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This article reviews the review articles and research papers related to biomanufacturing driven by engineered organisms published in the Chinese Journal of Biotechnology from 2023 to 2024. The content covers 26 aspects, including chassis cells; gene (genome) editing; facilities, tools and methods; biosensors; protein design and engineering; peptides and proteins; screening, expression, characterization and modification of enzymes; biocatalysis; bioactive substances; plant natural products; microbial natural products; development of microbial resources and biopesticides; steroidal compounds; amino acids and their derivatives; vitamins and their derivatives; nucleosides; sugars, sugar alcohols, oligosaccharides, polysaccharides and glycolipids; organic acids and monomers of bio-based materials; biodegradation of polymeric materials and biodegradable materials; intestinal microorganisms, live bacterial drugs and synthetic microbiomes; microbial stress resistance engineering; biodegradation and conversion utilization of lignocellulose; C1 biotechnology; bioelectron transfer and biooxidation-reduction; biotechnological environmental protection; risks and regulation of biomanufacturing driven by engineered organisms, with hundreds of technologies and products commented. It is expected to provide a reference for readers to understand the latest progress in research, development and commercialization related to biomanufacturing driven by engineered organisms.

Proteins with specific functions and characteristics play a crucial role in biomedicine and nanotechnology. De novo protein design enables the customization of sequences to produce proteins with desired structures that do not exist in the nature. In recent years, with the rapid development of artificial intelligence (AI), deep learning-based generative models have increasingly become powerful tools, enabling the design of functional proteins with atomic-level precision. This article provides an overview of the evolution of de novo protein design, with focus on the latest algorithmic models, and then analyzes existing challenges such as low design success rates, insufficient accuracy, and dependence on experimental validation. Furthermore, this article discusses the future trends in protein design, aiming to provide insights for researchers and practitioners in this field.

With the development of antibody engineering, a diverse range of structures for T cell-dependent bispecific antibodies targeting CD3 have been devised to meet the needs of various clinical therapies. T cell-dependent bispecific antibodies can be categorized into IgG-like and non-IgG-like formats based on the presence or absence of an Fc domain. The most common difficulty in manufacturing bispecific antibodies is the mispairing between light and heavy chains. To overcome this challenge, several techniques, such as knob-into-hole and CrossMab technologies, have been implemented for structural design. Given that the mechanism of action of T cell-dependent bispecific antibodies is to redirect T cells to tumor cells to induce a killing effect, abolishment of Fc domain effector function, the affinity design of the target, and the distance design of immune synapse formation are all focal points in the structural design of T cell-dependent bispecific antibodies. This review aims to provide an overview of recent advances in the structural design of T cell-dependent bispecific antibodies.

Natural enzymes are often difficult to meet the needs of application and research in terms of activity, enantiomer selectivity or thermal stability. Therefore, it is an important task of enzyme engineering to explore efficient molecular modification technologies to improve the properties of such enzymes. The molecular modification technologies of enzymes mainly include rational design, directed evolution, and artificial intelligence-assisted design. Directed evolution and rational design are experiment-driven molecular modification approaches of enzymes and have been successfully applied to enzyme engineering. However, due to the huge space sizes of protein sequences and the lack of experimental data, the current modification methods still face major challenges. With the development of next-generation sequencing, high-throughput screening, protein databases, and artificial intelligence (AI), data-driven enzyme engineering is emerging as a promising solution to these challenges. The AI-assisted statistical learning method has been used to establish a model for predicting the sequence/structure-properties of enzymes in a data-driven manner. Excellent mutant enzymes can be selected according to the prediction results, which greatly improve the efficiency of molecular modification. Considering the application requirements of molecular modification of enzymes, this paper reviews the data acquisition methods and application examples of AI-assisted molecular modification of enzymes, with focuses on the convolutional neural network method for predicting protein thermostability, aiming to provide reference for researchers in this field.

Directed evolution was conducted to improve the thermostability of lipase from Rhizopus chinensis CCTCC M201021. Mutations were introduced by two rounds of error-prone PCR and mutant lipase was selected by fast-blue RR top agar screening. Two positive variants were selected in the first-round and four in the second-round screening process. Ep2-4 was proved as the most thermostable lipase and its DNA sequencing revealed three amino acid substitutions: A129S, P168L and V329A. Compared with the parent, its half-life at 60 degrees C was 5.4- times longer and T50 was 7.8 degrees higher. Purified lipase of Ep2-4 was characterized and the result shows that its thermostability improved without compromising enzyme activity. According to the mimicked protein structure, mutation A129S formed a hydrogen bond with Gln133 and improved the thermostability by increasing the hydrophilicity and polarity of protein; mutation P168L by forming a hydrophobic bond with the nearby Leu164.

It has become a hotspot and keystone in gene engineering and bioengineering to produce recombinant proteins through heterologous expression systems. Unfortunately, not all the genes could be successfully and effectively expressed in heterologous hosts. The role of gene itself in regulating translation process through its intrinsic sequence characteristics such as codon bias, codon pair bias, GC content, mRNA secondary structure and mRNA stability, has been gradually elucidated. Here we review these factors that influence the translation processes and their corresponding optimization methods in the process of gene design. We emphatically discussed codon bias and codon pair bias and their optimization methods. In particular, the latest theories of codon harmonization and codon pair harmonization were discussed and compared with the traditional codon and codon pair optimization strategies in gene design.

Objective: To reveal the correlation between thermostability of xylanase EvXyn11(TS) and its N-terminal disulfide bridge, an EvXyn11(TS)-encoding gene (Syxyn11) was synthesized and subjected to site-directed mutagenesis., Methods: Multiple homology alignment of protein primary structures between the EvXyn11(TS) and several GH family 11 xylanases displayed that, in their N-termini, only EvXyn11(TS) contained a disulfide bridge (Cys5-Cys32), whose effect on the xylanase thermostability was predicted by molecular dynamics simulation. We constructed a gene Syxyn11(M), encoding the mutated xylanase (EvXyn11(M)) without N-terminal disulfide bridge. Then, Syxyn11 and Syxyn11(M) were expressed in Pichia pastoris GS115, and temperature and pH properties of the expressed enzymes were analyzed., Results: The analytical results displayed that the temperature optimum of EvXyn11(M) was 70 degrees C, which was 15 degrees C lower than that of EvXyn11(TS). The half-life (t1/2(90)) of EvXyn11(TS) at 90 degrees C was 32 min, while the t1/2(70) of EvXyn11(M) at 70 degrees C was only 8.0 min., Conclusion: The important role of the N-terminal disulfide bridge on the thermostability of EvXyn11(TS) was first predicted by molecular dynamics simulation, and confirmed by site-directed mutagenesis. This work provided a novel strategy to improve thermostabilities of the mesophilic family 11 xylanases with high specific activities.

