Efficient production of l-glutathione by whole-cell catalysis with ATP regeneration from adenosine.

l-glutathione (GSH) is an important tripeptide compound with extensive applications in medicine, food additives, and cosmetics industries. In this work, an innovative whole-cell catalytic strategy was developed to enhance GSH production by combining metabolic engineering of GSH biosynthetic pathways with an adenosine-based adenosine triphosphate (ATP) regeneration system in Escherichia coli. Concretely, to enhance GSH production in E. coli, several genes associated with GSH and  l-cysteine degradation, as well as the branched metabolic flow, were deleted. Additionally, the GSH bifunctional synthase (GshFSA) and GSH ATP-binding cassette exporter (CydDC) were overexpressed. Moreover, an adenosine-based ATP regeneration system was first introduced into E. coli to enhance GSH biosynthesis without exogenous ATP additions. Through the optimization of whole-cell catalytic conditions, the engineered strain GSH17-FDC achieved an impressive GSH titer of 24.19 g/L only after 2 h reaction, with a nearly 100% (98.39%) conversion rate from the added  l-Cys. This work not only unveils a new platform for GSH production but also provides valuable insights for the production of other high-value metabolites that rely on ATP consumption.

Advances in biotin biosynthesis and biotechnological production in microorganisms.

Biotin, also known as vitamin H or B7, acts as a crucial cofactor in the central metabolism processes of fatty acids, amino acids, and carbohydrates. Biotin has important applications in food additives, biomedicine, and other fields. While the ability to synthesize biotin de novo is confined to microorganisms and plants, humans and animals require substantial daily intake, primarily through dietary sources and intestinal microflora. Currently, chemical synthesis stands as the primary method for commercial biotin production, although microbial biotin production offers an environmentally sustainable alternative with promising prospects. This review presents a comprehensive overview of the pathways involved in de novo biotin synthesis in various species of microbes and insights into its regulatory and transport systems. Furthermore, diverse strategies are discussed to improve the biotin production here, including mutation breeding, rational metabolic engineering design, artificial genetic modification, and process optimization. The review also presents the potential strategies for addressing current challenges for industrial-scale bioproduction of biotin in the future. This review is very helpful for exploring efficient and sustainable strategies for large-scale biotin production.

Retron-mediated multiplex genome editing and continuous evolution in Escherichia coli.

While there are several genome editing techniques available, few are suitable for dynamic and simultaneous mutagenesis of arbitrary targeted sequences in prokaryotes. Here, to address these limitations, we present a versatile and multiplex retron-mediated genome editing system (REGES). First, through systematic optimization of REGES, we achieve efficiency of ∼100%, 85 ± 3%, 69 ± 14% and 25 ± 14% for single-, double-, triple- and quadruple-locus genome editing, respectively. In addition, we employ REGES to generate pooled and barcoded variant libraries with degenerate RBS sequences to fine-tune the expression level of endogenous and exogenous genes, such as transcriptional factors to improve ethanol tolerance and biotin biosynthesis. Finally, we demonstrate REGES-mediated continuous in vivo protein evolution, by combining retron, polymerase-mediated base editing and error-prone transcription. By these case studies, we demonstrate REGES as a powerful multiplex genome editing and continuous evolution tool with broad applications in synthetic biology and metabolic engineering.

CRISPR-Cas12a-based genome editing and transcriptional repression for biotin synthesis in Pseudomonas mutabilis.

AIMS: To establish a dual-function clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system combined genome editing and transcriptional repression for multiplex metabolic engineering of Pseudomonas mutabilis. MATERIALS AND RESULTS: This CRISPR-Cas12a system consisted of two plasmids that enabled single gene deletion, replacement, and inactivation with efficiency >90% for most targets within 5 days. With the guidance of truncated crRNA containing 16 bp spacer sequences, a catalytically active Cas12a could be employed to repress the expression of the reporter gene eGFP up to 66.6%. When bdhA deletion and eGFP repression were tested simultaneously by transforming a single crRNA plasmid and Cas12a plasmid, the knockout efficiency reached 77.8% and the expression of eGFP was decreased by >50%. Finally, the dual-functional system was demonstrated to increase the production of biotin by 3.84-fold, with yigM deletion and birA repression achieved simultaneously. CONCLUSIONS: This CRISPR-Cas12a system is an efficient genome editing and regulation tool to facilitate the construction of P. mutabilis cell factories.

High-level secretory production of leghemoglobin in Pichia pastoris through enhanced globin expression and heme biosynthesis.

