New technologies and industrialization progress in healthy sugar biomanufacturing based on in vitro synthetic biology

doi:10.12211/2096-8280.2025-054

Synthetic Biology Journal

New technologies and industrialization progress in healthy sugar biomanufacturing based on in vitro synthetic biology

SHI Ting¹^,², CHEN Xuemei², ZHANG Yi-Heng P. Job¹^,²

^1.Key Laboratory of Engineering Biology for Low-Carbon Manufacturing，Tianjin Institute of Industrial Biotechnology，Chinese Academy of Sciences，Tianjin 300308，China
^2.In vitro Synthetic Biology Center，Tianjin Institute of Industrial Biotechnology，Chinese Academy of Sciences，Tianjin 300308，China

Received:2025-06-04 Revised:2025-08-05
Contact: ZHANG Yi-Heng P. Job

基于体外合成生物学的健康糖生物制造新技术与产业化进展

石婷¹^,², 陈雪梅², 张以恒¹^,²

^1.低碳合成工程生物学全国重点实验室，中国科学院天津工业生物技术研究所，天津 300308
^2.中国科学院天津工业生物技术研究所体外合成生物学中心，天津 300308

通讯作者: 张以恒
作者简介:石婷（1984—），女，博士，中国科学院天津工业生物技术研究所正高级工程师。2008年本科毕业于合肥工业大学生物与食品工程学院，2010年和2014年分别获得天津大学化工学院生物化工专业硕士和博士学位。主要研究方向为体外合成生物学、酶工程与微生物代谢工程。E-mail：shi_ting@tib.cas.cn
张以恒（1971—），低碳合成工程生物学全国重点实验室主任，中国科学院天津工业生物技术研究所体外合成生物学与生物制造中心主任，曾经任美国弗吉尼亚理工大学终身正教授。他是体外合成生物学的奠基人之一与产业化领跑者，创建体外生物转化（ivBT）技术平台，率先提出“人工多酶分子机器”概念并实现万吨级产业化。在秸秆制粮、体外呼吸作用（无氧呼吸作用产氢、有氧呼吸作用制电）、人工合成淀粉、淀粉制塔格糖与肌醇等方向取得了一系列原创性（0-1）突破。E-mail：zhang_xw@tib.cas.cn
基金资助:
科技部重点专项(2022YFA0912300);国家自然科学基金面上项目(NSFC32271544);合成生物学海河实验室颠覆性创新项目(22HHSWSS000155);天津市合成生物技术创新能力提升行动项目(TSBICIP-CXRC-067)

Abstract

Abstract:

With the increasing global awareness of health, the demand for natural sugars that are low in calories, have wonderful sweetness and healthful functions continues to grow. However, the traditional sugar industry faces significant constraints related to health concerns, cost, and scalability. Enzymatic biocatalysis and in vitro BioTransformation (ivBT) are two pivotal technological platforms in biomanufacturing, playing an increasingly vital role in biomanufacturing of healthy sugars. The successful commercialization of high-fructose corn syrup opened the door of the starchy sweetener industry. Later, the Izumoring strategy opened the door to the rare sugars industry that is based on by enzymatic isomerization and oxidization-reduction of sugars. However, the key products made by the Izumoring strategy suffer from high reliance on limited feedstocks, low product yields due to equilibria of isomerization, high separation costs, and/or the use of costly NAD(P) for the oxidization-reduction of sugars. Based on the ivBT platform, we have developed the first new-to-nature pentose 4-epimerase, enabling the direct conversion of D-type to L-type pentoses without sugar alcohol intermediates, thereby establishing an innovative route for the biotransformation of D-xylose to L-arabinose. The successful industrial-scale production of myo-inositol from starch has validated the biomanufacturing advantages of Zhang strategy ("ATP-free Phosphorylation-Isomerization-Dephosphorylation" strategy) as applied to ivBT. This approach has since been extended to the synthesis of a few healthy sugars, such as D-tagatose, D-allulose, and D-mannose, from abundant polysaccharides including starch, cellulose, and sucrose, showing great potential for industrial application. Furthermore, the "carbon dioxide to sugar" ivBT platform overcomes the low energy efficiency and slow productivity bottlenecks of natural plant photosynthesis, opening up a new door for the synthesis of healthy sugars from the third-generation feedstock of CO₂. This ivBT platform has achieved multiple breakthroughs, such as the reconstruction of artificial enzymatic pathways for the biosynthesis of healthy sugars, low-cost biomanufacturing of multi-enzyme molecular machines equipped with thermostable enzyme bricks, great compatibility of multi-enzyme co-immobilization for prolonging enzyme life-time, promotion of the PE value (Product-to-Enzyme Weight Ratio) of multi-enzyme molecular machines, and low-cost separation and purification of healthy sugars. As a disruptive innovation in biomanufacturing, ivBT motivates the rapid development in the biomanufacturing of "affordable, accessible, and effective" healthy sugars and holds promise to drive a new wave of transformation in the sweetener industry.

