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Monoterpenoids are an important subclass of terpenoids that play important roles in the energy, cosmetics, pharmaceuticals, and fragrances fields. With the development of biotechnology, microbial synthesis of monoterpenoids has received great attention. Yeasts such Saccharomyces cerevisiae and Yarrowia lipolytica are emerging as potential hosts for monoterpenoids production because of unique advantages including rapid growth cycles, mature gene editing tools, and clear genetic background. Recently, advancements in metabolic engineering and fermentation engineering have significantly enhanced the accumulation of monoterpenoids in cell factories. First, this review introduces the biosynthetic pathway of monoterpenoids and comprehensively summarizes the latest production strategies, which encompass enhancing precursor flux, modulating the expression of rate-limited enzymes, suppressing competitive pathway flux, mitigating cytotoxicity, optimizing substrate utilization, and refining the fermentation process. Subsequently, this review introduces four representative monoterpenoids. Finally, we outline the future prospects for efficient construction cell factories tailored for the production of monoterpenoids and other terpenoids.

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Industrial biotechnology is regarded as the most promising technology for sustainable industrial development. The advancement of synthetic biology creates new opportunities and infinite possibilities for the progress of industrial biotechnology. Fermentation engineering is the grab and foothold of the industrialization of all the biotechnologies. Our teaching team optimized the teaching content and innovated the teaching mode to establish a teaching system of synthetic biology matching fermentation engineering. We highlighted the teaching characteristics (telling fermentation story cultivated the craftsmanship spirit; bioeconomic education strengthened the engineering thinking; bioethics and safety education fostered a sense of responsibility), then we summarized and prospected the teaching reform of this course. We believe that the teaching reform of synthetic biology will improve the learning performance of postgraduates, provide a reference for the teaching of synthetic biology in related fields, and promote the development of industrial biotechnology (strengthening the innovation capability in biological manufacturing and cultivating new momentum for bioeconomy).

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The increasing applications for eicosapentaenoic acid (EPA) and the potential shortfall in supply due to sustainability and contamination issues related with its conventional sources (i.e., fish oils; seafood) led to an extensive search for alternative and sustainable sources, as well as production processes. The present mini-review covers all the steps involved in the production of EPA from microorganisms, with a deeper focus on microalgae. From production systems to downstream processing, the most important achievements within each area are briefly highlighted. Comparative tables of methodologies are also provided, as well as additional references of recent reviews, so that readers may deepen their knowledge in the different issues addressed. KEY POINTS: • Microorganisms are more sustainable alternative sources of EPA than fish. • Due to the costly separation from DHA, species that produce only EPA are preferable. • EPA production can be optimised using non-genetic and genetic tailoring engineering.

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Polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and arachidonic acid (ARA), are beneficial for reducing blood cholesterol and enhancing memory. Traditional PUFA production relies on extraction from plants and animals, which is unsustainable. Thus, using microorganisms as lipid-producing factories holds promise as an alternative way for PUFA production. Several oleaginous microorganisms have been successfully industrialized to date. These can be divided into universal and specialized hosts according to the products range of biosynthesis. The Yarrowia lipolytica is universal oleaginous host that has been engineered to produce a variety of fatty acids, such as γ-linolenic acid (GLA), EPA, ARA and so on. By contrast, the specialized host are used to produce only certain fatty acids, such as ARA in Mortierella alpina, EPA in Nannochloropsis, and DHA in Thraustochytrids. The metabolic engineering and fermentation strategies for improving PUFA production in universal and specialized hosts are different, which is the subject of this review. In addition, the widely applicable strategies for microbial lipid production that are not specific to individual hosts were also reviewed.

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L-methionine, also known as L-aminomethane, is one of the eight essential amino acids required by the human body and has important applications in the fields of feed, medicine, and food. In this study, an L-methionine high-yielding strain was constructed using a modular metabolic engineering strategy based on the M2 strain (Escherichia coli W3110 ΔIJAHFEBC/PAM) previously constructed in our laboratory. Firstly, the production of one-carbon module methyl donors was enhanced by overexpression of methylenetetrahydrofolate reductase (methylenetetrahydrofolate reductase, MetF) and screening of hydroxymethyltransferase (GlyA) from different sources, optimizing the one-carbon module. Subsequently, cysteamine lyase (hydroxymethyltransferase, MalY) and cysteine internal transporter gene (fliY) were overexpressed to improve the supply of L-homocysteine and L-cysteine, two precursors of the one-carbon module. The production of L-methionine in shake flask fermentation was increased from 2.8 g/L to 4.05 g/L, and up to 18.26 g/L in a 5 L fermenter. The results indicate that the one carbon module has a significant impact on the biosynthesis of L-methionine, and efficient biosynthesis of L-methionine can be achieved through optimizing the one carbon module. This study may facilitate further improvement of microbial fermentation production of L-methionine.

