Exploring L-isoleucine riboswitches for enhancing 4-hydroxyisoleucine production in Corynebacterium glutamicum.

OBJECTIVES: To explore an L-isoleucine (Ile)-induced biosensor for down-regulation of Ile synthesis pathway and enhancement of 4-hydroxyisoleucine (4-HIL) production in Corynebacterium glutamicum SN01. RESULTS: Four Ile-induced riboswitches (IleRSN) with different strength were screened from mutation library based on TPP riboswitch. Firstly, IleRSN were integrated into the chromosome of strain SN01 immediately upstream of ilvA gene. The 4-HIL titer of strains carrying PtacM-driven IleRS1 or IleRS3 (14.09 ± 1.07, 15.20 ± 0.93 g 4-HIL L-1) were similar with control strain S-D5I (15.73 ± 2.66 g 4-HIL L-1). Then, another copy of IleRS3-ilvA was integrated downstream of the chromosomal cg0963 gene in SN01-derived strain D-RS with down-regulated L-lysine (Lys) biosynthesis. The Ile supply and 4-HIL titer increased in ilvA two-copy strains KIRSA-3-D5I and KIRSA-3-9I, and Ile concentration was maintained less than 35 mmol L-1 under the control of IleRS3 during fermentation. The resulting strain KIRSA-3-9I produced 22.46 ± 0.96 g 4-HIL L-1. CONCLUSION: The screened IleRS was effective in the dynamic down-regulation of Ile synthesis pathway in C. glutamicum, and IleRSN with different strength can be applied in various conditions.

Establishment of CRISPR-Cpf1-assisted gene editing tool and engineering of 4-hydroxyisoleucine biosynthesis in Corynebacterium glutamicum.

Corynebacterium glutamicum, an important industrial producer, is a model microorganism. However, the limited gene editing methods and their defects limit the efficient genome editing of C. glutamicum. To improve the screening efficiency of second-cross-over strains of traditional SacB editing system, a universal pCS plasmid which harbors CRISPR-Cpf1 system targeting kan gene of SacB system was designed and established to kill the false positive single-cross-over strains remained abundantly after the second-cross-over events. The lethality of pCS plasmid to C. glutamicum carrying kan gene on its genome was as high as 98.6%. In the example of PodhA::PilvBNC replacement, pCS plasmid improved the screening efficiency of second-cross-over bacteria from 5% to over 95%. Then this pCS-assisted gene editing system was applied to improve the supply of precursors and reduce the generation of by-products in the production of 4-hydroxyisoleucine (4-HIL). The 4-HIL titer of one edited strain SC01-TD5IM reached 137.0 ± 33.9 mM, while the weakening of lysE by promoter engineering reduced Lys content by 19.0-47.7% and 4-HIL titer by 16.4-64.5%. These editing demonstrates again the efficiency of this novel CRISPR-Cpf1-assisted gene editing tool, suggesting it as a useful tool for improving the genome editing and metabolic engineering in C. glutamicum.

Quorum sensing-mediated dynamic regulation of 4-hydroxyisoleucine biosynthesis in Corynebacterium glutamicum.

With the development of synthetic biology, some quorum sensing (QS) systems have been studied and applied to coordinate growth and production. Recently, a novel ComQXPA-PsrfA system with different response strengths was constructed in Corynebacterium glutamicum. However, the plasmid-harbored ComQXPA-PsrfA system lacks genetic stability, which restricts the application of this QS system. In this study, the comQXPA expression cassette was integrated into the chromosome of C. glutamicum SN01, resulting in QSc chassis strain. The green fluorescence protein (GFP) was expressed by the natural and mutant PsrfA promoters (PsrfAM) with various strengths in QSc. All the expressions of gfp were activated to the related level in a cell density-dependent manner. Therefore, ComQXPA-PsrfAM circuit was applied for modulating the dynamic biosynthesis of 4-hydroxyisoleucine (4-HIL). First, the expression of ido encoding α-ketoglutarate (α-KG)-dependent isoleucine dioxygenase was dynamically regulated by PsrfAM promoters, resulting in QSc/NI. The 4-HIL titer (125.18 ± 11.26 mM) increased by 45.1% compared to static ido expression strain. Then, to coordinate the α-KG supply between TCA cycle and 4-HIL synthesis, the activity of α-KG dehydrogenase complex (ODHC) was dynamically inhibited by regulating the expression of ODHC inhibitor gene odhI under QS-responsive PsrfAM promoters. The highest 4-HIL titer of QSc-11O/20I (145.20 ± 7.80 mM) increased by 23.2% compared to QSc/20I. This study modulated two critical genes expression in both cell growth and 4-HIL de novo synthesis pathways by the stable ComQXPA-PsrfAM system, and 4-HIL was produced responsively with the cell density. This strategy enhanced the 4-HIL biosynthesis efficiently without additional genetic regulation.

