多形汉逊酵母细胞工厂实现甲醇生物转化合成3-羟基丙酸

doi:10.1016/S1872-2067(22)64195-0

催化学报 ›› 2023, Vol. 46: 84-90.DOI: 10.1016/S1872-2067(22)64195-0

多形汉逊酵母细胞工厂实现甲醇生物转化合成3-羟基丙酸

禹伟^a^,^d, 高教琪^a^,^b^,^c, 姚伦^a^,^b^,^c, 周雍进^a^,^b^,^c^,^*()

^a中国科学院大连化学物理研究所, 生物技术研究部, 辽宁大连 116023
^b中国科学院大连化学物理研究所, 中国科学院分离分析化学重点实验室, 辽宁大连 116023
^c中国科学院大连化学物理研究所, 能源生物技术大连市重点实验室, 辽宁大连 116023
^d中国科学院大学, 北京100049

收稿日期:2022-09-15 接受日期:2022-11-14 出版日期:2023-03-18 发布日期:2023-02-21
通讯作者: *电子信箱: zhouyongjin@dicp.ac.cn (周雍进)
基金资助:
国家重点研发计划(2021YFC2103500);国家重点研发计划(2021YFC2104200);国家自然科学基金(21922812);国家自然科学基金(22161142008);中国科学院大连化学物理研究所创新基金(DICP I201920)

Bioconversion of methanol to 3-hydroxypropionate by engineering Ogataea polymorpha

Wei Yu^a^,^d, Jiaoqi Gao^a^,^b^,^c, Lun Yao^a^,^b^,^c, Yongjin J. Zhou^a^,^b^,^c^,^*()

^aDivision of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, Liaoning, China
^bCAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, Liaoning, China
^cDalian Key Laboratory of Energy Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, Liaoning, China
^dUniversity of Chinese Academy of Sciences, Beijing 100049, China

Received:2022-09-15 Accepted:2022-11-14 Online:2023-03-18 Published:2023-02-21
Contact: *E-mail: zhouyongjin@dicp.ac.cn (Y. J. Zhou)
Supported by:
National Key Research and Development Program of China(2021YFC2103500);National Key Research and Development Program of China(2021YFC2104200);The National Natural Science Foundation of China(21922812);The National Natural Science Foundation of China(22161142008);DICP innovation grant (DICP I201920) from Dalian Institute of Chemical Physics

摘要/Abstract

摘要：

3-羟基丙酸(3-HP)是公认的高附加值化学品之一, 可用于合成多种化学品, 例如丙烯酰胺、丙烯酸、1,3-丙二醇以及可降解塑料. 目前3-HP的生物合成过程均以糖为原料, 且需求不断增长, 亟需寻找不依赖耕地的替代原料. 碳一资源来源广泛, 是理想的生物制造原料. 其中, 甲醇可由煤、天然气制得, 也可由CO₂加氢制备. 多形汉逊酵母作为典型的甲基营养型酵母, 能够以甲醇为唯一碳源和能源进行生长, 并且能够耐受高温、高渗透压和低pH等条件, 是非常优秀的工业菌株. 近年来基因编辑技术的发展, 大大加快了多形汉逊酵母代谢工程改造, 有望构建高效细胞工厂, 实现甲醇生物转化.

本文以多形汉逊酵母为宿主, 系统改造其细胞代谢实现甲醇高效转化为3-HP. 由于汉逊酵母自身不能合成3-HP, 因此需要表达来源于Chloroflexus aurantiacus的3-HP合成途径关键基因MCR, 该途径以乙酰-CoA和丙二酰-CoA为前体, 以还原力NADPH为辅因子. 首先优化MCR表达, 采用融合蛋白并结合甲醇诱导型启动子P_AOX基因组整合MCR表达, 3-HP合成效率最高; 其次, 强化供应前体乙酰-CoA以及丙二酰-CoA, 使3-HP产量提高26%; 进一步改造氧化磷酸戊糖途径以增强辅因子NADPH的供应, 使3-HP产量提高30%; 最后, 将增强前体和辅因子供应的有效策略结合, 工程菌HP15相比出发菌HP07摇瓶发酵3-HP产量提高135%, 达到1.45 g/L. 通过摇瓶补料分批发酵, 3-HP产量达到7.10 g/L, 得率达到0.14 g/g甲醇, 为目前报道碳一资源合成3-HP的较高产量, 表明多形汉逊酵母作为甲醇细胞工厂合成化学品具有很好的潜力. 预期结合“液态阳光”CO₂制备甲醇技术以及甲醇生物转化过程, 有望将CO₂制备成高附加值化学品, 有助于实现碳中和.

关键词: 多形汉逊酵母, 甲醇生物转化, 代谢工程, 3-羟基丙酸, 碳中和

Abstract:

Methanol bioconversion toward chemical production can be helpful for carbon neutrality. Here metabolic engineering an industrial methylotrophic yeast Ogataea polymorpha was conducted for overproduction of 3-hydroxypropionate (3-HP), an important platform chemical, which can be used for production of special chemicals including acrylamide, acrylic acid, 1,3-propanediol, as well as biodegradable plastics. We developed several metabolic engineering strategies to optimize the 3-HP biosynthetic pathway and enhance the supply of precursors acetyl-CoA and malonyl-CoA, and cofactor NADPH, which enabled the 3-HP production of 1.45 g/L under batch fermentation and 7.10 g/L under fed-batch fermentation at shake flask scale, with a yield of 0.14 g/g methanol. This was, to our knowledge, the highest 3-HP titer from methanol and even one-carbon sources. This study demonstrated the potential of O. polymorpha as a cell factory for chemical production from methanol.

