Corynebacterium glutamicum cell factory design for the efficient production of cis, cis-muconic acid.

Cis, cis-muconic acid (MA) is widely used as a key starting material in the synthesis of diverse polymers. The growing demand in these industries has led to an increased need for MA. Here, we constructed recombinant Corynebacterium glutamicum by systems metabolic engineering, which exhibit high efficiency in the production of MA. Firstly, the three major degradation pathways were disrupted in the MA production process. Subsequently, metabolic optimization strategies were predicted by computational design and the shikimate pathway was reconstructed, significantly enhancing its metabolic flux. Finally, through optimization and integration of key genes involved in MA production, the recombinant strain produced 88.2 g/L of MA with the yield of 0.30 mol/mol glucose in the 5 L bioreactor. This titer represents the highest reported titer achieved using glucose as the carbon source in current studies, and the yield is the highest reported for MA production from glucose in Corynebacterium glutamicum. Furthermore, to enable the utilization of more cost-effective glucose derived from corn straw hydrolysate, we subjected the strain to adaptive laboratory evolution in corn straw hydrolysate. Ultimately, we successfully achieved MA production in a high solid loading of corn straw hydrolysate (with the glucose concentration of 83.56 g/L), resulting in a titer of 19.9 g/L for MA, which is 4.1 times higher than that of the original strain. Additionally, the glucose yield was improved to 0.33 mol/mol. These provide possibilities for a greener and more sustainable production of MA.

Metabolic Engineering for Effective Synthesis of 2-Hydroxyadipate.

Adipic acid is an important monomer in the synthesis of nylon-6,6. In recent years, the biosynthesis of adipic acid has received more and more attention. The pathway with l-lysine as a precursor has potential for adipic acid synthesis, and 2-hydroxyadipate is a key intermediate metabolite in this pathway. In this Letter, the biosynthesis pathway of 2-hydroxyadipate was constructed in Escherichia coli. Through enhancement of precursor synthesis and cofactors regulation, 7.11 g/L of 2-hydroxyadipate was produced in the 5 L bioreactor, which verified the scale-up potential of 2-hydroxyadipate production. Furthermore, 11.1 g/L of 2-hydroxyadipate was produced in the 5 L bioreactor on the basis of potential optimization strategies via transcriptome analysis. This is the first time for the biosynthesis of 2-hydroxyadipate. The results lay a solid foundation for the biosynthesis of adipic acid and the production of bionylon.

First Report of Tomato mottle mosaic virus infecting Chinese snake gourd (Trichosanthes kirilowii) in China.

Tomato mottled mosaic virus (ToMMV) was first identified in tomato in Mexico (Li et al. 2013). It belongs to the genus Tobamovirus and family Virgaviridae, and is a positive-sense single-stranded RNA virus. The viral genome contains about 6400 nucleotides, encoding four proteins, including the 126 K protein, 183 K protein, movement protein (MP) and coat protein (CP) (Tu et al. 2021). ToMMV mainly poses a serious risk to solanaceous crops. The virus-infected plants appear stunted growth and top necrosis, and the disease leaves show mottled, shrinkage and necrosis symptoms, resulting in a significant decline in tomato fruit yield and quality (Li et al. 2017; Tu et al. 2021). Chinese snake gourd (Trichosanthes kirilowii Maxim) is a perennial climbing herb in the family Cucurbitaceae, and the fruit, seed, peel and root can all be used as traditional Chinese medicine. In May of 2021, twenty-seven symptomless seedlings (developed from tissue culture plantlets) were randomly collected from nursery in Fengyang, Anhui Province. Total RNA of each sample was extracted, and RT-PCR was performed using degenerate tobamovirus primers Tob-Uni1 (5'-ATTTAAGTGGASGGAAAAVCACT-3') and Tob-Uni2 (5'-GTYGTT GATGAGTTCRTGGA-3') (Letschert et al. 2002). Amplicons with expected size were obtained from 6 of 27 samples and sequenced. Alignment results showed that the nucleotide sequence identities ranged from 98.7 to 100% with all ToMMV isolates deposited in NCBI GenBank. Then, ToMMV coat protein (CP) gene was amplified using specific primers CP-F (5'-ATGTCTTACGCTATTACTT CTCCG-3') and CP-R (5'-TTAGGACGCTGGCGCAGAAG-3'). The CP fragment was obtained and sequenced. Sequence alignment indicated that CP sequence of isolate FY (GenBank accession no. ON924176) exhibited a 100% identity with ToMMV isolate LN (MN853592.1). The anti-ToMMV polyclonal antibody (PAb) was prepared by the author (S.L.) by immunizing rabbit with purified virus from Nicotiana benthamiana, and serological tests (dot-enzyme linked immunosorbent assay, Dot-ELISA) of RNA-positive T. kirilowii leaf samples using anti-ToMMV PAb were also positive. To fulfill a Koch's postulate, a pure culture of ToMMV was obtained from N. benthamiana using infectious cDNA clone of ToMMV (Tu et al. 2021), and then healthy T. kirilowii plants were mechanically inoculated with a prepared inoculum from ToMMV-infected N. benthamiana, as described previously (Sui et al. 2017). T. kirilowii seedlings showed chlorosis and leaf tip necrosis symptoms at 10 and 20 day post-inoculation respectively, and ToMMV infection on symptomatic plants was also verified by RT-PCR detection using primers CP-F and CP-R. These results demonstrated that T. kirilowii is a host of ToMMV under natural conditions, which might threaten the production of this medicinal plant. The seedlings from nursery appeared to be asymptomatic, but the plants showed chlorosis and necrosis symptoms after indoor inoculation. In qRT-PCR analysis, viral accumulation level in greenhouse-inoculated plants was a 25.6-fold of that in field-collected samples, which may be the reason of different symptom expression between field samples and inoculated samples. ToMMV has now been detected from the solanaceous (tomato, pepper and eggplant) and leguminous (pea) crops in the field (Li et al. 2014; Ambrós et al. 2017; Zhang et al. 2022). To our knowledge, this is the first report of natural infection of ToMMV in T. kirilowii as well as its natural infection on Cucurbitaceae plants.

