CN112703251A - 控制发酵进料速率的方法 - Google Patents
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- CN112703251A CN112703251A CN201980046477.4A CN201980046477A CN112703251A CN 112703251 A CN112703251 A CN 112703251A CN 201980046477 A CN201980046477 A CN 201980046477A CN 112703251 A CN112703251 A CN 112703251A
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Abstract
本发明涉及用于提高发酵的生产率或产量的方法和组合物。所述方法和组合物提供对培养细胞生成的一种或多种化合物进行监测,作为发酵进料速率的反馈控制。
Description
1.技术领域
本发明涉及使用废气监测来控制或调节发酵培养基的进料速率以及用于所述发酵培养基的设备。
2.发明背景
合成生物学的出现带来了以工业规模和质量由可再生资源发酵微生物生成生物燃料、化学品和生物材料的希望。譬如,已在微生物宿主中成功构建了功能性非天然生物学途径,用于生成抗疟药青蒿素的前体类(参见例如Martin et al.,Nat Biotechnol 21:796-802(2003));脂肪酸衍生的燃料类和化学品类(例如,脂肪酯类、脂肪醇类和蜡类;参见例如Steen et al.,Nature 463:559-562(2010));制备降胆固醇药物的聚酮合酶类(参见例如Ma et al.,Science 326:589-592(2009));和聚酮类化合物(参见例如Kodumal,ProcNatl Acad Sci USA 101:15573-15578(2004))。然而,合成生物学的商业成功在很大程度上将取决于可再生成品的生成成本是否可竞争或优于与其相应的不可再生成品的生成成本。
合成生物学的一些最大成本发生在发酵过程中。所述这些成本大部分用于发酵培养基的成分,包括碳源类、氮源类、水、盐类和营养物类。在常规发酵中,将脉冲糖进料至培养物中,直至检测到溶解氧的峰值,这表明消耗了过量的碳。然后,将新的脉冲糖进料至培养物以开始新的循环。这些脉冲进料和检测溶解氧峰值的循环在整个发酵过程中重复进行,从而导致对进料的低效消耗以及从业人员劳动效率低下。提高发酵产量的方法和组合物将降低总成本,从而使可再生化合物的生成更加有效并更具竞争力。
3.发明摘要
本发明提供了用于向发酵培养基中的微生物培养物提供进料的方法。在一些实施方案,以初始速率提供进料至发酵培养基中的微生物培养物。测定所述发酵培养基的所述废气中挥发性细胞产物的浓度。在一些实施方案,当所述挥发性细胞产物的所述浓度增加时,所述进料速率降低;而当所述挥发性细胞产物的所述浓度降低时,所述进料速率增加。在一些实施方案,当所述挥发性细胞产物的所述浓度降低时,所述进料速率降低;和当所述挥发性细胞产物的所述浓度增加时,所述进料速率增加。有利的是,在特定实施方案,可对所述挥发性细胞产物进行快速或连续测定,和可对所述进料速率进行快速或连续调节。所述这些方法步骤可采用本领域技术人员显而易见的技术和组分来进行。本发明详细描述了特定的技术和组分。
如下文详细描述的,本发明提供的方法和组合物可使微生物菌株的生产率提高多达15%或更多。生产率提高15%可直接改善此种发酵的成本和效率。
4.附图简要说明
图1提供了进料速率(g/L/hr)、乙醇(ppm)、温度(℃)、pH值、体积(L)、摄氧速率(mM/L-min)、搅拌速率(rpm)、pO2(饱和度%)、空气流量(slpm)和平均pO2(饱和度%)的历程图。
图2提供了两种不同菌株的生产率(g/L/hr)与平均废气乙醇浓度(ppm)的函数关系。
图3提供了根据本发明提供的方法用于酵母菌株发酵的乙醇废气浓度。
5.具体实施方式
5.1定义
本发明使用的术语“经遗传修饰的”是指包含异源核苷酸序列的宿主细胞。
本发明使用的术语“异源的/异源性/异源”是指通常在自然界中不存在的物质。术语“异源核苷酸序列”是指自然界中在给定细胞中通常不存在的核苷酸序列。因此,异源核苷酸序列可以是:(a)相对于其宿主细胞是外源的(即,对所述细胞而言是“外源的”);(b)天然存在于所述宿主细胞中(即“内源性/內源的/內源”),但在所述细胞中以非天然量存在(即,比所述宿主细胞中天然存在的量更多或更少);或(c)天然存在于所述宿主细胞中,但位于其天然基因座之外。术语“异源酶”是指自然界中通常在给定细胞中不存在的酶。所述术语包括以下酶:(a)对给定细胞而言是外源的(即,由非天然存在于所述宿主细胞中或不在所述宿主细胞的给定环境中天然存在的核苷酸序列编码);和(b)天然存在于所述宿主细胞中(例如,所述酶由对细胞而言是内源性的核苷酸序列编码),但在所述宿主细胞中以非天然量(例如,大于或小于所述天然存在的量)生成。
另一方面,本发明使用的术语“天然的/原生的(native)”或“内源的/内源性/內源”涉及分子,特别是酶和核酸,表示在它们起源或在自然界中发现的生物体中表达的分子,与表达水平无关,所述表达水平可低于、等于或高于天然微生物体中分子的表达水平。应理解,天然酶或天然多核苷酸的表达可在重组微生物中进行修饰。
