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Guo Y.,Heze University | Guo Y.,Jiangnan University | Xu J.,Jiangnan University | Han M.,Jiangnan University | And 2 more authors.
World Journal of Microbiology and Biotechnology | Year: 2015

Isoleucine synthesis is strongly regulated by its end product (isoleucine) in Corynebacterium glutamicum, especially at threonine dehydratase (TD) node. Multiple alignments of TD sequences of C.glutamicum and other sources were performed. According to the structural analysis, three TD variants were constructed by site-directed mutagenesis. These TD variants improved the performance of the holoenzyme. The specific activity of V140M variant was 1.5-fold higher than that of the wild-type TD, whereas F383A variant showed complete resistance to feedback inhibition by isoleucine. V140M-F383A variant had all the advantages of V140M and F383A variants and displayed 1.5-fold specific activity and complete resistance to isoleucine. In C. glutamicum, overexpression of V140M, F383A, and V140M-F383A variants accumulated 0.55, 0.63, and 0.73 g/l isoleucine, and overexpression of wild-type TD produced 0.47 g/l isoleucine. Thus, these novel TD variants, particularly V140M-F383A, showed great potential in isoleucine synthesis. © 2015, Springer Science+Business Media Dordrecht.

Zhai Y.-G.,Jiangnan University | Han M.,Technology Center Bright Dairy and Food Co. | Zhang W.-G.,Jiangnan University | Qian H.,Jiangnan University
Preparative Biochemistry and Biotechnology | Year: 2014

Industrial waste substrates, sugarcane molasses, and corn steep liquor were used for production of carotenes by Arthrobacter globiformis in this study. At the first stage, a one-factor-at-a-time approach was used for optimization of different media components such as carbon, nitrogen, MgSO4 · 7H2O, and KH2PO4, as well as pH, temperature, liquid medium volume, and inoculums level. The response surface method was further applied to determination of optimum values of process variables for maximum carotenes concentration. Results showed that the optimum combination for carotenes formation was as follows (g/L): sugarcane molasses, 40.00; corn steep liquor, 50.00; MgSO4 · 7H2O, 0.75; KH 2PO4, 1.00. The maximum carotene concentration of 1.19 ± 0.02 mg/g dry biomass, about 113% of 1.05 ± 0.02 mg/g dry biomass growing in basal medium, was demonstrated by confirmatory experiments to be the optimum in liquid medium at 100 rpm, 30°C, initial pH of 7.5, and cultivation for 60 hr. In a second stage, detailed studies showed about 1.64-fold and 1.43-fold increase in carotene concentration (mg/g dry biomass) in the presence of addition of ethanol (4%, v/v) and addition of hydrogen peroxide (4%, v/v) at 40 hr, and 32 hr in liquid medium, separately. Copyright © 2014 Taylor & Francis Group, LLC.

Guo Y.,Jiangnan University | Han M.,Jiangnan University | Han M.,Technology Center Bright Dairy and Food Co. | Yan W.,Jiangnan University | And 2 more authors.
Biotechnology and Bioprocess Engineering | Year: 2014

In Corynebacterium glutamicum, acetohydroxy acid synthase (AHAS, encoded by ilvBN) is regulated by the end products in biosynthesis pathway, which catalyzes the first common reaction in the biosynthesis of branched-chain amino acids (BCAAs). In this study, conserved A42, A89 and K136 residues in AHAS regulatory subunit were chosen for site-directed mutagenesis, and the resulting mutations A42V, A89V and K136E exhibited higher resistance to inhibition by BCAAs than wild type AHAS. Furthermore, double-mutation was carried out on A42V, A89V and K136E mutations. Expectedly, A42V-A89V mutation exhibited nearly complete resistance to inhibition by all three BCAAs, which retained above 93% enzyme activity even at 10 mM. Strains were further studied to investigate the effects of over-expressing different mutant ilvBN on the biosynthesis of BCAAs. It was found that production of BCAAs was increased with the increase of resistance to BCAAs. However, the increase of isoleucine and leucine was slower than valine which showed a significant increase (up to 86.30 mM). Furthermore, strains harboring plasmids with different mutant ilvBN could significantly decrease production of alanine (main byproduct). This work gives additional understanding of roles of A42, A89 and K136 residues and makes the A42V, A89V, K136E and A42V-A89V mutations a good starting point for further development by protein engineering. © 2014 The Korean Society for Biotechnology and Bioengineering and Springer-Verlag.

Guo Y.,Jiangnan University | Han M.,Jiangnan University | Han M.,Technology Center Bright Dairy and Food Co. | Xu J.,Jiangnan University | Zhang W.,Jiangnan University
Protein Expression and Purification | Year: 2015

Acetohydroxy acid synthase (AHAS) controls carbon flux through the branch point and determines the relative rates of the synthesis of isoleucine, valine and leucine, respectively. However, it is strongly regulated by its end products. In this study, we characterized AHAS variants from five branched-chain amino acids-producing strains. Amino acid substitution occurred in both catalytic subunit and regulatory subunit. Interestingly, AHAS variants reduced sensitivity to feedback inhibition by branched-chain amino acids (BCAAs). Although AHAS with amino acid substitution in regulatory subunit showed higher resistance, amino acid substitution in catalytic subunit could also endow AHAS with resistance to feedback inhibition. In addition, AHAS variants from V2 and L5 displayed about 1.4-fold higher specific activity compared to other AHAS variants. On the other hand, AHAS variant from V1 exhibited the highest resistance to BCAAs, 87% of original activity left even in the presence of 10 mM BCAAs. Recombinant Corynebacterium glutamicum strains were further constructed to investigate the effects of expressing AHAS variants on the synthesis of BCAAs and alanine (main by-product) in C. glutamicum. BCAAs production was increased with the increase of resistance to feedback inhibition, although valine showed a significant increase. For instance, C. g-1BN could accumulate 9.51 g/l valine, 0.450 g/l leucine and 0.180 g/l isoleucine, and alanine was reduced to 0.477 g/l. These AHAS variants are important for further improving performance of BCAAs-producing strain. © 2015 Elsevier Inc.

Han M.,Jiangnan University | Han M.,Technology Center Bright Dairy and Food Co. | Xu Z.-Y.,Technology Center Bright Dairy and Food Co. | du C.,Jiangnan University | And 2 more authors.
Bioprocess and Biosystems Engineering | Year: 2016

Nitrogen limited but carbon excess condition was used to obtain high cellular lipid content and production. The maximum lipid production was 51 g/L, the lipid content in the dry cell was 60 %, and the lipid productivity was 0.53 g/L/h. In the fermentation, the content of lipid was raised from 20 % of dry cell weight to 60 %, and the proportion of oleic acid was raised from 66.8 to 72.5 %. Meanwhile, the metabolism of carotenoids switched to torulene, and its proportion was raised from 30 to 58 %. This was according to torulene had the better antioxidant ability than β-carotene to protect the strain from oxidative damage proved by their ABTS* radical scavenging activity and lipid peroxidation inhibition ability. Sporidiobolus pararoseus lipid was a good source of lipid not only because of its high oleic acid composition, but also the antioxidant ability of carotenoids in the lipid. © 2016 Springer-Verlag Berlin Heidelberg

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