Artificial designer nucleases targeting specific DNA sequences open up a new field for reverse genetics study. The rapid development of engineered endonucleases (EENs) enables targeted genome modification theoretically in any species. The construction of transcription activator-like effector nucleases (TALENs) is simpler with higher specificity and less toxicity than zinc-finger nucleases (ZFNs). Here, we summarized the recent progresses and prospects of TALEN technology, with an emphasis on its structure, function, and construction strategies, as well as a collection of species and genes that have been successfully modified by TALENs, especially the application in zebrafish.

TALEN (Transcription activator-like effector nuclease) is a newly developed family of artificially engineered sequence-specific endonucleases, which consists of a highly specific and repetitive DNA-binding domain derived from TALE (Transcription activator-like effector) and fused with the non-specific endonuclease domain of FokⅠ. TALENs have been reported to be able to induce site specific genome modification in quite a few species and in vitro cultured cells. Here, we introduced the principles for target site selection and confirmation and described a brief experimental protocol for the easy construction of customized TALENs using unit assembly (UA) method and also for generation of and screening for TALEN-mediated targeted zebrafish mutants through microinjection of TALEN mRNAs into zebrafish embryos. Theoretically, the principles and methods we described here are also applicable to gene targeting in other species.

Molecular engineering of cellulases can improve enzymatic activity and efficiency. Recently, the Carbohydrate-Active enZYmes Database (CAZy), including glycoside hydrolase (GH) families, has been established with the development of Omics and structural measurement technologies. Molecular engineering based on GH families can obviously decrease the probing space of target sequences and structures, and increase the odds of experimental success. Besides, the study of cellulase active-site architecture paves the way toward the explanation of catalytic mechanism. This review focuses on the main GH families and the latest progresses in molecular engineering of catalytic domain. Based on the combination of analysis of a large amount of data in the same GH family and their conservative active-site architecture information, rational design will be an important direction for molecular engineering and promote the rapid development of the conversion of biomass.

A mesophilic xylanase from Aspergillus oryzae, abbreviated to AoXyn11A, belongs to glycoside hydrolase family 11. Using AoXyn11A as the parent, the thermotolerant hybrid xylanase, we constructed AEx11A by substituting its N-terminus with the corresponding region of a hyperthermostable family 11 xylanase, EvXyn11(TS). AoXyn11A- and AEx11A-encoding genes were expressed in Pichia pastoris GS115 separately, and effects of temperatures on expressed products were determined and compared. The optimum temperature (T(opt)) of AEx11A was 75 degrees C and its half-life at 70 degrees C (t1/2(70)) was 197 min, improved as compared with those (T(opt) = 50 degrees C, t1/2(70) = 1.0 min) of AoXyn11A. Homology modeling of the AEx11A's structure and comparison between structures of AEx11A and AoXyn11A revealed that one disulfide bridge (Cys5-Cys32) was introduced into AEx11A resulted from N-terminus substitution. To explore the effect of the disulfide bridge on the thermostability of AEx11A, it was removed from AEx11A by site-directed mutagenesis (C5T). Analytical results show that the T(opt) of the mutant AEx11A (AEx11A(C5T)) dropped to 60 degrees C from 75 degrees C of AEx11A, and its t1/2(70) and t1/2(80) also decreased to 3.0 and 1.0 min from 197 and 25 min.

Aromatic L-Amino acids are important chiral building blocks for the synthesis of many drugs, pesticides, fine chemicals and food additives. Due to the high activity and steroselectivity, enzymatic synthesis of chiral building blocks has become the main research direction in asymmetric synthesis field. Guided by the phylogenetic analysis of transaminases from different sources, two representative aromatic transaminases TyrB and Aro8 in type I subfamily, from the prokaryote Escherichia coli and eukaryote Saccharomyces cerevisia, respectively, were applied for the comparative study of asymmetric transamination reaction process and catalytic efficiency of reversely converting keto acids to the corresponding aromatic L-amino acid. Both TyrB and Aro8 could efficiently synthesize the natural aromatic amino acids phenylalanine and tyrosine as well as non-natural amino acid phenylglycine. The chiral HPLC analysis showed the produced amino acids were L-configuration and the e.e value was 100%. L-alanine was the optimal amino donor, and the transaminase TyrB and Aro8 could not use D-amino acids as amino donor. The optimal molar ratio of amino donor (L-alanine) and amino acceptor (aromatic alpha-keto acids) was 4:1. Both of the substituted group on the aromatic ring and the length of fatty acid carbon chain part in the molecular structure of aromatic substrate alpha-keto acid have the significant impact on the enzyme-catalyzed transamination efficiency. In the experiments of preparative-scale transamination synthesis of L-phenylglycine, L-phenylalanine and L-tyrosine, the specific production rate catalyzed by TryB were 0.28 g/(g x h), 0.31 g/(g x h) and 0.60 g/(g x h) and the specific production rate catalyzed by Aro8 were 0.61 g/(g x h), 0.48 g/(g x h) and 0.59 g/(g x h). The results obtained here were useful for applying the transaminases to asymmetric synthesis of L-amino acids by reversing the reaction balance in industry.

As an efficient and promising protein engineering strategy, directed evolution includes the construction of mutant libraries and screening of desirable mutants. A rapid and high-throughput screening method has played a critical role in the successful application of directed evolution strategy. We reviewed several high-throughput screening tools which have great potential to be applied in directed evolution. The development of powerful high-throughput screening tools will make great contributions to the advancement of protein engineering.

