Isolation And Identification Of Halophilic Microorganisms And Construction Of Salt Tolerant Engineered Zymomonas Mobilis

With many excellent metabolic characteristics,Zymomonas mobilis is a desirable ethanologenic host. However, Z. mobilis is sensitive to inorganic salt inhibitors added to or produced from fermentable lignocellulosic hydrolysates and biodiesel westes,resulting in low growth rate and reduced ethonal yield. In order to develop salt tolerant Z. mobilis, salt resistant functional genes from halophilic strains were introduced into Z. mobilis in this study. Main conclusions were as follows:(1) A new species of halophilic Lentibacillus was isolated from sea sediment of Bohai Sea at Yantai City and identified by polyphase taxonomy methods, which was designated as Lentibacillus amyloliquefaciens. The 16 S rDNA sequence of type strain LAM0015 T was deposited in NCBI with the access number KJ002449. The type strain is available in Agricultural Culture Collection of China(ACCC 06401) and Japan Collection of Microorganisms(JCM 19838).(2) The complete genome sequence of L. amyloliquefaciens LAM0015 T was deposited in NCBI(access number CP013862) and published. The genome harbors 3,858,520 baseparis(bp), with an average G+C content of 42.12% and contains 2496 predicted CDSs and 169 ncRNA genes. It was found that multiple gene products related to osmotic balance are existed in the genome, such as aspartate, alanine, betaine, glutamate, glycine betaine, glycerol, mechanosensitive ion channel protein, choline, etc. This information will pave the way for further elucidation of halophilic mechanism and wider exploitation of functional genes.(3) Encoding genes for betaine and ectoine were cloned from strain L. amyloliquefaciens LAM0015 T. Several potential genes for salt tolerance were obtained from the genome library under NaCl stress, such as ABC transporter protein gene, TetR family transcription regulatory factor gene, penicillin binding protein gene, which were verified the function of improving the salt resistance in E. coli.(4) One mutant with improved salt tolerance was isolated from random mutation library of Z. mobilis ZM4 via Tn5 transposon system. The mutant was designated as Z. mobilis ZMT2(access number CGMCC No. 11888). The transposon insertion site was located in ZMO1122(himA) which was identified by genome walking method. Previous reports did not show that ZMO1122 played a role in responding to salt stress. In order to understand the relationship between himA and salt tolerance, gene himA was knocked out from ZM4, resulting strain designated as Δ1122.(5) Data of fermentation showed that ZMT2 had higher ethanol product, higher growth rate and faster glucose utilization than wild strain under NaCl stress. ZMT2,Δ1122 and ZM4 exhibited no obvious difference under low salt stress(<1.5% NaCl) in RM medium. When growing in RM medium containing 2% glucose and 1.5% NaCl for 10 h, ZMT2 utilized almost all the glucose, meanwhile Δ1122 and ZM4 only used 67.15% and 69.70% of total glucose, respectively, and used up all the glucose till 20 h. In comparison, after the cultivation for 40 h in RM medium containing 10% glucose and 1.5% NaCl, OD600 values of ZMT2 and Δ1122 were 1.62 and 1.74 times higher, glucose utilizations of ZMT2 and Δ1122 were 1.46 and 1.54 times higher, and ethanol products were 2.21 and 2.35 times higher than that of ZM4, respectively. When 2% NaCl was supplemented, however, OD600 values of ZMT2 and Δ1122 were 1.15 and 1.13 times higher than that of ZM4 in 2% glucose RM medium while 1.17 and 1.13 times higher than ZM4 in 10% glucose RM medium.(6) Real-time RT-PCR results showed that the transcription of ZMO1122 gene in ZM4 under 2% glucose and 1.5% NaCl stress was about 6 times than that without salt(0.56±0.17),1%(0.55±0.39) and 2%(0.54±0.28) NaCl. While no expression was detected in ZMT2 and Δ1122 with or without salt stress, which was in agreement with the conclusion that ZMO1122 gene was disrupted. With NaCl concentration increasing in a certain range, pdc and adhB gene expressions were up- and down-regulated respectively. PDC and ADHB activities showed similar trend with corresponding genes expression. Quantification of NADH/NAD+ showed that a low NADH/NAD+ ratio provided salt stress tolerance when exposed in salt stress, while higher ratio may facilitate to counteract the disruption of himA in ZMT2 and Δ1122 without salt stress.