Key words: in vitro BioTransformation (ivBT), Healthy sugars, Izumoring, D-type-L-type sugar epimerase, Zhang strategy (ATP-free Phosphorylation-Isomerization-Dephosphorylation)

摘要：

随着全球健康意识的提升，消费者对低热量、功能性的天然糖（简称健康糖）需求增长。酶催化与体外生物转化（in vitro BioTransformation，ivBT）是健康糖的工业生物制造的两大平台技术。酶法生产果葡糖浆的商业化成功开启淀粉糖产业，随后Izumoring策略推动酶法生产稀有糖，但受原料依赖、转化率低、分离成本高、辅酶循环利用等因素使该策略难以大规模生产健康糖（如D-塔格糖、L-阿拉伯糖）。基于ivBT平台，我们开发了首个非天然戊单糖4-差向异构酶，实现D型与L型戊糖的直接转换，无需经过糖醇中间体，打通D-木糖向L-阿拉伯糖转化新路径；淀粉制肌醇已成功实现产业化，验证了Zhang策略（“无ATP磷酸化-异构-脱磷酸”策略）的生物制造优势，该策略拓展至廉价多糖原料合成D-塔格糖、D-阿洛酮糖、D-甘露糖等健康糖；“二氧化碳合成糖类”技术突破了植物光合作用的能效低与速度慢的瓶颈，为以第三代原料合成健康糖及其衍生物开辟新途径。ivBT平台取得了系列突破性进展，有望为开发“低成本、高可及性、高功能性”的健康糖提供核心支撑，引领甜味剂产业的新变革。

关键词: 体外生物转化（ivBT）, 健康糖, Izumoring, D型-L型糖异构酶, Zhang策略（“无ATP磷酸化-异构-脱磷酸”策略）

CLC Number:

SHI Ting, CHEN Xuemei, ZHANG Yi-Heng P. Job. New technologies and industrialization progress in healthy sugar biomanufacturing based on in vitro synthetic biology[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2025-054.

石婷, 陈雪梅, 张以恒. 基于体外合成生物学的健康糖生物制造新技术与产业化进展[J]. 合成生物学, DOI: 10.12211/2096-8280.2025-054.

