Bioconversion of methanol to 3-hydroxypropionate by engineering Ogataea polymorpha

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

Abstract

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

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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URL: https://www.cjcatal.com/EN/10.1016/S1872-2067(22)64195-0

https://www.cjcatal.com/EN/Y2023/V46/I3/84

Figures/Tables 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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