Cofactor Engineering for Efficient Production of α-Farnesene by Rational Modification of NADPH and ATP Regeneration Pathway in Pichia pastoris.

α-Farnesene, an acyclic volatile sesquiterpene, plays important roles in aircraft fuel, food flavoring, agriculture, pharmaceutical and chemical industries. Here, by re-creating the NADPH and ATP biosynthetic pathways in Pichia pastoris, we increased the production of α-farnesene. First, the native oxiPPP was recreated by overexpressing its essential enzymes or by inactivating glucose-6-phosphate isomerase (PGI). This revealed that the combined over-expression of ZWF1 and SOL3 increases α-farnesene production by improving NADPH supply, whereas inactivating PGI did not do so because it caused a reduction in cell growth. The next step was to introduce heterologous cPOS5 at various expression levels into P. pastoris. It was discovered that a low intensity expression of cPOS5 aided in the production of α-farnesene. Finally, ATP was increased by the overexpression of APRT and inactivation of GPD1. The resultant strain P. pastoris X33-38 produced 3.09 ± 0.37 g/L of α-farnesene in shake flask fermentation, which was 41.7% higher than that of the parent strain. These findings open a new avenue for the development of an industrial-strength α-farnesene producer by rationally modifying the NADPH and ATP regeneration pathways in P. pastoris.

Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis.

BACKGROUND: Menaquinone-7 (MK-7), which is associated with complex and tightly regulated pathways and redox imbalances, is produced at low titres in Bacillus subtilis. Synthetic biology provides a rational engineering principle for the transcriptional optimisation of key enzymes and the artificial creation of cofactor regeneration systems without regulatory interference. This holds great promise for alleviating pathway bottlenecks and improving the efficiency of carbon and energy utilisation. RESULTS: We used a bottom-up synthetic biology approach for the synthetic redesign of central carbon and to improve the adaptability between material and energy metabolism in MK-7 synthesis pathways. First, the rate-limiting enzymes, 1-deoxyxylulose-5-phosphate synthase (DXS), isopentenyl-diphosphate delta-isomerase (Fni), 1-deoxyxylulose-5-phosphate reductase (DXR), isochorismate synthase (MenF), and 3-deoxy-7-phosphoheptulonate synthase (AroA) in the MK-7 pathway were sequentially overexpressed. Promoter engineering and fusion tags were used to overexpress the key enzyme MenA, and the titre of MK-7 was 39.01 mg/L. Finally, after stoichiometric calculation and optimisation of the cofactor regeneration pathway, we constructed two NADPH regeneration systems, enhanced the endogenous cofactor regeneration pathway, and introduced a heterologous NADH kinase (Pos5P) to increase the availability of NADPH for MK-7 biosynthesis. The strain expressing pos5P was more efficient in converting NADH to NADPH and had excellent MK-7 synthesis ability. Following three Design-Build-Test-Learn cycles, the titre of MK-7 after flask fermentation reached 53.07 mg/L, which was 4.52 times that of B. subtilis 168. Additionally, the artificially constructed cofactor regeneration system reduced the amount of NADH-dependent by-product lactate in the fermentation broth by 9.15%. This resulted in decreased energy loss and improved carbon conversion. CONCLUSIONS: In summary, a "high-efficiency, low-carbon, cofactor-recycling" MK-7 synthetic strain was constructed, and the strategy used in this study can be generally applied for constructing high-efficiency synthesis platforms for other terpenoids, laying the foundation for the large-scale production of high-value MK-7 as well as terpenoids.

Impact of overexpressing NADH kinase on glucoamylase production in Aspergillus niger.

Glucoamylase has a wide range of applications in the production of glucose, antibiotics, amino acids, and other fermentation industries. Fungal glucoamylase, in particular, has attracted much attention because of its wide application in different industries, among which Aspergillus niger is the most popular strain producing glucoamylase. The low availability of NADPH was found to be one of the limiting factors for the overproduction of glucoamylase. In this study, 3 NADH kinases (AN03, AN14, and AN17) and malic enzyme (maeA) were overexpressed in aconidial A. niger by CRISPR/Cas9 technology, significantly increasing the size of the NADPH pool, resulting in the activity of glucoamylase was improved by about 70%, 50%, 90%, and 70%, respectively; the total secreted protein was increased by about 25%, 22%, 52%, and 26%, respectively. Furthermore, the combination of the mitochondrial NADH kinase (AN17) and the malic enzyme (maeA) increased glucoamylase activity by a further 19%. This study provided an effective strategy for enhancing glucoamylase production of A. niger.

