Abstract: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 influenc 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.

Abstract:5-aminolevulinic acid is the key intermediate of the tetrapyrrole biosynthesis pathway in organisms and has broad application potentials. This review summarized and discussed recent progress in microbial production of 5-aminolevulinic acid, including screening, isolation and mutation of microbes to produce 5-aminolevulinic acid; microbial whole-cell transformation to synthesize 5-aminolevulinic acid depending on the C4 pathway; construction of high-yield 5-aminolevulinic acid producing strains by metabolic engineering. Finally, future research directions in microbial production of 5-aminolevulinic acid were addressed.

Abstract:Mannosylerythritol lipids (MELs), mainly produced by Ustilago and Pseudozyma, are surface active compounds that belong to the glycolipid class of biosurfactants. MELs have potential application in food, pharmaceutical and cosmetics industries due to their excellent surface activities and other peculiar bioactivities. In recent years, the research field of MELs has regained much attention abroad. However, MELs are rarely studied in China. In this review, the producing microorganisms and production conditions, diverse structures, biochemical properties, structure-function relationship and biosynthetic pathways of MELs are described. Some research problems and prospects are summarized and discussed as well.

Abstract:We studied the mutation effect of subsites -3(Lys47), -7(146-152), and cyclization center (Tyr195) in active domain on product specificity of α-cyclodextrin glucanotransferase (α-CGTase) from Paenibacillus macerans sp. 602-1. The Lys47 was replaced by Thr47 and Tyr195 by Ile195, and the amino acids from 146 to 152 were replaced by Ile (named as Δ6). All these mutant α-CGTases were actively expressed in E. coli BL21. Compared with the wild-type α-CGTase, the starch-degrading activities of all the mutant enzymes were declined. For mutant Y195I, the percentage of α-CD was decreased from 68% to 30%, and β-CD was raised from 22.2% to 33.3%. Interestingly, γ-CD was increased from 8.9% to 36.7% and became the main product, while the actual yield was increased from 0.4 g/L to 1.1 g/L. Mutant K47T and Δ6 still produced α-CD as main product though the percentage of β- and γ-CD increased. Purified Y195I CGTase showed similar optimum temperature with the wild-type α-CGTase, but its optimum pH shifted from 5.0 to 6.0 with better pH stability. In summary, mutant Y195I CGTase has the potential to produce γ-CD as the main product.

Abstract:Exploring new β-glucosidase genes is of great importance to industrialize β-glucosidase. The genomes of Aspergillus fumigatus contain a bgl gene, which encodes a 65 kDa putative β-glucosidase. The bgl gene was cloned into an expression plasmid and transformed to Escherichia coli BL21 (DE3). The bgl was expressed upon induction of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant protein was purified by GST-tag affinity chromatography. The purified recombinant Bgl was characterized using Esculin as substrate. The optimum temperature and pH were 45 ℃ and 5.0?6.0, respectively. The Km for Esculin was 17.7 mmol/L. The enzyme was stable in the range of pH 4?7. After incubation at 70 ℃ for 2 h, the recombinant Bgl remained 60% of its activity. Metal ions and chemical reagents had different influences on the activity of β-glucosidase. Ca2+ (1 mmol/L) could increase enzyme activity slightly. On the contrary, the enzyme activity was greatly inhibited by 5 mmol/L Sodium dodecyl sulfate (SDS). Based on our results, the A. fumigatus Bgl was thermostable β-glucosidase.

Abstract:Constructing ethanologenic strains with cellulose activity is important to achieve consolidated bioprocessing of lignocellulose for ethanol production. In this study, we integrated the pyruvate decarboxylase gene pdc and alcohol dehydrogenase gene adhB from Zymomonas mobilis ZM4 into Escherichia coli JM109 by Red recombination method to generatea recombinant strain E. coli P81that could produce ethanol from glucose. Aβ-glucosidase gene bglB from Bacillus polymyxa 1.794 was cloned into the recombinant E. coli P81 and β-glucosidase was expressed to give a new recombinant strain E. coli P81 (pUC19-bglB) with dual functions of cellobiose degradation and ethanol production. The extracellularβ-glucosidaseactivity was 84.78 mU/mL broth and the extracellular cellobiase activity of E. coli P81 (pUC19-bglB) was 32.32 mU/mL broth. E. coli P81(pUC19-bglB) fermented cellobiose to ethanol with a yield of 55.8% of the theoretical value, and when glucose and cellobiose were co-fermented, the ethanol yield reached 46.5% of thetheoretical value. The construction of consolidated bioprocessing strain opens the possibility to convert cellobiose to ethanol in a single bioprocess.

