Metabolic engineering最新文献

Complex phenylethanoid glycosides (PhGs), such as verbascoside and echinacoside, comprise a vital family of natural products with renowned nutraceutical and pharmaceutical significance. Despite the high demand for these compounds across various industries, traditional plant extraction methods yield insufficient quantities, highlighting the need for alternative production methods. Therefore, this paper reports the successful engineering of Saccharomyces cerevisiae cell factories for the efficient production of complex PhGs from glucose. First, key pathway enzymes with enhanced catalytic activities in yeast were primarily screened from various verbascoside-producing plants. Second, intermediate osmanthuside B was produced with a titer of 21.5 ± 1.5 mg/L from glucose by overexpressing several enzymes, including glucosyltransferase RrUGT33 from Rhdiola rosea, acyltransferase SiAT, and 1,3-rhamnosyltransferase SiRT from Sesamum indicum, UDP-L-rhamnose synthase AtRHM2, and 4-coumarate: coenzyme A ligase At4CL1 from Arabidopsis thaliana in a p-coumaric acid-overproducing S. cerevisiae strain. Third, the production of osmanthuside B was further enhanced by increasing the copy number of SiAT and AtRHM2 in genome and diverting L-tyrosine into tyrosol biosynthesis by introducing an aromatic aldehyde synthase PcAAS from Petroselinum crispum with a titer of 320.6 ± 59.3 mg/L. Fourth, the biosynthesis of verbascoside was accomplished by integrating genes CYP98A20 and AtCPR1 into the chromosomes of the osmanthuside B-producing strain, the titer reached 184.7 ± 5.7 mg/L. Furthermore, the overexpression of the glucose-6-phosphate dehydrogenase (ZWF1) led to significantly enhanced verbascoside production to 230.6 ± 11.8 mg/L. The strains were further engineered to produce echinacoside with a titer of 184.2 ± 11.2 mg/L. Finally, the fed-batch fermentation in a 5-L bioreactor yielded 4497.9 ± 285.2 mg/L of verbascoside or 3617.4 ± 117.4 mg/L of echinacoside. This work provides a crucial foundation for the green, industrial, and sustainable production of verbascoside and echinacoside and sets an initial point for the microbial production of other complex PhG derivatives.

Precise and predictable genetic elements are required to address various issues, such as suboptimal metabolic flux or imbalanced protein assembly caused by the inadequate control of polycistronic gene expression in bacteria. Here, we devised a synthetic biopart based on the translational coupling to control polycistronic gene expression. This module links the translation of genes within a polycistronic mRNA, maintaining their expression ratios regardless of coding sequences, transcription rate, and upstream gene translation rate. By engineering the Shine-Dalgarno sequences within these synthetic bioparts, we adjusted the expression ratios of polycistronic genes. We created 41 bioparts with varied relative expression ratios, ranging from 0.03 to 0.92, enabling precise control of pathway enzyme gene expression in a polycistronic manner. This led to up to a 7.6-fold increase in the production of valuable biochemicals such as 3-hydroxypropionic acid, poly(3-hydroxybutyrate), and lycopene. Our work provides genetic regulatory modules for precise and predictable polycistronic gene expression, facilitating efficient protein assembly, biosynthetic gene cluster expression, and pathway optimization.

The growing depletion of petroleum resources and the increasing demand for sustainable alternatives have spurred advancements in microorganism-based biofactories. Among high-value compounds, carotenoids are widely sought after in pharmaceuticals, cosmetics, and nutrition, making them prime candidates for microbial production. In this study, we engineered an efficient biosynthetic pathway in Escherichia coli for the production of the C₂₀-carotenoid crocetin-dialdehyde. By bypassing traditional oxidative cleavage reactions mediated by carotenoid cleavage dioxygenases (CCDs), our approach reduces the enzymatic complexity of the pathway. Using the crystal structure of a CrtMLIKE enzyme identified in this study, we developed a mutant enzyme capable of condensing two C₁₀-geranyl pyrophosphate molecules to form C₂₀-phytoene. This intermediate was then desaturated and oxidized by CrtN and CrtP to produce crocetin-dialdehyde, achieving a yield of 1.13 mg/L. By reducing enzyme requirements from six to three and eliminating the need for CCDs, this pathway alleviates metabolic stress on the host and enhances the scalability of production for industrial applications. Overall, our research presents a streamlined and innovative approach to carotenoid biosynthesis, advancing sustainable production methods for short-chain carotenoids.

