Huimin Yu

Mutagenesis of the Bacterial RNA Polymerase Alpha Subunit for Improvement of Complex Phenotypes

ABSTRACT Combinatorial or random methods for strain engineering have been extensively used for the improvement of multigenic phenotypes and other traits for which the underlying mechanism is not fully understood. Although the preferred method has traditionally been mutagenesis and selection, our laboratory has successfully used mutant transcription factors, which direct the RNA polymerase (RNAP) during transcription, to engineer complex phenotypes in microbial cells. Here, we show that it is also possible to impart new phenotypes by altering the RNAP core enzyme itself, in particular through mutagenesis of the alpha subunit of the bacterial polymerase. We present the use of this tool for improving tolerance of Escherichia coli to butanol and other solvents and for increasing the titers of two commercially relevant products, l-tyrosine and hyaluronic acid. In addition, we explore the underlying physiological changes that give rise to the solvent-tolerant mutant.

A high-throughput screen for hyaluronic acid accumulation in recombinant Escherichia coli transformed by libraries of engineered sigma factors

Hyaluronic acid (HA) is an important biomaterial with functional medical and cosmetic applications. As its synthesis has been recently reported in recombinant bacteria, it is of interest to develop a high throughput screening method for the rapid isolation of HA accumulating strains transformed by combinatorial libraries. Here we report a novel two‐step screening strategy to select for better HA‐producing recombinant Escherichia coli strains transformed by mutation libraries of rpoD and rpoS, coding for the σD and σS factors of the RNA polymerase, respectively. The first screen, based on translucent colony morphology identification, was used to qualitatively distinguish HA‐producing strains on agar plates from non‐HA producing strains that exhibit dense colony morphology. The second screen was based on the photometric measurement of an alcian blue staining solution that precipitates with HA, creating an inverse relationship between HA concentration and alcian blue absorbance. The color attenuation fitted a second‐order polynomial between HA concentration and OD540 absorbance. Using the alcian blue absorbance quantification, 74 translucent colonies from the HA‐rpoD library and 78 translucent colonies from the HA‐rpoS library were isolated and cultured for optimal strain selection. Three representative superior recombinants with high, medium and low increase of HA accumulation, respectively, were identified by the screen from the HA‐rpoD and HA‐rpoS mutant library. Further flask culture confirmed that results of the library screen were reliable and the superior recombinant D72 highly accumulated HA of 561.4 mg/L with a productivity of ∼265 mg HA/g dry cell. Sequencing results showed that the mutant rpoD gene in D72 is in a truncated protein that lacks the conserved regions 3 and 4 of the σD. Generally, this two‐step high throughput screen presents a promising strategy for selecting superior HA‐producing strains from large scale mutation libraries. Biotechnol. Bioeng. 2008;101: 788–796.

Insights into thermal stability of thermophilic nitrile hydratases by molecular dynamics simulation.

Thermal stability is of great importance for industrial enzymes. Here we explored the thermal-stable mechanism of thermophilic nitrile hydratases (NHases) utilizing a molecular dynamic simulation. At a nanosecond timescale, profiles of root mean square fluctuation (RMSF) of two thermophilic NHases, 1UGQ and 1V29, under enhancing thermal stress were carried out at 300 K, 320 K, 350 K and 370 K, respectively. Results showed that the region A1 (211-231 aa) and A2 (305-316 aa) in 1UGQ, region B1 (186-192 aa) in 1V29, and most of terminal ends in both enzymes are hyper-sensitive. Salt-bridge analyses revealed that in one hand, salt-bridges contributed to maintaining the rigid structure and stable performance of the thermophilic 1UGQ and 1V29; in the other hand, salt-bridges involved in thermal sensitive regions are relatively weak and prone to be broken at elevated temperature, thereby cannot hold the stable conformation of the spatial neighborhood. In 1V29, region A1 was stabilized by a well-organized hook-hook like cluster with multiple salt-bridge interactions, region A2 was stabilized by two strong salt-bridge interactions of GLU52-ARG332 and GLU334-ARG332. In 1UGQ, the absence of a charged residue decreased its thermal sensitivity of region B1, and the formation of a small beta-sheet containing a stable salt-bridge in C-beta-terminal significantly enhanced its thermal stability. By radius of gyration calculation containing or eliminating the thermal sensitive regions, we quantified the contribution of thermal sensitive regions for thermal sensitivity of 1UGQ and 1V29. Consequently, we presented strategies to improve thermal stability of the industrialized mesophilic NHase by introducing stable salt-bridge interactions into its thermal sensitive regions.