本发明使用的术语“生成量”通常是指由本发明提供的宿主细胞生成的化合物的量。在一些实施方案,生成量表示为由所述宿主细胞生成的化合物的产量。在其他实施方案,生成量表示为生成所述化合物时所述宿主细胞的生产率。
本发明使用的术语“生产率/生产力”是指由宿主细胞生成化合物的量,表示为每单位量的发酵液或发酵培养基生成的化合物的量(按重量计),在所述发酵液或发酵培养基中所述宿主细胞根据时间(每小时)进行培养(按体积计)。
本发明使用的术语“产量/产率”是指由宿主细胞生成的化合物的量,表示为所述宿主细胞消耗的每单位量的碳源生成的化合物的量,按重量计。
5.2发明详述
一方面,本发明提供了用于向微生物培养物提供进料化合物的方法。在某些实施方案,所述方法包括下列步骤:将进料化合物以初始速率进料至在发酵培养基中生长的微生物培养物;测定所述发酵培养基废气中挥发性细胞产物的浓度;和调节所述进料化合物向所述发酵培养基的进料速率。在某些实施方案,当所述挥发性细胞产物的所述浓度增加时,所述进料速率降低;而当所述挥发性细胞产物的所述浓度降低时,所述进料速率增加。在一些实施方案,当所述挥发性细胞产物的所述浓度降低时,所述进料速率降低;而当所述挥发性细胞产物的所述浓度增加时,所述进料速率增加。有利的是,在特定实施方案,可对所述挥发性细胞产物进行快速或连续测定,和可对所述进料速率进行快速或连续调节。在某些实施方案,对所述挥发性细胞产物进行连续测定,和对所述进料速率进行连续调节。
所述进料化合物可以是从业者认为对进料发酵培养基有用的任何化合物。在某些实施方案,所述进料化合物选自碳源类、氮源类、盐类、营养物类、及其组合。在特定实施方案,所述进料化合物是碳源。在某些实施方案,所述进料化合物由选自由糖蜜类、玉米浆、蔗汁、和甜菜汁组成的组的来源提供。在某些实施方案,所述进料化合物是糖。在一些实施方案,所述进料化合物是单糖(简单糖类)、二糖、多糖、不可发酵的碳源、或其一种或多种组合。合适的单糖类的非限制性实例包括葡萄糖、半乳糖、甘露糖、果糖、木糖、核糖、及其组合。合适的二糖类的非限制性实例包括蔗糖、乳糖、麦芽糖、海藻糖、纤维二糖、及其组合。合适的多糖类的非限制性实例包括淀粉、糖原、纤维素、几丁质、及其组合。合适的不可发酵碳源类的非限制性实例包括乙酸盐和甘油。
所述挥发性细胞产物可以是由从业者认为合适的微生物培养物生成的任何废气化合物。在特定实施方案,所述挥发性细胞产物是指示生成其的细胞的代谢状态的化合物。在某些实施方案,在所述废气中测定乙醇。在特定实施方案,当所述废气中的所述乙醇浓度增加时,所述进料速率降低,而当所述废气中的所述乙醇浓度降低时,所述进料速率增加。在某些实施方案,在所述废气中测定乙醇,和所述进料化合物是糖。在特定实施方案,当所述废气中的所述乙醇浓度增加时,所述糖的进料速率降低,而当所述废气中的所述乙醇浓度降低时,所述糖的进料速率增加。
本发明提供的方法利用了以下观察的优势:发酵中的乙醇可以是微生物培养物中细胞进料消耗的简便标志物。有利的是,可通过标准技术和组分对废气中的乙醇进行常规测定。根据本发明提供的发现,通过调节进料速率以保持废气中乙醇浓度恒定或接近恒定,与标准技术相比,微生物培养物可以更佳的速率进行生长。此举可提高效率、产量和生产率。如下文实施例所示,采用本发明提供的方法,其产量得到了定量提高。
在某些实施方案,所述进料速率保持在所述初始进料速率的范围内。所述范围可以是本领域技术人员认为合适的任何范围。在某些实施方案,所述进料速率保持在所述初始进料速率的±75%、±50%、±25%、±15%、±10%、或±5%以内。所述进料速率可根据标准技术进行调节。在某些实施方案,所述进料速率保持在0.01至25g/L/hr内。在某些实施方案,所述进料速率保持在0.1至25g/L/hr内。在某些实施方案,所述进料速率保持在1至25g/L/hr内。所述进料速率通常表示为每单位时间每升发酵培养基中进料化合物的质量。在典型的实施方案中,增加或降低进料溶液流入发酵培养基的流量以增加或降低所述进料速率。
在某些实施方案,所述微生物培养物的所述摄氧速率保持在所述初始摄氧速率的范围内。所述范围可以是本领域技术人员认为合适的任何范围。在某些实施方案,所述摄氧速率保持在所述初始摄氧速率的±75%、±50%、±25%、±15%、±10%、或±5%以内。在某些实施方案,所述微生物培养物的摄氧速率保持在1-150mmolO2/L/hr内。在某些实施方案,所述微生物培养物的摄氧速率保持在10-150mmol O2/L/hr内。在某些实施方案,所述微生物培养物的摄氧速率保持在25-150mmolO2/L/hr内。摄氧速率可通过标准技术来保持,例如鼓泡和/或搅拌。
在某些实施方案,所述废气中的所述挥发性细胞产物保持在一定范围内。所述范围可以是本领域技术人员认为合适的任何范围。在某些实施方案,所述挥发性细胞产物保持在目标量的±75%、±50%、±25%、±15%、±10%、或±5%以内。在某些实施方案,所述挥发性细胞产物是乙醇,和所述废气中的乙醇保持在约600ppm。在某些实施方案,所述废气中的乙醇保持在50-750ppm。在某些实施方案,所述废气中的乙醇保持在100-200ppm。在某些实施方案,所述废气中的乙醇保持在200-300ppm。在某些实施方案,所述废气中的乙醇保持在250-350ppm。在某些实施方案,所述废气中的乙醇保持在550-650ppm。在某些实施方案,所述废气中的目标乙醇浓度选自100ppm、150ppm、200ppm、250ppm、300ppm、350ppm、400ppm、450ppm、500ppm、550ppm、600ppm、650ppm、700ppm、和750ppm。