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Introduction: L-lysine is a bulk product. In industrial production using high-biomass fermentation, the high density of bacteria and the intensity of production require sufficient cellular respiratory metabolism for support. Conventional bioreactors often have difficulty meeting the oxygen supply conditions for this fermentation process, which is not conducive to improving the sugar-amino acid conversion rate. In this study, we designed and developed an oxygen-enhanced bioreactor to address this problem. Methods: This bioreactor optimizes the aeration mix using an internal liquid flow guide and multiple propellers. Results: Compared with a conventional bioreactor, it improved the kLa from 367.57 to 875.64 h-1, an increase of 238.22%. The results show that the oxygen supply capacity of the oxygen-enhanced bioreactor is better than that of the conventional bioreactor. Its oxygenating effect increased the dissolved oxygen in the middle and late stages of fermentation by an average of 20%. The increased viability of Corynebacterium glutamicum LS260 in the mid to late stages of growth resulted in a yield of 185.3 g/L of L-lysine, 74.57% conversion of lysine from glucose, and productivity of 2.57 g/L/h, an increase of 11.0%, 6.01%, and 8.2%, respectively, over a conventional bioreactor. Oxygen vectors can further improve the production performance of lysine strains by increasing the oxygen uptake capacity of microorganisms. We compared the effects of different oxygen vectors on the production of L-lysine from LS260 fermentation and concluded that n-dodecane was the most suitable. Bacterial growth was smoother under these conditions, with a 2.78% increase in bacterial volume, a 6.53% increase in lysine production, and a 5.83% increase in conversion. The different addition times of the oxygen vectors also affected the final yield and conversion, with the addition of oxygen vectors at 0 h, 8 h, 16 h, and 24 h of fermentation increasing the yield by 6.31%, 12.44%, 9.93%, and 7.39%, respectively, compared to fermentation without the addition of oxygen vectors. The conversion rates increased by 5.83%, 8.73%, 7.13%, and 6.13%, respectively. The best results were achieved by adding oxygen vehicles at the 8th hour of fermentation, with a lysine yield of 208.36 g/L and a conversion rate of 83.3%. In addition, n-dodecane significantly reduced the amount of foam produced during fermentation, which is beneficial for fermentation control and equipment. Conclusion: The new oxygen-enhanced bioreactor improves oxygen transfer efficiency, and oxygen vectors enhance the ability of cells to take up oxygen, which effectively solves the problem of insufficient oxygen supply during lysine fermentation. This study provides a new bioreactor and production solution for lysine fermentation.

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ß-Carotene is a kind of high-value tetraterpene compound, which shows various applications in medical, agricultural, and industrial areas owing to its antioxidant, antitumor, and anti-inflammatory activities. In this study, Yarrowia lipolytica was successfully metabolically modified through the construction and optimization of ß-carotene biosynthetic pathway for ß-carotene production. The ß-carotene titer in the engineered strain Yli-C with the introduction of the carotenogenesis genes crtI, crtE, and crtYB can reach 34.5 mg/L. With the overexpression of key gene in the mevalonate pathway and the enhanced expression of the fatty acid synthesis pathway, the ß-carotene titer of the engineered strain Yli-CAH reached 87 mg/L, which was 152% higher than that of the strain Yli-C. Through the further expression of the rate-limiting enzyme tHMGR and the copy number of ß-carotene synthesis related genes, the ß-carotene production of Yli-C2AH2 strain reached 117.5 mg/L. The final strain Yli-C2AH2 produced 2.7 g/L ß-carotene titer by fed-batch fermentation in a 5.0-L fermenter. This research will greatly speed up the process of developing microbial cell factories for the commercial production of ß-carotene. ONE-SENTENCE SUMMARY: In this study, the ß-carotene synthesis pathway in engineered Yarrowia lipolytica was enhanced, and the fermentation conditions were optimized for high ß-carotene production.

