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Additional file 1: Figure S1. Extracellular accumulation of heme caused by supplement of ALA and FeSO4. Figure S2. Expression of the native hemoglobin genes without codon optimization in C. glutamicum. Figure S3. Linear relationship between the hemoglobin proportion of the total protein and GFP fluorescence/OD600. Table S1. Gene sequences used in this study. Table S2. NCS variants screened in this study. Table S3. Promoter variants screened in this study. Table S5. Strains and plasmids used in this study. Table S6. Primers used for plasmid and library construction in this study.

We summarized two cases of congenital factor V deficiency (FVD) associated with a novel F5 mutation, and analyzed the relationship of the clinical features and genetic characteristics in congenital FVD. Case 1 was a female newborn infant with remarkable bleeding who died of severe intracranial hemorrhage on day 42 after birth. She had significant prolongation of prothrombin time (PT) and activated partial thromboplastin time (APTT). The percentage activity of FV (PFV) was lower than 3% in case 1. The mother of case 1 showed no tendency to bleed. She had mild prolongation of PT and APTT. The PFV was only 43%. Both cases were found to have the same novel mutation in F5, which was c.5419G>A (p.Ala1807Thr) in exon 16. The variant in case 1 was inherited from the mother of case 1. Whole-exome sequencing (WES) also found a splice site mutation: a 103 Mb maternal uniparental disomy (UPD) of 1q21.1-qter in case 1, in which the F5 gene is located in this segment. So case 1 was homozygote and the mother of case 1 was heterozygote. The novel mutation of F5 was predicted to be harmful by bioinformatics software including Sorting Intolerant From Tolerant (SIFT), Polyphen2, LRT, and Mutation Taster. In summary, c.5419G>A (p.Ala1807Thr) in exon 16 of F5 is a pathogenic mutation, which causes severe congenital FVD in homozygote patients.

The introduction of the key non-oxidative glycolytic (NOG) pathway enzyme, phosphoketolases (PKTs), into heterologous hosts can improve the yield of a variety of acetyl CoA-derived products of interest. However, the low specific activity of existing PKTs compared with that of 6-phosphofructokinase (PFK), the key EMP pathway enzyme, largely limits their potential applications. To improve PKT activity, previous attempts have focused on increasing intracellular PKT concentration via the use of strong promoters. Herein, we report the establishment of a growth-coupled evolution strategy for the enrichment and selection of PKT mutants with improved specific activity in Corynebacterium glutamicum hosts with defective PFK. Five mutants from 9 Bifidobacterium adolescentis-source PKT (BA-PKT) mutant libraries were obtained. Site-directed mutagenesis analysis revealed 11 mutant sites which contributed to improved BA-PKT specific activity. Further structural analysis revealed that the mutant sites were located far away from the enzyme active site, which makes them almost unpredictable using a rational design approach. Mutant site recombination led to the construction of a novel mutant, PKTT2A/I6T/H260Y, with Vmax 29.77 ± 1.58 U/mg and Kcat/Km 0.32 ± 0.01 s−1/mM, which corresponds to 73.27 ± 3.25% and 80.16 ± 3.38% improvements, respectively, compared with the wildtype (Vmax; 17.17 ± 0.59 U/mg, Kcat/Km; 0.17 ± 0.01 s−1/mM). Expression of PKTT2A/I6T/H260 in C. glutamicum Z188 resulted in 16.67 ± 2.24% and 18.19 ± 0.53% improvement in l-glutamate titer and yield, respectively, compared with the wildtype BA-PKT. Our findings provide an efficient approach for improving the activity of PKTs. Furthermore, the novel mutants could serve as useful tools in improving the yield of l-glutamate and other acetyl CoA-associated products.

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Whole-cell amino acid biosensors can sense the concentrations of certain amino acids and output easily detectable signals, which are important for construction of microbial producers. However, many reported biosensors have poor specificity because they also sense non-target amino acids. Besides, biosensors for many amino acids are still unavailable. In this study, we proposed a new strategy for constructing whole-cell biosensors based on aminoacyl-tRNA synthetases (aaRSs), which take the advantage of their universality and intrinsically specific binding ability to corresponding amino acids. Taking isoleucine biosensor as an example, we first mutated the isoleucyl-tRNA synthetase in Escherichia coli to dramatically decrease its affinity to isoleucine. The engineered cells specifically sensed isoleucine and output isoleucine dose-dependent cell growth as an easily detectable signal. To further expand the sensing range, an isoleucine exporter was overexpressed to enhance excretion of intracellular isoleucine. Since cells equipped with the optimized whole-cell biosensor showed accelerated growth when cells produced higher concentrations of isoleucine, the biosensor was successfully applied in high-throughput selection of isoleucine overproducers from random mutation libraries. This work demonstrates the feasibility of engineering aaRSs to construct a new kind of whole-cell biosensors for amino acids. Considering all twenty proteinogenic and many non-canonical amino acids have their specific aaRSs, this strategy should be useful for developing biosensors for various amino acids.

