Gene manipulation is essential for metabolic engineering and synthetic biology, but the current general gene manipulation methods are not applicable to the non-model strain Corynebacterium glutamicum (C. glutamicum) ATCC14067, which is used for amino acid production. Here, we report an effective and sequential deletion method for C. glutamicum ATCC14067 using the exonuclease-recombinase pair RecE + RecT (RecET) for recombineering via a designed self-excisable linear double-strand DNA (dsDNA) cassette, which contains the Cre/loxP system, to accomplish markerless deletion. To the best of our knowledge, this is the first effective and simple strategy for recombination with markerless deletion in C. glutamicum ATCC14067. This strategy provides a simple markerless deletion strategy for C. glutamicum and builds a solid basis for producer construction.

Corynebacterium glutamicum is an essential industrial strain that has been widely harnessed for the production of all kinds of value-added products. Efficient multiplex gene editing and large DNA fragment deletion are essential strategies for industrial biotechnological research. Cpf1 is a robust and simple genome editing tool for simultaneous editing of multiplex genes. However, no studies on effective multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1 system in C. glutamicum have been reported. Here, we developed a multiplex gene editing method by optimizing the CRISPR/Cpf1-RecT system and a large chromosomal fragment deletion strategy using the CRISPR/Cpf1-RecET system in C. glutamicum ATCC 14067. The CRISPR/Cpf1-RecT system exhibited a precise editing efficiency of more than 91.6% with the PAM sequences TTTC, TTTG, GTTG or CTTC. The sites that could be edited were limited due to the PAM region and the 1–7 nt at the 5′ end of the protospacer region. Mutations in the PAM region increased the editing efficiency of the − 6 nt region from 0 to 96.7%. Using a crRNA array, two and three genes could be simultaneously edited in one step via the CRISPR/Cpf1-RecT system, and the efficiency of simultaneously editing two genes was 91.6%, but the efficiency of simultaneously editing three genes was below 10%. The editing efficiency for a deletion of 1 kb was 79.6%, and the editing efficiencies for 5- and 20 kb length DNA fragment deletions reached 91.3% and 36.4%, respectively, via the CRISPR/Cpf1-RecET system. This research provides an efficient and simple tool for C. glutamicum genome editing that can further accelerate metabolic engineering efforts and genome evolution.

Corynebacterium glutamicum (C. glutamicum), an important industrial workhorse, is capable of efficiently producing a variety of value-added chemicals and fuels beyond amino acids. C. glutamicum has a broad natural substrate spectrum and can simultaneously utilize various carbon sources in blends. The substrate spectrum of C. glutamicum has been further extended by detailed knowledge of carbon core metabolism and well-established genetic tools and engineering strategies. At present, many pathways have been successfully engineered in C. glutamicum for access to alternative renewable sources to produce natural or non-natural products, making C. glutamicum a promising and favorable microbial cell factory. In this review, we mainly focus on synthetic biology and metabolic engineering strategies for developing synthetic strains that grow on renewable sources to produce the target products. At the same time, we also explore the promotion and future challenges of existing synthetic biology platforms for industrial platform microorganism metabolic engineering efforts.

Diamines serve as major platform chemicals that can be employed to a variety of industrial scenarios, particularly as monomers for polymer synthesis. High-throughput sensors for diamine biosynthesis can greatly improve the biological production of diamines. Here, we identified and characterized a transcription factor-driven biosensor for putrescine and cadaverine in Corynebacterium glutamicum. The transcriptional TetR-family regulatory protein CgmR (CGL2612) is used for the specific detection of diamine compounds. This study also improved the dynamic range and the sensitivity to putrescine by systematically optimizing genetic components of pSenPut. By a single cell-based screening strategy for a library of CgmR with random mutations, this study obtained the most sensitive variant CgmRI152T, which possessed an experimentally determined limit of detection (LoD) of ≤0.2 mM, a K of 11.4 mM, and a utility of 720. Using this highly sensitive putrescine biosensor pSenPutI152T, we demonstrated that CgmRI152T can be used as a sensor to detect putrescine produced biologically in a C. glutamicum system. This high sensitivity and the range of CgmR will be an influential tool for rewiring metabolic circuits and facilitating the directed evolution of recombinant strains toward the biological synthesis of diamine compounds.

We explored the ability of an Aspergillus niger α-glucosidase displayed on P. pastoris to act as a whole-cell biocatalyst (Pp-ANGL-GCW61) system to synthesize isomalto-oligosaccharides (IMOs). IMOs are a mixture that includes isomaltose (IG), panose (P), and isomaltotriose (IG). In this study, the IMOs were synthesized by a hydrolysis-transglycosylation reaction in an aqueous system of maltose. In a 2 mL reaction system, the IMOs were synthesized with a conversion rate of approximately 49% in 2 h when 30% maltose was utilized under optimal conditions by Pp-ANGL-GCW61. Additionally, the 0.5-L reaction system was conducted in a 2-L stirred reactor with a conversion rate of approximately 44% in 2 h. Moreover, the conversion rate was relatively stable after the whole-cell catalyst was reused three times. In conclusion, Pp-ANGL-GCW61 has a high reaction efficiency and operational stability, which makes it a powerful biocatalyst available for industrial scale synthesis.

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VLDLR gene was chosen as a candidate gene infl uencing abdominal fat trait and body weight due to previous studies in human and mouse. Herein, the objectives of this study were to identify genetic polymorphisms of duck VLDLR gene, and to analyze association between combined haplotypes and abdominal fat trait in Gaoyou domestic duck. A total of 207 individuals, including the elite reservation farm Gaoyou duck (FG, n = 50), the newly formed Gaoyou duck (NG, n = 54), the reserve area Gaoyou duck (AG, n = 50), and Hybrid duck (HY, n = 53) were used for study. In this paper, one genomic fragment was sequenced in all ducks encompassing a region from exon 14 to exon 16. Five single nucleotide polymorphisms (SNPs) and three insertions/deletions (indels) were identifi ed. Based on the above eight SNPs, 11 haplotypes were identifi ed. Th e H1 was the most common haplotype in AG, FG, and HY populations, which occurred at a frequency of more than 41%. Statistical analysis indicated that four combined haplotypes were associated with body weight at 10 weeks (BW10) (P < 0.05) and abdominal fat percentage (AFP) (P < 0.01) in Gaoyou FG and AG joint population. Th is result suggested that the VLDLR gene could be a potential gene infl uencing abdominal fat trait and body weight and may be used in marker-assisted selection (MAS).

With the rapid development of precision medicine industry, DNA sequencing becomes increasingly important as a research and diagnosis tool. For clinical applications, medical professionals require a platform which is fast, easy to use, and presents clear information relevant to definitive diagnosis. We have developed a single molecule desktop sequencing platform, GenoCare 1600. Fast library preparation (without amplification) and simple instrument operation make it friendlier for clinical use. Here we presented sequencing data of E. coli sample from GenoCare 1600 with consensus accuracy reaches 99.99%. We also demonstrated sequencing of microbial mixtures and COVID-19 samples from throat swabs. Our data show accurate quantitation of microbial, sensitive identification of SARS-CoV-2 virus and detection of variants confirmed by Sanger sequencing.