Improving stability of nitrile hydratase by bridging the salt-bridges in specific thermal-sensitive regions.

The regions and types suitable mutations for bridging salt-bridges to intensify enzyme stability are identified in this study. Using nitrile hydratase (NHase) as the model enzyme, three deformation-prone thermal-sensitive regions (A1, A2 and A3 in β-subunit), identified by RMSF calculations of the thermophilic NHase 1V29 from Bacillus SC-105-1 and 1UGQ from Pseudonocardia thermophila JCM3095, were determined and the stabilized salt-bridge interactions were transferred into the corresponding region of industrialized mesophilic NHase-TH from Rhodococcus ruber TH. Three types of salt bridges-active-center-adjacent (in A1), internal neighboring-residue-bridged (in A2) and C-terminal-residue-bridged (A3)-were constructed in NHase-TH. The engineered NHase-TH-A1 showed reduced expression of β-subunit, reduced activity and irregular stability. NHase-TH-A2 exhibited a enhanced expression of β-subunit but complete loss of activity; while NHase-TH-A3 exhibited not only a slightly enhanced expression of β-subunit and enzyme activity, but also a 160% increase in thermal stability, a 7% enhanced product tolerance and a 75% enhanced resistance to cell-disruption by ultrasonication. The molecular dynamic (MD) simulation revealed that NHase-TH-A3, with a moderate RMSD value, generates 10 new salt bridges in both internal-subunit and interfacial-subunit, confirming that a C-terminal salt-bridge strategy is powerful for enzyme stability intensification through triggering global changes of the salt bridge networks.

Identification of lipopeptide isoforms by MALDI-TOF-MS/MS based on the simultaneous purification of iturin, fengycin, and surfactin by RP-HPLC.

A three-stage linear gradient strategy using reverse-phase high-performance liquid chromatography (HPLC) was optimized for rapid, high-quality, and simultaneous purification of the lipopeptide isoforms of iturin, fengycin, and surfactin, which may differ in composition by only a single amino acid and/or the fatty acid residue. Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) was applied to detect the lipopeptides harvested from each reversed-phase HPLC peak. Amino acid analysis based on phenyl isothiocyanate derivatization was further used for confirmation of the amino acid species and molar ratio in a certain HPLC fraction. By this MALDI-TOF-MS/MS coupled with amino acid analysis, it was revealed that iturin at m/z 1,043 consists of a circular Asn-Tyr-Asn-Gln-Pro-Asn-Ser peptide and C14 β-OH fatty acid. Surfactin homologs from Bacillus subtilis THY-7 at m/z 1,030, 1,044, 1,058, and 1,072 possess a circular Glu-Leu-Leu-Val-Asp-Leu-Leu peptide and the β-OH fatty acid with a different length (C13–C16). Fengycin species at m/z 1,463 and 1,477 are homologs possessing the circular peptide Glu-Orn-Tyr-Thr-Glu-Ala-Pro-Gln-Tyr-Ile linked to a C16 or C17 γ-OH fatty acid, whereas fengycin at m/z 1,505 contains a Glu-Orn-Tyr-Thr-Glu-Val-Pro-Gln-Tyr-Ile sequence with a Val instead of Ala at position 6. The method developed in this work provided an efficient approach for characterization of diverse lipopeptide isoforms from the iturin, fengycin, and surfactin families.

Identification of nitrile hydratase-producing Rhodococcus ruber TH and characterization of an amiE-negative mutant.

Microbial transformation of acrylonitrile to acrylamide by nitrile hydratase is of great interest to green chemistry. During the transformation, acrylic acid is generally accumulated as a by-product through biocatalysis of amidase. A novel strain with high nitrile hydratase activity was isolated from soil and identified as Rhodococcus ruber TH by morphology and 16S rRNA gene analysis. An amidase-negative recombinant, R. ruber TH3 was constructed. Its nitrile hydratase activity was 25% higher than that of the wild type, reaching 490+/-29.2U/mg dry cell weight, while the amidase activity was 60% lower. After 6 h hydration of acrylonitrile using free cells as biocatalysts at 18 degrees C, acrylamide production by TH3 was 23% higher and the production of the by-product, acrylic acid, was 87% lower than that of the wild type. This result demonstrates that the strain TH3 could be valuable for industrial applications.