Redox Engineering by Ectopic Overexpression of NADH Kinase in Recombinant Pichia pastoris (Komagataella phaffii): Impact on Cell Physiology and Recombinant Production of Secreted Proteins.

High-level expression and secretion of heterologous proteins in yeast cause an increased energy demand, which may result in altered metabolic flux distributions. Moreover, recombinant protein overproduction often results in endoplasmic reticulum (ER) stress and oxidative stress, causing deviations from the optimal NAD(P)H regeneration balance. In this context, overexpression of genes encoding enzymes catalyzing endogenous NADPH-producing reactions, such as the oxidative branch of the pentose phosphate pathway, has been previously shown to improve protein production in Pichia pastoris (syn. Komagataella spp.). In this study, we evaluate the overexpression of the Saccharomyces cerevisiaePOS5-encoded NADH kinase in a recombinant P. pastoris strain as an alternative approach to overcome such redox constraints. Specifically, POS5 was cooverexpressed in a strain secreting an antibody fragment, either by directing Pos5 to the cytosol or to the mitochondria. The physiology of the resulting strains was evaluated in continuous cultivations with glycerol or glucose as the sole carbon source, as well as under hypoxia (on glucose). Cytosolic targeting of Pos5 NADH kinase resulted in lower biomass-substrate yields but allowed for a 2-fold increase in product specific productivity. In contrast, Pos5 NADH kinase targeting to the mitochondria did not affect growth physiology and recombinant protein production significantly. Growth physiological parameters were in silico evaluated using the recent upgraded version (v3.0) of the P. pastoris consensus genome-scale metabolic model iMT1026, providing insights on the impact of POS5 overexpression on metabolic flux distributions.IMPORTANCE Recombinant protein overproduction often results in oxidative stress, causing deviations from the optimal redox cofactor regeneration balance. This becomes one of the limiting factors in obtaining high levels of heterologous protein production. Overexpression of redox-affecting enzymes has been explored in other organisms, such as Saccharomyces cerevisiae, as a means to fine tune the cofactor regeneration balance in order to obtain higher protein titers. In the present work, this strategy is explored in P. pastoris In particular, one NADH kinase enzyme from S. cerevisiae (Pos5) is used, either in the cytosol or in mitochondria of P. pastoris, and its impact on the production of a model protein (antibody fragment) is evaluated. A significant improvement in the production of the model protein is observed when the kinase is directed to the cytosol. These results are significant in the field of heterologous protein production in general and in particular in the development of improved metabolic engineering strategies for P. pastoris.

A mitochondrial NAD/NADH kinase governs fungal virulence through an oxidative stress response and arginine biosynthesis in Fusarium graminearum.

NADP/NADPH plays an indispensable role in cellular metabolism, serving as a pivotal cofactor in numerous enzymatic processes involved in anabolic pathways, antioxidant defense, and the biosynthesis of essential cellular components. NAD/NADH kinases (NADKs) phosphorylate NAD/NADH, constituting the sole de novo synthetic pathway for NADP/NADPH generation. Despite the pivotal role of NADP/NADPH in cellular functions, the physiological role of NADK remains largely unexplored in filamentous fungi. In this study, we identified three putative NADKs in Fusarium graminearum-FgNadk1, FgNadk2, and FgNadk3-responsible for NAD/NADH phosphorylation. NADK-mediated formation of intracellular NADPH proved crucial for vegetative growth, sexual reproduction, and virulence. Specifically, FgNadk2, the mitochondrial NADK, played a role in oxidative stress resistance and the maintenance of mitochondrial reactive oxygen species levels. Moreover, the deletion of FgNADK2 resulted in arginine auxotrophy, contributing to the reduced fungal virulence. These findings underscore the necessity of mitochondrial NADK in fungal virulence in F. graminearum, revealing its involvement in mitochondrial redox homeostasis and the arginine biosynthetic pathway. This study provides critical insights into the interconnectedness of metabolic pathways essential for fungal growth, stress response, and pathogenicity.

