Biotechnology and Bioengineering最新文献

Photosynthesis, the most important biological process on Earth, converts light energy into chemical energy with essential pigments like chlorophylls and bacteriochlorophylls. The ability to reconstruct photosynthesis in heterotrophic organisms could significantly impact solar energy utilization and biomass production. In this study, we focused on constructing light-dependent biosynthesis pathways for bacteriochlorophyll (BChl) a and bacteriochlorophyllide (BChlide) d and c in the model strain Escherichia coli. The production of the starting compound, Mg protoporphyrin monomethylester, was optimized by screening the ribosome binding sites for the expression of each of the five genes. By fusing a maltose-binding protein and apolipoprotein A-I domain with the membrane protein BchF, the yield of 3-hydroxyethyl-Chlide a was increased by five-fold. Anaerobic cultivation of the engineered E. coli strains facilitated the reduction of the C7=C8 double bond by chlorophyllide a oxidoreductase, a critical step in BChl a synthesis. We further enhanced BChl a production by adjusting the isopropyl-β-d-thiogalactopyranoside concentration to optimize enzyme production and introducing an exogenous superoxide dismutase to combat oxidative stress. Additionally, fusing BciC with a RIAD tag resulted in an eight-fold increase in the production of 3-vinyl BChlide d. This study lays the foundation for the reconstitution of BChl-based photosynthetic apparatus in heterotrophic model organisms, offering promising avenues for future research and applications in biotechnology.

The repair process of bone tissue includes the early inflammatory response period and the late tissue repair period. It has been widely approved to be beneficial to the repair of bone injury by procedurally inhibiting the inflammatory response in the early stage and promoting bone regeneration in the late stage. In this study, the nano-hydroxyapatite/Poly(glycolide-co-caprolactone) (n-HA/PGCL) scaffold loaded with icariin was fabricated by fused deposition modeling technique, and the quercetin-loaded GelMA was further filled into the scaffold pores via light-curing methods to form a biphasic scaffold loaded with dual molecules (PHI + GQ scaffold). The releases of icariin and quercetin were sequential due to different degradation rates of GelMA and PGCL. In vitro, the scaffold not only scavenged reactive oxygen species production, but also promoted osteogenic differentiation of the MC-3T3-E1 cells. Furthermore, in vivo bone reconstruction of PHI + GQ scaffold was better than other groups by assessment of micro-CT data. In addition, the immunofluorescence staining of Arg-1 and iNOS indicated that PHI + GQ scaffold created an immune microenvironment conducive to bone repair due to the release of quercetin in the early stage, and HE and Masson staining suggested that PHI + GQ scaffold induced more new bone formation. These results demonstrated that the biphasic scaffold loaded with icariin and quercetin had both antioxidants in the early stage and osteogenesis properties in the late stage, obtaining satisfactory bone repair outcomes. Thus, the biphasic scaffold loaded with icariin and quercetin for sequential release could provide a promising solution for the restoration of bone defects and represent a potential strategy for bone regeneration.

Vascularization is a key issue facing the construction of functional three-dimensional (3D) tissues, which is critical for the long-term survival and stability of tissue construct transplantation. In this study, a photocurable hydrogel material carboxymethyl chitosan (CHIMA) was successfully prepared and integrated with methacryloyl gelatin (GelMA) to construct the bioink GelMA/CHIMA, which was subsequently used 3D printing technology to prepared a bioactive scaffold with angiogenesis-inducing functionality. The results showed that the cross-linked GelMA/CHIMA bioink had a porous structure that supported cell growth and metabolism. The incorporation of CHIMA could significantly improve the hydrophilicity, swelling rate, pressure resistance and mechanical strength of the bioink. GelMA/CHIMA bioink supported the survival and continued proliferation of human umbilical vein endothelial cells (HUVECs) in the scaffold. In particular, the bioink composed of 8 wt% GelMA and 2 wt% CHIMA could stimulate the expression of angiogenesis genes. 3D printed bioactive scaffolds supported the survival of HUVECs and had abundant protein deposition including CD31 and VEGF. Therefore, this study constructed a bioactive scaffold with angiogenesis induction function, which provides a feasible strategy for the construction of vascularized complex tissues.