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More than 200 million tons of plant oils and animal fats are produced annually worldwide from oil, crops, and the rendered animal fat industry. Triacylglycerol, an abundant energy-dense compound, is the major form of lipid in oils and fats. While oils or fats are very important raw materials and functional ingredients for food or related products, a significant portion is currently diverted to or recovered as waste. To significantly increase the value of waste oils or fats and expand their applications with a minimal environmental footprint, microbial biomanufacturing is presented as an effective strategy for adding value. Though both bacteria and yeast can be engineered to use oils or fats as the biomanufacturing feedstocks, the yeast Yarrowia lipolytica is presented as one of the most attractive platforms. Y. lipolytica is oleaginous, generally regarded as safe, demonstrated as a promising industrial producer, and has unique capabilities for efficient catabolism and bioconversion of lipid substrates. This review summarizes the major challenges and opportunities for Y. lipolytica as a new biomanufacturing platform for the production of value-added products from oils and fats. This review also discusses relevant cellular and metabolic engineering strategies such as fatty acid transport, fatty acid catabolism and bioconversion, redox balances and energy yield, cell morphology and stress response, and bioreaction engineering. Finally, this review highlights specific product classes including long-chain diacids, wax esters, terpenes, and carotenoids with unique synthesis opportunities from oils and fats in Y. lipolytica.

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This study aimed to investigate the teaching effect of the blended BOPPPS based on an online and offline mixed teaching model ("B + BOPPPS") in the course of fermentation engineering in applied universities. The participants were 142 undergraduates majoring from the course of fermentation engineering in Food Science and Engineering in 2019 and 2020 in Huanghuai University, Zhumadian city, Henan province, China. The students in the control group (68 students) were taught in 2019, and the students in the experimental group (74 students) were taught in 2020. The traditional teaching method and "B + BOPPPS" were implemented, respectively. The teaching effect was evaluated using the questionnaire survey of course satisfaction and theoretical knowledge test. The results showed that the scores of the theoretical knowledge test in the experimental group adopting "B + BOPPPS" were significantly higher than those in the control group, and the difference was statistically significant (p < 0.01). The students had a good evaluation of the "B + BOPPPS" in many aspects, which included achieving learning goals, providing in-depth understanding of knowledge points, stimulating interest in learning, training in the ability to analyze and think about problems, and so on. The results suggested that "B + BOPPPS" could stimulate students' interest in learning and improve their subjective initiative. They could also improve students' ability to master and apply knowledge, which was conducive to improving the theoretical teaching quality of the course of fermentation engineering.

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In this study, glycerate was produced from glucose using engineered Escherichia coli BW25113. Plasmid pSR3 carrying gpd1 and gpp2 encoding two isoforms of glycerol-3-phosphate dehydrogenase from Saccharomyces cerevisiae and plasmid pLB2 carrying aldO encoding alditol oxidase from Streptomyces violaceoruber were introduced into E. coli to enable the production of glycerate from glucose via glycerol. Disruptions of garK and glxK genes in the E. coli genome were performed to minimize the consumption of glycerate produced. As a result, E. coli carrying these plasmids could produce nearly three times higher concentration of glycerate (0.50 ± 0.01 g/L) from 10 g/L glucose compared to E. coli EG_2 (0.14 ± 0.02 g/L). In M9 medium, disruption of garK and glxK resulted in an impaired growth rate with low production of glycerate, while supplementation of 0.5 g/L casamino acids and 0.5 g/L manganese sulfate to the medium replenished the growth rate and elevated the glycerate titer. Further disruption of glpF, encoding a glycerol transporter, increased the glycerate production to 0.80 ± 0.00 g/L. MR2 medium improved the glycerate production titers and specific productivities of E. coli EG_4, EG_5, and EG_6. Upscale production of glycerate was carried out in a jar fermentor with MR2 medium using E. coli EG_6, resulting in an improvement in glycerate production up to 2.37 ± 0.46 g/L with specific productivity at 0.34 ± 0.11 g-glycerate/g-cells. These results indicate that E. coli is an appropriate host for glycerate production from glucose.