应当根据本发明所述的技术来保持所述挥发性细胞产物的量。换言之,当挥发性细胞产物的量偏离目标值时,应调节发酵的进料速率。在某些实施方案,当挥发性细胞产物的量增加时,应降低进料速率,而当挥发性细胞产物的量降低时,应增加进料速率。在其他实施方案,当挥发性细胞产物的量增加时,应增加进料速率,而当挥发性细胞产物的量降低时,应降低进料速率。在特定实施方案,所述挥发性细胞产物是乙醇。在这些实施方案中,当废气中乙醇的量增加时,应降低进料速率,而当废气中乙醇的量降低时,应提高进料速率。
所述废气可以从业者认为合适的任何频率进行测定。在有利的实施方案中,所述废气以高频进行测定。在特定实施方案,对所述废气进行连续测定。在某些实施方案,以每小时至少一次,每半小时至少一次,每一刻钟至少一次,每10分钟至少一次,每5分钟至少一次,每分钟至少一次,每分钟至少两次,每分钟至少三次,每分钟至少四次,或每分钟至少十次,对所述废气进行测定。在优选实施方案,对所述废气进行连续测定。
所述进料速率可以从业者认为合适的任何频率进行调节。在有利的实施方案中,所述进料速率以高频率进行调节。在特定实施方案,对所述进料速率进行连续调节。通常,在对废气中挥发性细胞产物进行测定之后调节所述进料速率。在某些实施方案,以每小时至少一次,每半小时至少一次,每一刻钟至少一次,每10分钟至少一次,每5分钟至少一次,每分钟至少一次,每分钟至少两次,每分钟至少三次,每分钟至少四次,或每分钟至少十次,对所述进料速率进行调节。在优选实施方案,对所述进料速率进行连续调节。
本发明提供的方法为微生物培养物(例如生成目的化合物的微生物培养物)的发酵,提供了改善的生产率和产量。在某些实施方案,生产率提高了5%、10%、15%、20%、25%、30%、35%、40%、45%、50%、或更多。在某些实施方案,产量提高了5%、10%、15%、20%、25%、30%、35%、40%、45%、50%、或更多。在某些实施方案,生产率和产量提高了。在这些实施方案中,生产率或产量提高是相对于在常规条件下,即未采用本发明所述方法的情况下生长的相同细胞株而言。
本发明所述方法在整个发酵过程中循环了几次。在一些实施方案,本发明所述方法进行3至20天的时间。在一些实施方案,本发明所述方法进行1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20天或超过20天的时间。
5.3细胞培养物、培养基和条件
用于微生物培养物的维持和生长的物料和方法是微生物学或发酵科学领域的技术人员所熟知的(参见,例如Bailey et al.,Biochemical Engineering Fundamentals,second edition,McGraw Hill,New York,1986)。根据细胞培养物、发酵和过程/方法的特定要求,必须考虑适当的培养基,pH值,温度,以及需氧、微需氧或厌氧条件的要求。
本发明提供的方法可在合适的容器(包括但不限于细胞培养板、烧瓶或发酵罐)中在合适的培养基(例如,含或不含泛酸补充)中进行。此外,所述方法可以本领域已知的任何发酵规模进行,以支持微生物产物的工业生产。可使用任何合适的发酵罐,包括搅拌槽发酵罐,气升式发酵罐,气泡发酵罐或其任何组合。在利用酿酒酵母(Saccharomycescerevisiae)作为宿主细胞的特定实施方案中,菌株可在发酵罐中生长,详细记载如Kosaric,et al,Ullmann's Encyclopedia of Industrial Chemistry,Sixth Edition,Volume 12,pages 398-473,Wiley-VCH Verlag GmbH&Co.KDaA,Weinheim,Germany中所述。
在一些实施方案,所述培养基是其中细胞培养物可存活,即保持生长和活力的任何培养基。在一些实施方案,所述培养基是包含可同化的碳源、氮源和磷源(phosphatesources)的水性培养基。此种培养基还可包括适当的盐类、矿物质类、金属类和其他营养物类。
用于培养微生物的合适条件和合适的培养基是本领域熟知的。在一些实施方案,所述合适的培养基补充有一种或多种附加试剂,例如诱导物(例如,当编码基因产物的一个或多个核苷酸序列受诱导型启动子的控制时),阻抑物(例如,当编码基因产物的一个或多个核苷酸序列受阻抑型启动子的控制时),或选择剂(例如,选择包含所述遗传修饰的微生物的抗生素)。
所述培养基中碳源(例如葡萄糖)的浓度应促进细胞生长,但不能高到抑制所用微生物的生长。在优选实施方案,所述碳源在发酵培养基中处于不可检测水平(例如,小于约<0.1g/L)。在此类实施方案中,培养物是碳受限的,并且培养细胞应当在碳源被递送时立即消耗所述碳源。应当注意,对培养组分浓度的提及可以指初始和/或正在进行的组分浓度。在一些情况下,可能需要在培养期间使所述培养基耗尽碳源。
