Problems and corresponding strategies for converting CO2 into value-added products in Cupriavidus necator H16 cell factories.

Elevated CO2 emissions have substantially altered the worldwide climate, while the excessive reliance on fossil fuels has exacerbated the energy crisis. Therefore, the conversion of CO2 into fuel, petroleum-based derivatives, drug precursors, and other value-added products is expected. Cupriavidus necator H16 is the model organism of the "Knallgas" bacterium and is considered to be a microbial cell factory as it can convert CO2 into various value-added products. However, the development and application of C. necator H16 cell factories has several limitations, including low efficiency, high cost, and safety concerns arising from the autotrophic metabolic characteristics of the strains. In this review, we first considered the autotrophic metabolic characteristics of C. necator H16, and then categorized and summarized the resulting problems. We also provided a detailed discussion of some corresponding strategies concerning metabolic engineering, trophic models, and cultivation mode. Finally, we provided several suggestions for improving and combining them. This review might help in the research and application of the conversion of CO2 into value-added products in C. necator H16 cell factories.

Co-conversion of lignocellulose-derived glucose, xylose, and aromatics to polyhydroxybutyrate by metabolically engineered Cupriavidus necator.

Utilization of all major components of lignocellulose is essential for biomass biorefining. Glucose, xylose, and lignin-derived aromatics can be generated from cellulose, hemicellulose, and lignin of lignocellulose degradation through pretreatment and hydrolysis. In present work, Cupriavidus necator H16 was engineered to utilize glucose, xylose, p-coumaric acid, and ferulic acid simultaneously by multi-step genetic engineering. Firstly, genetic modification and adaptive laboratory evolution were performed to promote glucose transmembrane transport and metabolism. Xylose metabolism was then engineered by integrating genes xylAB (xylose isomerase and xylulokinase) and xylE (proton-coupled symporter) in the locus of ldh (lactate dehydrogenase) and ackA (acetate kinase) on the genome, respectively. Thirdly, p-coumaric acid and ferulic acid metabolism was achieved by constructing an exogenous CoA-dependent non-ß-oxidation pathway. Using corn stover hydrolysates as carbon sources, the resulting engineered strain Reh06 simultaneously converted all components of glucose, xylose, p-coumaric acid, and ferulic acid to produce 11.51 g/L polyhydroxybutyrate.

The Overexpression of Phasin and Regulator Genes Promoting the Synthesis of Polyhydroxybutyrate in Cupriavidus necator H16 under Nonstress Conditions.

Cupriavidus necator H16 is an ideal strain for polyhydroxybutyrate (PHB) production from CO2. Low-oxygen stress can induce PHB synthesis in C. necator H16 while reducing bacterial growth under chemoautotrophic culture. The optimum growth and PHB synthesis of C. necator H16 cannot be achieved simultaneously, which restricts PHB production. The present study was initiated to address the issue through comparative transcriptome and gene function analysis. First, the comparative transcriptome of C. necator H16 chemoautotrophically cultured under low-oxygen stress and nonstress conditions was studied. Three types of genes were discovered to have differential levels of transcription: those involving PHB enzymatic synthesis, PHB granulation, and regulators. Under low-oxygen stress conditions, acetoacetyl-coenzyme A (CoA) reductase gene phaB2, PHB synthase gene phaC2, phasins genes phaP1 and phaP2, and regulator genes uspA and rpoN were upregulated 3.0-, 2.5-, 1.8-, 2.7-, 3.5-, and 1.6-fold, respectively. Second, the functions of upregulated genes and their applications in PHB synthesis were further studied. It was found that the overexpression of phaP1, phaP2, uspA, and rpoN can induce PHB synthesis under nonstress conditions, while phaB2 and phaC2 have no significant effect. Under the optimum conditions, the PHB percentage content in C. necator H16 was increased by 37.2%, 28.4%, 15.8%, and 41.0%, respectively, with overexpression of phaP1, phaP2, uspA, and rpoN, and the corresponding PHB production increased by 49.8%, 42.9%, 47.0%, and 77.5%, respectively, under nonstress chemoautotrophic conditions. Similar promotion by phaP1, phaP2, uspA, and rpoN was observed in heterotrophically cultured C. necator H16. The PHB percentage content and PHB production were increased by 54.4% and 103.1%, respectively, with the overexpression of rpoN under nonstress heterotrophic conditions. IMPORTANCE Microbial fixation of CO2 is an effective way to reduce greenhouse gases. Some microbes, such as C. necator H16, usually accumulate PHB when they grow under stress. Low-oxygen stress can induce PHB synthesis when C. necator H16 is autotrophically cultured with CO2, H2, and O2, while under stress, growth is restricted, and total PHB yield is reduced. Achieving the optimal bacterial growth and PHB synthesis at the same time is an ideal condition for transforming CO2 into PHB by C. necator H16. The present study was initiated to clarify the molecular basis of low-oxygen stress promoting PHB accumulation and to realize the optimal PHB production by C. necator H16. Genes upregulated under nonstress conditions were identified through comparative transcriptome analysis and overexpression of phasin, and regulator genes were demonstrated to promote PHB synthesis in C. necator H16.

