Microbial production of vitamin K2: current status and future prospects

Journal Pre-proof Microbial production of vitamin K2: current status and future prospects

Lujing Ren, Cheng Peng, Xuechao Hu, Yiwen Han, He Huang PII:

S0734-9750(19)30153-3

DOI:

https://doi.org/10.1016/j.biotechadv.2019.107453

Reference:

JBA 107453

To appear in:

Biotechnology Advances

Received date:

17 June 2019

Revised date:

24 August 2019

Accepted date:

17 September 2019

Please cite this article as: L. Ren, C. Peng, X. Hu, et al., Microbial production of vitamin K2: current status and future prospects, Biotechnology Advances (2018), https://doi.org/ 10.1016/j.biotechadv.2019.107453

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© 2018 Published by Elsevier.

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Microbial production of vitamin K2: current status and future prospects Lujing Ren1 , Cheng Peng1 , Xuechao Hu1 , Yiwen Han1 , He Huang1,3,* [email protected] 1

College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University,

School of Pharmaceutical Sciences, Nanjing Tech University, No. 30 South Puzhu

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2

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No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China

School of Food Science and Pharmaceutical Engineering, Nanjing Normal University,

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3

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Road, Nanjing 211816, People’s Republic of China

Corresponding author.

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Abstract

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*

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Wenyuan Road, Nanjing 210023, People’s Republic of China

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Vitamin K2, also called menaquinone, is an essential lipid-soluble vitamin that plays a critical role in blood clotting and prevention of osteoporosis. It has become a focus of research in recent years and has been widely used in the food and pharmaceutical industries. This review will briefly introduce the functions and applications of vitamin K2 first, after which the biosynthesis pathways and enzymes will be analyzed in-depth to highlight the bottlenecks facing the microbial vitamin K2 production on the industrial scale. Then, various strategies, including strain mutagenesis and genetic modification, different cultivation modes, fermentation and

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separation processes, will be summarized and discussed. The future prospects and perspectives of microbial menaquinone production will also be discussed finally. Keywords: Vitamin K2, Menaquinone, Bacillus, Strain improvement, Process engineering, Downstream processing 1 Introduction Osteoporosis is a disease that affects the health of all age and race groups. It has

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become an invisible burden and a threat to national health and security (Ganji et al.,

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2019). It is estimated that osteoporosis was contributed to approximately 9 million

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cases of bone fractures worldwide in 2010 (Balasubramanian et al., 2019). The number of osteoporosis patients is expected to increase in the future, especially in the

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Asia-Pacific region, owing to aging populations, sedentary lifestyles, and widespread

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vitamin D deficiency (Antunez et al., 2007; Si et al., 2015). Therefore, the

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development of highly effective products for the prevention of osteoporosis is

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urgently needed to enhance the quality of life and reduce the pain of elderly people. Vitamin K2 is an essential lipid-soluble vitamin that plays an important role in blood clotting and the prevention of osteoporosis (Frandsen and Gordeladze, 2017; Li et al., 2018). It is considered as a member of the fourth generation of anti-osteoporosis drugs (Shea and Holden, 2012; Vermeer, 2012). In 1943, Henrik Dam and Edward Doisy shared the Nobel Prize in physiology or medicine for their discovery and contribution of vitamin K (Dam, 2010; Dam, 1967). Dam firstly discovered the existence of vitamin K in chicks after feeding a very low- fat diet in 1929, and Diosy uncovered the chemical nature of vitamin K, which is characterized

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by the presence of a 2-methyl-1,4-naphthoquinone ring. According to the structure of the side chain at the third position of the naphthoquinone ring, vitamin K can be divided into different subtypes, such as K1, K2, K3 and K4. Vitamin K2 is also called menaquinoe and its naphthoquinone ring is attached with a variable side chain of 4-13 isoprene units, generating s a series of isoforms referred to MK-n. Menaquinone-7 is

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the topic of this review (Mahdinia et al., 2017a).