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L-methionine, also known as L-aminomethane, is one of the eight essential amino acids required by the human body and has important applications in the fields of feed, medicine, and food. In this study, an L-methionine high-yielding strain was constructed using a modular metabolic engineering strategy based on the M2 strain (Escherichia coli W3110 ΔIJAHFEBC/PAM) previously constructed in our laboratory. Firstly, the production of one-carbon module methyl donors was enhanced by overexpression of methylenetetrahydrofolate reductase (methylenetetrahydrofolate reductase, MetF) and screening of hydroxymethyltransferase (GlyA) from different sources, optimizing the one-carbon module. Subsequently, cysteamine lyase (hydroxymethyltransferase, MalY) and cysteine internal transporter gene (fliY) were overexpressed to improve the supply of L-homocysteine and L-cysteine, two precursors of the one-carbon module. The production of L-methionine in shake flask fermentation was increased from 2.8 g/L to 4.05 g/L, and up to 18.26 g/L in a 5 L fermenter. The results indicate that the one carbon module has a significant impact on the biosynthesis of L-methionine, and efficient biosynthesis of L-methionine can be achieved through optimizing the one carbon module. This study may facilitate further improvement of microbial fermentation production of L-methionine.

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Ergothioneine is a multifunctional physiological cytoprotector, with broad application in foods, beverage, medicine, cosmetics and so on. Biosynthesis is an increasingly favored method in the production of ergothioneine. This paper summarizes the new progress in the identification of key pathways, the mining of key enzymes, and the development of natural edible mushroom species and high-yield engineering strains for ergothioneine biosynthesis in recent years. Through this review, we aim to reveal the molecular mechanism of ergothioneine biosynthesis and then employ the methods of fermentation engineering, metabolic engineering, and synthetic biology to greatly increase the yield of ergothioneine.

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Artificial intelligence (AI) technology is booming up a new industrial revolution, and its successful application is rapidly spreading from the information industry to many other fields. The traditional fermentation industry also faces more opportunities and great challenges for reforming. First of all, the rapid development of synthetic biotechnology has greatly enhanced the availability and efficiency for obtaining high-performance strains, which poses great opportunities to the traditional fermentation optimization and scale-up technology. It is urgent to upgrade fermentation optimization technology to cope with the requirement for high-throughput verification of strain performance. Secondly, the development of fermentation equipment technology has laid a good foundation for advancing fermentation optimization technology. The application of AI technology, especially the digital twin and knowledge graph technology, will further boost the upgrade of the traditional fermentation technology. This review summarizes the challenges of fermentation optimization technology in the era of synthetic biology, the core technology of fermentation optimization and scale-up, the equipment technology of high-throughput fermentation, data visualization technology, as well as the application of digital twin and knowledge graph in fermentation optimization and scale-up. This review also prospects future industrial fermentation technology, and the associated new requirements for personnel training.

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l-cysteine is an important sulfur-containing α-amino acid. It exhibits multiple physiological functions with diverse applications in pharmaceutical cosmetics and food industry. Here, a strategy of coordinated gene expression between carbon and sulfur modules in Escherichia coli was proposed and conducted for the production of l-cysteine. Initially, the titer of l-cysteine was improved to (0.38±0.02) g/L from zero by enhancing the biosynthesis of l-serine module (serAf, serB and serCCg) and overexpression of CysB. Then, promotion of l-cysteine transporter, increased assimilation of sulfur, reduction or deletion of l-cysteine and l-serine degradation pathway and enhanced expression of cysEf (encoding serine acetyltransferase) and cysBSt (encoding transcriptional dual regulator CysB) were achieved, resulting in an improved l-cysteine titer (3.82±0.01) g/L. Subsequently, expressions of cysM, nrdH, cysK and cysIJ genes that were involved in sulfur module were regulated synergistically with carbon module combined with utilization of sulfate and thiosulfate, resulting in a strain producing (4.17±0.07) g/L l-cysteine in flask shake and (11.94±0.1) g/L l-cysteine in 2 L bioreactor. Our results indicated that efficient biosynthesis of l-cysteine could be achieved by a proportional supply of sulfur and carbon in vivo. This study would facilitate the commercial bioproduction of l-cysteine.

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Ergothioneine is a multifunctional physiological cytoprotector, with broad application in foods, beverage, medicine, cosmetics and so on. Biosynthesis is an increasingly favored method in the production of ergothioneine. This paper summarizes the new progress in the identification of key pathways, the mining of key enzymes, and the development of natural edible mushroom species and high-yield engineering strains for ergothioneine biosynthesis in recent years. Through this review, we aim to reveal the molecular mechanism of ergothioneine biosynthesis and then employ the methods of fermentation engineering, metabolic engineering, and synthetic biology to greatly increase the yield of ergothioneine.

