Engineering 2,4-Diacetylphloroglucinol-Hyper-Tolerant Escherichia coli via Adaptive Evolution for Efficient Biosynthesis and an Organic Solvent-Free Downstream Process.
2,4-Diacetylphloroglucinol (DAPG) is a valuable antimicrobial compound with significant agricultural potential, suffers bioproduction limitations from host toxicity and inefficient downstream processing. This study engineered DAPG-hyper-tolerant Escherichia coli via adaptive laboratory evolution (ALE) starting from a phloroglucinol-tolerant strain. Optimized shake-flask fermentation of evolved Bdt03 yielded 330.52 mg/L DAPG (17.84-fold of the wild type), and the yield from the whole fermentation broth could be further increased to 391.36 mg/L. A novel organic solvent-free extraction method recovered DAPG from fermentation broth with over 98% yield via acidification, cold incubation and centrifugation, simplifying downstream processing. Genomic resequencing identified several key mutations underlying DAPG tolerance, which were validated and stacked to precisely construct strain Bb03 with enhanced production and tolerance. This work addresses the critical bottlenecks in DAPG biosynthesis by enhancing host tolerance and developing a sustainable downstream processing strategy, and also offers valuable genetic insights for constructing high-yield DAPG-producing strains and advancing the application of DAPG-responsive genetic circuits in synthetic biology.
Muconic acid is an industrially valuable molecule that can be biologically produced from diverse biogenic and waste-derived feedstocks, including sugars and lignin- and plastic-derived aromatic compounds. However, accumulation of protocatechuate (PCA) has been observed in multiple microbes engineered for muconate production when the PCA decarboxylase, AroY, is used. This raises the question of whether PCA decarboxylation represents a rate-limiting step and how this bottleneck might be alleviated, especially given the toxicity and reactivity of PCA and catechol intermediates. To address this, we performed adaptive laboratory evolution (ALE) on a strain of Pseudomonas putida originally engineered for muconate production from aromatic compounds, but with catBC restored, to select for improved conversion of PCA and, in separate lineages, 4-hydroxybenzoate. Contrary to our expectations, the predominant beneficial mutations localized to the catA1 cassette encoding catechol 1,2-dioxygenase, rather than aroY or its associated cofactor biosynthesis genes. Transcriptomic analysis revealed elevated catA1 expression in evolved isolates from ALE, and introduction of these mutations improved productivity in strains designed for muconate production from both aromatic and sugar substrates. Quantitative proteomics and biochemical assays demonstrated that the mutations also led to increased CatA1 protein abundance and modest enhancements in catalytic efficiency, respectively, with strain phenotypes largely driven by high CatA1 levels and potentially synergistic kinetic improvements. Additional reverse-engineering studies identified variants with modest effects on muconate accumulation, including those with potential to enhance biosynthesis of the prenylated FMN cofactor of AroY. Collectively, these results indicate that catechol, not PCA, is the principal bottleneck in muconate production via the PCA decarboxylation route originally demonstrated by Draths et al., refining our understanding of pathway limitations and offering new strategies for improving rate, yield, and strain resilience in muconate bioproduction. Highlights Accumulation of metabolic intermediates was alleviated by adaptive laboratory evolution Sequencing, proteomics, and enzyme kinetics revealed mechanisms for adaptation Increased CatA1 expression reduced bottlenecks and improved muconate production
Alissa C. Bleem, Tracy L. Hodges, Torrey M Lind et al.· bioRxiv· 2 citations
2-Pyrone-4,6-dicarboxylic acid (PDC) is a promising pseudo-aromatic bio-based monomer for the production of biodegradable polyesters. Here, we combined metabolic engineering with fermentation process optimization to establish efficient de novo PDC biosynthesis in Corynebacterium glutamicum. To enhance the availability of protocatechuic acid (PCA), the direct precursor of PDC, endogenous PCA degradation was blocked, and precursor supply through the shikimate pathway was strengthened. Subsequently, the ligABC cluster from Sphingomonas paucimobilis SYK-6 was heterologously introduced to enable PDC formation from glucose. Medium screening identified a corn steep liquor-containing formulation that increased the PDC titer by 1.4-fold in shake-flask cultures. Further optimization of corn steep liquor concentration, dissolved oxygen, and glucose feeding rate in a 5-L bioreactor increased the PDC titer to 71.1 g/L at 90 h, with an average productivity of 0.8 g/L/h, the highest productivity for C. glutamicum-based PDC production reported to date.
