营养条件对光滑球拟酵母积累α-酮戊二酸的影响

以光滑拟球酵母为研究模型,研究α-酮戊二酸的浓度情况。通过单因素实验得到α-酮戊二酸积累最佳浓度的各单因素条件为：葡萄糖浓度140g／L,NH4Cl浓度5g／L。在碳源（30g/L葡萄糖初始浓度）匮乏条件下加入丙酮酸30g／L,在此条件下丙酮酸转化为α－酮戊二酸的转化率最高达53．7％。以30g／L丙酮酸为唯一碳源时在7L发酵罐中光滑拟球酵母可生成浓度为10．7g/Lα-酮戊二酸,外源丙酮酸的转化率可达66．9％。这一结果表明,T．glabrata具有将丙酮酸转化为α-KG的能力。     

过程优化提高解脂亚洛酵母积累α-酮戊二酸

目前,应用解脂亚洛酵母发酵生产α-酮戊二酸由于产量和底物转化率低、生产周期长等问题,仍未大规模工业化生产。为了解决这些问题,以研究室诱变选育获得的1株高产α-酮戊二酸的解脂亚洛酵母Yarrowia lipolytica WSH-Z06 C3为出发菌株,考察了该菌株在50 L发酵罐中转速、碳酸钙浓度、溶氧以及补料方式(多节点补料、恒速补料)等因素对α-酮戊二酸积累的影响。结果表明,当转速为300 r/min时,α-酮戊二酸和丙酮酸的产量分别为32.4 g/L和19.66 g/L;碳酸钙质量浓度为20 g/L时,α-酮戊二酸的产量提高至38.55 g/L,丙酮酸降低至8.28 g/L;控制溶氧水平在50%时,α-酮戊二酸产量为42.39 g/L,此时丙酮酸为6.22 g/L。比较高初始甘油浓度和不同的补料发酵策略,发现恒速补料效果最好,发酵144 hα-酮戊二酸产量达到66.27 g/L,丙酮酸产量为20.82 g/L。通过上述发酵过程参数的优化,α-酮戊二酸的产量和底物的转化率比未优化前分别提高了67.3%和4.56%,为解脂亚洛酵母工业化生产α-酮戊二酸提供一定参考。     

羰基还原酶产生菌SW2026的产酶条件及其不对称催化还原4＇-氯苯乙酮

以4＇-氯苯乙酮为模型底物,对筛选得到的Candida krusei SW2026的羰基还原酶产酶条件进行研究。结果表明：适宜的发酵培养基组成为甘油50g/L,玉米浆20g／L,KH2PO4 4g／L,MgSO4·7H2O 1．5g／L;适宜的培养条件为温度30℃,初始pH6,摇床转速200r／min,发酵周期48h。在产酶发酵条件下培养的湿细胞对4＇-氯苯乙酮进行不对称还原反应,产物（S）-4＇-氯-α-苯乙醇的产率最高达88．56％,e．e．值稳定在87％左右。     

产酸丙酸杆菌生长特性及5L罐发酵动力学研究

论文在摇瓶水平对产酸丙酸杆菌基本生长特性（温度、pH、摇床转速、接种量、种龄等）、碳源、氮源利用情况、产物抑制及5 L罐发酵动力学进行了研究。结果表明,该菌在32℃,初始pH 6．5,摇床转速150 r/min,接种24 h的种子液,接种量为5％条件下,产酸丙酸杆菌生长及产酸水平达最高值;该菌可利用碳源十分广泛,但对氮源要求比较高,只可利用有机氮源;在不同初始葡萄糖浓度下,产酸丙酸杆菌生长及产酸水平差异不大,无明显底物抑制现象;在2g/L的初始丙酸盐浓度下,该菌生长受到明显抑制;在5L发酵罐中,初始葡萄糖浓度为58．8 g/L,发酵72 h,葡萄糖消耗完全,丙酸终浓度达22．4 g/L,丙酸得率和产率分别达0．381 g/g和0．295 g/（L·h）,丙酸占总酸比例达72．10％。     

