To explore the characteristics of PAH gene variants among 113 phenylketonuria patients from Henan Province.The 13 exons of the PAH gene were subjected to PCR amplification and direct sequencing. Large fragment deletion and duplication of the PAH gene were detected with a multiple ligation-dependent probe amplification (MLPA) assay.In total 195 point variants and 3 large fragment deletions were detected among the 226 alleles, with the detection rates being 86.28% and 1.33%, respectively. Variants of p.Arg243Gln (18.14%), p.Arg111X (6.19%), p.Arg53His (5.31%), EX6-96A>G (5.31%), p.Tyr356X (4.87%) and p.Val399Val (4.42%) were relatively common. Most of the variants were located in exons 7, 11, 3 and 6. Missense variations were most common. Four novel variations were detected, which included c.1016C>A (p.Ser339Tyr), c.1000T>C (p.Cys334Arg), c.1110G>T (p.Glu370Asp), and IVS6+1G>T.The PAH gene variations in Henan Province have featured extensive allelic heterogeneity and variety.
Acidithiobacillus caldus is an important sulphur-oxidizing bacterium that plays crucial roles in the bioleaching industry. This study aims to analyse the optimal reference gene for real-time quantitative PCR (RT-qPCR) under different conditions and investigate the transcription levels of the sigma factor genes in the stress response.We selected six housekeeping genes and analysed them via RT-qPCR using two energy resources, under four stress conditions. Three statistical approaches BestKeeper, geNorm, and NormFinder were utilized to determine transcription stability of these reference genes. The gapdH gene was the best internal control gene using elemental sulphur as an energy resource and under heat stress, map was the best internal control gene under pH and osmotic stress, era was the best internal control gene for the K2 S4 O6 energy resource, and rpoC was the best internal control gene under Cu2+ stress. Furthermore, the expressional levels of 11 sigma factors were analysed by RT-qPCR in the stress response.Stable internal control genes for RT-qPCR analysis of A. caldus were determined, and the expression patterns of sigma factor genes of A. caldus were investigated.The identification of the optimal reference gene and analysis of transcription levels of sigma factors in A. caldus can provide clues for reference gene selection and the study of sigma factor function.
Metallothioneins (MTs) are a family of low molecular weight, cysteine rich, metal binding proteins, and exist in a variety of organisms ranging from bacteria to mammals (Moffatt & Denizeau, 1997). MTs play significant roles in clearance of free radical against oxidative damage (Masters et al., 1994), trace element metabolism and heavy metal detoxification (Feldman & Cousins, 1976), cell metabolism (Cherian & Apostolova, 2000), disease processes (Sens et al., 2001; Velazquez et al., 1999), rare metal accumulation (Nordberg, 1998), heavy metal removal and environment protection et al. (Nordberg, 1998; Elinder et al., 1981). The important applications stimulate the interest of large scale production of MT. Compared with the conventional method by extraction from animal organs, MT production by using recombinant E. coli has obvious advantages in reducing cost, increasing efficiency and product quality. However, recombinant MT is easily hydrolyzed and unstable for the thiol group of cysteine when produced by recombinant microorganism (Saito & Hunziker, 1996). By integrating the MT gene with fusion tags, MT was successfully expressed in E. coli (Hong et al., 2001; Yang et al., 2007). On the other hand, rapid measurement of MT production is important for optimization of fermentation process. MT was reported to have light absorption at the wave length of 250 nm after binding with Cd (Onosaka & Cherian, 1982), which could be used for MT measurement. But, according to our experience, this method has low accuracy and poor reproducibility. In order to detect MT production in an easy and rapid way during fermentation process, in this study, MT gene is fused with red fluorescent protein (RFP) gene and coexpressed in recombinant E. coli. Modeling and simulation is a powerful tool for the analysis of the mechanism and dynamic responses of the fermentation process, and for process optimization and automatic control. The production of recombinant protein using recombinant E. coli involves the regulations of aimed gene transcription and translation, which process can be well described by using the white box structured model involing operon regulation and is done in this research. But, the structured model has the disadvantage of extreme complex not suitable for the purpose of process optimization.
As a kind of rare sugar alcohol, allitol has important application values in food and medication. In addition, it can be used as a key substrate to produce other d/l-rare sugars. Allitol can be effectively produced by the resting-cell biotransformation method. Two recombinant Escherichia coli strains, one simultaneously expressing ribitol dehydrogenase (RDH) and formate dehydrogenase (FDH) in fusion (fusion expression strain for short) and the other expressing the above two enzymes individually (individual expression strain for short), were respectively constructed and used for allitol bioproduction. The produced allitol was confirmed by HPLC, mass spectrometry, and polarimetry. The individual expression strain had higher activity, which produced 58.5 g/L allitol from 90 g/L d-allulose (also named d-psicose) in 1 h with an allitol productivity of 58.5 g/L/h under optimized conditions. The constructed individual expression strain had the highest allitol productivity among the reports. The production process developed in this study was simple, highly efficient, and had the potential for mass production of allitol. How to cite: Wen X, Lin H, Ren Y, et al. Allitol bioproduction by recombinant Escherichia coli with NADH regeneration system co-expressing Ribitol Dehydrogenase (RDH) and Formate Dehydrogenase (FDH) in individual or in fusion. Electron J Biotechnol 2022;55. https://doi.org/10.1016/j.ejbt.2021.11.007
Bioprocess optimization is important in order to make the bioproduction process more efficient and economic. The conventional optimization methods are costly and less efficient. On the other hand, modeling and computer simulation can reveal the mechanisms behind the phenomenon to some extent, to assist the deep analysis and efficient optimization of bioprocesses. In this chapter, modeling and computer simulation of microbial growth and metabolism kinetics, bioreactor dynamics, bioreactor feedback control will be made to show the application methods and the usefulness of modeling and computer simulation methods in optimization of the bioprocess technology.
Summary Polylactide ( PLA ) is a bio‐based plastic commonly synthesized by chemical catalytic reaction using lactic acid ( LA ) as a substrate. Here, novel LA ‐containing terpolyesters, namely, P[ LA ‐ co ‐3‐hydroxybutyrate (3 HB )‐ co ‐3‐hydroxypropionate (3 HP )], short as PLBP , were successfully synthesized for the first time by a recombinant Escherichia coli harbouring polyhydroxyalkanoate ( PHA ) synthase from Pseudomonas stutzeri (PhaC1 Ps ) with 4‐point mutations at E130D, S325T, S477G and Q481K, and 3‐hydroxypropionyl‐CoA (3 HP ‐CoA) synthesis pathway from glycerol, 3‐hydroxybutyryl‐CoA (3 HB ‐CoA) as well as lactyl‐CoA ( LA ‐CoA) pathways from glucose. Combining these pathways with the PHA synthase mutant phaC1 Ps (E130D S325T S477G Q481K), the random terpolyester P( LA ‐ co ‐3 HB ‐ co ‐3 HP ), or PLBP , was structurally confirmed by nuclear magnetic resonance to consist of 2 mol% LA , 90 mol% 3 HB , and 8 mol% 3 HP respectively. Remarkably, the PLBP terpolyester was produced from low‐cost sustainable glycerol and glucose. Monomer ratios of PLBP could be regulated by ratios of glycerol to glucose. Other terpolyester thermal and mechanical properties can be manipulated by adjusting the monomer ratios. More PLBP applications are to be expected.
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