可用于合适培养基的可同化氮的来源包括但不限于简单氮源、有机氮源和复合氮源。此类氮源包括无水氨,铵盐类,以及动物、植物和/或微生物来源的物质。合适的氮源包括但不限于,蛋白质水解产物类,微生物生物质水解产物类,蛋白胨,酵母提取物,硫酸铵,尿素和氨基酸类。通常,所述培养基中所述氮源的浓度大于约0.1g/L,优选大于约0.25g/L,更优选大于约1.0g/L。然而,向所述培养基中添加氮源超过一定浓度对于微生物的生长是不利的。因此,所述培养基中所述氮源的浓度小于约20g/L,优选小于约10g/L,更优选小于约5g/L。此外,在一些实施例,可能需要在培养期间使所述培养基耗尽所述氮源。
有效的培养基可含有其他化合物,例如无机盐类、维生素类、痕量金属类、或生长促进剂类。此类其他化合物亦可存在于有效培养基中的碳源、氮源或矿物源中,或者可特异性地添加至所述培养基中。
所述培养基还可含有合适的磷源。此类磷源包括无机磷源和有机磷源。优选的磷源包括但不限于磷酸盐类,例如单或二元磷酸钠和磷酸钾、磷酸铵、和其混合物。通常,所述培养基中磷酸盐的浓度大于约1.0g/L,优选大于约2.0g/L,更优选大于约5.0g/L。然而,向所述培养基中添加磷酸盐超过一定浓度对于微生物的生长是不利的。因此,所述培养基中所述磷酸盐的浓度通常小于约20g/L,优选小于约15g/L,更优选小于约10g/L。
合适的培养基还可包括镁源,优选地以生理学上可接受的盐的形式,例如七水合硫酸镁,尽管也可使用浓度为贡献相似量的镁的其他镁源。通常,所述培养基中镁的浓度大于约0.5g/L,优选大于约1.0g/L,更优选大于约2.0g/L。然而,向所述培养基中添加镁超过一定浓度对于微生物的生长是不利的。因此,所述培养基中镁的浓度通常小于约10g/L,优选小于约5g/L,更优选小于约3g/L。此外,在一些实施例,可能需要在培养期间使所述培养基耗尽镁源。
在一些实施方案,所述培养基还可包含生物学上可接受的螯合剂,例如二水合柠檬酸三钠。在此类实施例中,所述培养基中螯合剂的浓度大于约0.2g/L,优选大于约0.5g/L,更优选大于约1g/L。然而,向所述培养基中添加螯合剂超过一定浓度对于微生物的生长是不利的。因此,所述培养基中螯合剂的浓度通常小于约10g/L,优选小于约5g/L,更优选小于约2g/L。
所述培养基最初还可包括生物学上可接受的酸或碱以维持所述培养基的所需pH值。生物学上可接受的酸包括但不限于,盐酸、硫酸、硝酸、磷酸、和其混合物。生物学上可接受的碱包括但不限于,氢氧化铵、氢氧化钠、氢氧化钾、和其混合物。在一些实施方案,使用的碱是氢氧化铵。
所述培养基还可包括生物学上可接受的钙源,包括但不限于氯化钙。通常,所述培养基中所述钙源(例如氯化钙二水合物)的浓度在约5mg/L至约2000mg/L的范围内,优选在约20mg/L至约1000mg/L的范围内,更优选在约50mg/L至约500mg/L的范围内。
在一些实施方案,所述培养基还可包含痕量金属。此类痕量金属可作为储备溶液添加至所述培养基中,为方便起见,可将其与培养基的其余部分分开制备。通常,添加至所述培养基中的此痕量金属溶液的量大于约1mL/L,优选大于约5mL/L,更优选大于约10mL/L。然而,向所述培养基中添加痕量金属超过一定浓度对于微生物的生长是不利的。因此,添加至所述培养基中的此痕量金属溶液的量通常小于约100mL/L,优选小于约50mL/L,更优选小于约30mL/L。应注意的是,除了在储备溶液中添加痕量金属之外,各个组分亦可单独进行添加,各自在与上述痕量金属溶液范围所规定的组分的量相对应的范围内。
所述培养基可包括其他维生素类,例如泛酸、生物素、钙、泛酸盐、肌醇、吡哆醇-HCl和硫胺素-HCl。此类维生素可作为储备溶液添加至所述培养基中,为方便起见,可将其与培养基的其余部分分开制备。然而,向所述培养基中添加维生素类超过一定浓度对于微生物的生长是不利的。
本发明所述的发酵方法可以常规培养模式进行,所述培养模式包括但不限于分批、补料分批、细胞再循环、连续和半连续。在一些实施方案,所述发酵以补料分批模式进行。在此种情况下,所述培养基中的一些组分在培养期间被耗尽,所述组分包括在发酵的生成阶段期间的泛酸。在一些实施方案,所述培养物可在开始时(例如,在生成阶段)补充相对高浓度的此类组分,使得在需要添加之前支持生长和/或类异戊二烯生成一段时间。所述这些组分的优选范围在整个培养过程中通过添加来维持,所述添加以培养物耗尽的水平进行添加。可通过例如定期对培养基取样并测定浓度来监测所述培养基中组分的水平。或者,一旦开发出标准培养程序,所述添加可在整个培养期间的特定时间以对应于已知水平的时间间隔进行。如本领域技术人员将认识到的,随着所述培养基的细胞密度增加,培养期间营养物的消耗速率亦将增加。此外,为了避免将外来微生物引入培养基中,可使用本领域已知的无菌添加方法进行添加。此外,在培养期间可加入少量消泡剂。
所述培养基的温度可以是适于经遗传修饰的细胞生长和/或类异戊二烯生成的任何温度。譬如,在用接种物接种培养基之前,所述培养基可置于并保持在约20℃至约45℃的温度范围,优选置于并保持在约25℃至约40℃的温度范围,更优选置于并保持在约28℃至约32℃的温度范围。
可通过向所述培养基中添加酸或碱来控制所述培养基的pH值。在此种情况下,当使用氨来控制pH值时,其在所述培养基中也可方便地作为氮源。优选地,所述pH值保持在约3.0至约8.0,更优选保持在约3.5至约7.0,最优选保持在约4.0至约6.5。