Abstract:High-efficient conversion of glycerol to L-lactate is beneficial for the development of both oil hydrolysis industry and biodegradable materials manufacturing industry. In order to construct an L-lactate producer, we first cloned a coding region of gene BcoaLDH encoding an L-lactate dehydrogenase from Bacillus coagulans CICIM B1821 and the promoter sequence (PldhA) of the D-lactate dehydrogenase (LdhA) from Escherichia coli CICIM B0013. Then we assembled these two DNA fragments in vitro and yielded an expression cassette, PldhA-BcoaLDH. Then, the cassette was chromosomally integrated into an ldhA mutant strain, Escherichia coli CICIM B0013-080C, by replacing lldD encoding an FMN-dependent L-lactate dehydrogenase. An L-lactate higher-producer strain, designated as E. coli B0013-090B, possessing genotype of lldD::PldhA-BcoaLDH, Dack-pta Dpps DpflB Ddld DpoxB DadhE DfrdA and DldhA, was generated. Under the optimal condition, 132.4 g/L L-lactate was accumulated by B0013-090B with the lactate productivity of 4.90?g/L?h and the yield of 93.7% in 27 h from glycerol. The optical purity of L-lactate in broth is above 99.95%.

Abstract:To enhance biohydrogen production of Klebsiella sp. HQ-3, the global transcriptional factor (Fnr), formate dehydrogenase H (FDHH) and the pncB gene encoding the nicotinic acid phosphoribosyltransferase (NAPRTase) were for the first time over-expressed in Klebsiella sp. HQ-3. The fnr, fdhF, pncB genes were cloned from the genomic DNA of Klebsiella sp. HQ-3 by 3 pairs of universal primers, and introduced into the corresponding sites of the modified pET28a-Pkan, resulting in the plasmids pET28a-Pkan-fnr, pET28a-Pkan-fdhF and pET28a-Pkan-pncB. The 4 plasmids were then electroported into wild Klebsiella sp. HQ-3 to create HQ-3-fnr, HQ-3-fdhF, HQ-3-pncB and HQ-3-C, respectively. Hydrogen production was measured using a gas chromatograph and the metabolites were analyzed with a high-performance liquid chromatograph (HPLC). The results indicate that over-expression of fnr, fdhF and pncB significantly enhanced hydrogen production in the three recombinant strains. Hydrogen production per mol glucose for HQ-3 fnr, HQ-3 pncB, HQ-3 fdhF was 1.113, 1.106 and 1.063 mol of hydrogen/mol glucose, which was respectively increased by 12.26%, 11.62% and 7.28% compared with that of the control strain HQ-3-C (0.991 mol of hydrogen/mol glucose). Moreover, the analysis of HPLC showed that the concentrations of formate and lactate were markedly decreased, but succinate remained unchanged in culture media compared with those of the control strain HQ-3-C.

Abstract:2,3-butanediol (2,3-BD) is a major byproduct of 1,3-propandediol (1,3-PDO) fermentation by Klebsiella pneumoniae. To decrease the formation of 2,3-BD, the budC and budA gene, coding two key enzymes of 2,3-BD synthetic pathway in K. pneumoniae, were knocked out using Red recombination technology. The growth of the two mutants were suppressed in different level. The budC deficient strain fermentation results showed that 1,3-PDO concentration increased to 110% and 2,3-butanediol concentration dropped to 70% of the parent strain. However, the budA deficient strain did not produce 1,3-PDO and 2,3-BD, and the final titer of lactic acid, succinic acid, ethanol and acetic acid increased remarkably compared with the parent strain. Further analysis of budC deficient strain fermentation inferred that K. pneumoniae possessed the 2,3-BD cycle as a replenishment pathway. The consequence provided a new evidence for reforming low-byproduct K. pneumoniae.

