衣康酸生产菌种的定向选育和产酸条件的研究*

通过紫外线 高温复合诱变处理衣康酸生产菌株土曲霉AspergillusterreusAs 3 2 81 1 ,用以琥珀酸为唯一碳源的选择性平板定向筛选高产菌株 ,获得产酸率较其亲株提高了 5倍以上的突变株。用正交试验的方法对突变株的适宜产酸条件进行了研究 ,通过分批补糖发酵可提高其产酸率高达 39 92 %。     

烷烃发酵产生1，13-十三碳二元酸及其诱导作用的研究

从能利用正十二烷产生1,12-十二碳二元酸的热带假丝酵母突变株D28出发,经两次紫外线照射诱变,选育到一株从正十三烷产生1,13-十三碳二元酸较高的突变株2—23号菌。该突变株较出发菌株提高产酸率20％,达40．4g／L。突变株2—23也能将一定链长的长链烷烃以较高的产率转变成相应的单一二元酸。此外,在产酸摇瓶条件试验中观察到烷烃的诱导作用,使突变株产酸能力得以提高。用烷烃预培养的种子发酵正十三烷,其产生1,13一十三碳二元酸的量较糖质碳源培养的种子发酵时要提高30％。     

烷烃发酵产生1，13-十三碳二元酸及其诱导作用的研究

从能利用正十二烷产生1,12-十二碳二元酸的热带假丝酵母突变株D28出发,经两次紫外线照射诱变,选育到一株从正十三烷产生1,13-十三碳二元酸较高的突变株2—23号菌。该突变株较出发菌株提高产酸率20％,达40．4g／L。突变株2—23也能将一定链长的长链烷烃以较高的产率转变成相应的单一二元酸。此外,在产酸摇瓶条件试验中观察到烷烃的诱导作用,使突变株产酸能力得以提高。用烷烃预培养的种子发酵正十三烷,其产生1,13一十三碳二元酸的量较糖质碳源培养的种子发酵时要提高30％。     

L-谷氨酸温度敏感突变株的选育

采用黄色短杆菌TJ1为出发菌株,根据代谢控制发酵原理,利用紫外线、硫酸二乙酯进行诱变,定向选育出具有寡霉素抗性、谷氨酸氧肟酸盐抗性的温度敏感突变株TMGO106。然后,以温度敏感突变株TMGO106和产酸率高（10.5%以上）的天津短杆菌TG961为新株,通过原生质体融合技术,成功地选育出了产酸率高的融合子CN1021（13．6g/dl,糖酸转化率达60％）,在6m^3发酵罐上中试其L－谷氨酸产量达14．6％,糖酸转化率达62．8％,并且该菌株系温度敏感型菌株,可用于谷氨酸强度发酵。     

离子注入结合紫外线及~(60)Co-γ射线诱变选育碱性果胶酸裂解酶高产菌株的研究

以碱性果胶酸裂解酶产生菌芽孢杆菌WZ008为出发菌株,经形态鉴定和16S鉴定为类芽孢杆菌,命名为Paenibacillus sp.WZ008,通过N~+注入诱变、紫外线诱变、~(60)Co-γ射线诱变等多次反复诱变,选育得到一株产碱性果胶酸裂解酶性能稳定且酶活明显提高的突变株,其酶活为97.8U/mL,比出发菌株产碱性果胶酸裂解酶能力提高了1.04倍。     

L-丝氨酸高产菌株的选育和摇瓶发酵条件优化

采用B revibacterium flavmC-11A为出发菌株,经紫外线照射和亚硝基胍诱变处理,选育出一株L-丝氨酸高产菌株C32为目的突变株,使摇瓶产酸率由12.1 g.L-1增加到16.4 g.L-1,然后对其进行摇瓶发酵条件优化,使菌株C32的L-丝氨酸产率提高到30.1 g.L-1。     