在一些实施方案,在培养期间监测所述培养基的碳源浓度,例如葡萄糖浓度。可使用已知技术监测所述培养基的葡萄糖浓度,例如,采用葡萄糖氧化酶试验或高压液相色谱,其可用于监测上清液(例如,所述培养基的无细胞组分)中的葡萄糖浓度。如本发明其他地方所述,根据本发明提供的方法来调节所述碳源的进料速率。使用原始培养基的等分试样可能是合乎需要的,因为可同时维持培养基中某些营养物(例如,氮源类和磷源类)的浓度。同样,通过添加痕量金属溶液的等份试样,亦可在所述培养基中维持所述痕量金属浓度。
5.4细胞
细胞培养物可包含本领域技术人员认为有用的任何细胞。在本发明提供的组合物和方法中有用的细胞包括古细菌细胞、原核细胞或真核细胞。在某些实施方案,所述细胞是重组的,其包含一种或多种异源核酸。在某些实施方案,所述细胞是宿主细胞,其包含一种或多种异源核酸,所述一种或多种异源核酸编码能够催化生成目的化合物的一种或多种酶。
合适的原核细胞包括但不限于,多种革兰氏阳性、革兰氏阴性或革兰氏变种细菌中的任一种。实例包括但不限于,属于以下属的细胞:土壤杆菌属(Agrobacterium),脂环酸芽孢杆菌属(Alicyclobacillus),鱼腥藻属(Anabaena),蓝细菌属(Anacystis),节细菌属(Arthrobacter),固氮菌属(Azobacter),芽孢杆菌属(Bacillus),短杆菌属(Brevibacterium),着色菌属(Chromatium),梭菌属(Clostridium),棒状杆菌属(Corynebacterium),肠杆菌属(Enterobacter),欧文氏菌属(Erwinia),埃希氏杆菌属(Escherichia),乳酸杆菌属(Lactobacillus),乳球菌属(Lactococcus),中慢生根瘤菌属(Mesorhizobium),甲基杆菌属(Methylobacterium),细杆菌属(Microbacterium),席藻属(Phormidium),假单胞菌属(Pseudomonas),红细菌属(Rhodobacter),红假单胞菌属(Rhodopseudomonas),红螺菌属(Rhodospirillum),红球菌属(Rhodococcus),沙门氏菌属(Salmonella),栅藻属(Scenedesmun),沙雷氏菌属(Serratia),志贺氏菌属(Shigella),葡萄球菌属(Staphlococcus),链霉菌属(Strepromyces),聚球蓝细菌属(Synnecoccus)和发酵单胞菌属(Zymomonas)。原核菌株的实例包括但不限于:枯草芽孢杆菌(Bacillussubtilis),解淀粉芽孢杆菌(Bacillus amyloliquefacines),产氨短杆菌(Brevibacterium ammoniagenes),嗜氨短杆菌(Brevibacterium immariophilum),拜氏梭菌(Clostridium beigerinckii),阪崎肠杆菌(Enterobacter sakazakii),大肠杆菌(Escherichia coli),乳酸乳球菌(Lactococcus lactis),百脉根根瘤菌(Mesorhizobiumloti),绿脓假单胞菌(Pseudomonas aeruginosa),迈氏假单胞菌(Pseudomonasmevalonii),普迪卡假单胞菌(Pseudomonas pudica),荚膜红细菌(Rhodobactercapsulatus),类球红细菌(Rhodobacter sphaeroides),深红红螺菌(Rhodospirillumrubrum),肠道沙门氏菌(Salmonella enterica),伤寒沙门氏菌(Salmonella typhi),鼠伤寒沙门氏菌(Salmonella typhimurium),痢疾志贺氏菌(Shigella dysenteriae),福氏志贺菌(Shigellaflexneri),宋内志贺菌(Shigella sonne)和金黄色葡萄球菌(Staphylococcus aureus)。在特定实施方案,所述细胞是大肠杆菌(Escherichia coli)细胞。
合适的古细菌细胞包括但不限于属于以下属的细胞:气火菌属(Aeropyrum),古球菌属(Archaeglobus),盐杆菌属(Halobacterium),产甲烷球菌属(Methanococcus),甲烷杆菌属(Methanobacterium),火球菌属(Pyrococcus),硫化叶菌属(Sulfolobus),和热原体属(Thermoplasma)。古细菌菌株的实例包括但不限于:闪烁古生球菌(Archaeoglobusfulgidus),盐杆菌属(Halobacterium sp.),詹氏甲烷球菌(Methanococcus jannaschii),嗜热自养甲烷杆菌(Methanobacterium thermoautotrophicum),嗜酸热原体(Thermoplasma acidophilum),火山热原体(Thermoplasma volcanium),掘越氏热球菌(Pyrococcus horikoshii),深海火球菌(Pyrococcus abyssi),和敏捷气热菌(Aeropyrumpernix)。
