Renewable biosynthesis of isoprene from wastewater through a synthetic biology approach:the role of individual organic compounds

The biosynthesis of isoprene offers a more sustainable alternative to fossil fuel-based approaches,yet its success has been largely limited to pure organic compounds and the cost remains a challenge.This study proposes a waste-to-wealth strategy for isoprene biosynthesis utilizing genetically engineered E.coli bacteria to convert organic waste from real food wastewater.The impact of organic compounds present in wastewater on E.coli growth and isoprene production was systematically investigated.The results demonstrated that with filtration pretreatment of wastewater,isoprene yield,and production achieved 115 mg/g COD and 7.1 mg/(L·h),respectively.Moreover,even without pretreatment,isoprene yield only decreased by~24%,indicating promising scalability.Glucose,maltose,glycerol,and lactate are effective substrates for isoprene biosynthesis,whereas starch,protein,and acetate do not support E.coli growth.The optimum C/N ratio for isoprene production was found to be 8:1.Furthermore,augmenting essential nutrients in wastewater elevated the isoprene yield increased to 159 mg/g COD.The wastewater biosynthesis significantly reduced the cost(44%–53%decrease,p-value<0.01)and CO_(2)emission(46%–55%decrease,p-value<0.01)compared with both sugar fermentation and fossil fuel–based refining.This study introduced a more sustainable and economically viable approach to isoprene synthesis,offering an avenue for resource recovery from wastewater.

Recent advances in polymeric biomaterials-based gene delivery for cartilage repair

Untreated articular cartilage damage normally results in osteoarthritis and even disability that affects millions of people.However,both the existing surgical treatment and tissue engineering approaches are unable to regenerate the original structures of articular cartilage durably,and new strategies for integrative cartilage repair are needed.Gene therapy provides local production of therapeutic factors,especially guided by biomaterials can minimize the diffusion and loss of the genes or gene complexes,achieve accurate spatiotemporally release of gene products,thus provideing long-term treatment for cartilage repair.The widespread application of gene therapy requires the development of safe and effective gene delivery vectors and supportive gene-activated matrices.Among them,polymeric biomaterials are particularly attractive due to their tunable physiochemical properties,as well as excellent adaptive performance.This paper reviews the recent advances in polymeric biomaterial-guided gene delivery for cartilage repair,with an emphasis on the important role of polymeric biomaterials in delivery systems.

A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting in Pichia pastoris

The methylotrophic budding yeast Pichia pastoris has been utilized to the production of a variety of heterologous recombinant proteins owing to the strong inducible alcohol oxidase promoter(pAOX1).However,it is difficult to use P.pastoris as the chassis cell factory for high-valuable metabolite biosynthesis due to the low homologous recombination(HR)efficiency and the limitation of handy selective markers,especially in the condition of multistep biosynthetic pathways.Hence,we developed a novel CRISPR/Cas9 system with highly editing efficiencies and recyclable auxotrophic selective marker(HiEE-ReSM)to facilitate cell factory in P.pastoris.Firstly,we improved the HR rates of P.pastoris through knocking out the non-homologous-end-joining gene(Δku70)and overexpressing HR-related proteins(RAD52 and RAD59),resulting in higher positive rate compared to the basal strain,achieved 97%.Then,we used the uracil biosynthetic genes PpURA3 as the reverse screening marker,which can improve the recycling efficiency of marker.Meanwhile,the HR rate is still 100%in uracil auxotrophic yeast.Specially,we improved the growth rate of uracil auxotrophic yeast strains by overexpressing the uracil transporter(scFUR4)to increase the uptake of exogenous uracil from medium.Meanwhile,we explored the optimal concentration of uracil(90 mg/L)for strain growth.In the end,the HiEE-ReSM system has been applied for the inositol production(250 mg/L)derived from methanol in P.pastoris.The systems will contribute to P.pastoris as an attractive cell factory for the complex compound biosynthesis through multistep metabolic pathway engineering and will be a useful tool to improve one carbon(C1)bio-utilization.