产生二羧酸的耐高温酵母菌的研究

野生株热带假丝酵母(Candida tropicalis)1230能在40℃生长,而其产二羧酸的突变株U3-21则不能在这样高的温度下生长和产酸。用高温(40℃)直接处理的方法从突变株U3-21．获得能在该温度下产酸的耐高温株,其机率为亿分之三。这些菌株在高温下的产酸能力绝大多数相近,经筛选获得在40℃产酸为3％以上的耐高温株MHT39-90该菌株经室温转接保存三年,高温产酸性能稳定。     

D-核糖生产菌的选育

将枯草芽胞杆菌通过紫外线诱变得到了莽草酸缺陷突变株,在２８株突变株中有１０株积累Ｄ－核糖。这些菌株均属戊糖磷酸途径的非氧化支路缺失突变株。对这些菌株的产核糖能力进行了验证、培养基中芳香族氨基酸的浓度影响Ｄ－核糖的积累     

L-乳酸高产菌株的选育和产酸条件的研究

为了获得更适于工业生产的产乳酸菌株,采用低能离子诱变方法,对出发菌米根霉(Rhizopus oryzae)PW352进行改良,获得高产L( )．乳酸菌株RE3303,其产酸比亲株提高75％。用正交试验方法对突变株适宜产酸条件进行了研究,在最优条件下,其产酸量可达131～136g/L,最高可达140g/L,糖转化率为86％-90％。     

L-乳酸高产菌株的选育和产酸条件的研究*

为了获得更适于工业生产的产乳酸菌株 ,采用低能离子诱变方法 ,对出发菌米根霉(Rhizopusoryzae)PW35 2进行改良 ,获得高产L ( + ) 乳酸菌株RE330 3,其产酸比亲株提高75 %。用正交试验方法对突变株适宜产酸条件进行了研究 ,在最优条件下 ,其产酸量可达1 31～ 1 36g L ,最高可达 1 40g L ,糖转化率为 86%～ 90 %。     

定向选育衣康酸高产菌株的研究

以衣康酸生产菌土曲霉Ａ９００２为出发菌株,经紫外线及亚硝基胍复合诱变后再行定向选育,即在以衣康酸为唯一碳源的培养基中富集不能同化衣康酸的菌种,将将它们涂布在含乌头酸酶抑制剂（单氟醋酸）的高糖、高衣康酸平板培养基上,最后从中而筛选出一支衣康酸氧化酶弱,乌头酸酶活强,并耐自身代谢产物的高产突变株Ａ９００３。此菌株在摇瓶培养７２ｈ后,产酸为９．２％,转化率为５８．１％,在１００ｍ＾３发酵罐生产性试验中,     

以淀粉为碳源衣康酸高产菌株的筛选

对土曲霉出发菌株进行紫外线诱变、LiCl诱变以及代谢终产物抗性菌株选育。代谢终产物抗性菌株选育是一种有效的遗传育种方法,能显著提高产酸量。得到一株代号为At394的菌株,以玉米淀粉部分水解糖为碳源,产酸量为53.9g/L,比出发菌株提高了42.6%。糖酸转化率为61.5%,为所有筛选菌株最高。用红外光谱进行结构分析证实所得产物为衣康酸。     

Temperature‐dependent dynamic control of the TCA cycle increases volumetric productivity of itaconic acid production by Escherichia coli