Anilinopyrimidine Resistance in Botrytis cinerea Is Linked to Mitochondrial Function.

Crop protection anilinopyrimidine (AP) fungicides were introduced more than 20 years ago for the control of a range of diseases caused by ascomycete plant pathogens, and in particular for the control of gray mold caused by Botrytis cinerea. Although early mode of action studies suggested an inhibition of methionine biosynthesis, the molecular target of this class of fungicides was never fully clarified. Despite AP-specific resistance having been described in B. cinerea field isolates and in multiple other targeted species, the underlying resistance mechanisms were unknown. It was therefore expected that the genetic characterization of resistance mechanisms would permit the identification of the molecular target of these fungicides. In order to explore the widest range of possible resistance mechanisms, AP-resistant B. cinerea UV laboratory mutants were generated and the mutations conferring resistance were determined by combining whole-genome sequencing and reverse genetics. Genetic mapping from a cross between a resistant field isolate and a sensitive reference isolate was used in parallel and led to the identification of an additional molecular determinant not found from the characterized UV mutant collection. Together, these two approaches enabled the characterization of an unrivaled diversity of resistance mechanisms. In total, we report the elucidation of resistance-conferring mutations within nine individual genes, two of which are responsible for almost all instances of AP resistance in the field. All identified resistance-conferring genes encode proteins that are involved in mitochondrial processes, suggesting that APs primarily target the mitochondria. The functions of these genes and their possible interactions are discussed in the context of the potential mode of action for this important class of fungicides.

Effect of NADH kinase on poly-3-hydroxybutyrate production by recombinant Escherichia coli.

The cofactor NADPH participates in a variety of anabolic reactions and its availability is considered to play a critical role in biotransformation processes. NADH kinase (Pos5) from Saccharomyces cerevisiae catalyzes the phosphorylation of NADH to generate NADPH. To investigate the effect of NADH kinase on poly-3-hydroxybutyrate (PHB) production, pos5 was co-expressed with PHB synthetic operon phbCAB in Escherichia coli. The recombinant strain carrying pos5 and phbCAB co-expression plasmid reached 5.96 g/L cell dry weight with 64.1% PHB accumulation in 72 h shake flask cultivation, while the control strain without pos5 yielded 3.93 g/L cell dry weight with 58.5% PHB content. PHB production titer was enhanced from 2.30 g/L to 3.82 g/L. Intracellular cofactor concentration analysis revealed that the ratio of NADP/NAD in pos5 overexpression strain was two times more compared with that of the control without pos5. The results showed that NADH kinase could be employed as an effective metabolic manipulation target to improve PHB synthesis.

Evidence that the antiproliferative effects of auranofin in Saccharomyces cerevisiae arise from inhibition of mitochondrial respiration.

Auranofin is a gold based drug in clinical use since 1985 for the treatment of rheumatoid arthritis. Beyond its antinflammatory properties, auranofin exhibits other attractive biological and pharmacological actions such as a potent in vitro cytotoxicity and relevant antimicrobial and antiparasitic effects that make it amenable for new therapeutic indications. For instance, auranofin is currently tested as an anticancer agent in four independent clinical trials; yet, its mode of action is highly controversial. With the present study, we explore the effects of auranofin in Saccharomyces cerevisiae and its likely mechanism. Notably, auranofin is reported to induce remarkable yeast growth inhibition. Solid evidence is provided that growth inhibition is the consequence of a direct cytotoxic insult occurring at the mitochondrial level; a profound depression of cell respiration is indeed clearly documented as the main cause of cell death while induction of ROS plays only a secondary role. More in detail, the mitochondrial NADH kinase Pos5 is identified as a primary target for auranofin. The implications of these results are discussed in the frame of current mechanistic knowledge on the cellular effects of auranofin and of its role as a prospective anticancer drug.