With the developments in metabolic engineering and the emergence of synthetic biology,many breakthroughs in medicinal,biological and chemical products as well as biofuels have been achieved in recent decades.As an important barrier to traditional metabolic engineering,however,the identification of ratelimiting step(s)for the improvement of specific cellular functions is often difficult.Meanwhile,in the case of synthetic biology,more and more BioBricks could be constructed for targeted purposes,but the optimized assembly or engineering of these components for high-efficiency cell factories is still a challenge.Owing to the lack of steady-state kinetic data for overall flux,balancing many multistep biosynthetic pathways is time-consuming and needs vast resources of labor and materials.A strategy called targeted engineering is proposed in an effort to solve this problem.Briefly,a targeted biosynthetic pathway is to be reconstituted in vitro and then the contribution of cofactors,substrates and each enzyme will be analyzed systematically.Next is in vivo engineering or de novo pathway assembly with the guidance of information gained from in vitro assays.To demonstrate its practical application,biosynthesis pathways for the production of important products,e.g.chemicals,nutraceuticals and drug precursors,have been engineered in Escherichia coli and Saccharomyces cerevisiae.These cases can be regarded as concept proofs indicating targeted engineering might help to create high-efficiency cell factories based upon constructed biological components.
作为一种快速高效的体外蛋白合成手段,无细胞蛋白表达体系(Cell-free Protein Synthesis,CFPS)一直以来就被广泛应用于基础生物学领域的研究。与传统的基于细胞的体内表达体系相比,CFPS突破了细胞的生理限制,其可调控性强、对毒性蛋白的耐受力高,使得许多很难在体内合成的复杂蛋白在体外顺利表达。近年来随着研究人员不断对CFPS进行优化,通过简化制备工艺、开发价格低廉的能量再生系统、稳定底物供应、促进蛋白正确折叠等方式,成功研发出生产效率高、成本低廉、反应体积大的表达体系。凭借其高通量和大规模的蛋白表达优势,CFPS为解决生物制药领域中面临的难题提供了新的解决思路,并成功地应用于高通量药物筛选、大规模生产重组蛋白药物、个体化定制肿瘤疫苗等领域,显示出其在生物制药领域的重要应用潜力。
Cell-free synthetic biology system organizes multiple enzymes(parts)from different sources to implement unnatural catalytic functions.Highly adaption between the catalytic parts is crucial for building up efficient artificial biosynthetic systems.Protein engineering is a powerful technology to tailor various enzymatic properties including catalytic efficiency,substrate specificity,temperature adaptation and even achieve new catalytic functions.However,altering enzymatic pH optimum still remains a challenging task.In this study,we proposed a novel sequence homolog-based protein engineering strategy for shifting the enzymatic pH optimum based on statistical analyses of sequence-function relationship data of enzyme family.By two statistical procedures,artificial neural networks(ANNs)and least absolute shrinkage and selection operator(Lasso),five amino acids in GH11 xylanase family were identified to be related to the evolution of enzymatic pH optimum.Site-directed mutagenesis of a thermophilic xylanase from Caldicellulosiruptor bescii revealed that four out of five mutations could alter the enzymatic pH optima toward acidic condition without compromising the catalytic activity and thermostability.Combination of the positive mutants resulted in the best mutant M31 that decreased its pH optimum for 1.5 units and showed increased catalytic activity at pH<5.0 compared to the wild-type enzyme.Structure analysis revealed that all the mutations are distant from the active center,which may be difficult to be identified by conventional rational design strategy.Interestingly,the four mutation sites are clustered at a certain region of the enzyme,suggesting a potential“hot zone”for regulating the pH optima of xylanases.This study provides an efficient method of modulating enzymatic pH optima based on statistical sequence analyses,which can facilitate the design and optimization of suitable catalytic parts for the construction of complicated cell-free synthetic biology systems.
Fuqiang MaYuan XieManjie LuoShuhao WangYou HuYukun LiuYan FengGuang-Yu Yang
Microbial-derived natural products are important in both the pharmaceutical industry and academic research.As the metabolic potential of original producer especially Streptomyces is often limited by slow growth rate,complicated cultivation profile,and unfeasible genetic manipulation,so exploring a Streptomyces as a super industrial chassis is valuable and urgent.Streptomyces sp.FR-008 is a fast-growing microorganism and can also produce a considerable amount of macrolide candicidin via modular polyketide synthase.In this study,we evaluated Streptomyces sp.FR-008 as a potential industrial-production chassis.First,PacBio sequencing and transcriptome analyses indicated that the Streptomyces sp.FR-008 genome size is 7.26 Mb,which represents one of the smallest of currently sequenced Streptomyces genomes.In addition,we simplified the conjugation procedure without heat-shock and pre-germination treatments but with high conjugation efficiency,suggesting it is inherently capable of accepting heterologous DNA.In addition,a series of promoters selected from literatures was assessed based on GusA activity in Streptomyces sp.FR-008.Compared with the common used promoter ermE*-p,the strength of these promoters comprise a library with a constitutive range of 60e860%,thus providing the useful regulatory elements for future genetic engineering purpose.In order to minimum the genome,we also target deleted three endogenous polyketide synthase(PKS)gene clusters to generate a mutant LQ3.LQ3 is thus an“updated”version of Streptomyces sp.FR-008,producing fewer secondary metabolites profiles than Streptomyces sp.FR-008.We believe this work could facilitate further development of Streptomyces sp.FR-008 for use in biotechnological applications.
Qian LiuLiping XiaoYuanjie ZhouKunhua DengGaoyi TanYichao HanXinhua LiuZixin DengTiangang Liu