发酵法生产L-异亮氨酸的溶氧控制策略

研究了溶氧对Brewibacterium lactofermentation分批发酵生产L-异亮氨酸（Ile）的影响,提出了前10h恒700d/min以维持溶氧在35％以上,10h后调至600r／min以维持溶氧在15％～20％的两阶段供氧控制模式。与对照相比,获得了较高的产率（0．094g／g）和糖耗速度（4．76/L·h）,在较短时间内（52h）获得较高的Ile产量（23．3g／L）,比结果最好的单一搅拌转速（600r/min）提高11．6％。生产强度（0．448d/L·h）比恒定搅拌转速（500、600、700、800r/min）控制下的过程分别提高了83．6％、28．7％、44．9％、35．7％。最后采用代谢通量分析对该结果产生的原因进行了定量解释。     

杆菌JSM1601发酵产核酸的研究

确定了杆菌（Brevibacterium ammoniagenes）JMS1601发酵产核酸的最佳发酵培养基：葡萄糖12％,酵母浸膏1．5％,磷酸二氢钾0．3％。最佳摇瓶培养条件：温度32℃,摇床转速160r／min,接种量（v／v）5％,装液量100ml／500ml。     

提高光滑球拟酵母乙酰辅酶A水平促进α－酮戊二酸合成

[目的]为了了解光滑球拟酵母中乙酰辅酶A含量对其碳代谢及其通量的影响.[方法]将来源于酿酒酵母中编码乙酰辅酶A合成酶ACS2基因过量表达于发酵法生产丙酮酸的生产菌株Torulopsis glabrata中,获得了一株乙酰辅酶A合成酶活性提高9.2倍(1.20 U/mg protein)的重组菌T. glabrataACS2-1.[结果]与出发菌株WSH-IP303相比,重组菌T glabrataACS2-1:(1)能以乙酸为唯一碳源在胞内积累0.94 mmol/(L·g DCW)的L酰辅酶A;(2)以葡萄糖为唯一碳源时胞内乙酰辅A浓度、α-酮戊二酸产量和Cα-KG,Cpyr是出发菌株WSH-IP303的3.22、2.05和2.52倍;(3)在葡萄糖培养基中添加4 g/L 乙酸,使乙酰辅酶A浓度、α-酮戊二酸产量和CαKG>>/Cpyr是出发菌株WSH-IP303的4.55、2.47和3.75倍,α-酮戊二酸浓度达到17.8 g/L.[结论]这一结果表明,改变细胞内关键辅因子的浓度能使碳代谢流的流向与通量发生改变,从积累丙酮酸转向过量积累α-酮戊二酸.     

高浓度糖发酵制备乙醇

以树干毕赤酵母为发酵菌株,混合糖（木糖、葡萄糖）为发酵底物,通过培养基和培养条件的改变来确定树干毕赤酵母高糖浓度发酵时所需的条件。研究结果表明：在24h发酵周期内初始木糖质量浓度为63．0g／L较适宜;在36h发酵周期内初始木糖质量浓度为72．0g/L较适宜。24h发酵周期内,在36．0g／L木糖中添加的葡萄糖质量浓度以54．0g/L为最佳,发酵结束乙醇质量浓度达32．9g／L;36h发酵周期内,添加的葡萄糖质量浓度以72．0g／L为最佳,发酵结束乙醇质量浓度为36．9g／L。以（NH4）2SO4为N源时较适合戊糖发酵制备乙醇,（NH2）2SO4的最佳质量浓度为1．1g／L。发酵前8h摇床转速为90r／min,后16h为150r／min,乙醇质量浓度较高,可达17．5g/L。     

过表达氨基葡萄糖脱氨酶对大肠杆菌氨基葡萄糖合成及中心碳代谢的影响

考察过表达氨基葡萄糖脱氨酶对氨基葡萄糖合成及大肠杆菌（Escherichia coli）中心碳代谢的影响。实验结果表明：过表达氨基葡萄糖脱氨酶使得在36 g/L葡萄糖,pH为9.0的发酵条件下,发酵24 h后,重组菌发酵液中氨基葡萄糖、丙酮酸和乙酸的量分别是对照菌Rosetta的2.1、1.48和1.74倍;而乳酸的量为2.53 g/L,对照菌Rosetta发酵液中的乳酸含量未检测到,重组菌发酵液中柠檬酸及α-酮戊二酸的含量分别是Rosetta的2.99和2.73倍。     