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In recent years, preparation and industrialization of vitamin K2 has become a focus

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of research. Several microorganisms were found to produce different forms of vitamin

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K2 (Bentley and Meganathan, 1982; Dairi, 2012a). Notably, Bacillus subtilis has

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the potential to produce menaquinone-7 (MK-7) on a large scale (Sato et al., 2001). The industrialization of vitamin K2 production started since the 1990s. In 1995, a

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Japanese company called Honen Corp. disclosed a process for producing lipids with

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high content of natural menaquinone-7 by extraction from food raw material

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fermented by Bacillus natto. To enhance the productivity of menaquinone-7, Gnosis subsequently proposed a liquid fermentation process for menaquinone-7 production using B. subtilis DSM 17766 in 2007 (Alberto, 2010; Valoti, 2007). Meanwhile, a chemical route to synthesize menaquinone-7 was also invented by Kappa Bioscience Company in 2008 (Inger, 2013; Lars, 2008). In the late years, Chinese companies such as Sungen Bioscience have also built the industrial plants focusing on the production of microbial MK-7 and other nutrition chemicals like nattokinase. Under the impetus of these companies, vitamin K2 has been approved for use as a novel food ingredient in the USA, European Union, China, and Australia, among others.

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It is obvious that vitamin K2 will play a more critical role in the future, and microbial production will be the most effective strategy for its industrialization. In this review, a brief introduction of the functions and applications of vitamin K2 will be given first. Then, different sources of vitamin K2 especially different producing microorganisms and their biosynthesis pathways will be illustrated for a better

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understanding of menaquinone biosynthesis. Furthermore, techniques to enhancing

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vitamin K2 production from the perspective of strain improvement, process

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optimization and metabolic engineering will be discussed in detail. We hope this

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review will be helpful to expand the development and applications of vitamin K2 and

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facilitate the industrialization of microbial vitamin K2 in the near further. 2 Types, functions and application of vitamin K2

K

is

a

group

of

structurally

similar,

lipid-soluble

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Vitamin

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2.1 Types and functions

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2-methyl-1,4-naphthoquinone-3-derivatives. Vitamin K1 and K2 are two natural forms of vitamin K (Fig. 1). Vitamin K1 (phylloquinone) has a mono-unsaturated side chain and is usually produced by plants and algae involving in photosynthesis (Shearer, 1992). Vitamin K2 is synthesized mainly by certain bacteria and the most well-known are menaquinone-4 (MK-4) and menaquinone-7. Vitamin K3 also belongs to the vitamin K series, but it is a synthetic form and may be toxic, so it is seldom used to treat vitamin K deficiency. Vitamin K2, acting as the key electron-delivery vector of the respiratory chain (Azarkina and Konstantinov, 2002; Melo et al., 2004) (Fig.2), plays important roles

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in electron transport, blood coagulation and calcium homeostasis (Iwamoto et al., 2011; Theuwissen et al., 2014). It is also an important coenzyme involved in the biosynthesis of many nature products (Law et al, 2018). In the early years following the discovery of vitamin K2, researchers concentrated solely on its function in blood coagulation. It wasn’t until 1960 that Bouckaert and Said reported that vitamin K2

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could also promote fracture healing (Willems et al., 2014), which opened the study of

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vitamin K2 in bone research. It’s reported that vitamin K2 is an essential cofactor for

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the γ-carboxylation of osteocalcin, and the carboxylated osteocalcin plays roles in

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skeleton development. Therefore, it is helpful in transferring calcium from blood to

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the bone. Accordingly, vitamin K2 is used clinically in Japan to treat osteoporosis either alone or in conjunction with other medications. In addition, the function of

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preventing cerebrovascular disease has been proved based on its role in activating

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and Holden, 2012).

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matrix Gla protein, a calcification inhibitor that is expressed in vascular tissue (Shea

Furthermore, many additional functions of vitamin K2 were also discovered in recent years, such as cancer prevention by inducing cell cycle arrest to inhibit proliferation (Fan et al., 2018; Liu et al., 2016a; Nimptsch et al., 2010), prevention of Parkinson's disease by transferring electrons to rescue mitochondrial dysfunction (Vos and Verstreken, 2012), promoting functional recovery of the liver (Lin et al., 2014) and reducing the risk of type 2 diabetes mellitus (Li et al., 2018). Considering all these biological activities and functions of vitamin K2, it is regarded as having broad application prospects in the future.