Extremophilic yeasts have favorable metabolic and tolerance traits for biomanufacturing- like lipid biosynthesis, flavinogenesis, and halotolerance – yet the connection between these favorable phenotypes and strain genotype is not well understood. To this end, this study compares the phenotypes and gene expression patterns of biotechnologically relevant yeasts Yarrowia lipolytica, Debaryomyces hansenii, and Debaryomyces subglobosus grown under nitrogen starvation, iron starvation, and salt stress. To analyze the large data set across species and conditions, two approaches were used: a “network-first” approach where a generalized metabolic network serves as a scaffold for mapping genes and a “cluster-first” approach where unsupervised machine learning co-expression analysis clusters genes. Both approaches provide insight into strain behavior. The network-first approach corroborates that Yarrowia upregulates lipid biosynthesis during nitrogen starvation and provides new evidence that riboflavin overproduction in Debaryomyces yeasts is overflow metabolism that is routed to flavin cofactor production under salt stress. The cluster-first approach does not rely on annotation; therefore, the coexpression analysis can identify known and novel genes involved in stress responses, mainly transcription factors and transporters. Therefore, this work links the genotype to the phenotype of biotechnologically relevant yeasts and demonstrates the utility of complementary computational approaches to gain insight from transcriptomics data across species and conditions.

Measurement of antibody and antibody fusion protein concentration is vital for process development and manufacturing. Continuous, in-line monitoring of antibody concentration could be useful in a variety of applications, such as controlling the loading of protein A columns to prevent breakthrough, monitoring bioreactor titer, and detecting leaks past ultrafiltration/diafiltration membranes. Molecule-specific monitoring techniques are advantageous for antibody detection in cell culture fluid in the presence of complex process impurities. Here we report a continuous in-line, real-time IgG monitoring platform using a fiber-optic biosensor with a replaceable sensor tip covalently functionalized with a fluor-labeled protein consisting of a pentamer of the Z domain (a more stable form of the B domain) of protein A. The sensor demonstrates concentration-dependent fluorescence enhancement in the presence of human IgG (0.01–0.75 g/L), with consistent signals during five runs each with 1 and 0.1 g/L IgG, and maintains its specificity in the presence of Chinese hamster ovary (CHO) cell culture fluid. A 5% breakthrough of a typical 10 g/L load would be detected in less than 20 s in a flowing stream emerging from a protein A column without prior sample preparation. This sensor platform may be suitable for monitoring IgG and fragment, crystallizable (Fc) fusion proteins in diverse upstream and downstream bioprocess applications.

β-caryophyllene is a plant-derived sesquiterpene and is regarded as a promising ingredient for aviation fuels. Microalgae can convert CO₂ into energy-rich bioproducts through photosynthesis, making them potential platforms for the sustainable production of sesquiterpenes. However, heterologous sesquiterpene engineering in microalgae is still in its infancy, and β-caryophyllene production in eukaryotic photosynthetic microorganisms has not been reported. In this study, we succeeded in producing β-caryophyllene in the model eukaryotic microalga Chlamydomonas reinhardtii by heterologously expressing a β-caryophyllene synthase (QHS). Furthermore, overexpressing the key enzyme of the 2-C-methyl-D-erythritol 4-phosphate pathway in the QHS-expressing strain (QHS-DXS-HDR−18) resulted in a 17-fold higher β-caryophyllene production compared to the single expression of QHS (QHS−28). Additionally, when isopentenyl diphosphate isomerase (CrIDI) was overexpressed, the β-caryophyllene production was up to 480.6 μg/L in QHS-DXS-HDR-CrIDI−16 and increased by 1.8-fold compared to the parental strain QHS-DXS-HDR−18. Under photoautotrophic and photomixotrophic conditions in photobioreactors, the β-caryophyllene production in QHS-DXS-HDR-CrIDI−16 reached 854.7 and 1016.8 μg/L, respectively. Noticeably, all the β-caryophyllene-producing strains generated in this study did not exhibit adverse effects on cell growth and photosynthesis activity compared to the untransformed strain. This study demonstrates the first successful attempt to produce β-caryophyllene in the eukaryotic microalga C. reinhardtii and develops a novel strategy for increasing sesquiterpene production in eukaryotic photosynthetic microorganisms.