瑞拉菌素产生菌GAO-1-GR-2发酵工艺条件优化研究

对瑞拉菌GAO-1-GR-2的发酵条件进行了单因素研究,通过正交试验设计对该菌株的培养基进行了筛选。并对其发酵条件进行了研究,结果表明：有机氮源以酵母粉和黄豆饼粉为宜;碳源以葡萄糖和饴糖最好,但考虑到发酵成苓,应选用葡萄糖2％。发酵培养基应呈中性,以采用自然pH为宜,pH超过8,或小于7,则会抑制发酵产物活性;通气量为1：0．5最好,能降低20％耗氧量;摇床转速140r／min,培养温度30℃,发酵周期72h。抗生素发酵原粉效价能达到4000μg／mL。     

Manipulating the pyruvate dehydrogenase bypass of a multi-vitamin auxotrophic yeast Torulopsis glabrata enhanced pyruvate production

AIMS: To investigate the relationship between the activity of pyruvate dehydrogenase (PDH) bypass and the production of pyruvate of a multi-vitamin auxotrophic yeast Torulopsis glabrata. METHODS AND RESULTS: Torulopsis glabrata CCTCC M202019, a multi-vitamin auxotrophic yeast that requires acetate for complete growth on glucose minimum medium, was selected after nitrosoguanidine mutagenesis of the parent strain T. glabrata WSH-IP303 screened in previous study [Li et al. (2001) Appl. Microbiol. Biotechnol. 55, 680-685]. Strain CCTCC M202019 produced 21% higher pyruvate than the parent strain and was genetically stable in flask cultures. The activities of the pyruvate metabolism-related enzymes in parent and mutant strains were measured. Compared with the parent strain, the activity of pyruvate decarboxylase (PDC) of the mutant strain CCTCC M202019 decreased by roughly 40%, while the activity of acetyl-CoA synthetase (ACS) of the mutant increased by 103.5 or 57.4%, respectively, in the presence or absence of acetate. Pyruvate production by the mutant strain CCTCC M202019 reached 68.7 g l(-1) at 62 h (yield on glucose of 0.651 g g(-1)) in a 7-l jar fermentor. CONCLUSIONS: The increased pyruvate yield in T. glabrata CCTCC M202019 was due to a balanced manipulation of the PDH bypass, where the shortage of cytoplasmic acetyl-CoA caused by the decreased activity of PDC was properly compensated by the increased activity of ACS. SIGNIFICANCE AND IMPACT OF THE STUDY: Manipulating the PDH bypass may provide an alternative approach to enhance the production of glycolysis-related metabolites.     

一种基于途径分析提高丙酮酸产量的育种策略

为进一步提高光滑球拟酵母发酵生产丙酮酸的水平 ,在途径分析的基础上提出了一种组成型降低丙酮酸脱酸酶、但增强乙酰辅酶A合成酶活性的育种策略。通过亚硝基胍诱变 ,获得 1株乙酸需求型突变株CCTCCM2 0 2 0 19,在外加乙酸的培养基中表现出高于出发株 2 1%的丙酮酸生产能力和良好的遗传稳定性。检测突变株CCTCCM2 0 2 0 19中丙酮酸代谢相关酶的活性发现 :(1)丙酮酸脱羧酶活性降低了 4 0 % ;(2 )外加乙酸与否的条件下 ,乙酰辅酶A合成酶的活性分别提高了 10 3 5 %和 5 7 4 % ;(3)添加乙酸和突变对丙酮酸羧化酶、丙酮酸脱氢酶系、乙醇脱氢酶和乙醛脱氢酶的活性没有显著影响。在含有乙酸的培养基中突变株细胞干重比出发株高 2 1 7% ,可能是因为乙酰辅酶A合成酶活性的提高 ,补充了因丙酮酸脱羧酶活性降低而引起的胞质乙酰辅酶A短缺。在 7L罐中含有 6g L乙酸钠的培养基中发酵 6 2h ,丙酮酸产量达到 6 8 7g L ,对葡萄糖的产率为 0 6 5 1g g。     