Key words: Ogataea polymorpha, Methanol bioconversion, Metabolic engineering, 3-Hydroxypropionate, Carbon neutrality

禹伟, 高教琪, 姚伦, 周雍进. 多形汉逊酵母细胞工厂实现甲醇生物转化合成3-羟基丙酸[J]. 催化学报, 2023, 46: 84-90.

Wei Yu, Jiaoqi Gao, Lun Yao, Yongjin J. Zhou. Bioconversion of methanol to 3-hydroxypropionate by engineering Ogataea polymorpha[J]. Chinese Journal of Catalysis, 2023, 46: 84-90.

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图/表 6

Fig. 1. Methanol bioconversion for production of the platform chemical 3-HP in O. polymorpha cell factory and its comparison with glucose bioconversion, which require large amount of arable land and might result in feed-food competition. The overexpressed genes were marked with red.

Fig. 2. Construction of 3-HP synthetic pathway using methanol as sole carbon source in O. polymorpha. (a) Malonyl-CoA reductase (encoded by MCR) based 3-HP production from methanol. (b) Scheme of MCR expression on episomal plasmid or genome neutral site. (c) The effect of MCR expression under the control of strong constitutive promoter PGAP or methanol induced promoter PAOX on 3-HP synthesis. (d) The effect of plasmid expression, genome integration of MCR, and LEU2 recovery on 3-HP synthesis.

Fig. 3. Optimization of Mcr expression for 3-HP synthesis. (a) Scheme of Mcr catalyzing reaction from malonyl-CoA to 3-HP, with malonate semialdehyde (MSA) as the intermediate. (b) Three different types of Mcr expression: fusion, separation and affinity expression. (c) 3-HP production and biomass yield in 10 g/L methanol minimal medium. Linker 1: GPGRPPPGRG; Linker 2: ASGAGGSEGGGSEGGTSGAT; SZ3: NEVTTLENDAAFIENENAYLEKEIARLRKEKAALRNRLAHKK; SZ4: MQKVAELKNRVAVKLNRNEQLKNKVEELKNRNAYLKNELATLENEVARLENDVAE.

Fig. 4. Enhancing the supply of precursors acetyl-CoA and malonyl-CoA, and cofactor NADPH to improve 3-HP production from methanol. (a) Metabolic engineering strategies for enhancing the supply of acetyl-CoA and malonyl-CoA, as well as NADPH. (b) Effect of EcPDH, MmACL and ACC1 overexpression on 3-HP production. (c) Effect of overexpression of ScIDP2 or ZWF1 and GDN1, as well as ACC1, on 3-HP production. AOX: alcohol oxidase gene; DAS: dihydroxyacetone synthase gene; DAK: dihydroxyacetone kinase gene; ZWF1: glucose-6-phosphate dehydrogenase gene; GND1: 6-phosphogluconate dehydrogenase gene; EcPDH: pyruvate dehydrogenase gene from E. coli; ACC1: acetyl-CoA carboxylase gene; MmACL: ATP-citrate lyase from Mus musculus; ScIDP2: NADP+-dependent isocitrate dehydrogenase gene from S. cerevisiae. XuMP cycle: xylulose monophosphate cycle. DHA: dihydroxyacetone; DHAP: dihydroxyacetone phosphate; G3P: glyceraldehyde-3-phosphate; FBP: fructose-1,6-phosphate; F6P: fructose-6-phosphate; E4P: erythrose-4-phosphate; S7P: sedoheptulose-7-phosphate; R5P: ribose-5-phosphate; Xu5P: xylulose-5-phosphate; Ru5P: ribulose-5-phosphate; α-KG: α-ketoglutarate; OAA: oxalacetate.

Table 1 3-HP production from C1 sources in microbes

Host	Carbon source	Cultivation condition	Titer (g/L)	Conversion rate (%)	Productivity (g/(L·d))	Ref.
Synechococcus elongatus	CO₂	MM, shake flask	0.665	2.6	0.07	[11]
Synechocystis sp.	CO₂	MM, shake flask	0.837	8.0	0.14	[36]
Methylosinus trichosporium	methane	MM, bioreactor	0.061	2.4	0.03	[10]
Methylobacterium extorquens	methanol	MM, shake flask	0.070	2.0	0.04	[37]
Methylobacterium extorquens	methanol	MM, bioreactor	0.857	3.1	0.21	[38]
Ogataea polymorpha	methanol	MM, shake flask	7.10	14.2	1.2	This study

Fig. 5. Fed-batch fermentation of the engineered strain HP18 in 250 mL shake flasks. 3-HP titer, OD600 and methanol consumption were measured to monitor the production process. The pH was adjusted with 2 mol/L KOH every 24 h to keep 5?6, so as to maintain normal cell growth when producing 3-HP.

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多形汉逊酵母细胞工厂实现甲醇生物转化合成3-羟基丙酸

Bioconversion of methanol to 3-hydroxypropionate by engineering Ogataea polymorpha

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