合适的真核细胞包括但不限于真菌细胞、藻类细胞、昆虫细胞和植物细胞。在一些实施方案,可用于本发明方法的酵母包括已被微生物保藏中心(例如,IFO、ATCC等)保藏并属于以下属的酵母:芽孢酵母属(Aciculoconidium),神食酵母属(Ambrosiozyma),节束酵母属(Arthroascus),Arxiozyma,阿舒囊霉属(Ashbya),Babjevia,本森顿酵母属(Bensingtonia),Botryoascus,Botryozyma,酒香酵母属(Brettanomyces),布勒掷孢酵母属(Bullera),布勒担孢酵母属(Bulleromyces),假丝酵母属(Candida),固囊酵母属(Citeromyces),棒孢酵母属(Clavispora),隐球菌属(Cryptococcus),产黑色素酵母属(Cystofilobasidium),德巴利氏酵母属(Debaryomyces),德克酵母属(Dekkara),拟双足囊菌属(Dipodascopsis),双足囊菌属(Dipodascus),Eeniella,Endomycopsella,Eremascus,假囊酵母属(Eremothecium),担孢酵母属(Erythrobasidium),Fellomyces,线黑粉酵母属(Filobasidium),耐碱酵母属(Galactomyces),地丝菌属(Geotrichum),季氏酵母属(Guilliermondella),有孢汉逊酵母属(Hanseniaspora),汉逊酵母属(Hansenula),Hasegawaea,胶珊瑚属(Holtermannia),Hormoascus,生丝毕赤酵母属(Hyphopichia),伊萨酵母属(Issatchenkia),克勒克酵母属(Kloeckera),孢克勒克酵母属(Kloeckeraspora),克鲁维酵母属(Kluyveromyces),Kondoa,Kuraishia,克氏担孢酵母属(Kurtzmanomyces),白冬孢酵母属(Leucosporidium),油脂酵母属(Lipomyces),路德酵母属(Lodderomyces),马拉色氏霉菌属(Malassezia),梅奇酵母属(Metschnikowia),木拉克酵母属(Mrakia),油脂酵母属无性属(Myxozyma),拿逊酵母属(Nadsonia),Nakazawaea,针孢酵母属(Nematospora),甲醇诱导型酵母属(Ogataea),卵孢酵母属(Oosporidium),管囊酵母属(Pachysolen),厚壁孢酵母(Phachytichospora),法夫酵母属(Phaffia),毕赤酵母属(Pichia),红冬孢酵母属(Rhodosporidium),红酵母属(Rhodotorula),酵母属(Saccharomyces),类酵母属(Saccharomycodes),覆膜孢酵母属(Saccharomycopsis),齐藤酵母属(Saitoella),Sakaguchia,Saturnospora,裂芽酵母孢子菌属(Schizoblastosporion),裂殖酵母属(Schizosaccharomyces),许旺酵母属(Schwanniomyces),锁掷酵母属(Sporidiobolus),掷孢酵母属(Sporobolomyces),原孢酵母属(Sporopachydermia),冠孢酵母属(Stephanoascus),梗孢酵母属(Sterigmatomyces),拟梗孢酵母属(Sterigmatosporidium),Symbiotaphrina,合轴酵母属(Sympodiomyces),Sympodiomycopsis,有孢圆酵母属(Torulaspora),Trichosporiella,毛孢子菌属(Trichosporon),三角酵母属(Trigonopsis),Tsuchiyaea,Udeniomyces,Waltomyces,威克酵母属(Wickerhamia),拟威克酵母属(Wickerhamiella),拟威尔酵母属(Williopsis),Yamadazyma,耶氏酵母属(Yarrowia),接合囊酵母属(Zygoascus),接合酵母属(Zygosaccharomyces),接合拟威尔酵母属(Zygowilliopsis),和Zygozyma等等。
在一些实施方案,所述宿主是酿酒酵母(Saccharomyces cerevisiae),巴斯德毕赤酵母(Pichia pastoris),粟酒裂殖酵母(Schizosaccharomyces pombe),布鲁赛尔德克酵母(Dekkera bruxellensis),乳酸克鲁维酵母(Kruyveromyces lactis,此前称为乳酸酵母(Saccharomyces lactis)),马克斯克鲁维酵母(Kluveromyces marxianus),食腺嘌呤芽生葡萄孢酵母(Arxula adeninivorans),或多形汉逊酵母(Hansenula polymorpha)(现称为安格斯毕赤酵母(Pichia angusta))。在一些实施方案,所述细胞是假丝酵母属(Candida)的菌株,例如解脂假丝酵母(Candida lipolytica),吉利蒙假丝酵母(Candidaguilliermondii),克鲁斯假丝酵母(Candida krusei),假热带假丝酵母(Candidapseudotropicalis)或产朊假丝酵母(Candida utilis)的菌株。