过量表达NADH氧化酶加速光滑球拟酵母合成丙酮酸

[目的]进一步提高光滑球拟酵母(Torulopsis glabrata)发酵生产丙酮酸的生产强度.[方法]将来源于乳酸乳球菌(Lactococcus lactis)中编码形成水的NADH氧化酶noxE基因过量表达于丙酮酸工业生产菌株T. glabrata CCTCC M202019中,获得了一株NADH氧化酶活性为34.8 U/mg蛋白的重组菌T. glabrata-PDnoxE.[结果]与出发菌株T. glabrata CCTCC M202019相比,细胞浓度、葡萄糖消耗速率和丙酮酸生产强度分别提高了168%、44.9%和12%,发酵进行到36 h葡萄糖消耗完毕.补加50 g/L葡萄糖继续发酵20 h,则使丙酮酸浓度提高到67.2 g/L.葡萄糖消耗速度和丙酮酸生产强度增加的原因在于形成水的NADH氧化酶过量表达,导致NADH和ATP含量分别降低了18.1%和15.8%.而NAD<' 增加了11.1%.[结论]增加细胞内NAD<' 含量能有效地提高酵母细胞葡萄糖的代谢速度及目标代谢产物的生产强度.     

Increasing glycolytic flux in Torulopsis glabrata by redirecting ATP production from oxidative phosphorylation to substrate-level phosphorylation

AIMS: This study aimed at further increasing the pyruvate productivity of a multi-vitamin auxotrophic yeast Torulopsis glabrata by redirecting ATP production from oxidative phosphorylation to substrate-level phosphorylation. METHODS AND RESULTS: We examined two strategies to decrease the activity of F0F1-ATPase. The strategies were to inhibit F0F1-ATPase activity by addition of oligomycin, or to disrupt F0F1-ATPase by screening neomycin-resistant mutant. The addition of 0.05 mmol l(-1) oligomycin to the culture broth of T. glabrata CCTCC M202019 resulted in a significantly decreased intracellular ATP level (35.7%) and a significantly increased glucose consumption rate (49.7%). A neomycin-resistant mutant N07 was screened and selected after nitrosoguanidine mutagenesis of the parent strain T. glabrata CCTCC M202019. Compared with the parent strain, the F0F1-ATPase activity of the mutant N07 decreased about 65%. As a consequence, intracellular ATP level of the mutant N07 decreased by 24%, which resulted in a decreased growth rate and growth yield. As expected, glucose consumption rate and pyruvate productivity of the mutant N07 increased by 34% and 42.9%, respectively. Consistently, the activities of key glycolytic enzymes of the mutant N07, including phosphofructokinase, pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase, increased by 63.7%, 28.8% and 14.4%, respectively. In addition, activities of the key enzymes involved in electron transfer chain of the mutant N07 also increased. CONCLUSIONS: Impaired oxidative phosphorylation in T. glabrata leads to a decreased intracellular ATP production, thereby increasing the glycolytic flux. SIGNIFICANCE AND IMPACT OF THE STUDY: The strategy of redirecting ATP production from oxidative phosphorylation to substrate-level phosphorylation provides an alternative approach to enhance the glycolytic flux in eukaryotic micro-organisms.     

光滑球拟酵母新霉素抗性株加速葡萄糖代谢

为进一步提高光滑球拟酵母发酵生产丙酮酸的生产强度,在能量代谢分析的基础上提出了降低ATP合成酶活性、但不影响NADH氧化的育种策略。通过亚硝基胍诱变,获得一株新霉素抗性突变株N07,该菌株F1ATPase活性降低65%、丙酮酸产量高于48gL且单位细胞消耗葡萄糖能力提高38%。添加双环己基碳二亚胺(DCCD)、叠氮钠(NaN3)、新霉素显著降低出发株F1ATPase活性但不影响突变株F1ATPase活性。突变菌株胞内ATP含量下降23.7%导致生长速率和最终菌体浓度(为出发菌株的76%)均低于出发菌株,但葡萄糖消耗速度和丙酮酸生产速度分别提高34%和42.9%,发酵周期缩短12h。进一步研究发现,突变株糖酵解途径中关键酶磷酸果糖激酶、丙酮酸激酶和磷酸甘油醛激酶的活性提高了63.7%、28.8%和14.4%,电子传递链关键酶活性提高10%。结果表明降低真核微生物F1ATPase活性有效地提高了糖酵解关键酶活性而加速葡萄糖代谢。     

Enhancement of pyruvate productivity in Torulopsis glabrata: Increase of NAD+ availability

