Systematic metabolic engineering of Escherichia coli for high-level production of pseudouridine via pathway optimization and precursor enhancement.

l-Homoserine, which is one of the few amino acids that is not produced on a large scale by microbial fermentation, plays a significant role in the synthesis of a series of valuable chemicals. In this study, systematic metabolic engineering was applied to target Escherichia coli W3110 for the production of l-homoserine. Initially, a basic l-homoserine producer was engineered through the strategies of overexpressing thrA (encoding homoserine dehydrogenase), removing the degradative and competitive pathways by knocking out metA (encoding homoserine O-succinyltransferase) and thrB (encoding homoserine kinase), reinforcing the transport system, and redirecting the carbon flux by deleting iclR (encoding the isocitrate lyase regulator). The resulting strain constructed by these strategies yielded 3.21 g/liter of l-homoserine in batch cultures. Moreover, based on CRISPR-Cas9/dCas9 (nuclease-dead Cas9)-mediated gene repression for 50 genes, the iterative genetic modifications of biosynthesis pathways improved the l-homoserine yield in a stepwise manner. The rational integration of glucose uptake and recovery of l-glutamate increased l-homoserine production to 7.25 g/liter in shake flask cultivation. Furthermore, the intracellular metabolic analysis further provided targets for strain modification by introducing the anaplerotic route afforded by pyruvate carboxylase to oxaloacetate formation, which resulted in accumulating 8.54 g/liter l-homoserine (0.33 g/g glucose, 62.4% of the maximum theoretical yield) in shake flask cultivation. Finally, a rationally designed strain gave 37.57 g/liter l-homoserine under fed-batch fermentation, with a yield of 0.31 g/g glucose.IMPORTANCE In this study, the bottlenecks that sequentially limit l-homoserine biosynthesis were identified and resolved, based on rational and efficient metabolic-engineering strategies, coupled with CRISPR interference (CRISPRi)-based systematic analysis. The metabolomics data largely expanded our understanding of metabolic effects and revealed relevant targets for further modification to achieve better performance. The systematic analysis strategy, as well as metabolomics analysis, can be used to rationally design cell factories for the production of highly valuable chemicals.

Light-Scattering Submicroscopic Particles as Highly Fluorescent Analogs and Their Use as Tracer Labels in Clinical and Biological Applications: II. Experimental Characterization

Fungal azaphilones have attracted considerable interest as they exhibit great potential in food and pharmacological industries. However, there is a severe bottleneck in the low production in wild strains and the ability to genetically engineer azaphilone-producing fungi. Using Monascus azaphilones (MAs) as an example, we demonstrate a systematic metabolic engineering strategy for improving the production of MAs. In this study, Monascus purpureus HJ11 was systematically engineered through a combination of promoter engineering, gene knockout, rate-limiting enzyme overexpression, repression of the competing pathway, enzyme engineering, and metabolic rebalance. The maximum yield and titer of MAs successfully increased to 906 mg/g dry cell weight (DCW) and 14.6 g/L, respectively, 2.6 and 3.7 times higher than those reported in the literature. Our successful model not only offers a practical and efficient way to improve the azaphilone production but also sheds light on the potential of systematic metabolic engineering in nonmodel fungi as a chassis for the production of high-value chemicals.

Metabolic engineering of Pichia pastoris for overproduction of cis-trans nepetalactol

Enhanced production of l-histidine in Escherichia coli through systematic metabolic engineering and CER controlled fermentation

Lipid engineering combined with systematic metabolic engineering of Saccharomyces cerevisiae for high-yield production of lycopene

Systematic metabolic engineering of Photobacterium sp. TLY01 for high-yield biosynthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate)

Interleukin–17E (also known as Interleukin–25) is the distinct member of Interleukin–17 cytokine family, induces the expression of IL–4, IL–5 and IL–13. Economical production of interleukin – 17E has lot of importance in the current day research in several clinical applications. The objective of the study was to optimize the physico–chemical parameters i.e., dissolved oxygen (DO) and nutritional factors i.e., carbon, nitrogen and phosphate sources on production of Interleukin–17E using industrially important salt inducible Escherichia coli GJ1158. The expression levels were not increased beyond 30 % DO in batch fermentation, but expression levels were increased beyond 30 % DO in fed-batch fermentation. The threshold level of dissolved oxygen ranges was 50 % in respect to the IL–17E production. Pulses of nutritional factors i.e., glucose, yeast extract and K2HPO4 enhanced the expression levels in fed batch fermentation at 40 % and 50 % dissolved oxygen ranges. When OD600 of the culture reaches to 74 in fed batch fermentation, culture was induced with 100 mM sterile NaCl and further incubated for next 15 hr. Purification was carried out using Ni – NTA spin column. A final concentration of 98 mg/L of purified IL–17E was obtained using cost effective medium viz., modified M9ON medium. The IL–17E thus produced is tested for its activity. In this study, fed-batch fermentation emphasizes the highest concentration of codon optimized recombinant human interleukin–17E using salt inducible expression host till to date, which manages to satisfy the industrial and clinical requirements.