Abstract:Engineering the existing or manual assembling biosynthetic pathways involves two important issues: the activity and expression level of key enzymes in the pathway. Concerning the enzyme expression study, the conventional approach is to use strong promoter to initiate the overexpression of the target protein. The excessive expression of the target protein usually result in the intracellular accumulation of a large number of inactive inclusion bodies, thereby seriously affect the physiological state of the cell and the effective functioning of the relevant biological pathways. To solve this problem, we would like to design a molecular switch to precisely manipulate the expression level of key enzymes in the biosynthetic process, which has important practical value for the study of metabolic rhythm of the biosynthetic pathway and to promote the efficiency of the biosynthetic pathway. Based on the basic principles of quorum sensing existing in the bacterial community and combining the dynamic characteristics of the enzymatic catalysis, we first established cell-cell communication mechanisms mediated by signal molecule homoserine lactone (AHL) in the E. coli community and target protein EGFP was expressed under the control of the promoter PluxI. In the process of cell growth, AHL accumulated to a certain concentration to start the expression of target gene egfp. At the different cell growth stages, AHL-degrading enzyme AiiA was induced and resulted in the degradation of AHL molecule in a controlled environment, thereby controlling the transcription efficiency of target gene egfp and ultimately achieve the precise control of the level of expression of the target protein EGFP. The detection of cell growth state, the mRNA level and protein expression level of the target gene showed the artificially designed molecular switch can control the level of expression of a target gene in a convenient and efficient manner with a spatial and temporal regulation of rigor. The molecular switch is expected to be widely used in the field of metabolic engineering and synthetic biology research areas.

Abstract:Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by reduced expression levels of the frataxin gene (FXN) due to expansion of triplet nucleotide GAA repeats in the first intron of FXN. FXN is a mitochondrial protein which plays an important role in the regulation of intracellular iron trafficking, biogenesis of iron-sulfur cluster and heme, and removal of reactive oxygen species. Our previous work showed that tissue-specific expression of FXN in cerebellum and heart generates two novel isoforms. In order to find the isoforms in mouse tissues, we tried to obtain a polyclonal antibody against mouse Fxn with high specificity and sensitivity. Thus, the recombinant plasmid pET24(+)-mFxn was constructed to express his-tagged Fxn in BL21(DE3) cells. The expressed protein is a mature form with 130 amino acids (aa, 14.38 kDa) without the N-terminal signal peptide (77 aa), purified on Ni-NTA column and further dialyzed with Centrifugal Filtration Device. The polyclonal antibody against Fxn was produced by immunizing rabbits with highly purified protein. The collected antiserums were preliminarily purified by precipitation with (NH4)2SO4. Western blotting analysis and cell immunofluorescence showed that the obtained antibody was able to detect both purified and endogenous Fxn. It also worked well in immunoprecipitation with mouse tissues. This is the first time, to our knowledge, to report that mouse Fxn was used as immunogen to generate antibody with high specificity and sensitivity. This work provides a powerful tool for our further research on mouse Fxn isoforms.

Abstract:A repetitive DNA fragment, named P1, was amplified by PCR with the full-length Minor Ampullate Spidroin gene sequence of Araneus ventricosus as template. P1 was ligated with pPic3.5 and PKT expression vectors and transferred into GS115 and BL21(DE3) competence cells, respectively. SDS-PAGE and Western blot were used to analyze the recombinant his-tag fusion protein. With expressed in different expression systems, soluble P1 induced proteins could be obtained as the same size. Furthermore, the expression level and purification recovery efficiency were also higher in GS115 than that of BL21(DE3). Additionally, the expression level could be improved after optimizing the incubation and induction conditions of GS115. In this research, Pichia pastoris expression system is more suitable for the native repetitive Gly/Ala-rich spider spidroin gene sequence expression than Escherichia coli system. The data can help the native full-length MiSp gene expression and large-scale exploitation of recombinant of spider silk proteins.

Abstract:For metabolic engineering of cyanobacteria, there is an urgent need to construct a group of efficient heterologous gene expression platforms and to evaluate their expression efficiencies. Here we constructed three integrative vectors, the pKW1188-derived pFQ9F, pFQ9R and pFQ20, for integration of heterologous genes into the genome of the model cyanobacteria strain Synechocystis sp. strain PCC6803. The pFQ16, an RSF1010-derived broad host range shuttle vector, was constructed for conjugative transfer of genes to various cyanobacteria strains. All the four platforms constructed here applied the rbc (encodes Ribulose-1, 5-bisphosphate carboxylase/oxygenase) and the rbc terminator to promote and terminate the gene transcription. Besides, a “Shine-Dalgarno -AUG” fusion translation strategy was used to keep the high protein translation efficiency. Using lacZ as a reporter gene, the expression efficiency of pFQ20 was evaluated and showed a strong β-galactosidase expression (109 Miller). Furthermore, the platform pFQ20 was used to express the E. coli tesA’ gene and showed significant protein bands through the Western Blot test. The expression platforms constructed in this study offer useful molecular tools for metabolic engineering of cyanobacteria in the future.