Microbial cell factories (MCFs) have emerged as a sustainable tool for the production of value-added biochemicals. However, developing high-performance MCFs remains a major challenge to fulfill the burgeoning demands of global markets. This study aimed to establish the B. licheniformis cell factory for the cost-effective production of glutamate-derived chemicals by modular metabolic engineering. Initially, the glutamate decarboxylase from E. coli was introduced into B. licheniformis DW2 to construct the artificial γ-aminobutyric acid (GABA) pathway. By systematically optimizing the central metabolic pathway, boosting the L-Glu synthesis pathway and improving the cofactor NADPH supply, the strain G35/pHY-P_r5u12-gadB^E89Q/H465A achieved a remarkable yield of 62.9 g/L of GABA in a 5-L bioreactor, representing the highest yield of 0.5 g/g glucose with a significant 49.3-fold increase. Remarkably, bioinformatics analyses and function verification identified the putative glyoxylate to glycolic acid synthesis pathway and KipR, an inhibitor of the glyoxylate cycle, as the rate-limiting steps in GABA production. Additionally, a versatile and robust platform using engineered B. licheniformis for efficient production of diverse glutamate-derived chemicals was established and the titer of 5-aminolevulinic acid, heme and indigoidine was improved by 5.3-, 4.7- and 1.9-fold, respectively. This study not only facilitates extensive application of B. licheniformis for chemical production, but also sheds light on research to improve the performance of other MCFs.

Non-conventional yeasts have emerged as important sources of valuable products in bioindustries. However, tools for the control of expression are limited in these hosts. In this study, we aimed to excavate the tools for the regulation of translation that are often overlooked. 5'UTR analysis of genome-scale annotated genes of four yeast species revealed a distinct decreasing 'G' frequency in -100 ∼ -1 region from 5040 5'UTRs in Komagataella phaffii. New 5'UTRs were regenerated by base substitutions in defined regions, and replacement of 'G' by 'A' or 'T' in the -50 ∼ -1 region highly facilitated gene expression. Preference analysis of all nucleotide triplets in 5'UTRs revealed a KZ₃ (-3 ∼ -1) that dominantly affected gene expression. A total of 128 KZ₃ variants were constructed to work with promoters of methanol-inducible P_AOX1 and constitutive P_GAP, of which 58 KZ₃ variants increased gene expression and maximum difference in strength was 15-fold among all variants. Polysome profiling analysis clarified that 5'UTR-KZ₃ enhanced gene expression at translational but not transcriptional levels. Finally, improved production of three industrial proteins and one platform compound were achieved by ready-made 5'UTR-KZ₃ or in situ modification of the 5'UTR. This study provides new references and tools for the fine-tuning of translational regulation in yeast and other fungi.

2'-Fucosyllactose (2'-FL) is the most abundant human milk oligosaccharide and plays significant roles in gut microbiome balance, neural development, and immunoregulation. However, current fermentation schemes using multiple carbon sources increase production cost and metabolism burden. This study reported the development of an engineered Bacillus subtilis strain that produces 2'-FL using glucose as the sole carbon source. First, a lactose biosynthesis module was constructed by expressing β-1,4-galactosyltransferase gene from Neisseria meningitidis. A 2'-FL titer of 2.53 ± 0.07 g/L was subsequently achieved using glucose as the sole carbon source by the combination of lactose and GDP-L-fucose (GDP-Fuc) biosynthesis modules. Introducing an exogenous nonphosphorylated transport system enhanced the supply of intracellular nonphosphorylated glucose, and the 2'-FL titer increased to 4.94 ± 0.35 g/L. Next, a transcription factor screening platform was designed. Based on this platform, the ligand of the transcription factor LacI was changed from isopropyl β-D-thiogalactoside to lactose. A lactose-responsive genetic circuit was then constructed and used for the dynamic regulation of metabolic fluxes between lactose and GDP-Fuc biosynthesis modules. Ultimately, the 2'-FL titer of the dynamically regulated strain improved by 107% to 9.67 ± 0.65 g/L in shake-flask, and the titer and yield in a 3-L bioreactor reached 30.1 g/L and 0.15 g/g using glucose as the sole carbon source. By using multidimensional engineering strategies, this study constructed a B. subtilis strain capable of efficiently producing 2'-FL with glucose as the sole carbon source, paving the way for the industrial production of 2'-FL with low cost in the future.