Based on the recently constructed Escherichia coli itaconic acid production strain ita23, we aimed to improve the productivity by applying a two‐stage process strategy with decoupled production of biomass and itaconic acid. We constructed a strain ita32 (MG1655 ΔaceA Δpta ΔpykF ΔpykA pCadCs), which, in contrast to ita23, has an active tricarboxylic acid (TCA) cycle and a fast growth rate of 0.52 hr^?1 at 37°C, thus representing an ideal phenotype for the first stage, the growth phase. Subsequently we implemented a synthetic genetic control allowing the downregulation of the TCA cycle and thus the switch from growth to itaconic acid production in the second stage. The promoter of the isocitrate dehydrogenase was replaced by the Lambda promoter (p_R) and its expression was controlled by the temperature‐sensitive repressor CI857 which is active at lower temperatures (30°C). With glucose as substrate, the respective strain ita36A grew with a fast growth rate at 37°C and switched to production of itaconic acid at 28°C. To study the impact of the process strategy on productivity, we performed one‐stage and two‐stage bioreactor cultivations. The two‐stage process enabled fast formation of biomass resulting in improved peak productivity of 0.86 g/L/hr (+48%) and volumetric productivity of 0.39 g/L/hr (+22%) in comparison to the one‐stage process. With our dynamic production strain, we also resolved the glutamate auxotrophy of ita23 and increased the itaconic acid titer to 47 g/L. The temperature‐dependent activation of gene expression by the Lambda promoters (p_R/p_L) has been frequently used to improve protein or, in a few cases, metabolite production in two‐stage processes. Here we demonstrate that the system can be as well used in the opposite direction to selectively knock‐down an essential gene (icd) in E. coli to design a two‐stage process for improved volumetric productivity. The control by temperature avoids expensive inducers and has the potential to be generally used to improve cell factory performance.

     

Production of (S)-(+)-citramalic acid from itaconic acid by resting cells of Alcaligenes denitrificans strain MCI2775

(S)-(+)-Citramalic-acid-producing activity in microorganisms was studied with resting cells in a reaction mixture containing itaconic acid. Itaconic-acid-utilizing bacteria were found to produce (S)-(+)-citramalic acid from itaconic acid. The strain, which showed the best productivity among those studied, was identified taxonomically as Alcaligenes denitrificans strain MCI2775. (S)-(+)-Citramalic acid produced by this strain was present in a 99.9% enantiometric excess. The culture and reaction conditions for the production were optimized for this strain. Addition of Mn²⁺, d-pantothenic acid and l-leucine to the culture medium enhanced the (S)-(+)-citramalic acid-producing activity. Under optimal conditions, 27 g (S)-(+)-citramalic acid/l was produced in 30 h. The yield to itaconic acid added was 69.0 mol%. Correspondence to: Y. Asano     

Process development of itaconic acid production by a natural wild type strain of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> to reach industrially relevant final titers

Itaconic acid is a promising organic acid and is commercially produced by submerged fermentation of Aspergillus terreus. The cultivation process of the sensitive filamentous fungus has been studied intensively since 1932, with respect to fermentation media components, oxygen supply, shearing rate, pH value, or culture method. Whereas increased final titers were achieved over the years, the productivity has so far remained quite low. In this study, the impact of the pH on the itaconic acid production was investigated in detail. The pH during the growth and production phase had a significant influence on the final itaconic acid concentration and pellet diameter. The highest itaconic acid concentration of 160 g/L was achieved at a 1.5-L scale within 6.7 days by raising and controlling the pH value to pH 3.4 in the production phase. An ammonia solution and an increased phosphate concentration were used with an itaconic acid yield of 0.46 (w/w) and an overall productivity of 0.99 g/L/h in a fed-batch mode. A cultivation with a lower phosphate concentration resulted in an equal final concentration with an increased yield of 0.58 (w/w) after 11.8 days and an overall productivity of 0.57 g/L/h. This optimized process was successfully transferred from a 1.5-L scale to a 15-L scale. After 9.7 days, comparable pellet morphology and a final concentration of 150 g/L itaconic acid was reached. This paper provides a process strategy to yield a final titer of itaconic acid from a wild-type strain of A. terreus which is in the same range as the well-known citric acid production.     