在特定实施方案,所述细胞是酿酒酵母(Saccharomyces cerevisiae)。在一些实施方案,所述细胞是酿酒酵母(Saccharomyces cerevisiae)的菌株,所述酿酒酵母的菌株是选自由贝克氏(Baker’s)酵母、CBS7959、CBS7960、CBS7961、CBS7962、CBS7963、CBS7964、IZ-1904、TA、BG-1、CR-1、SA-1、M-26、Y-904、PE-2、PE-5、VR-1、BR-1、BR-2、ME-2、VR-2、MA-3、MA-4、CAT-1、CB-1、NR-1、BT-1、和AL-1组成的组。在一些实施方案,所述细胞是酿酒酵母(Saccharomyces cerevisiae)的菌株,所述酿酒酵母的菌株是选自由PE-2、CAT-1、VR-1、BG-1、CR-1、和SA-1组成的组。在特定实施方案,酿酒酵母(Saccharomyces cerevisiae)的菌株是PE-2。在另一特定实施方案,酿酒酵母(Saccharomyces cerevisiae)的菌株是CAT-1。在另一特定实施方案,酿酒酵母(Saccharomyces cerevisiae)的菌株是BG-1。
在一些实施方案,所述细胞是适于工业发酵的微生物。在特定实施方案,所述微生物适应于在高溶剂浓度、高温、扩大的底物利用率、营养限制、由糖和盐引起的渗透应力、酸度、亚硫酸盐和细菌污染、或其组合下存活,所述这些是公认的工业发酵环境的应激条件。
在一些实施方案,所述细胞经工程化以生成C5类异戊二烯。所述这些化合物衍生自一个异戊二烯单元,也被称为半萜。半萜的示例性实例有异戊二烯。在其他实施方案,所述类异戊二烯是C10类异戊二烯。所述这些化合物衍生自两个异戊二烯单元,也被称为单萜。单萜的示例性实例有柠檬烯、香茅醇(citranellol)、香叶醇、薄荷醇、紫苏醇、芳樟醇、侧柏酮(thujone)和月桂烯。在其他实施方案,所述类异戊二烯是C15类异戊二烯。所述这些化合物衍生自三个异戊二烯单元,也被称为倍半萜。倍半萜的示例性实例有蜚蠊酮B、银杏苦内酯B、紫穗槐二烯、青蒿素、青蒿酸、瓦伦烯、诺卡酮、表-雪松醇、表-马兜铃烯、法呢醇、棉酚、sanonin、蜚蠊酮、毛喉素和广藿香醇(也被称为天竺薄荷醇)。在其他实施方案,所述类异戊二烯是C20类异戊二烯。所述这些化合物衍生自四个异戊二烯单元,也被称为二萜。二萜的示例性实例有蓖麻烯、五加素、紫杉醇、prostratin、伪蕨素和紫杉二烯(taxadiene)。在其他实施例,所述类异戊二烯是C20+类异戊二烯。所述这些化合物衍生自四个以上的异戊二烯单元,包括:三萜类(由6个异戊二烯单元衍生的C30类异戊二烯化合物),如阿布糖苷E(arbrusideE)、鸦胆丁、睾酮、黄体酮、可的松、洋地黄毒甙和角鲨烯;四萜类(衍生自8个类异戊二烯的C40类异戊二烯化合物),如β-胡萝卜素;和多萜类(衍生自多于8个异戊二烯单元的C40+类异戊二烯化合物),如聚异戊二烯。在一些实施方案,所述类异戊二烯选自由以下组成的组:松香二烯(abietadiene)、紫穗槐二烯、蒈烯、α-法呢烯、β-法呢烯、法呢醇、香叶醇、香叶基香叶醇、异戊二烯、芳樟醇、柠檬烯、月桂烯、橙花叔醇、罗勒烯、广藿香醇、β-蒎烯、桧烯、γ-萜品烯、萜品油烯和瓦伦烯。类异戊二烯化合物还包括但不限于类胡萝卜素类(如番茄红素、α-和β-胡萝卜素、α-和β-隐黄素、胭脂素、玉米黄质、虾青素和叶黄素),类固醇化合物类,和由其他化学基团如混合萜类生物碱和辅酶Q-10修饰的类异戊二烯组成的化合物类。
在一些实施方案,所述细胞经工程化以生成聚酮化合物。在某些实施方案,所述聚酮化合物选自由聚酮化合物大环内酯类化合物、抗生素类化合物、抗真菌化合物、抑制细胞生长化合物、抗胆甾醇血的化合物、抗寄生物化合物、抗球虫化合物、动物生长促进剂、和杀虫剂组成的组。
在一些实施方案,所述细胞经工程化以生成脂肪酸。
有用的细胞记载在WO 2015/095804、WO 2015/020649和WO 2014/144135中,其各自内容通过引用其全文并入本发明。
5.5化合物的回收
一旦通过细胞培养物生成了化合物,便可采用本领域已知的任何合适的分离和纯化方法将其回收或分离以供后续使用。在一些实施方案,通过离心从发酵中分离出包含所述化合物的有机相。在其他实施方案,包含所述化合物的有机相自发地从发酵中分离。在其他实施方案,通过向发酵反应中加入破乳剂和/或成核剂,进而从发酵中分离出包含类异戊二烯的有机相。破乳剂的示例性实例包括絮凝剂和凝结剂。成核剂的示例性实例包括所述类异戊二烯本身的液滴以及有机溶剂类如十二烷、肉豆蔻酸异丙酯和油酸甲酯。
在所述这些细胞中生成的化合物可存在于培养基上清液中和/或与细胞结合。在所述化合物与细胞结合的实施方案中,所述类异戊二烯的回收可包括透化或裂解细胞的方法。另外或同时,培养基中的所述化合物可使用包括但不限于色谱法、提取法、溶剂萃取法、膜分离法、电渗析法、反渗透法、蒸馏法、化学衍生法和结晶法的回收方法进行回收。
在一些实施方案,将所述化合物与有机相中可能存在的其他产物进行分离。在一些实施方案,采用吸附法、蒸馏法、气-液萃取(汽提法)、液-液萃取(溶剂萃取法)、超滤法和标准色谱技术来实现分离。
6.实施例
6.1实施例1
本实施例提供了方法,所述方法示出了增加的测定酵母细胞培养物的细胞密度(OD600)的示例性方法。