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2.2 Safety certification and applications of Vitamin K2 Vitamin K2 has been treated as a nutritional enhancer for use in foods and medications, relying on the safety approval of vitamin K2 by different safety supervision and administration organizations (Marles et al., 2017; Pucaj et al., 2011). Japan was the first country to approve the sale of vitamin K2 as a drug for the

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treatment of osteoporosis in 2001. Subsequently, the US Food and Drug

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Administration and the European Food Safety Authority approved its use as food and

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enhancer food additive in 2008 and 2009 (Approval in Europa and EFSA, 2009),

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respectively. In 2009, the European Food Safety Authority further announced that the

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MK-7 form of vitamin K2 can be claimed as a vitamin K with high bioavailability that can help patients maintain bone and cardiovascular health. However, the approval

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in China was not published until 2016 by the Health and Family Planning

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Commission (Approval in China, 2016).

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3 Current production methods and industrialization Human cells cannot synthesize MK-7 and humans can only obtain it from food or dietary supplements. However, the amount of vitamin K2 in normal food is quiet low. For example, there is only 1 mg of vitamin K2 from 6.25 kg pork or 7.14 kg of eggs (Walther and Chollet, 2017). For this reason vitamin K2 is also sometimes called the “platinum vitamin”. Currently, there are three sources of menaquinone-7: (1) extraction from plants, animals, or foodstuffs; (2) chemical synthesis; (3) microbial production. 3.1 Extraction

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Japan was the first country to develop and use vitamin K2, which is mainly attributed to their traditional food of natto. This traditional Japanese soybean product has a characteristic stringy texture and a unique flavor. Moreover, there is about 800-900 µg of vitamin K2 per 100g natto (Tarvainen et al., 2019). Besides vitamin K2, natto also contains many other bioactive compounds like nattokinase and γ

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-polyglutamic acid. In addition, minute amounts of vitamin K2 analogs were also

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found in different foods such as cheese (Vermeer et al., 2018), honey (Brudzynski

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and Maldonado, 2018), meat, yogurt, milk and dairy products (Walther and Chollet,

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2017). However, low productivity is the main problem of extraction for vitamin K2

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production. Recently, natto is still the main source of extraction, but other sources could provide new information for the dietary supplement of vitamin K2 and find

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3.2 Chemical synthesis

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structural analogues with new activities and physiological functions.

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Due to the low concentration of menaquinone in natural sources, there have been many attempts to synthesize it chemically. In 1958, Isler (1954) firstly completed the synthesis of vitamin K2 via Friedel-Crafts alkylation . In spite of further optimization of this method in subsequent years, the selectivity of the reaction was still poor (Lipshutz, 1998; Yoji., 1977). In 1995, Tso and Chen (1995) published a novel one-pot procedure to synthesize molecules of the vitamin K series containing vitamin K1, MK-1, MK-2 and MK-9 with a yield of more than 60%. Shimada et al. (1989) specifically synthesized MK-4 with more than 96% selectivity for the trans isomer using ethyl acetoacetate as the substrate. Researchers also tried different methods to

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synthesize all-trans MK-7. Baj et al (2016) obtained 99.9% high-purity MK-7 using menadione, isoprene and trans-farnesol as substrates. Meanwhile, different chemical routes for the synthesis of MK-7 were also invented by many companies (Kraje wski Krzysztof, 2017; Lars, 2009) which led to its industrialization. In China, MK-7 powder or commercial products could be obtained based on the selective reaction of

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chromatography, recrystallization, drying and sieving.

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7-terpineol and menadione after steps encompassing distillation, extraction, column

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However, chemical synthesis methods usually encompass complicated steps with

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low yields. At the same time, such methods often produce different cis isomers with

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low activity, generate large amounts of by-products, and cause environmental pollution. Although the chemical synthesis of vitamin K2 has been industrialized, new

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production methods still need to be explored due to the mentioned shortcomings of

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chemical processes.