Dynamic control of 4-hydroxyisoleucine biosynthesis by multi-biosensor in Corynebacterium glutamicum.

4-hydroxyisoleucine (4-HIL) has a potential value in treating diabetes. The α-ketoglutarate (α-KG)-dependent isoleucine dioxygenase (IDO) can catalyze the hydroxylation of L-isoleucine (Ile) to form 4-HIL by consuming O2. In our previous study, the ido gene was overexpressed in an Ile-producing Corynebacterium glutamicum strain to synthesize 4-HIL from glucose. Here, a triple-functional dynamic control system was designed to regulate the activity of IDO, the supply of α-KG, O2, and Ile and the synthesis of by-product L-lysine (Lys) for promoting 4-HIL synthesis. Firstly, the codon-optimized ido was positively regulated by seven Ile biosensors Lrp-PbrnFEN with different intensities, and the resulting seven D-NI strains produced 38.7-111.1 mM 4-HIL. Then on the basis of D-NI, odhI and vgb were simultaneously regulated by three PbrnFEN with different intensities to synergistically control α-KG and O2 supply. The 4-HIL titer of twelve D-NINONV strains was more than 90 mM, with D-0I7O7V generating the highest titer of 141.1 ± 15.5 mM. Thirdly, ilvA was negatively regulated by an Ile attenuator PilvBNC on the basis of D-NI strains and some D-NINONV strains to balance the synthesis and conversion of Ile. The resulting D-NIPA strains produced 73.6-123.2 mM 4-HIL, while D-7I7O1VPA accumulated 127.1 ± 20.2 mM 4-HIL. Finally, dapA was negatively regulated by a Lys-OFF riboswitch and Lys content decreased by approximately 70% in most D-RS-NIPA strains. A strain D-RS-5IPA with the highest 4-HIL titer (177.3 ± 8.9 mM) and the lowest Lys concentration (6.1 ± 0.6 mM) was successfully obtained. Therefore, dynamic regulation of main and branch pathway by three functional biosensors can effectively promote 4-HIL biosynthesis in C. glutamicum. KEY POINTS: • Three biosensors were coordinated for dynamic 4-HIL biosynthesis in C. glutamicum • Bidirectional regulation of Ile synthesis and conversion promoted 4-HIL synthesis • Negative regulation of Lys synthesis further increased 4-HIL production.

An ultrasensitive biosensor for dual-specific DNA based on deposition of polyaniline on a self-assembled multi-functional DNA hexahedral-nanostructure.

Kras and Braf are major oncogenes. The mutation of Kras codon 12 or Braf V600E can lead to ovarian carcinoma. The detection of oncogene-related DNAs and their mutations offers solution for early diagnosis of ovarian cancer. Herein, a size-tunable multi-functional DNA hexahedral-nanostructure (DHN) has been rationally designed and modified on the electrode to response to Kras and Braf DNA. The size of DHN is controlled via polyadenines (polyA). The complete self-assembly of DHN depends on the presence of both target DNAs and two assistant probes. Meanwhile, a HRP-mimicking DNAzyme forms in DHN, which catalyzes the polymerization of aniline. The produced polyaniline is utilized as the output signal through differential pulse voltammetry (DPV). The biosensor shows the linear range from 100 fM to 1 µM, with the detection limit of 48.7 fM for Kras gene; and the linear range from 100 fM to 100 nM, with the detection limit of 44.1 fM for Braf gene, respectively. Since the current response depends on both gene sequences, the high specificity of the biosensor endows it to operate in an "OR"-type logic gate to discriminate the mutation of both genes. When Kras codon 12 or Braf V600E mutation happens, the response decreases significantly due to the incomplete formation of DNAzyme in DHN. The practicability of the biosensor has been verified through challenging human serum samples. Thus, it has great potential for clinical diagnosis of ovarian cancer through simultaneous detection of Kras and Braf genes and their mutations.