Directed evolution was used to improve the performance of beta-1,3-1,4-glucanase (designated as PtLicl6A) from Paecilomyces thermophila J18 under acidic condition. A mutant library was constructed by error-prone PCR and DNA shuffling, and positive clones were screened by Congo red staining. More than 1 500 mutants were selected. One mutant (PtLic16AM1) exhibited an optimal activity at pH 5.5, while the optimal pH of the wild-type enzyme was 7.0. The mutant PtLic16AM1 kept the high specific activity and thermotolerence of the wild-type enzyme. Sequence analysis revealed that the mutant enzyme has four sense substitutions which caused four amino acid substitutions - namely T58S, Y110N, G195E and D221G.. Homology modeling showed that among the four amino acid substitutions, Y110N was near the active site of the enzyme, while the other three was distant. T58S and G195E may play key roles in the change of optimal pH. This study provided a new perspective of obtaining applicable 3-1,3-1,4-glucanase for industrial use.

Microbial transglutaminase, which could catalyze the cross-linking of many proteins or non-protein materials, has been widely used in food, pharmaceutical and textile industry. To enhance the yield of the enzyme and establish corresponding platform for molecular modification, the researchers of Japanese Ajinomoto began to construct the recombinant strain producing transglutaminase in the 1990s. So far, the enzyme has been successfully expressed in different expression systems. Some of the recombinant strains are more productive than wild strains. Recently, progress has been made in the molecular modification of microbial transglutaminase, and the activity, thermo-stability and specificity of the enzyme are improved. This review briefly summarized and analyzed the strategies involved in these studies, and noted its trends.

Aim: To construct recombinant co-expression adenovirus vector of SEA and CD80 genes regulated by mouse TERT(telomerase reverse transcriptase, TERT) promoter and to observe the expression of SEA and CD80 in the Hepa1-6 cells mediated by it., Methods: Using AdEasy adenovirus system, the core promoter region of mTERT was subcloned to shuttle plasmid pShuttle2 and Myc-Max response element was inserted upstream of it to regulate the expression of SEA and CD80. The recombinant co-expression adenovirus vector of SEA and CD80 genes was constructed and named as Ad-MMRE-mTERT-BIS. Hepatoma cell line Hepa1-6 and fibroblast cell line NIH3T3 were infected by recombinant adenovirus at MOI(multiplicity of infection)of 100, the expression of SEA and CD80 on the surface of cells was detected by indirect immunofluorescent staining., Results: SEA and CD80 was specifically co-expressed on the surface of infected Hepa1-6 cells but not on NIH3T3 cells., Conclusion: The recombinant co-expression adenovirus vector of SEA and CD80 gene regulated by mTERT promoter was sucessfully constructed and make targeting-expression of SEA and CD80 on the surface of hepatoma cells, which lays the foundation for further research on application of SEA and CD80 in targeted genetherapy for hepatoma.

Aim: To amplify the SSU1664 gene from Streptococcus suis serotype 2 strain 05ZY, to express the gene in E.coli, and to evaluate the activities of the recombinant protein., Methods: SSU1664 gene was amplified by PCR using primers according to 05ZY genome sequences and cloned into the expression vector. The recombinant protein was purified by affinity chromatography and its immunogen activities were tested by Western blot and ELISA., Results: SSU1664 gene could solublely express in E.coli BL21(DE3). Western blot analysis showed that the recombinant protein could react with rat serum immunized with Streptococcus suis, but not with non-immunized rat serum. ELISA assay showed that anti-SSU1664 IgM content in Streptococcus suis-infected patient was significantly higher than that in healthy donors., Conclusion: The recombinant SSU1664 protein has immunogen activity and might be one promising Streptococcus suis vaccine candidate and diagnosis marker of Streptococcus suis early infection.

Aim: Design and synthesis complementary DNA sequences of 3 pairs of short hairpin structure and a pair of negative control sequence by RNA interference technique and construct and identify a lentiviral interference vector with human BIRC5 gene as target gene., Methods: The designed and synthesised Single-Stranded primer were annealed to Double-Stranded oligo sequences and subcloned into linear pMAGic lentiviral plasmid vector digested by enzyme Age I and EcoR I. Screening positive clone after transformed into DH5α competent cells and identified by PCR amplification and DNA sequencing., Results: 335 bp straps of positive clone and 298 bp straps of negative clone form PCR amplification production have been obtained after gel electrophoresis, the designed and synthesised sequences have been contained in these clone straps confirmed by the result of DNA sequencing., Conclusion: Four pairs of BIRC5 shRNA recombinant lentiviral expression vector were constructed successfully, which laid the foundation for researching the inhibition of BIRC5 siRNA target against tumor cells proliferation, induction apoptosis and gene therapy.

Aim: To construct the eukaryotic expression vector of human CXCR4 and CCR5, transfect SKOV3 cells with them., Methods: The cDNA of CXCR4 and CCR5 was amplified by RT-PCR. After purification, the gene was cloned into a vector pEGFP. The sequence of inserted CXCR4 and CCR5 gene fragment was identified by enzyme digestion of EcoR I/Sal I and sequencing, and then the recombinant plasmid was transfected into SKOV3 cells which did not express CXCR4 and CCR5 by lipofectamine-mediated gene transfection method. The SKOV3 cells transfected with CXCR4 and CCR5 were examined by FCM., Results: Vectors pEGFP-CXCR4 and pEGFP-CCR5 were obtained. Cell clones which were screened by G418 were obtained. The results of FCM indicatedthat transfected SKOV3 cells expressed CXCR4 and CCR5., Conclusion: SKOV3 cells that can express CXCR4 and CCR5 protein stably have been established successfully, which facilitates the researches of epithelial ovarian.

Neuregulin-1 (NRG1), now in a phase II clinical trial, has beneficial effects on heart failure patients through the activation of ErbB2/ErbB4 receptor pair. To decrease the side effect of NRG1 on activating ErbB3, a mutation screen was carried out to get NRG1 mutants, which have more specific binding to ErbB2/ErbB4 receptor pair. Two CHO stable cell lines were constructed, which express ErbB2/ErbB3 or ErbB2/ErbB4 receptor pair, respectively. The ErbB2/ErbB4 cell line showed similar characteristics in ligand-binding activity and the activation of downstream signaling molecules, such as the AKT and PI3K to the primary neonatal rat ventricular myocytes (NRVM), which endogenously expresses ErbB2/ErbB4. Both cell lines have good dose-response. Thirty-one NRG1 mutants were successfully expressed in Escherichia coli and purified. Their ability to stimulate the downstream signaling was measured by detecting AKT phosphorylation. Some mutants showed more specific activation activity in ErbB2/ErbB4 cells. Further study on five of these mutants demonstrated that the change of the activation activity is associated with that of their binding activities to ErbB2/ErbB4 and ErbB2/ErbB3. Four of the candidates are more specific ligands for ErbB2/ErbB4 activation, and thus may serve as more potent drug candidates for heart failure.

