Novel Bioproduction of 1,6-Hexamethylenediamine from l-Lysine Based on an Artificial One-Carbon Elongation Cycle.

1,6-hexamethylenediamine (HMD) is an important precursor for nylon-66 material synthesis, while research on the bioproduction of HMD has been relatively scarce in scientific literature. As concerns about climate change, environmental pollution, and the depletion of fossil fuel reserves continue to grow, the significance of producing fundamental chemicals from renewable sources is becoming increasingly prominent. In recent investigations, the bioproduction of HMD from adipic acid has been reported but with lingering challenges concerning costly raw materials and low yields. Here, we have undertaken the reconstruction of the HMD synthetic pathway within Escherichia coli, which was constituted with l-lysine α-oxidase (Raip), LeuABCD, α-ketoacid decarboxylase (KivD), and transaminases (Vfl), leveraging a carbon chain extension module and a metabolic pathway of transaminase-decarboxylase cascade catalysis within the strain WD20, which successfully produce 46.7 ± 2.0 mg/L HMD. To increase the cascade activity and create a higher tolerance to external environmental disturbance for l-lysine to convert into HMD, another two enzymes d-alanine aminotransferase (Dat) and alpha-ketoacid decarboxylase (KdcA) were introduced into WD21 to provide flux flexibility for α-ketoacid metabolization, which was named "Smart-net metabolic engineering" in our research, and high-efficiency synthesis of HMD utilizing l-lysine as the substrate has been successfully achieved. Finally, we established a + 1C bioconversion multienzyme cascade catalyzing up to 65% conversion of l-lysine to HMD. Notably, our fermentation process yielded an impressive 213.5 ± 8.7 mg/L, representing the highest reported yield to date for the bioproduction of HMD from l-lysine.

SERS-based microdroplet platform for high-throughput screening of Escherichia coli strains for the efficient biosynthesis of D-phenyllactic acid.

D-Phenyllactic acid (D-PLA) is a potent antimicrobial typically synthesized through chemical methods. However, due to the complexity and large pollution of these reactions, a simpler and more eco-friendly approach was needed. In this study, a strain for D-PLA biosynthesis was constructed, but the efficiency was restricted by the activity of D-lactate dehydrogenase (DLDH). To address this issue, a DLDH mutant library was constructed and the Surface-Enhanced Raman Spectroscopy (SERS) was employed for the precise quantification of D-PLA at the single-cell level. The TB24 mutant exhibited a significant improvement in D-PLA productivity and a 23.03-fold increase in enzymatic activity, which was attributed to the enhanced hydrogen bonding and increased hydrophobicity within the substrate-binding pocket. By implementing multi-level optimization strategies, including the co-expression of glycerol dehydrogenase (GlyDH) with DLDH, chassis cell replacement, and RBS engineering, a significant increase in D-PLA yields was achieved, reaching 128.4 g/L. This study underscores the effectiveness of SERS-based microdroplet high-throughput screening (HTS) in identifying superior mutant enzymes and offers a strategy for large-scale D-PLA biotransformation.

Metabolic Engineering of Saccharomyces cerevisiae for High-Level Production of l-Pipecolic Acid from Glucose.

l-Pipecolic acid (L-PA), an essential chiral cyclic nonprotein amino acid, is gaining prominence in the food and pharmaceutical sectors due to its wide-ranging biological and pharmacological properties. Historically, L-PA has been synthesized chemically for commercial purposes. This study introduces a novel and efficient microbial production method for L-PA using engineered strain Saccharomyces cerevisiae BY4743. Initially, an optimized biosynthetic pathway was constructed within S. cerevisiae, converting glucose to L-PA with a yield of 0.60 g/L in a 250 mL shake flask in vivo. Subsequently, a multifaceted engineering strategy was implemented to enhance L-PA production: substrate-enzyme affinity modification, global transcription machinery engineering modification, and Kozak sequence optimization for enhanced L-PA production. Approaches above led to an impressive 8.6-fold increase in L-PA yield, reaching 5.47 g/L in shake flask cultures. Further scaling up in a 5 L fed-batch fermenter achieved a remarkable L-PA concentration of 74.54 g/L. This research offers innovative insights into the industrial-scale production of L-PA.