Production of L-lactic acid from methanol by engineered yeast Pichia pastoris

Scutellarin, a flavonoid glycoside with anti-inflammatory, antioxidant, and cerebral/myocardial ischemia-ameliorating activities, is currently sourced from plants; however, this method is limited by high cultivation costs and low yields. In this study, de novo biosynthesis of scutellarin was achieved through systematic metabolic engineering of Yarrowia lipolytica. First, fusion of the key enzyme SbF6H with ATR2 (Sl1A) enhanced catalytic efficiency by 12.57%. Next, adding a solubility-enhancing tag to FNS I increased scutellarin titer by 44.33%. Subsequently, a xylose-inducible system coupregulated FNS I and Sl1A, while precursor supply was enhanced via metabolic rewiring. The resulting strain produced 1,184.24 mg/L in shake flasks; fed-batch fermentation in a 5-L bioreactor reached 6,370.39 mg/L, which is the highest microbial titer reported to date, with only 7.26% byproduct. This work establishes a scalable platform for industrial scutellarin production and provides a generalizable framework for microbial synthesis of high-value flavonoids.

Metabolic engineering of Escherichia coli for d-pantothenic acid production

Manipulating DNA transcription using synthetic DNA binder has always been one of the ideal strategies for biological regulation and disease therapy, in the premise of considerable efficacy. Although it has been studied for decades, synthetic DNA binder is gradually less popular for researchers from the aspects of structural study, wide biological exploration and clinical application. The partial reasons arise from less sequence selectivity and difficulty to install advanced working moiety on the scaffold of synthetic DNA binder. In order to closely study its structural domain and potential optimization approaches, here I discuss the architecture engineering of DNA binding system and how to construct advanced DNA binding assembly based on the well-studied DNA binding system Pyrrole–imidazole polyamides (PIPs). The recent progress on structural assembly and biological application are extensively introduced.

Systems metabolic engineering of Corynebacterium glutamicum for efficient l-tryptophan production.

Protein bioassay is a critical tool for the screening and detection of protein biomarkers in disease diagnostics and biological applications. However, the detection sensitivity and system automation of current immunoassays do not meet the emerging demands of clinical applications. Here, we developed a dissolution-enhanced luminescence-enhanced digital microfluidics immunoassay (DEL-DMF), which significantly improves the sensitivity and automation of the protein bioassay. In DEL-DMF, the sample and reagent droplets are controlled to complete the processes of sample transport, immunoreaction, and buffer washing, which not only minimizes sample consumption to 2 μL and enhances the binding efficiency of immunoreaction but also streamlines all the procedures and simplifies the process of immunoassay. Moreover, dissolution-enhanced luminescence using NaEuF4 NPs as nanoprobes boosts the fluorescence and increases the sensitivity of the bioassay. We demonstrate the enhanced analytical performance of our DEL-DMF immunoassay to detect H5N1 hemagglutinin in human serum and saliva. A limit of detection of 1.16 pM was achieved in less than 0.5 h with only 2 μL sample consumption. Overall, our DEL-DMF immunoassay combines the merits of the microfluidics platform and dissolution-enhanced luminescence, thus affording superior detection sensitivity and system automation for protein biomarkers. This novel immunoassay microsystem holds great potential in clinical and biological applications.

(R)-3-Hydroxybutyric acid (R-3HB) is an important chiral chemical with extensive applications in the agricultural, food, and chemical industries. The synthesis of R-3HB by microbial fermentation is of interest due to its remarkable stereoselectivity and economy. However, the low production of R-3HB failed to meet the needs of large-scale industrial production. In this study, an engineered strain for the efficient biosynthesis of R-3HB was constructed through a three-pronged approach encompassing biosynthetic pathway optimization, engineering of NADPH regenerators, and central metabolism regulation. The engineered strain Q5081 produced 75.7 g/L R-3HB, with a productivity of 1.26 g/L/h and a yield of 0.34 g/g glucose in fed-batch fermentation, showing the highest reported titer and productivity of R-3HB to date. We also performed transcriptome sequencing and annotation to illustrate the mechanism underlying the enhanced R-3HB production. The systematic metabolic engineering by a three-pronged approach demonstrated the feasibility of improving the biosynthesis, and the engineered strain Q5081 has the potential for widespread applications in the industrial production of R-3HB.