Efficient Itaconic acid production via protein–protein scaffold introduction between GltA,AcnA, and CadA in recombinant Escherichia coli

Itaconic acid, which is a promising organic acid in synthetic polymers and some base-material production, has been produced by Aspergillus terreus fermentation at a high cost. The recombinant Escherichia coli that contained the cadA gene from A. terreus can produce itaconic acid but with low yield. By introducing the protein–protein scaffold between citrate synthesis, aconitase, and cis-aconitase decarboxylase, 5.7 g/L of itaconic acid was produced, which is 3.8-fold higher than that obtained with the strain without scaffold. The optimum pH and temperature for itaconic acid production were 8.5 and 30°C, respectively. When the competing metabolic network was inactivated by knock-out mutation, the itaconic acid concentration further increased, to 6.57 g/L.     

Enhanced production of itaconic acid from corn starch and market refuse fruits by genetically manipulated Aspergillus terreus SKR10

A potent itaconic acid producing strain, Aspergillus terreus SKR10, was isolated from horticulture waste. Market refuse, apple and banana, were explored as novel substrates for itaconic acid production with yields of 20+/-2.0 and 20.0+/-1.0 g l(-1), respectively. Itaconic acid yields of 28.5+/-2.2 and 31.0+/-1.7 g l(-1) were obtained with acid and alpha-amylase hydrolyzed corn starch. The efficiency of itaconic acid production by this wild type strain was improved by ultraviolet, chemical and mixed mutagenic treatments. Two high itaconic acid yielding mutants, N45 and UNCS1 were obtained by gradient plating. These two mutants were capable of producing twice the yield of itaconic acid as the parent strain.     

Itaconic acid production from wheat chaff by Aspergillus terreus

Biotechnologically produced itaconic acid is an important building block for the chemical industry and still based on pure carbon sources, detoxified molasses or starch hydrolysates. Changing these first generation feedstocks to alternative renewable resources of a second generation implies new challenges for the cultivation process of the industrial itaconic acid producer Aspergillus terreus, which is known to be very sensitive towards impurities. To select a suitable pretreatment method of a second generation feedstock, the influences of different hydrolysate components, like monosaccharides and sugar degradation products, were tested. Particular the impact of those components on itaconic acid yield, productivity, titer and morphology was investigated in detail. Wheat chaff was used as lignocellulosic biomass, which is an agricultural residue. An alkaline pretreatment method with sodium hydroxide at room temperature and a subsequent enzymatic saccharification at pH 4.8 at 50 °C with 10 FPU/g_Biomass Biogazyme 2x proved to be very suitable for a subsequent biotechnological production of itaconic acid. A purification by a cation exchanger of the wheat chaff hydrolysate resulted in a final titer of 27.7 g/L itaconic acid with a yield of 0.41 g/g_{total sugar}.     

离子束注入对衣康酸生产菌种的改良

用10keV、剂量5.2×10~(14)～5.2×10~(15)ions/cm~2的氮离子注入衣康酸生产菌株土曲霉A9003,在2％LiCl抗性平板筛选到一株能在39℃发酵的衣康酸高产菌株。该菌株在30L发酵罐发酵产酸7.5％,转化率60.1％,发酵周期50h。     

Characterizing MttA as a mitochondrial cis-aconitic acid transporter by metabolic engineering

The mitochondrial carrier protein MttA is involved in the biosynthesis of itaconic acid in Aspergillus terreus. In this paper, the transport specificity of MttA is analyzed making use of different metabolically engineered Aspergillus niger strains. Furthermore, the mitochondrial localization of this protein is confirmed using fluorescence microscopy. It was found that MttA preferentially transports cis-aconitic acid over citric acid and does not transport itaconic acid. The expression of MttA in selected A. niger strains results in secretion of aconitic acid. MttA can be used in further strain engineering strategies to transport cis-aconitic acid to the cytosol to produce itaconic acid or related metabolites. The microbial production of aconitic acid (9 g/L) is achieved in strains expressing this transport protein. Thus, metabolic engineering can be used for both the in vivo characterization of transport protein function like MttA and to make use of this protein by creating aconitic acid producing strains.