Combining directed evolution with high cell permeability for high-level cadaverine production in engineered Escherichia coli.

The biosynthesis of cadaverine from lysine is an environmentally promising technology, that could contribute to a more sustainable approach to manufacturing bio-nylon 5X. However, the titer of biosynthesized cadaverine has still not reached a sufficient level for industrial production. A powerful green cell factory was developed to enhance cadaverine production by regulating lipopolysaccharide (LPS) genes and improving membrane permeability. Firstly, 10 LPS mutant strains were constructed and the effect on the growth was investigated. Then, the lysine decarboxylase (CadA) was overexpressed in 10 LPS mutant strains of Escherichia coli MG1655 and the ability to produce cadaverine was compared. Using 20.0 g L-1 of L-lysine hydrochloride (L-lysine-HCl) as the substrate for the biotransformation reaction, Cad02 and Cad06 strains exhibited high production levels of cadaverine, with 8.95 g L-1 and 7.55 g L-1 respectively while the control strain Cad00 only 4.92 g L-1 . Directed evolution of CadA was also used to improve its stability under alkaline conditions. The cadaverine production of the Cad02-M mutant stain increased by 1.86 times at pH 8.0. Finally, the production process was scaled up using recombinant whole cells as catalysts, achieving a high titer of 211 g L-1 cadaverine (96.8%) by fed-batch bioconversion. This study demonstrates the potential role of LPS in enhancing the efficiency of mass transfer between substrate and enzymes in vivo by increasing cell permeability. The results indicate that the argumentation of cell permeability could not only significantly enhance the biotransformation efficiency of cadaverine, but also provide a universally applicable, straightforward, environment-friendly, and cost-effective method for the biosynthesis of other high-value chemicals.

A synthetic cell-free 36-enzyme reaction system for vitamin B12 production.

Adenosylcobalamin (AdoCbl), a biologically active form of vitamin B12 (coenzyme B12), is one of the most complex metal-containing natural compounds and an essential vitamin for animals. However, AdoCbl can only be de novo synthesized by prokaryotes, and its industrial manufacturing to date was limited to bacterial fermentation. Here, we report a method for the synthesis of AdoCbl based on a cell-free reaction system performing a cascade of catalytic reactions from 5-aminolevulinic acid (5-ALA), an inexpensive compound. More than 30 biocatalytic reactions are integrated and optimized to achieve the complete cell-free synthesis of AdoCbl, after overcoming feedback inhibition, the complicated detection, instability of intermediate products, as well as imbalance and competition of cofactors. In the end, this cell-free system produces 417.41 µg/L and 5.78 mg/L of AdoCbl using 5-ALA and the purified intermediate product hydrogenobyrate as substrates, respectively. The strategies of coordinating synthetic modules of complex cell-free system describe here will be generally useful for developing cell-free platforms to produce complex natural compounds with long and complicated biosynthetic pathways.

Optimization of Hydrogenobyrinic Acid Synthesis in a Cell-Free Multienzyme Reaction by Novel S-Adenosyl-methionine Regeneration.

Hydrogenobyrinic acid, a modified tetrapyrrole composed of eight five-carbon compounds, is a key intermediate and central framework of vitamin B12. Synthesis of hydrogenobyrinic acid requires eight S-adenosyl-methionine working as the methyl group donor catalyzed by 12 enzymes including six methyltransferases, causing the great shortage of S-adenosyl-methionine and accumulation of S-adenosyl-homocysteine, which is uneconomic and unsustainable for the cascade reaction. Here, we report a cell-free synthetic system for producing hydrogenobyrinic acid by integrating 12 enzymes using 5-aminolevulininate as a substrate and develop a novel S-adenosyl-methionine regeneration system to steadily supply S-adenosyl-methionine and avoid the accumulated inhibition of S-adenosyl-homocysteine by consuming a cheaper substrate (l-methionine and polyphosphate). By combination of the reaction system optimization and S-adenosyl-methionine regeneration, the titer of hydrogenobyrinic acid was improved from 0.61 to 29.39 mg/L in a 12 h reaction period, representing an increase of 48.18-fold, raising an efficient and rapidly evolutional alternative method to produce high-value-added compounds and intermediate products.