De novo biosynthesis of α-aminoadipate via multi-strategy metabolic engineering in Escherichia coli.

As a non-protein amino acid, α-aminoadipate is used in the fields of medicine, chemical engineering, food science, and others. For example, α-aminoadipate is an important precursor for the production of ß-lactam antibiotics. Currently, the synthesis of α-aminoadipate depends on chemical catalysis that has the disadvantages of high cost, low yield, and serious pollution. In this study, we construct a biosynthesis pathway of α-aminoadipate in Escherichia coli using lysine as a precursor. In addition, we regulate the cell metabolism to improve the titer of α-aminoadipate via multi-strategy metabolic engineering. First, a novel synthetic pathway was constructed to realize de novo synthesis of α-aminoadipate with titers of 82 mg/L. Second, the key enzymes involved in enhancing precursor synthesis were overexpressed and the CO2 fixation process was introduced, and these led to 80% and 34% increases in the α-aminoadipate concentration, reaching 147 and 110 mg/L, respectively. Third, cofactor regulation was used to maintain the coupling balance of material and energy, with the intracellular α-aminoadipate concentration reaching 140 mg/L. Fourth, the weakening of the synthesis of acetic acid was used to strengthen the synthesis of α-aminoadipate, and this resulted in the enhancement of the α-aminoadipate concentration by 2.2 times, reaching 263 mg/L. Finally, combination optimization was used to promote the production of α-aminoadipate. The titers of α-aminoadipate reached 368 mg/L (strain EcN11#) and 415 mg/L (strain EcN11##), which was 3.5 and 4 times higher than that of the parent strain. With these efforts, 1.54 g/L of α-aminoadipate was produced under fed-batch conditions by strain EcN11#. This study is the first to present the effective biosynthesis of α-aminoadipate in E. coli using multi-strategy metabolic engineering.

Non-targeted NIR spectroscopy and SIMCA classification for commercial milk powder authentication: A study using eleven potential adulterants.

A non-targeted detection method using near-infrared (NIR) spectroscopy combined with chemometric modeling was developed for the rapid screening of commercial milk powder (MP) products as authentic or potentially mixed with known and unknown adulterants. Two benchtop FT-NIR spectrometers and a handheld NIR device were evaluated for model development. The performance of SIMCA classification models was then validated using an independent test set of genuine MP samples and a set of gravimetrically prepared mixtures consisting of MPs spiked with each of eleven potential adulterants. Classification models yielded 100% sensitivities for the benchtop spectrometers. Better specificity, which was influenced by the nature of the adulterant, was obtained for the benchtop FT-NIR instruments than for the handheld NIR device, which suffered from lower spectral resolution and a narrower spectral range. FT-NIR spectroscopy and SIMCA classification models show promise for the rapid screening of commercial MPs for the detection of potential adulteration.