We engineered a disulphide bridge between two adjacent double-layered beta-sheet at the N-terminal region of Trichoderma reesei endo-1,4-beta-xylanase II(XYN II) by site-directed mutagenesis. The native xylanase XYN-OU and the mutated xylanase XYN-HA12 (T2C, T28C and S156F) were separately expressed in Pichia pastoris. Both xylanases were purified and characterized. The optimum temperature of XYN-HA12 was increased from 50 degrees C to 60 degrees C, relative to XYN-OU. At 70 degrees C, the halftime of inactivation for XYN-OU and XYN-HA12 were 1 min and 14 min, respectively. The optimum pH of XYN-HA12 was 5.0, similar to XYN-OU. However, XYN-HA12 could retain over 50% activity from pH 3.0 to 10.0 at 50 degrees C for 30 min. As for XYN-OU, it could retain over 50% activity from the pH value 4.0 to 9.0 at 50 degrees C in 30 min. The result of the mutated xylanase indicated that constructed disulphide bridge could improve its thermostability at relatively higher temperature.

3-ketosteroid-9alpha-hydroxylase (KSH), a key enzyme in the microbial steroid degradation, is highly valuable for the production of some steroid drugs. Degenerate primers were designed by comparing the ksh from Rhodococcus erythropolis SQ1 and its homologous sequences in the reported genome of Mycobacteria. Subsequently, a gene fragment of KSH was cloned from Mycobacterium sp. NwIB-01, a sterol-transforming bacterium isolated from soil in our lab. According to the conservative sequence, the full-length 1188 bp gene encoding ksh (designated as M.S.-ksh) was obtained by chromosome walking, which showed 85% identity with the ksh of M. smegmatis mc(2)155. The heterologous expression of KSH was achieved in Escherichia coli BL21(DE3) using the pET-32a-c(+) vector system. The expressed KSH protein was mostly in soluble form after IPTG induction at 30 degreesC and accounted for more than 30% of total bacterial proteins according to SDS-PAGE electrophoresis. The molecular mass of KSH was about 45 kD, which was exactly the size predicted. After Ni2+ affinity chromatography, the purity of the target protein was more than 90%. Our work will definitely contribute to the industrial production of some steroid drugs by developing KSH genetically engineered bacteria.

Wild-type biocatalysts usually show high activity and selectivity towards their native substrates. Since non-native substrates are often used in synthetically useful biocatalytic transformations, it is necessary to engineer enzymes for improved activity, stability and selectivity (chemo-, regio- and stereoselectivity). Herein we give an overview of the recent advances in engineering the enantioselectivity of biocatalysts, with an aim to stimulate further development of this important field in China.

Aim: To construct eukaryotic expression plasmids encoding Sox2 and Sox2 K247R and identify their expression and SUMOylation., Methods: With gift plasmid encoding Sox2 gene as a template, Sox2 K247R was obtained by overlapping extension PCR, followed by construction of pCMV-HA-Sox2 and pCMV-HA-Sox2 K247R. After enzyme digestion analysis and DNA sequencing, these two constructs were transfected or co-transfected with pCMV-Myc-SUMO1 into 293FT cells by lipofectin method. Western blot was employed to analyze expression and SUMO ylation of Sox2., Results: It was revealed that eukaryotic expression vectors were constructed with correct sequence, where in mutant Sox2, the AAG codon was switched to CGG codon. Western blot results showed that good expression of both wt and mut Sox2, of which the latter could not be modified by SUMO1., Conclusion: Successful construction and expression of Sox2 and Sox2 K247R. Sox2 could be SUMO lyated in vitro but Sox2 K247R not.

Plant can be used as bioreactor for heterogenous protein expression. We reviewed different expression systems of plant bioreactor as well as recent relevant developments. In addition, we discussed perspectives in combination with our own experience.

Based on carbon metabolic pathway analysis of Escherichia coli MG1655, an aerobic succinate fermentation platform was constructed by knocking out five genes (ptsG, poxB, pta, iclR and sdhA), which was named E. coli QZ1111. Flask cultivation results showed that E. coli QZ1111 could accumulate succinate with a concentration of 26.4 g/L under aerobic conditions. The byproduct acetate was only 2.3 g/L. The production ratio of succinate and acetate reached 11.5:1.

Objective: To overcome coenzyme restriction in the asymmetric reduction at high substrate concentration, we constructed a recombinant Escherichia coli with (R)-sepcificity carbonyl reductase coupled with formate dehydrogenase for cofactor regeneration., Methods: The R-carbonyl reductase gene (rcr) and formate dehydrogenase gene (fdh) were amplified from Candida parapsilosis and Candida boidinii genomes by PCR technique respectively. Then the purified PCR products were inserted into a co-expression vector pETDuet-1 to construct plasmid pETDuet-rcr-fdh. The positive plasmid was transformed into codon optimized E. coli Rosetta, and a recombinant strain E. coli Rosetta/pETDuet-rcr-fdh was obtained., Results: SDS-PAGE analysis showed that two enzymes were expressed simultaneously. Isopropyl-BETA-D-thiogalactopyranoside (1 mmol/L) induced at 30 degrees C the expression of both proteins encoded by rcr and fdh genes with the molecular weights of 37 kDa and 40 kDa. The biotransformation experiments were done using 2-hydroxyacetophenone and sodium formate as substrates. When the concentration of 2-hydroxyacetophenone was 6 g/L, the (R)-1-phenyl-1,2-ethanediol was produced with the high optical purity of 100% enantiomeric excess and a yield of 85.9%. Compared with the recombinant strain E. coli Rosetta/pETDuet-rcr withoutfdh gene for cofactor regeneration, the optical purity and yield of product from the asymmetric reduction of 2-hydroxyacetophenone by E. coli Rosetta/pETDuet-rcr-fdh were increased by 1.3 and 2.7 times, respectively., Conclusion: This method supplied a foundation for biosynthesis of (R)-1-phenyl-1,2-ethanediol for the cofactor regeneration by genetic engineering.