Engineering the next generation of theranostic biomaterials with synthetic biology

Biomaterials have evolved from inert materials to responsive entities,playing a crucial role in disease diagnosis,treatment,and modeling.However,their advancement is hindered by limitations in chemical and mechanical approaches.Synthetic biology enabling the genetically reprograming of biological systems offers a new paradigm.It has achieved remarkable progresses in cell reprogramming,engineering designer cells for diverse applications.Synthetic biology also encompasses cell-free systems and rational design of biological molecules.This review focuses on the application of synthetic biology in theranostics,which boost rapid development of advanced biomaterials.We introduce key fundamental concepts of synthetic biology and highlight frontier applications thereof,aiming to explore the intersection of synthetic biology and biomaterials.This integration holds tremendous promise for advancing biomaterial engineering with programable complex functions.

Injectable ECM hydrogel for delivery of BMSCs enabled full-thickness meniscus repair in an orthotopic rat model

Meniscal injuries have poor intrinsic healing capability and are associated with the development of osteoarthritis.Decellularized meniscus extracellular matrix(mECM)has been suggested to be efficacious for the repair of meniscus defect.However,main efforts to date have been focused on the concentration,crosslinking density and anatomical region dependence of the mECM hydrogels on regulation of proliferation and differentiation of adult mesenchymal stem cells(MSCs)in vitro 2D or 3D culture.A systematic investigation and understanding of the effect of mECM on encapsulated MSCs response and integrative meniscus repair by in vivo rat subcutaneous implantation and orthotopic meniscus injury model will be highly valuable to explore its potential for clinical translation.In this study,we investigated the in situ delivery of rat BMSCs in an injectable mECM hydrogel to a meniscal defect in a SD rat model.Decellularized mECM retained essential proteoglycans and collagens,and significantly upregulated expression of fibrochondrogenic markers by BMSCs versus collagen hydrogel alone in vitro 3D cell culture.When applied to an orthotopic model of meniscal injury in SD rat,mECM is superior than collagen I scaffold in reduction of osteophyte formation and prevention of joint space narrowing and osteoarthritis development as evidenced by histology and micro-CT analysis.Taken together,these results indicate mECM hydrogel is a highly promising carrier to deliver MSCs for long-term repair of meniscus tissue,while preventing the development of osteoarthritis.

Probing the growth and mechanical properties of Bacillus subtilis biofilms through genetic mutation strategies

Bacterial communities form biofilms on various surfaces by synthesizing a cohesive and protective extracellular matrix,and these biofilms protect microorganisms against harsh environmental conditions.Bacillus subtilis is a widely used experimental species,and its biofilms are used as representative models of beneficial biofilms.Specifically,B.subtilis biofilms are known to be rich in extracellular polymeric substances(EPS)and other biopolymers such as DNA and proteins like the amyloid protein TasA and the hydrophobic protein BslA.These materials,which form an interconnected,cohesive,three-dimensional polymer network,provide the mechanical stability of biofilms and mediate their adherence to surfaces among other functional contributions.Here,we explored how genetically-encoded components specifically contribute to regulate the growth status,mechanical properties,and antibiotic resistance of B.subtilis biofilms,thereby establishing a solid empirical basis for understanding how various genetic engineering efforts are likely to affect the structure and function of biofilms.We noted discrete contributions to biofilm morphology,mechanical properties,and survival from major biofilm components such as EPS,TasA and BslA.For example,EPS plays an important role in maintaining the stability of the mechanical properties and the antibiotic resistance of biofilms,whereas BslA has a significant impact on the resolution that can be obtained for printing applications.This work provides a deeper understanding of the internal interactions of biofilm components through systematic genetic manipulations.It thus not only broadens the application prospects of beneficial biofilms,but also serves as the basis of future strategies for targeting and effectively removing harmful biofilms.