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3.2 Microbial production

As menaquinone is the essential electron-delivery vector of the respiratory chain, various microbial cells, are able to produce menaquinone (Bentley et al., 1971; Bentley and Meganathan, 1982). After comparing the distribution of isoprenoid quinones among bacterial and fungal strains, Tani et al found bacteria are the main producers of menaquinone (Tani et al., 1984). Different strains can produce different types of menaquinone. B. subtilis is capable of producing MK homologues from MK-4 to MK-8 and the percentage of MK-7 can reach 96%, while Flavobacterium mainly produces MK-4 and MK-6. Some intestinal microbes, especially lactic acid

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bacteria (LAB) such as Lactococcus, Lactobacillus, Enterococcus, Leuconostoc and Streptococcus, were also found to produce wider range of MKs including MK-7, MK-8, MK-9 and MK-10 (Chollet et al., 2017; Takashi, 1999). Recently, more new strains were also found to synthesize many other MK series compounds such as MK-6, MK-8, MK-9, and MK-10 (Cao et al., 2008; Izumi et al., 2012; Kulichevskaya et

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al., 2009; Nishijima et al., 2012). On the other hand, the type of menaquinone was

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also used as the basis of bacterial classification. Among all these menaquinone

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producers, Bacillus species are considered as the dominant and most promising

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safe use and high content of MK-7.

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potential strains for microbial production of vitamin K2 due to their long history of

3.4 Current industrial production

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Only about 10 companies around the world are able to manufacture vitamin K2

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(Table 1). Kappa Bioscience and Gnosis are two prominent manufacturers that

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produce vitamin K2 by chemical synthesis and microbial fermentation, respectively. Additionally, NattoPharma, Viridis Biopharma and Sungen are typical manufacturers that use microbial fermentation to produce natural MK-7 products. These companies provided most of the vitamin K2 in the word at a relatively high price, which is about $1200 per kg of 0.1% formulation. According to a recent study by British market research firm Intel Mint, new products containing vitamin K2 in food and beverages worldwide have increased by 183% in the past five years. Shockingly, the annual cost of treating osteoporotic fractures and cardiovascular disease in the United States alone is $22 billion (Blume and Curtis, 2011) and $502 billion (Berenjian et al., 2015;

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Lloyd, 2010), respectively, showing an expanding market and a bright further for the the application of vitamin K2. 4 Biochemistry of vitamin K2 biosynthesis 4.1 Classic MK biosynthesis pathway To better understand the microbial production of vitamin K2, the biosynthesis

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pathway will be discussed first. Several carbon sources including monosaccharides

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and glycerol could be fermented to produce menaquinone. Bentley et al. briefly

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illustrated vitamin K2 biosynthesis pathway in bacteria in 1971 (Bentley et al., 1971;

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Bentley and Meganathan, 1982). The structure of menaquinone consists of a

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naphthoquinone ring and an isoprene side chain. The isoprenoid side chain is formed in the methylerythritol 4-phosphate (MEP) pathway and isopentenyl diphosphate

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biosynthesis pathway, while the naphthoquinone ring is synthesized through the MK

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pathway. As shown in Fig. 3, when using glycerol as the substrate, glycerol kinase

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(GK) will convert glycerol to glycerol 3-phosphate firstly and then to glyceraldehyde 3-phosphate entering into glycolytic pathway to generate pyruvate. Pyruvate will be decarboxylated to form acetyl-CoA, which enters the TCA cycle. At the same time, pyruvate

will

also

condense

with

glyceraldehyde

3-phosphate

to

form

1-deoxy-D- xylose-5-phosphate and enter the MEP pathway for IPP formation. For the biosynthesis of MK-7, HPP with seven isoprene units is needed. Additionally, glyceraldehyde 3-phosphate also enters the pentose phosphate (HMP) pathway to yield the important intermediate of 4-phosphate-erythritol, which can be used to synthesize shikimic acid by a series of ligation, dehydration, and dehydrogenation

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reactions. Shikimic acid, as the starting point of menaquinone biosynthesis, is used to form the quinone skeleton of 2- hydroxy-2-1,4-naphthalene formic acid (DHNA) by six enzymes encoded by the menFDHCEB genes (Dairi, 2012a; Meganathan, 2001a). Finally, the HPP unit is transferred to the carboxyl group of DHNA by 1,4-dihydroxy-2-naphthoate octaprenyl transferase encoded by menA, finally forming

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menaquinone via methylation by UbiE/menG (Dairi, 2012b; Meganathan and

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Kwon, 2011b).