We first cloned the dsz operon of Pseudomonas delafieldii R-8 into the expressing plasmid (pPR9TT) to construct the recombinant plasmid pPR-dsz, and then reintroduced it into strain R-8 to obtain a muti-copy dsz operon engineering strain R-8-1. Compared with the wild-type, strain R-8-1 showed a higher desulfurization activity for dibenzothiophene (DBT). Initial rates of DBT removal by strain R-8-1 were 6.25 micromol/g dry cell/h, about 2-fold higher than that for wild-type strain. The recombinant cells were also applied in the desulfurization of diesel. It resulted in a 68% reduction of total sulfur from 310.8 mg/L to 100.1 mg/L, whereas only 53% of sulfur was removed by strain R-8. The stability of pPR-dsz in strain R-8-1 was studied. The results revealed the first obtain a muti-copy dsz operon engineering strain are helpful for further development in biodesulfurization.

Objective: 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (EC 2.5.1.54;DS) is the key enzyme in tryptophan synthesis pathway. Cloning DS I gene from Corynebacterium pekinense and expression of DS I gene might facilitate testing the existence and function of DS I in Corynebacterium pekinense., Methods: According to the homology between Corynebacterium glutamicum ATCC13032 and Corynebacterium pekinense, we designed a pair of PCR primers to clone the DS I gene from wild-type C. pekinense AS1.299 and its mutant PD-67, then the mutant DS I gene was expressed in C. pekinense PD-67 by subcloning the the PCR fragment into plasmid pAK6., Results: Analysis of PCR fragments revealed that they contained the whole DS I gene. There was no base change all over the structure genes and regulatory sequences between C. pekinense AS1.299 and PD-67. An internal promoter was found in the upstream of the DS I gene from C. pekinense and it functioned in E. coli 3257. The DS I gene from C. pekinense PD-67 was expressed homogenously, and the specific enzyme activity of DS I in C. pekinense PD-67 (pAD1) was much higher than that of the control strain C. pekinense PD-67(pAK6)., Conclusion: This is the first report that DS I gene existed in Corynebaterium Pekinense, The amplification of the specific activity of DS I is expected to increase L-tryptophan accumulation of C. pekinense PD-67.

We studied the aggregation of a recombinant engineering antibody (chA21). Anti-ErbB2 antibody chA21 was produced by fusing single-chain Fv (scFv) with human IgG1 Fc fragment, and it was proved to be a drug candidate for cancer therapy. We characterized the aggregation of chA21 by high performance sized-exclusive chromatography (HPSEC), dynamic light scattering (DLS), SDS-PAGE, indirect ELISA assay, and compared the influence of temperature and additive on the level of aggregation and binding activity. Conformation changes of different levels of aggregation were also analyzed via circular dichroism (CD). Finally, we analyzed which part of chA21 was involved in aggregation by cleaving it into scFv and Fc fragments. The results showed that chA21 could form aggregates in the storage solution. The aggregates interacted through non-covalent bonds and remained binding activity. Temperature and additive could slightly affect the level of aggregation and binding activity, while the conformations of chA21 were stable. Aggregation propensity of scFv fragment was almost same as chA21, indicating that scFv may be the major part to form the aggregates. The research on aggregation may be helpful to develop a suitable formulation for chA21 clinical application as well as provide direction for future antibody design and reconstruction.

Acquired immune deficiency syndrome (AIDS) is increasing its negative influences on human society and economy. In the present paper, HIV-1 cell fusion peptide inhibitor C22 was expressed and purified based on the C-terminal sequence of HIV-1 membrane fusion glycoprotein gp41. The gene coding for C22 was totally synthesized using gp41 gene as a template and amplified by PCR The cloned C22 gene was confirmed by restriction endonuclease and sequence analysis and then cloned into plasmid pTMHa30-51. The prepared plasmid was transformed into E. coli BL21 (DE3) and the expressing products were analyzed on SDS-PAGE and tested with mass spectrum. The results indicated that C22 showed high HIV-1 fusion inhibiting capacity, meanwhile, with good thermal stability and water-solubility, and showed no cell toxicity in tested concentrations. The spectral characteristics were tested with circular dichroism (CD). When treated at different temperature in solution condition, the content of a helix of C22 increased at 37 degrees C while decreased sharply at 80 degrees C. The peak value changed significantly with different pH values. The content of a helix of C22 decreased as pH varied toward acid and alkali and the random coiling increased, which led to a relaxed structure. This result indicated that the C22 structure is stable with pH 6. This research may provide a theoretic foundation for the new type HIV-1 peptide inhibitor designing and spectral characteristics study.

The study was aimed to investigate the technological parameters of rhIL-11 preparation from fusion protein by using enterokinase. Fusion expression vector pET32a/IL-11 was transferred into E.coli BL21 (DE3) pLysS and its expression was induced by IPTG, the lysis supernatant of the engineering strain was purified by Ni-NTA resin and then the target rhIL-11 was digested by auto-cleavaged DsbA-EKL-(His)(8). The results showed that after affinity chromatography, Trx-IL-11 was obtained with the yield of 11.25mg/g, the purity of 89.2% and the recovery of 91.8%. Finally the target rhIL-11 was digested and purified to over 95%. In conclusion, the preparation method of rhIL-11 from fusion protein by using enterokinase is simple and feasible with good separation, which can meet industrial requirements.

This experiment was aimed to create A20 gene site-specific zinc finger DNA-binding protein. The sequence of A20 gene promoter was analyzed with bioinformatics means and submitted to ZF Tools Server at TSRI. Using the database of the web site, we determined the A20 gene valid target sites and designed the amino acid sequence of zinc finger protein predicted to be bound to the target site. And then, the structure of the protein sequence was analyzed and homology was modeled with various bioinformatics means. Based on the characteristic of this protein, the prokaryotic expression vector pTYB11-ZFP was constructed and expressed. Thus, the artificial zinc finger protein that recognized A20 specific sequence was designed, and expressed in Escherichia coli. The results indicate that it is feasible to design engineered artificial Zinc finger proteins by means of bioinformatics.