Recent Advances in the Biosynthesis of Farnesene Using Metabolic Engineering.

Farnesene, as an important sesquiterpene isoprenoid polymer of acetyl-CoA, is a renewable feedstock for diesel fuel, polymers, and cosmetics. It has been widely applied in agriculture, medicine, energy, and other fields. In recent years, farnesene biosynthesis is considered a green and economical approach because of its mild reaction conditions, low environmental pollution, and sustainability. Metabolic engineering has been widely applied to construct cell factories for farnesene biosynthesis. In this paper, the research progress, common problems, and strategies of farnesene biosynthesis are reviewed. They are mainly described from the perspectives of the current status of farnesene biosynthesis in different host cells, optimization of the metabolic pathway for farnesene biosynthesis, and key enzymes for farnesene biosynthesis. Furthermore, the challenges and prospects for future farnesene biosynthesis are discussed.

Metabolic engineering of Cupriavidus necator H16 for improved chemoautotrophic growth and PHB production under oxygen-limiting conditions.

The oxygen-limiting condition promotes the accumulation of ployhydroxybutyrate (PHB) in C. necator H16, while the growth of which is restricted. Under autotrophic culture using carbon dioxide, hydrogen, and oxygen as substrates, the oxygen concentration below 6.9% (v/v) in the mixture is considered as a safe condition. It also expected to achieve cell rapid growth and large accumulation of PHB simultaneously under the oxygen-limiting condition in C. necator H16. In this study, a metabolically engineered strain capable of both rapid growth and large accumulation of PHB under oxygen-limiting conditions was constructed based on the transcriptomic analysis. In the comparative transcriptomic analysis, the genes related to energy-generating of C. necator H16 at autotrophic culture were downregulated under oxygen-limiting conditions (3%, v/v). Besides, the genes related to the key intermediates (pyruvate and acetyl-CoA) metabolism in PHB biosynthetic pathway were analyzed. Most of which were downregulated, except the genes ldh, iclA, and ackA2 respectively encoding L-lactate dehydrogenase, isocitrate lyase, and acetate kinase were upregulated under oxygen-limiting conditions (3%, v/v). The Vitreoscilla hemoglobin (VHb) has the ability to promote aerobic metabolism and energy generation. To promote the bacterium growth and improve the energy generation in C. necator H16 under oxygen-limiting conditions, the VHb gene was introduced into C. necator H16 with the optimized promoter PphaC1-j5. Moreover, VHb was localized to the periplasmic space of the bacterium by the traction of membrane-bound hydrogenase (MBH) signal peptide. By optimizing the knockout of different genes, it was found that knockout of ldh can improve PHB production and reduce the by-products. Finally, a recombinant strain Reh01 (p2M-pj-v) was constructed by heterologous expression of vgb and ldh knockout in C. necator H16. Compared with the control (Reh (p2)) under oxygen-limiting conditions (3%, v/v), the dry cell weight (DCW), PHB content, and PHB production of Reh01 (p2M-pj-v) increased by 31.0%, 30.9%, and 71.5%, respectively. From the perspectives of transcriptome and metabolic engineering, the work provides new ideas to achieve rapid cell growth and large PHB accumulation in C. necator under oxygen-limiting and autotrophic conditions.

Corrosion behavior and mechanism of carbon steel influenced by interior deposit microflora of an in-service pipeline.

Investigation of carbon steel corrosion influenced by in-situ microbial communities can provide reliable information about microbiologically influenced corrosion (MIC) in the oil and gas field. Here, we investigated the 90-day corrosion behavior of Q235 carbon steel influenced by interior deposit microflora of an in-service pipeline using open circuit potential (OCP) and electrochemical impedance spectroscopy (EIS). Linear sweep voltammetry (LSV), 16S rRNA gene sequencing, and surface analysis were used to comprehensively analyze the corrosion mechanisms. The results indicated that OCP was decreased while the charge transfer resistance (Rct) was increased, and that steel corrosion was inhibited during the first 45 days. Subsequently, OCP was significantly increased while Rct was rapidly decreased, and steel corrosion was enhanced. After 90-day immersion, severe pitting corrosion with a maximum pit depth of 89.6 µm occurred on the steel surface. Viable microbes in the final biofilm significantly increased the cathodic current. Iron carbonate, chukanovite and cementite were identified as the main corrosion products on the steel surface. Methanobacterium dominated the final biofilm community. These observations indicate that the corrosion mechanism of the final biofilm can be explained by extracellular electron transfer MIC in which microbes corrode steel by direct electron uptake.