Volume 2, Issue 1 (March 2024) – 6 articles

Cover Story (View full-size image):
Two terpene synthases, from a plant (Plectranthus barbatus) combined with geranylgeranyl diphosphate synthases from a cyanobacterium, a yeast or a plant, were expressed in the cyanobacterium Synechocystis sp. PCC 6803, effectively boosting the channeling of carbon towards the production of diterpenoid 13-R-manoyl oxide (13-R-MO). The 13-R-MO titer was increased 11-fold and reached 9.7 mg/L. Application of a N-dodecane overlay to remove 13-R-MO from the culture medium provided a 2–3 fold increase of the 13-R-MO.  View this manuscript

Article

23 January 2024

Analysis of a σ54 Transcription Factor L420P Mutation in Context of Increased Organic Nitrogen Tolerance of Photofermentative Hydrogen Production in Cereibacter sphaeroides Strain 2.4.1 Substrain H2

Photofermentative hydrogen production with non-sulfur purple bacteria like Cereibacter sphaeroides (formerly Rhodobacter sphaeroides) is a promising and sustainable process to convert organic waste into the energy carrier hydrogen gas. However, this conversion is inhibited by elevated organic nitrogen concentrations in the substrate, which limits its applicability to nitrogen-poor organic waste. We present genomic and transcriptomic insights into a substrain of Cereibacter sphaeroides strain 2.4.1 that shows unexpected high levels of photofermentative hydrogen evolution when fed with glutamate. Genome sequencing revealed 222 single nucleotide variances (SNVs) between the reference genome of C. sphaeroides strain 2.4.1 and the analyzed substrain H2. These affect 61 protein coding genes. A leucine-proline exchange is present in the σ54 factor (rpoN2 gene), a global hydrogen and nitrogen metabolism regulator. We propose a model how this mutation alters DNA-binding properties that explain the unexpected organic nitrogen tolerance of hydrogen production. Transcriptomic analyses under varying glutamate concentrations support this finding. Thus, we present the first thorough genomic and transcriptomic analysis of a Cereibacter strain that shows promising metabolic characteristics for biotechnological hydrogen gas production from organic waste. These results suggest a potential target for strain optimization. Possibly, our key finding can be transferred to other hydrogen producing microorganisms.

Article

06 February 2024

Bio-Based Production of Uroporphyrin in Escherichia coli

Uroporphyrin (UP) is a porphyrin compound with medical applications and a key precursor for heme biosynthesis. However, there is no biosynthetic strategy for UP production. In this study, we present a novel bioprocess for enhanced production of UP in engineered Escherichia coli. We first implemented the Shemin/C4 pathway heterologously in an E. coli strain with an enlarged intracellular pool of succinyl-CoA. Using a plasmid with the trc promoter regulating the expression of a synthesized gene operon, the effects of key pathway genes, including hemA, hemB, hemC, and hemD, on UP biosynthesis were characterized. By cultivating the resulting engineered E. coli strains in a batch bioreactor with 30 g/L glycerol under aerobic conditions, up to 901.9 mg/L UP was produced. Most of the synthesized UP was extracellularly secreted with a high purity more than 80 wt%, facilitating its downstream purification. The study paves the way for large-scale bio-based production of UP using synthetic biology and metabolic engineering strategies.

Review

13 February 2024

Development and Perspective of Production of Terpenoids in Yeast

Terpenoids are a large class of secondary metabolites known for their remarkable diverse biological activities, making them widely utilized in the pharmaceutical, food, cosmetic, biofuel and agricultural fields. However, the current production of terpenoids heavily relies on plant extraction and chemical synthesis, which brings about concerns regarding infield, environmental and ecological issues. With the advancements in metabolic engineering and emerging synthetic biology tools, it is now possible to sustainably produce these high value-added terpenoids using microbial chassis. Among them, yeast has emerged as a promising candidate for the heterologous biosynthesis of terpenoids due to its inherent advantages, including robustness, safety, and the availability of sufficient precursor. This review focuses on the diverse strategies employed to enable terpenoids production in yeasts. These strategies encompass metabolic engineering approaches to optimize the mevalonate pathway, protein engineering techniques to improve terpenoid biosynthesis, the applications of organelles compartmentalization, high throughput screening and global approaches for the development of efficient cell factories. Furthermore, this review discusses the future prospects and challenges associated with yeast-based terpenoid production, while also emphasizing guidelines for future studies in this field.