In order to improve the thermostability of the Penicillium expansum Lipase (PEL), the lipase encoding genes was mutated by site-directed mutagenesis. A recombinant vector pAO815-ep8-K55R which contain double mutant genes was constructed by overlap extension PCR using the cDNA of a random-mutant lipase ep8 (a single site mutant) as the template and two special primers were used to generate another mutation site K55R. The recombinant vector was transformed into Pichia pastoris GS115 by electroporation and the recombinant mutant GS-pAO815-ep8- K55R can secret double-mutant lipase PEL-ep8-K55R-GS into the medium when it was induced by Methanol. The yield of the double-mutant lipase is 508 u/mL, which is 81% that of the wild type lipase PEL-GS (627 u/mL) and 55% that of random-mutant PEL-ep8-GS (924 u/mL). The specific activity of double-mutant lipase is 2309.1 u/mg, which is similar to random-mutant lipase PEL-ep8-GS and the wild type lipase PEL-GS. The optimum temperature of the double-mutant lipase is same with the wild type lipase PEL-GS and random-mutant lipase PEL-ep8-GS. While the Tm of the double-mutant lipase is 41.0 degrees C, 2.3 degrees C higher than the wild type lipase PEL-GS and 0.8% higher than the random-mutant lipase PEL-ep8-GS, indicating that the double-mutant lipase PEL-ep8-K55R-GS has higher thermostability.

Terpenoids are present in all organisms but are especially abundant in plants, with more than 30,000 compounds. Not only do they play an important role in the life of plant, but also have high commercial values. However, the content of many important terpenoids in plant is very low. Therefore, how to improve the inefficient production of terpenoids is an urgent task. Metabolic engineering has been one of the most potential technologies to improve terpenoids production in recent years, following the study of metabolic pathway and regulation mechanism of terpenoids. Although there are some breakthroughs, metabolic engineering of terpenoids is still full of challenges because of the lack of knowledge on metabolic control of most terpenoids. Functional genomics approaches, including transcriptomics, proteomics and metabolomics, are potential tools for exploring of metabolic engineering. Integrating transcriptomics and metabolomics is an effective way to discover new genes involved in metabolic pathway. In this paper, the representative research outcomes about the metabolic engineering of terpenoids in plant were reviewed concisely and then the application of functional genomics approaches to study metabolic pathway and regulation mechanism of terpenoids and the strategies for metabolic engineering of terpenoids were discussed.

S-layer protein CTC surface display system of Bacillus thuringiensis (Bt) was used to test the possibility of displaying avian influenza virus nucleoprotein (NP) on Bt cell surface. Four recombinant plasmids were constructed by replacing 3'-terminal or central part below the surface anchor sequence slh of S-layer protein gene ctc with full length of np gene or part np gene (npp). The four resulting plasmids were pSNP (harboring fusion gene ctc-np), pCSA-SNP (harboring fusion gene csa-ctc-up, csa represents csaAB operon which is very important to the anchoring of S-layer protein on the bacterial cell surface), pCTC-NPP (harboring fusion gene ctc-npp) and pCSNPP (harboring fusion gene csa-ctc-npp). Five recombinant Bt strains were constructed by electro-transferring recombinant plasmids to Bt plasmid-free derivative strain BMB171. The resulting strains were BN (harboring pSNP), BCN (harboring pSNP as well as the plasmid pMIL-CSA which carried csaAB operon), C-S (harboring pCSA-SNP), BCCN (harboring pCTC-NPP and pMIL-CSA) and CN (harboring pCSNPP). The vegetative cells of five recombinant strains were used as agglutinogens of slide agglutination assay. Slide agglutination assay showed recombinant NP proteins were successfully displayed on the surface of five recombinant strains, respectively. After immunizing mice with vegetative cells of five recombinant strains respectively, five recombinant strains all elicited humoral respones to NP and exhibited immunogenicity as assayed by enzyme-linked immunosorbent assay (ELISA). Meanwhile these assays showed recombinant strain CN (harboring fusion gene csa-ctc-npp) exhibited the highest immunogenicity among five recombinant strains. That means the best way of constructing S-layer fusion gene is csa-ctc-* (* denotes heterologous antigen gene) which means the central part of S-layer protein gene ctc replaced by the heterologous antigen gene and csaAB operon located on the upstream of fusion gene. The strategy developed in this study gives a possibility to generate heat stable,oral, veterinary vaccine with Bt S-layer protein CTC surface display system.

Aspergillus fumigatus wild-type phytase has many favorable properties, such as a good thermorstability and a broad pH optimum. However, the specific activity of the enzyme is relative low. A. fumigatus Q23L phytase resulted in a remarkable increase in specific activity around pH4.5 - 7.0, but the pH stability of Q23L was lower than A. fumigatus wild-type phytase. To increase the pH stability of Q23L, the mutant Q23LG272E was constructed by site-directed mutagenesis with PCR. The gene of A. fumigatus wild-type phytase and the mutant genes encoding the Q23LG272E and the Q23L were correctly expressed in Pichia pastoris GS115. Enzymes were purified and their enzymatic properties were determined. The results revealed that the specific activity of the Q23L improved remarkably, which increased from 51 u/mg of the wild type to 109 u/mg at pH5.5. Meanwhile, the pH stability of Q23L, decreased evidently, especially from pH3.0 to pH4.0.The pH stability of Q23LG272E in pH3.0 - 4.5 and pH6.5 - 7.0 has been improved compared with Q23L. The specific activity of Q23LG272E basically maintained at the level of Q23L. Analysis of 3-D structure and sequence similarity were used to reveal the presumable factors influencing the enzymatic properties of Q23LG272E, and discussion for the relationship between structure and function of phytase was given.

Objective: To prepare the engineering peptide Pheromonicin-P. aeruginosa (Ph-PA) and evaluate the antibactericidal activity of prepared Ph-PA against P. aeruginosa via in vitro/in vivo bactericidal assays., Methods: Ph-PA was purified by CM sepharose ion-exchange column. In vitro/in vivo bactericidal assays were performed to identify the antibactericidal activity of Ph-PA against P. aeruginosa., Results: In vitro and in vivo data showed that antibactericidal effects of Ph-PA, on molar basis, were approximately over 2200 times greater than that of piperacillin, and 220 times greater than that of ceftazidime against P. aeruginosa., Conclusion: The Ph-PA may have potentially applied value as a novel kind of peptide antibiotics against P. aeruginosa infection.