Soy leghemoglobin is a key food additive that imparts meaty flavor and color to meat analogs. Here, a Pichia pastoris strain capable of high-yield secretory production of functional leghemoglobin was developed through gene dosage optimization and heme pathway consolidation. First, multi-copy integration of LegH expression cassette was achieved via both post-transformational vector amplification and CRISPR/Cas9 mediated genome editing methods. A combination of inducible expression and constitutive expression resulted in the highest production of leghemoglobin. Then, heme biosynthetic pathway was engineered to address challenges in heme depletion and leghemoglobin secretion. Finally, the disruption of ku70 was complemented in engineered P. pastoris strain to enable high-density fermentation in a 10-L bioreactor. These engineering strategies increased the secretion of leghemoglobin by more than 83-fold, whose maximal leghemoglobin titer and heme binding ratio reached as high as 3.5 g/L and 93 %, respectively. This represents the highest secretory production of heme-containing proteins ever reported.

Effects of low molecular weight polysaccharides from Ulva prolifera on the tolerance of Triticum aestivum to osmotic stress.

Polysaccharides derived from seaweeds can be used as biostimulants to enhance plant resistance to different stressors. In this study, we investigated the effects of applying low molecular weight polysaccharides (LPU) derived from Ulva prolifera with 14.2 kDa on the responses of wheat (Triticum aestivum) to osmotic stress. The results showed that osmotic stress simulated using polyethylene glycol inhibited seedling growth, whereas we observed increases in the fresh weights and shoot lengths of seedlings treated with polysaccharide for 120 h. Furthermore, we observed enhanced activities of antioxidant enzymes, and significant reductions in malondialdehyde content of 23.13%, 19.82%, and 20.04% in response treatment for 120 h with 0.01%, 0.03%, and 0.05% LPU, respectively, relative to those in the group treated with polyethylene glycol alone. In all treatments, expression of the P5CS gene was upregulated to promote proline accumulation. Moreover, after 120 h, exogenously applied LPU induced the expression of stress-related genes, including SnRK2, Wabi5, Wrab18, and Wdhn13. Collectively, these findings indicate that LPU might have the effect of regulating the abscisic acid-dependent pathway in wheat, thereby increasing seedling antioxidant capacity and growth. Application of LPU may accordingly represent an effective approach for enhancing the resistance to osmotic stress in wheat.

Structural characterization of ulvan extracted from Ulva clathrata assisted by an ulvan lyase.

Rhamnan-rich sulfated polysaccharides extracted from green algae (ulvan) constitute potentially useful natural materials for drug development. However, the characterization of their complex structures poses a challenge for their application. In this study, the structure of ulvan extracted from Ulva clathrata was analyzed with the assistance of an ulvan lyase belonging to the PL25 family. According to mass spectrometry and nuclear magnetic resonance analysis of the degraded oligosaccharides, the backbone of such a polysaccharide mainly consisted of →4)-ß-d-GlcA-(1→4)-α-l-Rha3S-(1→ and →4)-ß-d-Xyl-(1→4)-α-l-Rha3S-(1→ disaccharide repeating units, and the ratio is approximately 4:1. In addition, about 4% of the xylose moieties bear sulfate groups. Minor amounts of branches containing hexose and unsaturated glucuronic acid were found during the sequence analysis of hexa- to octasaccharides. These results indicated the presence of a long branch in the ulvan. The clarification of the detailed structure provides a foundation for ulvan modification and its structure-activity relationship studies.

Cloning, Expression, and Characterization of a New PL25 Family Ulvan Lyase from Marine Bacterium Alteromonas sp. A321.

Ulvan lyases can degrade ulvan to oligosaccharides with potent biological activity. A new ulvan lyase gene, ALT3695, was identified in Alteromonas sp. A321. Soluble expression of ALT3695 was achieved in Escherichia coli BL21 (DE3). The 1314-bp gene encoded a protein with 437 amino acid residues. The amino acid sequence of ALT3695 exhibited low sequence identity with polysaccharide lyase family 25 (PL25) ulvan lyases from Pseudoalteromonas sp. PLSV (64.14% identity), Alteromonas sp. LOR (62.68% identity), and Nonlabens ulvanivorans PLR (57.37% identity). Recombinant ALT3695 was purified and the apparent molecular weight was about 53 kDa, which is different from that of other polysaccharide-degrading enzymes identified in Alteromonas sp. A321. ALT3695 exhibited maximal activity in 50 mM Tris-HCl buffer at pH 8.0 and 50 °C. ALT3695 was relatively thermostable, as 90% activity was observed after incubation at 40 °C for 3 h. The Km and Vmax values of ALT3695 towards ulvan were 0.43 mg·mL-1 and 0.11 µmol·min-1·mL-1, respectively. ESI-MS analysis showed that enzymatic products were mainly disaccharides and tetrasaccharides. This study reports a new PL25 family ulvan lyase, ALT3695, with properties that suggest its great potential for the preparation of ulvan oligosaccharides.