Figures/Tables 7

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Production method	Enzyme	Substrate	Titer (g/L)	Yield (%)	Productivity (g/L/h)	Temp (^oC)	Reference
Enzymatic isomerization	L-AI from Arthrobacter sp. 22c	100 g/L D-galactose	30	30	0.25	30	[104]
	L-AI from G. stearothermophilus	100 g/L D-galactose	30.6	30.6	1.9	60	[105]
	L-AI from G. thermodenitrificans	300 g/L D-galactose	158	52.7	8	60	[48]
	L-AI from Thermoanaerobacter mathranii	300 g/L D-galactose	126	42.0	2.6	65	[106]
	L-AI from Thermotaga maritima	1.8 g/L D-galactose	1.0	56.0	0.2	80	[107]
	Tagaturonate 3-epimerase from Thermotoga petrophila	700 g/L D-fructose	213	30.0	107	80	[49]
	Tagatose-4-epimerase from Thermotogae bacterium	200 g/L D-fructose	28	14.0	14	70	[108]
	D-tagaturonate epimerase from Thermotoga neapolitana	100 g/L D-fructose	21.8	21.8	7.3	65	[109]
	Tagatose 4-epimerase from Thermoprotei archaeon	100 g/L D-fructose	18.9	18.9	7.6	70	[50]
Whole-cell biosynthesis	E. coli BL21/pET28a-P3-LfaraAD390V/V468LlacZ	500 g/L lactose	115	23.1	2.4	50	[68]
	E. coli/pETDuet-αgp-pgm andpCDFDuet-pgi-gatz-pgp	20 g/L maltodextrin	3.2	16.0	0.13	60	[70]
	E. coli ER-2GatZ (ΔpΔz)	10 g/L maltodextrin	3.38	33.8	1.13	60	[72]
Fermentation	S. cerevisiae EJ2g_iXiG_pXpG	114 g/L lactose	37.7	33	0.13	30	[75]
Fermentation	B. subtilis BS-3CA4	72 g/L D-galactose	39.6	0.55	0.33	45	[76]
ivBT	αGP, PGM, PGI, TPE, and TPP from B. subtilis	100 g/L maltodextrin	78	78	2.0	37	[110]
	αGP, PGM, PGI, TPE, and TPP from E.coli BL21	20 g/L maltodextrin	17.7	88.5	0.74	50	[61]
	αGP, PGM, PGI, TPE, and TPP from E.coli BL21	50 g/L maltodextrin	37.6	75	1.57	50	[61]

Production method	Enzyme	Substrate	Titer (g/L)	Yield (%)	Productivity (g/L/h)	Temp (^oC)	Reference
Enzymatic isomerization	L-AI from Arthrobacter sp. 22c	100 g/L D-galactose	30	30	0.25	30	[104]
	L-AI from G. stearothermophilus	100 g/L D-galactose	30.6	30.6	1.9	60	[105]
	L-AI from G. thermodenitrificans	300 g/L D-galactose	158	52.7	8	60	[48]
	L-AI from Thermoanaerobacter mathranii	300 g/L D-galactose	126	42.0	2.6	65	[106]
	L-AI from Thermotaga maritima	1.8 g/L D-galactose	1.0	56.0	0.2	80	[107]
	Tagaturonate 3-epimerase from Thermotoga petrophila	700 g/L D-fructose	213	30.0	107	80	[49]
	Tagatose-4-epimerase from Thermotogae bacterium	200 g/L D-fructose	28	14.0	14	70	[108]
	D-tagaturonate epimerase from Thermotoga neapolitana	100 g/L D-fructose	21.8	21.8	7.3	65	[109]
	Tagatose 4-epimerase from Thermoprotei archaeon	100 g/L D-fructose	18.9	18.9	7.6	70	[50]
Whole-cell biosynthesis	E. coli BL21/pET28a-P3-LfaraAD390V/V468LlacZ	500 g/L lactose	115	23.1	2.4	50	[68]
	E. coli/pETDuet-αgp-pgm andpCDFDuet-pgi-gatz-pgp	20 g/L maltodextrin	3.2	16.0	0.13	60	[70]
	E. coli ER-2GatZ (ΔpΔz)	10 g/L maltodextrin	3.38	33.8	1.13	60	[72]
Fermentation	S. cerevisiae EJ2g_iXiG_pXpG	114 g/L lactose	37.7	33	0.13	30	[75]
Fermentation	B. subtilis BS-3CA4	72 g/L D-galactose	39.6	0.55	0.33	45	[76]
ivBT	αGP, PGM, PGI, TPE, and TPP from B. subtilis	100 g/L maltodextrin	78	78	2.0	37	[110]
	αGP, PGM, PGI, TPE, and TPP from E.coli BL21	20 g/L maltodextrin	17.7	88.5	0.74	50	[61]
	αGP, PGM, PGI, TPE, and TPP from E.coli BL21	50 g/L maltodextrin	37.6	75	1.57	50	[61]