Construction and selection of the novel recombinant Escherichia coli strain for poly(β-hydroxybutyrate) production

Heterogeneous cloning of Vitreoscilla hemoglobin gene (vgb), lytic genes of phage lambda with S amber mutation (S(-)RRz) and PHB biosynthetic genes (phbCAB) in the same host strain E. coli JM105 was carried out for production of poly(beta-hydroxybutyrate) (PHB). A superior novel strain, VG1 (pTU14), was constructed and selected, which contained the vgb gene in the chromosomal DNA and the plasmid pTU14 containing S(-)RRz and phbCAB genes. When cultured in 100 ml of LBG medium in a 300-ml flask, all of the exogenous genes in VG1 (pTU14) were expressed. The cell concentration of VG1 (pTU14) grown by batch culture in a flask reached 10.2 gl(-1); PHB concentration, PHB content and PHB yield, which is the ratio of the PHB accumulation to the glucose consumption, were 8.54 gl(-1), 84% and 0.43 gg(-1), respectively. When cultured by batch-feeding of glucose in a 300-ml shaking flask, the cell concentration and PHB content reached 26 gl(-1) and over 96%, respectively.

Hydration of acrylonitrile to produce acrylamide using biocatalyst in a membrane dispersion microreactor

In this work, a membrane dispersion microreactor was utilized for the hydration of acrylonitrile to produce acrylamide. Through observation using a microscopy, it was found that the acrylonitrile was dispersed into the continuous phase (the aqueous phase contains nitrile hydratase (NHase)) as droplets with a diameter ranged from 25 to 35 μm, hence the mass transfer specific surface area was significantly increased, and the concentration of acrylamide reached 52.5 wt% within 50 min. By contrast, in stirred tanks, the concentration of acrylamide only got 39.5 wt% within 245 min. Moreover, only a few amounts of acrylonitrile were accumulated in this microreactor system. Through optimizing the flow rate, the concentration of acrylamide reached 45.8 wt% within 35 min, the short reaction time greatly weakened the inhibition of acrylonitrile and acrylamide on the enzyme activity, which is suitable for prolonging the life of free cell.

Overexpression of synthesized cephalosporin C acylase containing mutations in the substrate transport tunnel.

Cephalosporin C (CPC) acylase converts CPC into 7-aminocephalosporanic acid (7-ACA) by single-step enzymatic catalysis. An optimized CPC acylase gene with substituted codons and a reduced GC content was artificially designed, synthesized and overexpressed in recombinant Escherichia coli. The synthetic CPC acylase (sCPCAcy) exhibited 2.3 times more CPC specific deacylation activity with substrate CPC than with substrate glutaryl-7-ACA (GL-7-ACA). Site-directed mutagenesis of the residues around the active center showed that not only the residues that were adjacent to the CPC D-α-aminoadipyl moiety, but also the residues that were in the substrate transport tunnel (Leu666, Ala675, Leu677), played crucial roles in catalysis as the ones locating in active center. Mutant sCPCAcy(Leu666Phe) and sCPCAcy(Leu677Ala) exhibited significantly reduced specific enzymatic activity, while mutant sCPCAcy(Ala675Gly) demonstrated enhanced activity. The specific activity of purified sCPCAcy and sCPCAcy(Ala675Gly) was 10.0 U/mg and 11.3 U/mg, respectively. The optimal CPC acylase productivity of mutant sCPCAcy(Ala675Gly) reached 5349 U/l after 24h in culture, which was a 35% increase over the activity of sCPCAcy.

Effect of poly(β-hydroxybutyrate) accumulation on the stability of a recombinant plasmid in Escherichia coli

Poly(beta-hydroxybutyrate) (PHB) production using a recombinant Escherichia coli VG1 (pTU14) was carried out. PHB granules were gradually biosynthesized in recombinant cells as inclusion bodies and the morphology of cells became linearly inflated with PHB accumulation from 6 h to 36 h of culture. The stability of plasmid pTU14 was inevitably affected by in-cell PHB accumulation. During repeated subcultures under both PHB-accumulating and non-PHB-accumulating conditions, plasmid DNAs of pTU14 were extracted and analyzed after more than 60 generations. The results showed that plasmid pTU14 is structurally stable but segregationally unstable, and that the segregational instability worsened with increasing accumulation of PHB granules. This is due to not only the serious metabolic burden of plasmid replication and gene expression on the host cell, but also the volume effects of PHB granules on the natural random distribution of plasmids during the binary fission of cells.