Design and application of artificial rare L-lysine codons in Corynebacterium glutamicum.

Background: L-lysine is widely used in the feed, food, and pharmaceutical industries, and screening for high L-lysine-producing strains has become a key goal for the industry. Methods: We constructed the rare L-lysine codon AAA by corresponding tRNA promoter replacement in C. glutamicum. Additionally, a screening marker related to the intracellular L-lysine content was constructed by converting all L-lysine codons of enhanced green fluorescent protein (EGFP) into the artificial rare codon AAA. The artificial EGFP was then ligated into pEC-XK99E and transformed into competent Corynebacterium glutamicum 23604 cells with the rare L-lysine codon. After atmospheric and room-temperature plasma mutation and induction culture, 55 mutants (0.01% of total cells) with stronger fluorescence were sorted using flow cytometry, and further screened by fermentation in a 96-deep-well plate and 500 mL shaker. Results: The fermentation results showed that the L-lysine production was increased by up to 9.7% in the mutant strains with higher fluorescence intensities, and that the highest screening positive rate was 69%, compared with that in the wild-type strain. Conclusion: The application of artificially constructed rare codons in this study represents an efficient, accurate, and simple method for screening other amino acid-producing microorganisms.

Study on the construction technology of ß-alanine synthesizing Escherichia coli based on cellulosome assembly.

Introduction: ß-Alanine is the only ß-amino acid in nature; it is widely used in food additives, medicines, health products, and surfactants. To avoid pollution caused by traditional production methods, the synthesis of ß-alanine has been gradually replaced by microbial fermentation and enzyme catalysis, which is a green, mild, and high-yield biosynthesis method. Methods: In this study, we constructed an Escherichia coli recombinant strain for efficient ß-alanine production using glucose as the raw material. The microbial synthesis pathway of L-lysine-producing strain, Escherichia coli CGMCC 1.366, was modified using gene editing by knocking out the aspartate kinase gene, lysC. The catalytic efficiency and product synthesis efficiency were improved by assembling key enzymes with cellulosome. Results: By-product accumulation was reduced by blocking the L-lysine production pathway, thereby increasing the yield of ß-alanine. In addition, catalytic efficiency was improved by the two-enzyme method to further increase the ß-alanine content. The key cellulosome elements, dockerin (docA) and cohesin (cohA), were combined with L-aspartate-α-decarboxylase (bspanD) from Bacillus subtilis and aspartate aminotransferase (aspC) from E.coli to improve the catalytic efficiency and expression level of the enzyme. ß-alanine production reached 7.439 mg/L and 25.87 mg/L in the two engineered strains. The ß-alanine content reached 755.465 mg/L in a 5 L fermenter. Discussion: The content of ß-alanine synthesized by constructed ß-alanine engineering strains were 10.47 times and 36.42 times higher than the engineered strain without assembled cellulosomes, respectively. This research lays the foundation for the enzymatic production of ß-alanine using a cellulosome multi-enzyme self-assembly system.

Exploration of microbiome diversity of stacked fermented grains by flow cytometry and cell sorting.

Sauce-flavor baijiu is one of the twelve flavor types of Chinese distilled fermented product. Microbial composition plays a key role in the stacked fermentation of Baijiu, which uses grains as raw materials and produces flavor compounds, however, the active microbial community and its relationship remain unclear. Here, we investigated the total and active microbial communities of stacked fermented grains of sauce-flavored Baijiu using flow cytometry and high-throughput sequencing technology, respectively. By using traditional high-throughput sequencing technology, a total of 24 bacterial and 14 fungal genera were identified as the core microbiota, the core bacteria were Lactobacillus (0.08-39.05%), Acetobacter (0.25-81.92%), Weissella (0.03-29.61%), etc. The core fungi were Issatchenkia (23.11-98.21%), Monascus (0.02-26.36%), Pichia (0.33-37.56%), etc. In contrast, using flow cytometry combined with high-throughput sequencing, the active dominant bacterial genera after cell sorting were found to be Herbaspirillum, Chitinophaga, Ralstonia, Phenylobacterium, Mucilaginibacter, and Bradyrhizobium, etc., whereas the active dominant fungal genera detected were Aspergillus, Pichia, Exophiala, Candelabrochaete, Italiomyces, and Papiliotrema, etc. These results indicate that although the abundance of Acetobacter, Monascus, and Issatchenkia was high after stacked fermentation, they may have little biological activity. Flow cytometry and cell sorting techniques have been used in the study of beer and wine, but exploring the microbiome in such a complex environment as Chinese baijiu has not been reported. The results also reveal that flow cytometry and cell sorting are convenient methods for rapidly monitoring complex microbial flora and can assist in exploring complex environmental samples.