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l-cysteine is an important sulfur-containing α-amino acid. It exhibits multiple physiological functions with diverse applications in pharmaceutical cosmetics and food industry. Here, a strategy of coordinated gene expression between carbon and sulfur modules in Escherichia coli was proposed and conducted for the production of l-cysteine. Initially, the titer of l-cysteine was improved to (0.38±0.02) g/L from zero by enhancing the biosynthesis of l-serine module (serAf, serB and serCCg) and overexpression of CysB. Then, promotion of l-cysteine transporter, increased assimilation of sulfur, reduction or deletion of l-cysteine and l-serine degradation pathway and enhanced expression of cysEf (encoding serine acetyltransferase) and cysBSt (encoding transcriptional dual regulator CysB) were achieved, resulting in an improved l-cysteine titer (3.82±0.01) g/L. Subsequently, expressions of cysM, nrdH, cysK and cysIJ genes that were involved in sulfur module were regulated synergistically with carbon module combined with utilization of sulfate and thiosulfate, resulting in a strain producing (4.17±0.07) g/L l-cysteine in flask shake and (11.94±0.1) g/L l-cysteine in 2 L bioreactor. Our results indicated that efficient biosynthesis of l-cysteine could be achieved by a proportional supply of sulfur and carbon in vivo. This study would facilitate the commercial bioproduction of l-cysteine.

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Fermentation engineering is an industrial process that uses the transformation of microorganisms or other cells to produce a specific product in a specific bioreactor. Fermentation engineering has developed from an ancient food fermentation relying solely on experience accumulation to an important production mode of food, agriculture, medicine, chemical industry and other means of production and life. It has become a key technology to support the sustainable development of human beings, and is inseparable from the continuous progress of interdisciplinary technology. The interdisciplinary integration and the continuous upward movement of China's global industrial chain will inevitably put forward higher requirements for the cultivation of fermentation engineering composite talents in the new situation. In order to constantly improve the interdisciplinary fermentation engineering compound talent training system, in recent years, the research lab has been refining and improving the concept of talent training, and actively deepening the reform of talent training system. Systematic research and practice have been carried out around the aspects of training program, enrollment system, teacher background, subject setting, scientific research practice, evaluation system, etc., which has promoted the technological progress of fermentation engineering and related supporting industries, and contributed an important force to the transformation of China from a big fermentation country to a powerful fermentation country.

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In this study, chicken peptone was produced by hydrolysing inedible parts derived from chickens using endo-protease and exo-protease. The usefulness of chicken peptone as a nutrient source for bacteria was evaluated in comparison with other commercially produced peptones (animal, soy and casein-derived peptone). Escherichia coli and Bacillus subtilis were used as test strains to determine the effect of peptones from different sources on their growth ability. Both bacteria were successfully cultured in chicken peptone solution, which is similar to peptone solution containing commercial peptones apart from animal peptone. In chemical analysis, chicken peptone contained 12·0% nitrogen; this was similar to the nitrogen content from other commercial peptone sources, except for the 9·0% nitrogen found in soy peptones. The molecular weight of the peptone was determined by gel filtration chromatography, and those of all peptone, except animal-derived peptone, were found to be <5000 Da. In addition, when B. subtilis was cultured in a medium containing chicken peptone, it was shown that the protease activity was highest as compared with other commercial peptones. From these results, it is suggested that chicken peptone can be utilized for microbial culture, and this is an effective method to reuse chicken waste.

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Fermentation engineering is an industrial process that uses the transformation of microorganisms or other cells to produce a specific product in a specific bioreactor. Fermentation engineering has developed from an ancient food fermentation relying solely on experience accumulation to an important production mode of food, agriculture, medicine, chemical industry and other means of production and life. It has become a key technology to support the sustainable development of human beings, and is inseparable from the continuous progress of interdisciplinary technology. The interdisciplinary integration and the continuous upward movement of China's global industrial chain will inevitably put forward higher requirements for the cultivation of fermentation engineering composite talents in the new situation. In order to constantly improve the interdisciplinary fermentation engineering compound talent training system, in recent years, the research lab has been refining and improving the concept of talent training, and actively deepening the reform of talent training system. Systematic research and practice have been carried out around the aspects of training program, enrollment system, teacher background, subject setting, scientific research practice, evaluation system, etc., which has promoted the technological progress of fermentation engineering and related supporting industries, and contributed an important force to the transformation of China from a big fermentation country to a powerful fermentation country.

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Enzyme engineering combines enzymology and engineering, and is one of the major fields of modern biotechnology. To promote enzyme engineering research in China, we present in this special issue with reviews and original articles focusing on recent relevant advances reported by Chinese scientists.

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