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Many enzymes that take part in the de- novo biosynthesis of menaquinone have

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been biochemically and structurally studied in the past few years (Table 2). The genes

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encoding enzymes of the shikimate pathway and menaquinone biosynthesis usually cluster together and are called the men gene cluster. This gene cluster was identified

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in different menaquinone producers (Fig. 4). In E. coli, five of the six genes

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responsible for the formation of DHNA (all except menA) cluster together at 51 min

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on the E. coli chromosome, while menA, encoding 1,4-dihydroxy-2-naphthoate octaprenyl transferase, is located at 88 min (Rowland et al., 1995; Suvarna et al., 1998). B. subtilis is arguably the best-studied MK-7 producer. Kobayashi et al. (2003) studied the essential genes of B. subtilis and they identified 22 genes involved in the respiration process, in which 16 are required for menaquinone synthesis, including menA-E, menH and so on (Rowland et al., 1995). Additionally, there are many rate- limiting enzymes that are not encoded in the men gene cluster. Polyprenyl pyrophosphate synthetase is a key enzyme determining the final product by changing the length of the isoprene side-chain. A mutant heptaprenyl diphosphate synthase

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obtained through site-directed mutagenesis could yield prenyl diphosphates with different chain lengths and form different menaquinone compounds. 4.2 The alternative pathway: futalosine pathway In addition to the classic MK biosynthesis pathway from shikimic acid, encompassing seven enzymes encoded by the men gene cluster in Bacillus spp. and E.

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coli, there is another alternative pathway for menaquinone biosynthesis, the futalosine

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pathway (Fig. 5) (Hirats uka et al., 2008). In early work leading to the discovery of

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this pathway, researchers were unable to find the men gene cluster in the genome of

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Streptomyces and some pathogenic species, such as Helicobacter pylori and

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Campylobacter jejuni. Further tracer experiment proved the existence of an important intermediate, 1,4-dihydroxy-6-naphthoate for menaquinone biosynthesis in S.

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coelicolor A3 (Hiratsuka et al., 2009) and more genes and enzymes were also

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identified by genetic studies and genome sequence comparisons (Jos hi et al., 2018).

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In the futalosine pathway, chorismate will be converted to futalosine (Arakawa et al., 2011) and then to 1,4-dihydroxy-6-naphthoate by four enzymes encoded by the mqnABCD gene cluster (Kim et al., 2014). Then, the prenyl moiety from trans-polyprenyl diphosphate will be attached by MqnP to form menaquinone (Cotrim et al., 2017). In addition, a modified futalosine pathway was also found in Campylobacter jejuni (Xu et al., 2011), which starts from 6-amino-6-deoxyfutalosine instead of futalosine as the first step. The mqn genes encoding the newly discovered futalosine pathway were found to be scattered throughout the genomes in most of the microorganisms that utilize it

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(Arakawa et al., 2011; Dairi, 2012b; Jos hi et al., 2018). The reason why some microorganisms use the new pathway to synthesize menaquinone is still unclear, but it provided new ideas for drug development (Choi et al., 2016; Paudel et al., 2016). In particular, considering that the futalosine-dependent menaquinone biosynthesis pathway is absent in humans, studying inhibitors targeting this pathway is a hot topic

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for the development of new antibiotics (Shimizu et al., 2018; Tanaka et al., 2011).

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4.3 Problems of vitamin K2 biosynthesis

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Usually, the menaquinone levels in microorganisms stay at micromolar level which

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is related to the complex and tightly regulated biosynthesis pathway. Both

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biosynthesis pathways contain many enzymes, cofactors and reactions, and this complexity determines the low biosynthesis efficiency of menaquinone. Furthermore,

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there are some tradeoffs in the pathway. For example, pyruvate is used to synthesize

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1-deoxy-dxylose-5-phosphate in the MEP pathway, while the precursor of pyruvate,

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phosphoenolpyruvate, is also needed for shikimate formation with the intermediate of the HMP pathway, D-erythrose 4-phosphate. Similarly, α-ketoglutaric acid, which is an intermediate of the citric acid cycle, is also a precursor in the menaquinone biosynthesis pathway. Consequently, menaquinone biosynthesis requires the cell to coordinate many different metabolic pathways. Furthermore, feedback-inhibition of key enzymes also restricts vitamin K2 biosynthesis. For example, pyruvate kinase is inhibited by sodium citrate and alanine. Similarly, DAHP synthase, which takes part in shikimate biosynthesis and determines the formation of the quinone skeleton, is feedback- inhibited by DHNA, and aromatic amino acids (Fernandez and Collins,

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1987; Tsukamoto et al., 2001). Polyprenyl pyrophosphate synthetase, which determines the formation of the isoprene side chain, is inhibited by DPA (Takahas hi et al., 1980; Zhang et al., 1998). Moreover, the low expression levels of the related enzymes for MK-7 biosynthesis might be another important reason for the low rates of menaquinone biosynthesis.