Objective: To construct the recombinant plasmid pET28a/hES and coexpress the human endostatin (hES) and predigested human plasminogen kringle 5 (predhPK-5) in E. coli., Methods: The mRNA was extracted from human liver tissue, and the endostatin gene was amplified by RT-PCR, which then was cloned into pET-28a(+). Under screening pressure by ampicillin and kanamycin simultaneously, E. coli BL21 (DE3) was cotransformed with pET28a/hES and pGEX-1lambdaT/predhPK-5 and induced with IPTG to express the recombinant proteins. The stability of cotransformants existing in E. coli was measured through two aspects in the serial culture time and passage number of bacterium., Results: The two incompatible plasmids could be coexisted under the pressure of two antibiotics (ampicillin and kanamycin). After induced with IPTG, both human endostation and predhPK-5 gene were coexpressed, and the recombinant proteins comprised about 20% and 21% of total cell proteins, respectively. The two incompatible plasmids could still be maintained in over 75% E. coli cells for at least 16 hours or after 120 passages under the pressure of two antibiotics., Conclusion: The two incompatible plasmids pET28a/hES and pGEX-1lambda T/predhPK-5 may coexpress the recombinant proteins in E. coli. A new method for coexpression of proteins in E. coli containing two incompatible plasmids is proved to be feasible.

Hepcidin is a small cystein-rich cationic peptide produced mainly by the liver. It was initially isolated from human plasma and exhibited antimicrobial activity. Recently, several lines of evidence have suggested that hepcidin is a key regulator of iron metabolism at the whole body level and is relative to inflammation, infection, hypoxia and anemia. Hepcidin, is implicated in duodenal iron absorption and iron mobilization from reticuloendothelial macrophages. The major mechanism of hepcidin function seems to be the regulation of transmembrane iron transport. As both iron deficiency and iron excess are associated with cellular dysfunction, so hepcidin or hepcidin-related therapeutics could find a place in the treatment of various diseases such as hemochromatosis and anemia of chronic disease. To elucidate biological function of hepcidin further and use it for other research, it is necessary to produce enough hepcidin through DNA recombinant technique. As a highly successful system for the production of a variety of heterologous proteins, the methylotrophic Pichia pastoris system has the probability for a high level production of hepcidin. The subject of this paper is to summarize the regulation of hepcidin gene expression and the understanding of functions of hepcidin. At last, giving a prospect of production hepcidin by gene engineer.

Human brain natriuretic peptide (hBNP) was used clinically for the treatment of acute decompensated congestive heart failure. In this paper, hBNP was expressed as a fusion protein with a histidine tag and Ssp dnaB mini-intein which was capable of self-cleavage. After affinity chromatography with Ni-Sepharose and renaturation, the fusion protein was enriched with CM-cellulose. Ssp dnaB mini-intein mediated peptide-bond hydrolysis was triggered by shifting the pH and temperature in the CM-cellulose column, which let to the release of hBNP from the fusion protein and the separation of hBNP from His-DnaB. The hBNP sample was further purified by C4 reverse phase HPLC, and 2.8mg of the peptide with homogeneity of 97% was obtained from one liter of culture medium. The biological activity was assayed in vitro, which indicated that hBNP had a potent vasodilatory effect on rabbit aortic strips with an EC50 of 1.94 x 10(-6) mg/mL.

Self-assembly of peptides is ubiquitous in the body of creatures. The molecules of peptides combine with each other to form proteins with different functions through self-assembly. The formation of a specific conformation of one type of protein is owing to the self-assembly of its compositive amino acids. So, researchers can design self-assembly of peptides at the molecular level and can control its formation and configuration. It has the potential for application in the preparation of new medicines and biomaterials. In recent years, self-assembling peptides have been increasingly high-lighted and used to simulate the function of natural biomolecules, to synthesize peptide-medicine, and to serve as the carriers of medicine.

The hybrid xylanase TB was constructed by the substitution of the N-terminus segment of the Streptomyces olivaceoviridis xylanase XYNB with corresponding region of Thermomonosporafusca xylanase TfxA. The hybrid gene tb, encoding the TB, was correctly expressed in Escherichia coli BL21 and Pichia pastoris GS115. TB was purified and its enzymatic properties were determined. The results revealed that the optimal temperature and optimal pH of TB were at 70 degrees C and 6.0, which have been obviously improved compared with those of XYNB. The thermostability of TB were all about six-fold of XYNB's after incubating the properly diluted enzyme solutions at 80 degrees C and 90 degrees C for 3min, respectively. The pH stability of TB was 5 to approximately 9, which was narrower than that of XYNB. Still, TB remains a high specific activity as XYNB does. Analysis of a homology modeling and sequence similarity were used to reveal the factors influencing the enzymatic properties of TB and the discussion for the relationship between structure and function of xylanase was given.

Objective: To study the specific cytotoxicity mediated by anti-P-gp/anti-CD(3) diabodies in multidrug resistant solid tumor using P-gp as target., Methods: The anti-P-gp/anti-CD(3) diabodies were secreted from E. coli strain 16C9 containing the expression plasmid PAYZDCP, grown at 30 degrees C in a shaker flask; the diabodies were purified by affinity chromatography and identified by SDS-PAGE; the effect of the anti-P-gp/anti-CD(3) diabody mediated lysis of P-gp-expressing tumor cells was assayed by (51)Cr release assay in vitro, and by human KB nude mouse xenograft models in vivo., Results: The diabodies were generated by bacteria as a soluble functional form and purified by one-step affinity chromatography with a yield > 4 mg/L culture medium. In (51)Cr release assay, the diabodies targeted human activated T cells to lyse P-gp(+)-KB/MDR cells in a dose-dependent manner. It suggested that the diabody was able to induce an efficient lysis of the target cells by human T cells in vitro. When combined with activated human T cells, the diabody significantly inhibited the growth of KB/MDR, but had no effect on KB xenografts., Conclusion: The anti-P-gp/anti-CD(3) bispecific antibody is a potent agent for targeting human T lymphocytes to lyse solid tumor cells overexpressing P-gp in vitro and in vivo.

