Improvement of optimum pH and specific activity of pectate lyase from Bacillus RN.1 using loop replacement.

Background: Alkaline pectate lyase plays an important role in papermaking, biological refining and wastewater treatment, but its industrial applications are largely limited owing to its low activity and poor alkali resistance. Methods: The alkaline pectate lyase BspPel from Bacillus RN.1 was heterologously expressed in Escherichia coli BL21 (DE3) and its activity and alkali resistance were improved by loop replacement. Simultaneously, the effect of R260 on enzyme alkaline tolerance was also explored. Results: Recombinant pectate lyase (BspPel-th) showed the highest activity at 60°C and pH 11.0, and showed significant stability over a wide pH range (3.0-11.0). The specific enzyme activity after purification was 139.4 U/mg, which was 4.4 times higher than that of the wild-type enzyme. BspPel-th has good affinity for apple pectin, since the V max and K m were 29 µmol/min. mL and 0.46 mol/L, respectively. Molecular dynamics simulation results showed that the flexibility of the loop region of BspPel-th was improved. Conclusion: The modified BspPel-th has considerable potential for industrial applications with high pH processes.

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.

Increasing Long-Chain Dicarboxylic Acid Production in Candida tropicalis by Engineering Fatty Transporters.

Candida tropicalis can metabolize alkanes or fatty acids to produce long-chain dicarboxylic acids (DCAs). Fatty acid transporters located on the cell or peroxisome membrane may play an important role in this process. Using amino acid sequence homologous alignment, two putative proteins, CtFat1p and CtPxa1p, located on the cell and peroxisome membrane were found, respectively. Moreover, single- and double-knockout homologous recombination technology was used to study ctfat1p and ctpxa1p gene effects on DCA synthesis. In comparison to the wild-type strain, long-chain DCA yield decreased by 65.14%, 88.38% and 56.19% after single and double-copy knockout of ctfat1p genes and double-copy knockout of ctpxa1p genes, respectively, indicating that the knockout of ctfat1p and ctpxa1p genes had a significant effect on the conversion of oils and fats into long-chain DCAs by C. tropicalis. However, the yield of long-chain DCAs increased by 21.90% after single-knockout of the ctpxa1p gene, indicating that the single-knockout of the ctpxa1p gene may reduce fatty acid transport to peroxisome for further oxidation. Moreover, to improve the intracellular transport rate of fatty acids, ctfat1p copy number increased, increasing DCA yield by 30.10%. These results may provide useful information for enhancing the production of long-chain DCAs by C. tropicalis.

Increasing L-lysine production in Corynebacterium glutamicum by engineering amino acid transporters.

Corynebacterium glutamicum has a long and successful history in the biotechnological production of L-lysine. Besides the adjustment of metabolic pathways, intracellular and extracellular transport systems are critical for the cellular metabolism of L-lysine or its by-products. Here, three amino acid transmembrane transporters, namely, GluE, BrnE/BrnF, and LysP, which are widely present in C. glutamicum strains, were each investigated by gene knockout. In comparison with that in the wild-type strain, the yield of L-lysine increased by 9.0%, 12.3%, and 10.0% after the deletion of the gluE, brnE/brnF, and lysP genes, respectively, in C. glutamicum 23,604. Moreover, the amount of by-product amino acids decreased significantly when the gluE and brnE/brnF genes were deleted. It was also demonstrated that there was no effect on the growth of the strain when the gluE or lysP gene was deleted, whereas the biomass of C. glutamicum WL1702 (ΔbrnE/ΔbrnF) in the fermentation medium was significantly reduced in comparison with that of the wild type. These results also provide useful information for enhancing the production of L-lysine or other amino acids by C. glutamicum.