Meng Chai, Rui-Tao Yu, Yi-Tong Li et al.· Fermentation· 0 citations
1,6-Hexanediol (1,6-HDO) is an important chemical platform widely used in polymer and pharmaceutical industries, while its toxicity remains a major bottleneck limiting efficient microbial production. This study demonstrates that 1,6-HDO exhibited strong inhibitory effects on Escherichia coli, reducing the maximum OD600 to approximately 50% of the control level under 10 g/L stress. Transcriptomic analysis was performed under 1,6-HDO stress, identifying 779 differentially expressed genes enriched in pathways related to energy metabolism, sulfur metabolism and amino acid metabolism. To systematically identify functional targets associated with tolerance, genome-wide screening using the ASKA overexpression library identified 93 tolerance-related genes that improved E. coli growth under 1,6-HDO stress by 0.06- to 3.17-fold. Notably, multiple targets were functionally associated with energy and amino acid metabolism, consistent with the transcriptomic analysis. Furthermore, co-expression of rumA and yhbO showed the greatest improvement in growth, reaching a biomass of 9.43 under 1,6-HDO stress, which was 2.61-fold higher than that of the control strain. Collectively, this study systematically elucidates the global stress response and identifies key determinants contributing to tolerance, providing valuable targets for developing robust microbial cell factories for efficient 1,6-HDO bioproduction.
Xinyi Zhang, Lisha Qin, Hongxu Li et al.· Synthetic and Systems Biotec...· 0 citations
This study demonstrates the combined optimization of isozyme combination and environmental stress to elevate the synthesis of astaxanthin and other carotenoids in D. salina, providing new research ideas and experimental evidence for the future construction of high-yield engineered algal strains.
Hao Zhang, Yifan Kong, Yaping Shao et al.· World Journal of Microbiolog...· 0 citations
Cyanophycin granule polypeptide (CGP), also known as multi-L-arginyl-poly(aspartic acid), is a biodegradable biopolymer composed primarily of aspartic acid and arginine. Due to its versatile functional properties, CGP has attracted increasing interest for potential applications in food, medicine, cosmetics, agriculture, and corrosion inhibition. The objective of this study was to biosynthesize a biologically derived corrosion-inhibiting biomaterial using recombinant Escherichia coli BL21(DE3) expressing cyanophycin synthetase (CphA), followed by cost-effective induction strategy. Specifically, this work aimed to establish a high-cell-density cultivation process capable of achieving improved CGP production while reducing dependence on costly inducers such as IPTG. Initial shake-flask experiments demonstrated that lactose induction resulted in higher CGP production and biomass formation compared to IPTG induction. In addition, supplementation with phosphate, ribose, trace elements, yeast extract, and tryptone further improved CGP accumulation. Based on these findings, a high-cell-density fed-batch fermentation strategy using lactose as both inducer and carbon source was developed, maximum gravimetrically recovered crude soluble and insoluble CGP-containing fractions of 35.4 and 17.8 g/L. Product characterization was further supported by FTIR, XRD, MALDI-MS. Furthermore, the recovered CGP-related material was evaluated in preliminary corrosion inhibition experiments under acidic conditions and showed significant reduction in corrosion rates compared with untreated control samples, indicating its potential for future corrosion-protection applications.
S. Kafle, Anirudh Mukunth, Arum Han et al.· Bioprocess and biosystems en...· 0 citations
1,4-Butanediamine is an important raw material for the synthesis of engineering plastics such as Polyamide 46 with excellent performance, which is widely used in automotive, electronics and machinery manufacturing industries. In this study, Escherichia coli BL21(DE3) was used as the starting strain to systematically modify the 1,4-butanediamine biosynthesis pathway using a modular strategy. 1,4-Butanediamine biosynthesis was re-divided into two modules: 1,4-butanediamine production module and α-ketoglutaric acid production module. By controlling the expression intensity of genes pykF, ppc, aceEF, gltA, icdA and gdhA, argD, argCB, argJ, ODC10, a recombinant producing 1,4-butanediamine strain PKT was obtained, achieving a yield of 862.82 mg/L of 1,4-butanediamine, which increased 8.62-fold compared to that of Escherichia coli BL21(DE3). Then, the fermentation medium of PKT strain was optimized. After 24 h of fermentation, the yield of 1,4-butanediamine reached 1843.60 mg/L, representing a 16.23-fold increase over the original strain. Furthermore, non-target metabolomics analysis was used to analyze the changes of metabolites during the efficient synthesis of 1,4-butanediamine by the recombinant strain PKT. The results showed that the total number of differential metabolites detected was 653. Differential metabolite pathway analysis showed that there were 9 differential metabolites in the 1,4-butanediamine synthesis pathway, with 5 involved in its synthesis pathway and 4 in its degradation branch. These differences in metabolites provide a theoretical basis for the modification of recombinant strains. The green production of 1,4- butanediamine by microbial cell factories through fermentation is the future direction. This research provides a theoretical guidance of building high yield 1,4-butanediamine engineering strains.
Xiangxiang Sun, Zongda Li, Yan-Ling Sun et al.· Journal of Genetic Engineeri...· 0 citations
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