在连续进料工艺方法中,使用废气中目标浓度为150、300和600ppm的乙醇对两种菌株进行评估。生物量(biomass)是采用典型的脉冲进料工艺方法产生。大约48小时后,开始连续进料的生成阶段。在生成期间,将摄氧速率(OUR)控制在110mmol/L/h,无需进行溶解氧峰值检测。下面在图1中示出了使用乙醇废气的连续进料方法的工艺流程趋势。
图1提供了连续进料工艺方法的MFCS(BioPAT)迹线。通过调节进料速率(绿色),将废气乙醇浓度(蓝色)保持在150ppm。通过调节搅拌(深绿色)使OUR(红色)保持恒定。溶解氧保持在接近零的水平(黑色)。
选择3-6天的时间间隔来阐明此工艺方法。如图3所示,随着废气乙醇浓度设定值的增加,两种菌株均表现出生产率增加的趋势,并且产量无差异。此附图表明,生产率随着废气中平均乙醇浓度的增加而增加。数据还表明,CFSC方法通常产生的平均废气浓度为220-320ppm。通过调节进料速率将乙醇废气浓度控制在较高水平,可使得两种菌株均能提供更高的生产率。
图3提供了标准CFSC方法和连续进料工艺方法中菌株A的平均乙醇废气浓度。CFSC方法在废气中生成平均200-225ppm的乙醇,其远低于连续进料实验的较高设定值。
Claims (24)
1.向微生物培养物提供糖的方法,其包含下列步骤:
a.将糖以初始速率进料至在发酵培养基中生长的微生物培养物;
b.测定所述发酵培养基废气中的乙醇浓度;
c.当所述废气中的所述乙醇浓度增加时,所述糖的进料速率降低;和
d.当所述废气中的所述乙醇浓度降低时,所述糖的进料速率增加。
2.根据权利要求1所述的方法,其中,将糖连续进料至所述发酵培养基。
3.根据权利要求1或2所述的方法,其中,对所述废气中的所述乙醇浓度进行连续测定。
4.根据前述权利要求任一项所述的方法,其中,所述糖的进料速率保持在所述初始进料速率的±25%以内。
5.根据前述权利要求任一项所述的方法,其中,所述糖的进料速率保持在所述初始进料速率的±15%以内。
6.根据前述权利要求任一项所述的方法,其中,所述糖的进料速率保持在所述初始进料速率的±10%以内。
7.根据前述权利要求任一项所述的方法,其中,所述进料速率为1至25g/L/hr。
8.根据前述权利要求任一项所述的方法,其中,保持所述微生物培养物的所述摄氧速率。
9.根据前述权利要求任一项所述的方法,其中,所述微生物培养物的所述摄氧速率通过搅拌来保持。
10.根据前述权利要求任一项所述的方法,其中,所述微生物培养物的所述摄氧速率为1-150mmol O2/L/hr。
11.根据前述权利要求任一项所述的方法,其中,所述废气中的所述乙醇浓度为50-750ppm。
12.根据前述权利要求任一项所述的方法,其中,所述废气中的所述乙醇浓度为100-200ppm。
13.根据前述权利要求任一项所述的方法,其中,所述废气中的所述乙醇浓度为200-300ppm。
14.根据前述权利要求任一项所述的方法,其中,所述废气中的所述乙醇浓度为250-350ppm。
15.根据前述权利要求任一项所述的方法,其中,所述废气中的所述乙醇浓度为550-650ppm。
16.根据前述权利要求任一项所述的方法,所述方法进行1-10、2-9、3-7或4-6天。
17.根据前述权利要求任一项所述的方法,其中,所述微生物培养物是原核的。
18.根据前述权利要求任一项所述的方法,其中,所述微生物培养物是真核的。
19.根据前述权利要求任一项所述的方法,其中,所述微生物培养物是酵母。
20.根据前述权利要求任一项所述的方法,其中,所述微生物培养物是酿酒酵母(S.cerevisiae)。
21.根据前述权利要求任一项所述的方法,其中,所述微生物培养物是重组的。
22.根据前述权利要求任一项所述的方法,其中,所述微生物培养物生成水不混溶性化合物。
23.根据前述权利要求任一项所述的方法,其中,所述微生物培养物生成类异戊二烯、聚酮化合物、或脂肪酸。
24.根据前述权利要求任一项所述的方法,其中,所述微生物培养物生成倍半萜。
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- 2019-07-11 CA CA3106250A patent/CA3106250A1/en not_active Abandoned
- 2019-07-11 CN CN201980046477.4A patent/CN112703251A/zh active Pending
- 2019-07-11 EP EP19745933.2A patent/EP3821022A1/en not_active Withdrawn
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BR112021000313A2 (pt) | 2021-04-06 |
EP3821022A1 (en) | 2021-05-19 |
MX2021000053A (es) | 2021-03-25 |
CA3106250A1 (en) | 2020-01-16 |
WO2020014513A1 (en) | 2020-01-16 |
US20210230640A1 (en) | 2021-07-29 |
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