According to the amino acid sequence of monellin, a single chain 294bp monellin gene was synthesized and inserted into vector pET-22b to yield the recombinant secretion plasmid pETMO. The single-chain monellin gene was designed based on the biased codons of E. coli so that its expression would be then optimized. Under the expressing conditions, monellin was produced accounting for 44.8% of total soluble proteins. The E. coli-expressed single-chain monellin is 3000 times sweeter than sucrose. The thermal-stability and acid-resistance of the protein are higher than the natural monellin.

Interleukin-2 (IL-2) was initially isolated as a T cell growth factor and had been shown to direct the expansion and differentiation of several hematopoietic cell types. Clinical studies using IL-2 in the treatment of AIDS have been encouraging, due to its critical role as a proliferative signal for activated T-lymphocytes. IL-2 has also undergone trials in the treatment of several types of cancer, based on its stimulation of cytotoxic, antitumor cells. Today, human IL-2 is produced completely by genetically engineered method, and it has been proved that genetically engineered recombinant human IL-2 has almost the same function and clinical effect as wild IL-2. In the former study, recombinant human IL-2 usually comes from E. coli, in this paper the mutant IL-2 was successfully expressed and purified in Pichia pastoris for the first time. As a eukaryote, Pichia pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification, while being as easy to manipulate as E. coli or Saccharomyces cerevisiae. It is faster, easier, and less expensive to use than other eukaryotic expression systems such as baculovirus or mammalian tissue culture, and generally gives higher expression level. Expression conditions of human mutant interleukin-2(the codon for cysteine-125 of human IL-2 with alanine; the codon for leucine-18 with methionine; the codon for leucine-19 with serine) in the recombinant Pichia pastoris strain were optimized via test of some factors such as the rate of aeration, the inductive duration, the initial pH and the concentration of methanol. The results from tests showed that the most important parameter for efficient expression of interleukin-2 in recombinant Pichia pastoris strain is adequate aeration during methanol induction, and the optimum inductive condition for interleukin-2 expression was: more than 80% aeration, 2 days for induction, the initial pH of 6.0, the final methanol concentration of 1.0%. With this condition, the expressed IL-2 was secreted into fermentation broth and reached a yield of 30%, approximately 200 mg/L. Expressed interleutin-2 (MvIL-2) was isolated and purified by centrifugation, millipore filtration to concentration, Econo-PacS strongly acidic cation exchanger cartridge and molecular sieve chromatography and the yield of MvIL-2 was 27%. MvIL-2 was purified to electrophoretic purity by SDS-PAGE and only one peak being loaded on HPLC. Purified MvIL-2 protein had stimulating activity similar to the wild type of IL-2 as assayed by IL-2-dependent CTLL-2 cells. However, the stability of MvIL-2 was superior than that of IL-2 at different temperatures. The activity of obtained MvIL-2 was 4 - 5 times of the wild type of IL-2, So MvIL-2 had an advantage over wild type of rhIL-2 in storage stability and activity.

Production of Hepatitis E Virus capsid protein by high cell density culture in recombinant E. coli has been studied in 10L and 30L fermentors. The effects of different factors on growth and producing recombinant protein of E. coli have been studied by batch culture, such as different media, the ratio of phosphate and Magnesium sulfate. Comparison of fermentation performance for recombinant E. coli in different fed-methods culture has been investigated by fed-batch culture. The effects of inducing at different stages of growth and time of inducing on growth and producing recombinant protein, also obtained by fed-batch culture. At last, the solubility of inclusion body in different urea concentrations also has been obtained by fed-batch culture. The results show that the concentration of phosphate and Magnesium sulfate in the optimal media is 80mmol/L and 20mmol/L in batch culture respectively, that induction with 1.0mmol/L IPTG at mid log phase (about 45 OD at 600nm) is suitable for growth and recombinant protein expression, the cells were approaching stationary growth phase and the maximum cell OD at 600nm of 80 was achieved in 5h of fed-batch culture, and the expression level is 29.74%. The results also indicate that the solubility of inclusion body in 4mol/L urea solution induced at 37 degrees C reaches 14mg/mL, over 80% inclusion body was resolved. The culture process achieved in 10L fermentor could be successfully scaled up to 30L fenmentor with good reproducibility.

Objective: To construct a phosphatidylinositol 4-kinase beta (PI4K-beta) mutant with the 325th to 373rd amino acid codons deleted, and try to develop a simple method for constructing middle fragment deletion mutant., Methods: In line with the mechanism of gene splicing by overlap extension(SOE), an additional PCR was used to get the PI4K-beta mutant in which the 325th to 373rd amino acid codons were deleted. Then the mutated gene was cloned into pCMV-Tag4A mammalian expression vector., Results: A mutant with the 325th to 373rd amino acid codons deleted was constructed successfully., Conclusion: The improved SOE is a very effective and reliable method to construct middle fragment deletion mutant. It is worthy to be popularized.

Protein splicing is a newly discovered posttranslational editing process that removes an internal protein fragment from the protein precursor. During the splicing process the internal protein fragment, intein, triggered the self-excision from the precursor protein and the concomitant ligation of the flanking protein fragments, exteins. The self-catalysis requires neither auxiliary enzymes nor cofactors and only involves four intramolecular reactions. A number of key catalytic residues in inteins and flanking fragments have been identified, which led to the development of the protein splicing process as a protein engineering tool. Controllable cleavage of the peptide bond at either the N or the C terminus of an intein has allowed the design of novel strategies for manipulation of protein and peptides. Affinity purification of recombinant proteins can be facilitated by fusion the target protein with an intein. The fusion also creates C-terminal thioester, which expands the scope of chemical ligation in protein. Inteins can be engineered in a "split and inverted" configuration to form a cyclic polypeptide consisting of the sequence linking two intein subdomains. This article summarizes the recent advance in the mechanism of protein splicing and its applications in protein purification, protein ligation and protein cyclization.

The developments of recombinant DNA technology and structural biology make it possible to modify enzyme in molecular level. Scientists show growing interests in the evolution or functional fusion of enzymes. Recent advances and applications of the molecular enzyme engineering are reviewed and discussed in this article.