Background 5-Aminolevulinic acid (5-ALA) is a promising biostimulant, feed nutrient, and photodynamic drug with wide applications in modern agriculture and therapy. Considering the complexity and low yield of chemical synthesis methods, bioproduction of 5-ALA has drawn intensive attention recently. However, the present bioproduction processes use refined glucose as the main carbon source and the production level still needs further enhancement. Results To lay a solid technological foundation for large-scale commercialized bioproduction of 5-ALA, an industrial workhorse Corynebacterium glutamicum was metabolically engineered for high-level 5-ALA biosynthesis from cheap renewable bioresources. After evaluation of 5-ALA synthetases from different sources, the 5-ALA biosynthetic pathway and anaplerotic pathway were rebalanced by regulating intracellular activities of 5-ALA synthetase and phosphoenolpyruvate carboxylase. The engineered biocatalyst produced 5.5 g/L 5-ALA in shake flasks and 16.3 g/L in 5-L bioreactors with a one-step fermentation process from glucose. To lower the cost of feedstock, cheap raw materials were used to replace glucose. Enzymatically hydrolyzed cassava bagasse was proven to be a perfect alternative to refined sugars since the final 5-ALA titer further increased to 18.5 g/L. Use of corn starch hydrolysate resulted in a similar 5-ALA production level (16.0 g/L) with glucose, whereas use of beet molasses caused seriously inhibition. The results obtained here represent a new record of 5-ALA bioproduction. It is estimated that replacing glucose with cassava bagasse will reduce the carbon source cost by 90.1%. Conclusions The high-level biosynthesis of 5-ALA from cheap bioresources will brighten the prospects for industrialization of this sustainable and environment-friendly process. The strategy for balancing metabolic flux developed in this study can also be used for improving the bioproduction of other value-added chemicals.

To enhance the thermostability and deregulate the hemin inhibition of 5-aminolevulinic acid (ALA) synthase from Rhodopseudomonas palustris (RP-ALAS) by a computer-aided rational design strategy. Eighteen RP-ALAS single variants were rationally designed and screened by measuring their residual activities upon heating. Among them, H29R and H15K exhibited a 2.3 °C and 6.0 °C higher melting temperature than wild-type, respectively. A 6.7-fold and 10.3-fold increase in specific activity after 1 h incubation at 37 °C was obtained for H29R (2.0 U/mg) and H15K (3.1 U/mg) compared to wild-type (0.3 U/mg). Additionally, higher residual activities in the presence of hemin were obtained for H29R and H15K (e.g., 64% and 76% at 10 μM hemin vs. 27% for wild-type). The ALA titer was increased by 6% and 22% in fermentation using Corynebacterium glutamicum ATCC 13032 expressing H29R and H15K, respectively. H29R and H15K showed high thermostability, reduced hemin inhibition and slightly high activity, indicating that these two variants are good candidates for bioproduction of ALA.

l-Threonine is an important amino acid for animal feed. Though the industrial fermentation technology of threonine achieved a very high level, there is still significant room to further improve the industrial strains. The biosensor-based high-throughput screening (HTS) technology has demonstrated its powerful applications. Unfortunately, for most of valuable fine chemicals such as threonine, a HTS system has not been established mainly due to the absence of a suitable biosensor. In this study, we developed a HTS method to gain high-yielding threonine-producing strains. Novel threonine sensing promoters including cysJp and cysHp were discovered by proteomic analyses of Escherichia coli in response to extracellular threonine challenges. The HTS method was constructed using a device composed of the fused cysJp and cysHp as a promoter and a linked enhanced green fluorescent protein gene as a reporter. More than 400 strains were selected with fluorescence activated cell sorting technology from a library of 20 million mutants and tested within 1 week. Thirty-four mutants have higher productivities than the starting industrial producer. One mutant produced 17.95 % more threonine in a 5-L jar fermenter. This method should play a functional role for continuous improvement of threonine industry. Additionally, the threonine sensor construction using promoters obtained by proteomics analyses is so convenient that it would be easily extended to develop HTS models for other biochemicals.

We disclosed the complete genome sequence of Corynebacterium glutamicum B253, an important lysine-producing strain in China. The genome consists a circular chromosome (3,159,203bp) and a plasmid (24,775bp), encoding 2767 protein coding genes in total. The genome contains all genes for lysine biosynthesis, and some mutations potentially relevant to lysine production were detected in comparison with sequence of other C. glutamicum.

α-Amylase was used as a heterologous model protein to investigate the effects of promoters, signal peptides and over-expression of an extra-cytoplasmic molecular chaperone, PrsA lipoprotein, on enhancing the secretion of α-amylase in Bacillus subtilis. Four promoters and six signal peptides were compared, successively, and the highest yield of α-amylase was achieved under the promotion mediated by PAprE, a strong constitutive promoter, and secretion by SPnprE, a signal peptide from B. subtilis. Moreover, under conditions of overexpressed PrsA lipoprotein, the secretion production and activity of α-amylase increased to 2.5-fold. The performance of the recombinant B. subtilis 1A751PL31 was evaluated with a fed-batch fermentation in a 7.5 l fermentor. Optimization of regulatory elements and over-expression of PrsA lipoprotein had a significant effect on enhancing the production of α-amylase in B. subtilis.