentation of bagasse hydrolysate and develop an evolutionary algorithm to integrate the transcriptomic data with the available metabolic model to identify potential targets for strain engineering. Results The novel integrated procedure matures our understanding of suboptimal citric acid production and reveals potential targets for strain engineering, including targets consistent with the literature such as the up-regulation of citrate export and pyruvate carboxylase as well as novel targets such as the down-regulation of inorganic diphosphatase. Conclusions In this study, we demonstrate the production of citric acid from lignocellulosic hydrolysate and show how transcriptomic data across multiple timepoints can be coupled with evolutionary and metabolic modelling to identify potential targets for further engineering to maximise productivity from a chosen feedstock. The in silico strategies employed in this study can be applied to other biotechnological goals, assisting efforts to harness the potential of microorganisms for bio-based production of valuable chemicals.

Omics analysis coupled with gene editing revealed potential transporters and regulators related to levoglucosan metabolism efficiency of the engineered Escherichia coli

Background Bioconversion of levoglucosan, a promising sugar derived from the pyrolysis of lignocellulose, into biofuels and chemicals can reduce our dependence on fossil-based raw materials. However, this bioconversion process in microbial strains is challenging due to the lack of catalytic enzyme relevant to levoglucosan metabolism, narrow production ranges of the native strains, poor cellular transport rate of levoglucosan, and inhibition of levoglucosan metabolism by other sugars co-existing in the lignocellulose pyrolysate. The heterologous expression of eukaryotic levoglucosan kinase gene in suitable microbial hosts like Escherichia coli could overcome the first two challenges to some extent; however, no research has been dedicated to resolving the last two issues till now. Results Aiming to resolve the two unsolved problems, we revealed that seven ABC transporters (XylF, MalE, UgpB, UgpC, YtfQ, YphF, and MglA), three MFS transporters (KgtP, GntT, and ActP), and seven regulatory proteins (GalS, MhpR, YkgD, Rsd, Ybl162, MalM, and IraP) in the previously engineered levoglucosan-utilizing and ethanol-producing E. coli LGE2 were induced upon exposure to levoglucosan using comparative proteomics technique, indicating these transporters and regulators were involved in the transport and metabolic regulation of levoglucosan. The proteomics results were further verified by transcriptional analysis of 16 randomly selected genes. Subsequent gene knockout and complementation tests revealed that ABC transporter XylF was likely to be a levoglucosan transporter. Molecular docking showed that levoglucosan can bind to the active pocket of XylF by seven H-bonds with relatively strong strength. Conclusion This study focusing on the omics discrepancies between the utilization of levoglucosan and non-levoglucosan sugar, could provide better understanding of levoglucosan transport and metabolism mechanisms by identifying the transporters and regulators related to the uptake and regulation of levoglucosan metabolism. The protein database generated from this study could be used for further screening and characterization of the transporter(s) and regulator(s) for downstream enzymatic/genetic engineering work, thereby facilitating more efficient microbial utilization of levoglucosan for biofuels and chemicals production in future.

Co-production of amino acid-rich xylooligosaccharide and single-cell protein from paper mulberry by autohydrolysis and fermentation technologies

Background Autohydrolysis is an extensively investigated pretreatment method due to its environmental friendliness. During autohydrolysis, most xylan from hemicellulose can be converted into xylooligosaccharides (XOS), and cellulose in the autohydrolyzed residues can be transformed into glucose after enzymatic hydrolysis. Both of these are value-added biochemicals in the biorefining process. In this work, paper mulberry (PM), which contains abundant protein, was utilized as a raw material to coproduce XOS and single-cell protein (SCP) through autohydrolysis and fermentation technologies. Results The results showed that 8.3 g of XOS and 1.8 g of amino acids could be recovered in the autohydrolysate (based on 100 g raw material) after autohydrolysis (170 °C, 1 h). Moreover, 5.7 g of low-DP XOS along with 1.8 g of amino acids could be further obtained from the autohydrolysate after hydrolysis with endo-β-1-4-xylanase. In addition, 20.1 g of fermentable monosaccharides was recovered after hydrolyzing the autohydrolyzed PM with cellulase, which can be used to produce 4.8 g of SCP after fermentation with Candida utilis. Conclusion As a valuable application of PM, a novel process is proposed to coproduce amino acid-rich XOS and SCP through autohydrolysis. The carbohydrate of PM is effectively converted to high value-added products.