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5 Strategies for enhancing vitamin K2 production

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To accelerate the industrialization of vitamin K2, many research efforts have

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focused on enhancing vitamin K2 production, including strain improvement to

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enhance biosynthesis capacity, process optimization to make the production more

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efficient, and new bioreactor designs to accelerate the industrialization process. Table 3 shows the fermentation strategies and corresponding vitamin K2 production yields

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obtained using different vitamin K2 producers. The detailed information will be

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discussed below.

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5.1 Strain improvement

5.1.1 Mutagenesis by structural analogs Strain performance is the key of fermentation and determines the final product concentration and productivity. The biosynthesis of menaquinone involves a variety of metabolic pathways and enzymes, several of which are inhibited or regulated by specific compounds. These phenomena affected the menaquinone biosynthesis and also provided clues for strain mutagenesis. Resistance to structural analogs was commonly used as a screening method to improve the biosynthesis of vitamin K2. The 1,4-dihydroxy-2-naphthoate analog, 1- hydroxy-2-naphthoate (HNA) is the

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most commonly used molecule for strain screening. The Yoshiki group was the first to report the use of structural analog resistance to improve vitamin K2 biosynthesis. They used HNA to screen for mutants of Flavobacterium meningosepticum (Tani, 1985; Tani, 1987a) and Flavobacterium sp. (Tani, 1987b). The biosynthesis of MK-6 and MK-5 as well as the production of vitamin K2 increased in all the HNA-resistant

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mutants, proving the effectiveness of this method. Their findings indicated that

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conventional mutagenesis might change the specificity of prenyltransferase

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determining the side-chain length of MK. Chorismate is an important intermediate for

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the synthesis of aromatic amino acids, folic acid and menaquinone. It is reported that

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sulfonamides can inhibit the biosynthesis of folic acid and increase chorismate accumulation in Brevibacterium flavum. Therefore, Yoshiki et al. further used

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derivatives of sulfonamides to increase MK-4 production in Flavobacterium sp.

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(Taguchi et al., 1989). The production capacity of the final mutant strain was 50%

type strain.

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higher than that of the HNA-resistant mutant and almost 20 times than that of the wild

In addition, the structural analog resistance strategy was also used in B. subtilis (Song et al., 2014). Vitamin K2 production of a menadione-resistant mutant increased by 30% (Toshiro et al, 2001a). In another study, resistance to analogs of compounds in the menaquinone biosynthetic pathway with mutations combined. Yoshinori et al. (Tsukamoto et al., 2001) constructed a mutant with analog resistance to six different compounds including 2-hydroxy-1-naphthaldehyde (HNA) and L-phenylalanine, and the productivity of the mutant increased by two- fold compared to the wild type.

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Diphenylamine (DPA) was known to inhibit the synthesis of menaquinone and carotenoids in Bacillus megaterium, Staphylococcus aureus and other strains (Bentley and Meganathan, 1982; Hammond and White, 1970; Salton and Schmitt, 1967), and a DPA-resistant mutant was also found to produce more MK-7 or MK-8 after mutagenesis with NTG or ARTP treatment (Xu et al, 2017a).

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Although the method of inducing resistance to structural analogs yielded some

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efficient producers of vitamin K2, this method is still time-consuming and

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labor-intensive. A high-throughput screening strategy was also proposed based on the

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fluorescence-activated cell sorting (Liu et al., 2016b). Using the rhodamine 123 to

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connect the value of membrane potential with the quantitate of MK content by a fluoresence signal, the vitamin K2 contents in 50 thousand cells could be assayed in

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5.1.2 Genetic modification

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less than 1h, greatly improving the screening efficiency.

