Strain engineering of Bacillus coagulans with high osmotic pressure tolerance for effective L-lactic acid production from sweet sorghum juice under unsterile conditions.

Open unsterile fermentation of the low-cost non-food crop, sweet sorghum, is an economically feasible lactic acid biosynthesis process. However, hyperosmotic stress inhibits microbial metabolism and lactic acid biosynthesis, and engineering strains with high osmotic tolerance is challenging. Herein, heavy ion mutagenesis combined with osmotic pressure enrichment was used to engineer a hyperosmotic-tolerant Bacillus coagulans for L-lactic acid production. The engineered strain had higher osmotic pressure tolerance, when compared with the parental strain, primarily owing to its improved properties such as cell viability, cellular antioxidant capacity, and NADH supply. In a pilot-scale open unsterile fermentation using sweet sorghum juice as a feedstock, the engineered strain produced 94 g/L L-lactic acid with a yield of 91 % and productivity of 6.7 g/L/h, and optical purity of L-lactic acid at the end of fermentation was 99.8 %. In short, this study provided effective and low-cost approach to produce polymer-grade L-lactic acid.

Isolation and Screening of High-Yielding α-Amylase Mutants of Bacillus subtilis by Heavy Ion Mutagenesis.

To improve fermentative production of α-amylase, heavy-ion mutagenesis technology was used to irradiate Bacillus subtilis (B. subtilis) to obtain the high yielding mutants in this study. After continuous cultivation for 12 generations, eight mutants exhibited positive mutation rate with greater H/C. The α-amylase production was stable and obviously exceeded that by the parent strain, which shows that the mutants have a good genetic stability. Among the mutants, the α-amylase activity of B. subtilis KC-180-2 was 72.26 U·mL-1, which was 82.34% higher than that of the original strain. After optimization of fermentation conditions and media, the α-amylase activity of B. subtilis KC-180-2 reached a maximum of 156.83 U·mL-1 at 36 h in a bioreactor. In addition, the optimized fermentation temperature of B. subtilis KC-180-2 was increased to 49℃, indicating B. subtilis KC-180-2 possesses high-temperature resistance, which has great application prospects for industrial fermentation for α-amylase production.

Heavy-ion mutagenesis significantly enhances enduracidin production by Streptomyces fungicidicus.

To improve fermentative production of enduracidin, heavy-ion beams generated by the Heavy Ion Research Facility in Lanzhou (HIRFL), China, were employed for the first time to generate mutations in Streptomyces fungicidicus. Initial screening detected 44 positive mutants with larger inhibition zone, which were subsequently tested based on flask fermentation. Finally, 20 mutants showed 20% increase in enduracidin production, when compared with the original strain. Among them, enduracidin production by the three mutants (M13, M30, and M34) was significantly higher than that by the original strain. In particular, mutant M30 exhibited highest enduracidin production, which was 114% higher than that obtained with the original strain. Following culture optimization, the maximal enduracidin yield obtained by M30 reached 918.5 mg/L in 10 days, which was 34% higher than that noted in the control.

Heavy ion mutagenesis combined with triclosan screening provides a new strategy for improving the arachidonic acid yield in Mortierella alpina.

BACKGROUND: Arachidonic acid (ARA), which is a ω-6 polyunsaturated fatty acid, has a wide range of biological activities and is an essential component of cellular membranes in some human tissues. Mortierella alpina is the best strain for industrial production of ARA. To increase its yield of arachidonic acid, heavy ion beam irradiation mutagenesis of Mortierella alpina was carried out in combination with triclosan and octyl gallate treatment. RESULTS: The obtained mutant strain F-23 ultimately achieved an ARA yield of 5.26 g L- 1, which is 3.24 times higher than that of the wild-type strain. In addition, quantitative real-time PCR confirmed that the expression levels of fatty acid synthase (FAS), Δ5-desaturase, Δ6-desaturase, and Δ9-desaturase were all significantly up-regulated in the mutant F-23 strain, especially Δ6- and Δ9-desaturase, which were up-regulated 3- and 2-fold, respectively. CONCLUSIONS: This study confirmed a feasible mutagenesis breeding strategy for improving ARA production and provided a mutant of Mortierella alpina with high ARA yield.

Effects of heavy-ion beam irradiation on avermectin B1a and its analogues production by Streptomyces avermitilis.

The biggest challenge in anabolism research is to improve the stability and safety of microbial metabolite production on an industrial scale. One class of metabolites, avermectins, are produced by Streptomyces avermitilis. In this study, an avermectin B1a-high-producing mutant was produced using heavy ion mutagenesis and selected based on LTQ-MS and HPLC-UV method. The mutants ZJAV-Y-147 and ZJAV-Y-HS, obtained after subjecting the spores of S. avermitilis to 70 Gy of 12C6+ heavy ion irradiation, were found to best improve the avermectin B1a production (4822.23 µg/mL and 4632.17 µg/mL, respectively). These two mutants' yielded of avermectin B1a were 2-fold high than the original strains. The DNA of the original and mutant strains were analyzed by RAPD technique with four random primers after irradiated with ion beam irradiation. The results show that different high-titer S. avermitilis strains contain different genetic modifications. In addition, the mutation position, mutation type and sequence context of all mutations of aveC, aveD, aveI, aveR gene in two mutants S.avermitilis were researched, and the production of avermectin B1a and its analogues of wild-type and mutants were analyzed by fermenting 240 h, which was suggested that the partial base deletion of aveI gene may be the key sites for increasing avermectin B1a production after the 12C6+-ion irradiation. All these modifications promote increased avermectin biosynthesis, leading to multiple high-titer S. avermitilis strains. The results demonstrate that this is an effective approach to engineer S. avermitilis as a host for the biological production of commercial analogs.

Enhanced production of l-lactic acid by Lactobacillus thermophilus SRZ50 mutant generated by high-linear energy transfer heavy ion mutagenesis.

The aim of this study was to improve l-lactic acid production of Lactobacillus thermophilus SRZ50. For this purpose, high efficient heavy-ion mutagenesis technique was performed using SRZ50 as the original strain. To enhance the screening efficiency for high yield l-lactic acid producers, a scale-down from shake flask to microtiter plate was developed. The results showed that 24-well U-bottom MTPs could well alternate shake flasks for L. thermophilus cultivation as a scale-down tool due to its a very good comparability to the shake flasks. Based on this microtiter plate screening method, two high l-lactic acid productivity mutants, A59 and A69, were successfully screened out, which presented, respectively, 15.8 and 16.2% higher productivities than that of the original strain. Based on fed-batch fermentation, the A69 mutant can accumulate 114.2 g/L l-lactic acid at 96 h. Hence, the proposed traditional microbial breeding method with efficient high-throughput screening assay was proved to be an appropriate strategy to obtain lactic acid-overproducing strain.