This study aimed at increasing the pyruvate productivity from a multi-vitamin auxotrophic yeast Torulopsis glabrata, by increasing the availability of NAD+. We examined two strategies for increasing availability of NAD+. To supplement nicotinic acid (NA), the precursor of NAD+; and to increase the activity of alcohol dehydrogenase integrating with addition acetaldehyde as exterior electron acceptor. The addition of 8 mg l(-1) NA to the fermentation medium resulted in a significant increase in the glucose consumption rate (48.4%) and the pyruvate concentration (29%). An ethanol-utilizing mutant WSH-13 was screened and selected after nitrosoguanidine mutagenesis of the parent strain T. glabrata CCTCC M202019. Compared with the parent strain, the alcohol dehydrogenase activity of the mutant WSH-13 increased about 110% and the mutant could utilize ethanol as the sole carbon source for growth (1.8 g l(-1) dry cell weight). When growing with glucose, the addition of 4 mg l(-1) acetaldehyde to the mutant WSH-13 culture broth led to a significant increase in the glucose consumption rate (26.3%) and pyruvate production (22.5%), but the ratio of NADH/NAD+ decreased to 0.22. Acetaldehyde did not affect the glucose and energy metabolism at high dissolved oxygen (DO) concentration. However, at lower DO concentration (20%), maintaining the acetaldehyde concentration in the mutant culture broth at 4 mg l(-1) caused an increased NAD+ concentration but a decreased NADH concentration. As a consequence, the pyruvate production rate, the pyruvate yield on glucose and the pyruvate concentration were 68, 44 and 45% higher, respectively, than the corresponding values of the control (without acetaldehyde). The strategy for increasing the glycolytic flux and the pyruvate productivity in T. glabrata by increasing the availability of NAD+ may provide an alternative approach to enhance the metabolites productivity in yeast.     

添加TCA循环中间产物加速光滑球拟酵母积累丙酮酸

在维生素限制的条件下,研究了添加TCA循环中间产物对光滑球拟酵母多重维生素营养缺陷型菌株CCTCC M202019生长和积累丙酮酸的影响。该菌株能以TCA循环中间产物为唯一碳源进行生长,且在以葡萄糖、乙酸和TCA循环中间产物为复合碳源的平板上菌落数高于分别以葡萄糖和乙酸或TCA循环中间产物为唯一碳源时的菌落数。与其它TCA循环中间产物相比,草酰乙酸更能促进细胞的生长、提高丙酮酸产量和对葡萄糖的得率。草酰乙酸能够促进细胞生长,是因为T. glabrata CCTCC M202019菌株能够利用乙酸作为乙酰辅酶A供体。在含有100 g/L葡萄糖和6 g/L乙酸钠的培养基中再添加10 g/L草酰乙酸进行分批发酵实验,可使菌体浓度从11.8 g/L提高到 13.6 g/L,增长幅度为15%;丙酮酸对葡萄糖的得率(0.66 g/g)以及生产强度(1.19 g·L^{-1<、sup>·h-1<、sup>)分别高出6%和24%,使发酵结束时间提前8～12h。}     

降低光滑球拟酵母电子传递链活性加速丙酮酸合成

光滑球拟酵母CCTCCM2 0 2 0 19经溴化乙锭诱变 ,挑选假阳性呼吸缺陷型菌株共 4 0株。对其中 7株丙酮酸产量提高的突变株进行发酵性底物 (葡萄糖 )和非发酵性底物 (甘油、乙酸 )的利用能力测试 ,鉴定得到 3株呼吸缺陷型突变株RD 16、RD 17和RD 18。相对于出发菌株 ,呼吸缺陷型突变株生长速率下降 ,最终菌体浓度降低 2 1%～2 9% ,胞内ATP含量下降 15 %～ 2 1% ,但单位细胞耗葡萄糖能力和单位细胞产丙酮酸能力分别提高了 2 0 7%～30 7%和 30 7%～ 5 5 5 %。进一步研究发现 ,呼吸缺陷型突变株线粒体复合体Ⅰ、Ⅰ Ⅲ、Ⅱ Ⅲ和Ⅳ的活性分别下降了 34%～ 4 1%、38 6 %～ 5 2 6 %、2 1%～ 2 5 %、15 0 %～ 6 30 % ,表明线粒体电子传递链氧化NADH的功能受到抑制。为使酵解产生的NADH正常氧化 ,在RD 18菌株的对数生长期流加 2 1mmol L外源电子受体乙醛。发现细胞合成丙酮酸能力提高 2 1 6 % ,且葡萄糖消耗速度明显加快 ,发酵周期缩短 14h。结果表明适当削弱能量代谢能够提高真核微生物中心代谢途径的速度