Review

21 February 2024

One-pot Multi-enzyme Cascade Synthesis of Bifunctional Compounds from Vegetable Oils

Green and efficient biocatalytic technology has become a complementary or alternative means of organic synthesis. Chemicals with two functional groups, such as α,ω-dicarboxylic acids, ω-amino fatty acids and ω-hydroxy fatty acids, are widely used in the synthesis of polymers such as polyesters and polyamides. In recent years, the production of biodegradable materials using renewable and abundant vegetable oils as green raw materials has attracted increasing attention, receiving an additional impetus from synthetic biology. This paper presents the recent research progress in the production of bifunctional chemicals with medium chain lengths of C8–C12 using multi-enzyme cascades. Recent studies have developed multilevel optimization strategies to improve the efficiency, economics, and sustainability of multi-enzyme cascades. Cofactor regeneration strategies were developed to avoid large additions of expensive coenzymes. Protein engineering strategies were applied to improve enzyme stability and catalytic performance. In addition, blocking the β-oxidation pathway, improving the efficiency of substrate transport across membranes and increasing cellular robustness are effective optimization strategies for whole-cell catalytic systems. In addition, we discuss the development prospects of producing high value-added fine chemicals from vegetable oils using one-pot multi-enzyme reaction systems.

Article

22 March 2024

Modulation of the MEP Pathway for Overproduction of 13-R-manoyl Oxide in Cyanobacteria

The cyanobacterium Synechocystis sp. PCC 6803 has gained scientific interest for its potential to use solar energy and atmospheric CO2 for the production of high-value chemicals like pharmaceuticals, flavors, and fragrances. Forskolin is a diterpenoid found in the root cork of the plant Plectranthus barbatus and its biosynthetic pathway is initiated by two terpene synthases that convert geranylgeranyl diphosphate (GGDP) into the precursor 13-R-manoyl oxide (13-R-MO). Using the cyanobacterium Synechocystis sp. PCC 6803 as host, we expressed the two terpene synthases resulting in the synthesis of 0.83 mg/L 13-R-MO. Three different geranylgeranyl diphosphate synthases (GGDPSs) were selected for screening; a prokaryotic (Synechococcus sp. JA-3-3Ab (Sj)), a yeast (Saccharomyces cerevisiae (Sc)), and a plant (P. barbatus (Pb)) derived GGDPS. Strains containing the prokaryotic Sj- or the yeast ScGGDPS consistently yielded more 13-R-MO than the base strain. By overexpression of 1-Deoxy-D-xylulose-5-phosphate synthase (DXS) positioned at the entry of the 2-C-methyl-d-erythritol 4-phosphate pathway (MEP) together with the prokaryotic SjGGDPS, the 13-R-MO titer was increased 11-fold to reach 9.7 mg/L by boosting the synthesis of GGDP, the direct substrate for the diterpenoid synthases. We further show that application of a n-dodecane overlay to remove 13-R-MO from the culture medium provided a 2–3 fold increase of the 13-R-MO in a separate cultivation system.

Article

25 March 2024

Proteomic Analysis of Pleurotus ostreatus Grown on Glucose and Xylose Mixtures in Submerged Fermentation Provides Insights into Differentiated Mycelial Composition

Pleurotus ostreatus, an edible white-rot fungus of great commercial and nutritional value, grows by metabolizing mainly glucose and xylose, the two major sugars in lignocellulosic biomass. In this study, a comparative proteomic analysis of P. ostreatus grown in submerged fermentation on a medium with glucose, xylose and mixtures of them as carbon sources was conducted. In the same conditions, the metabolic response of the fungus was evaluated in the production of the main nutritional components of the fungus such as proteins, lipids, and intracellular and extracellular polysaccharides. The proteomic analysis revealed that glucose and xylose upregulate different clusters of proteins. Glucose mainly up-regulates macromolecule metabolic processes, translation and glycolysis whereas xylose up-regulates, small molecule metabolic processes and tricarboxylic acid cycle (TCA). The mixtures show mostly similarities with the proteome response to glucose, although there are differential responses depending on xylose concentration. The carbon source type found to affect the basic macromolecule metabolic processes, with amino acids biosynthesis to differentiate mostly. An analysis of the upregulated proteins through the STRING database revealed that xylose upregulates mostly proteins related to amino acid biosynthesis. Leucine, Valine and Isoleucine biosynthesis pathways were found to be the most triggered pathway. All the branched-chain amino acids (BCAAs)-related enzymes intensities were gradually increased when xylose concentration was increased in the growth medium. BCAAs play an important role in the human diet so the enhancement of BCAAs biosynthesis pathway for P. ostreatus could convert it to a very remarkable protein substitute in human diet. These findings provide new insights into the proteomic and metabolic response of the fungus to the major sugars of lignocellulosic biomass, which are not well understood until now. 

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