Juvenile hormones (JHs) are farnesoic acid-derived sesquiterpenoids that play a crucial role in regulating various developmental processes in insects. Based on these reported biological activities, JHs and their synthetic analogs have been utilized as insecticides with significant commercial success over the past years. Here we describe the engineering of the JH pathway of the yellow fever mosquito (Aedes aegypti) by transient gene expression in the plant Nicotiana benthamiana. This approach led to the successful production of JH III in N. benthamiana leaves at a concentration of ca. 10 μg/g fresh weight. The co-expression of a feedback-insensitive version of 3-hydroxy-3-methylglutaryl coenzyme A reductase from Arabidopsis thaliana further increased the titer eight-fold from 10 to 80 μg/g fresh weight. Our efforts also revealed that the rich endogenous metabolic background of N. benthamiana can generate farnesoic acid, a key precursor to JH III, and thus, only 3 genes need to be expressed to provide high titers of this compound. Our study demonstrates the production of high titers of JH III in N. benthamina via heterologous expression of insect JH biosynthetic genes.

Poly (ethylene terephthalate) (PET) is one of the most ubiquitous plastics and can be depolymerized through biological and chemo-catalytic routes to its constituent monomers, terephthalic acid (TPA) and ethylene glycol (EG). TPA and EG can be re-synthesized into PET for closed-loop recycling or microbially converted into higher-value products for open-loop recycling. Here, we expand on our previous efforts engineering and applying Pseudomonas putida KT2440 for PET conversion by employing adaptive laboratory evolution (ALE) to improve TPA catabolism. Three P. putida strains with varying degrees of metabolic engineering for EG catabolism underwent an automation-enabled ALE campaign on TPA, a TPA and EG mixture, and glucose as a control. ALE increased the growth rate on TPA and TPA-EG mixtures by 4.1- and 3.5-fold, respectively, in approximately 350 generations. Evolved isolates were collected at the midpoints and endpoints of 39 independent ALE experiments, and growth rates were increased by 0.15 and 0.20 h^-1 on TPA and a TPA-EG, respectively, in the best performing isolates. Whole-genome re-sequencing identified multiple converged mutations, including loss-of-function mutations to global regulators gacS, gacA, and turA along with large duplication and intergenic deletion events that impacted the heterologously-expressed tphAB_II catabolic genes. Reverse engineering of these targets confirmed causality, and a strain with all three regulators deleted and second copies of tphAB_II and tpaK displayed improved TPA utilization compared to the base strain. Taken together, an iterative strain engineering process involving heterologous pathway engineering, ALE, whole genome sequencing, and genome editing identified five genetic interventions that improve P. putida growth on TPA, aimed at developing enhanced whole-cell biocatalysts for PET upcycling.

Ethylene glycol is a promising substrate for bioprocesses which can be derived from widely abundant CO₂ or plastic waste. In this work, we describe the construction of an eight-step synthetic metabolic pathway enabling carbon-conserving biosynthesis of threonine from ethylene glycol. This route extends the previously disclosed synthetic threose-dependent glycolaldehyde assimilation (STEGA) pathway for the synthesis of 2-oxo-4-hydroxybutyrate with three additional reaction steps catalyzed by homoserine transaminase, homoserine kinase, and threonine synthase. We first validated the functionality of the new pathway in an Escherichia coli strain auxotrophic for threonine, which was also employed for discovering a better-performing D-threose dehydrogenase enzyme activity. Subsequently, we transferred the pathway to producer strains and used ¹³C-tracer experiments to improve threonine biosynthesis starting from glycolaldehyde. Finally, extending the pathway for ethylene glycol assimilation resulted in the production of up to 6.5 mM (or 0.8 g L^-1) threonine by optimized E. coli strains at a yield of 0.10 mol mol^-1 (corresponding to 20 % of the theoretical yield).