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Traditional strain mutagenesis can improve the production ability but with low efficiency (Lee et al., 2005; Sergio and Arnold, 2002). Moreover, low expression levels of the key enzymes involved in menaquinone biosynthesis is another key reason for the low quantities of vitamin K2 in wild-type strains. As Bacillus has clear genomic annotation information and mature tools for genetic modification (Liu et al., 2017a), researchers attempted to change the metabolic pathways in Bacillus or construct new pathways in other model microorganisms. Enhancing the precursor supply and eliminating byproduct synthesis pathways are commonly used to improve the strain performance. MK-8 is the main

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menaquinone of E. coli, and its content was significantly enhanced by overexpressing the key enzymes relating to the supply of two precursors. Fivefold increase was observed when MenA or MenD overexpressed and further 30% increase was obtained by blocking the ubiquinone-8 pathway (Kong and Lee, 2011). Furthermore, ubiquinone is another important compound in the membrane sharing the same

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metabolic pathway for the isoprenoid tail chain formation with menaquinone. In

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another study about the strain of E. meningoseptica, the key enzyme of the

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ubiquinone-8 pathway, 4-hydroxybenzoate octaprenyl transferase, was inactivated by

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site-directed mutagenesis, generating 130% increase of MK content (Liu et al.,

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2017b). In addition, further co-expressing of rate-limiting enzymes encoded by dxr and menA and supplementing the substrate precursors such as sodium pyruvate and

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shikimic acid to the medium led to 11 times of increase in menaquinone content (Liu

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et al., 2018). Furthermore, in the strain of B. subtilis, Ma et al. (2019) overexpressed

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different combinations of the rate-limiting enzymes like Dxs, Dxr, Idi and MenA, resulting in the increase of MK-7 titer from 4.5 mg/L to 50 mg/L in the recombinant strain with a good genetic stability. Modular pathway engineering (Boock et al., 2015; Liu et al., 2019) is another effective method for improving the biosynthesis of specific chemicals. Using B. subtilis 168 as the chassis for MK-7 production, Yang et al. (2019b) categorized the MK-7 biosynthesis pathway into four modules and tried to enhance different modules to study the key limiting steps for MK-7 biosynthesis. 2.1 fold and 82% increase were obtained by overexpressing menA in MK-7 pathway and seven enzymes in MEP

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pathway. Unexpectedly, enhancing the shikimate pathway has a negative effect on MK-7 biosynthesis because of the feedback inhibition by chorismate. Final mutant strain could produce 69.5 mg/L of MK-7, which represented a more than 20- fold increase compared with the starting strain. Xu et al. paid more attention to the enzymes encoded by men gene cluster (Xu et al., 2017b). Similar increase was also

amyloliquefaciens

Y-2,

overexpressing

HepS

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Bacillus

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observed when overexpressing menA in B. subtilis 168. While in another strain of encoding

heptapreny

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pyrophosphate synthase led to a greater increase than that of other enzymes, which

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also provided the information about different rate-limiting steps in different MK-7

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producers. All of the above are belonging to static metabolic engineering strategies, but static metabolic engineering strategies are easy to overload or destroy the normal

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metabolic network and cause metabolic imbalances. Recently a kind of dynamic

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pathway regulation strategy has been successfully applied to improve the synthesis of

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MK-7. Cui et al. (2019) developed a bifunctional and modular Phr60-Rap60-Spo0A QS system to balance the relationship between efficient synthesis of the MK-7 and cell growth. Finally, their dynamic pathway regulation led to a 40- fold improvement of MK-7 production from 9 to 360 mg/L which is the highest reported titer of MK-7. 5.2 Process engineering 5.2.1 Exploring different cultivation mode Solid state fermentation (SSF) and liquid state fermentation (LSF) were two main processes for menaquinone production. Natto was traditionally made by cultivating B. subtilis natto using boiled soybeans. This traditional method is a simple

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solid stage fermentation mode. The earlier production mode of vitamin K2 was also based on solid-state cultivation. A number of different approaches to improve the fermentation yield have been described in the literature (Mandinia et al., 2017). For the solid state fermentation, many kinds of substrates such as legumes, cereals, and raw wheat, were used as the basis of the medium for the cultivation of B. subtilis natto

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(Xu and Zhang, 2017). Depending on different strains, medium components, initial

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moisture levels and environmental factors, the yield of MK-7 varied between 0.53 and

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140 mg/kg (Mahanama et al., 2011a; 2011b; 2012; Mahdinia et al., 2017a; Singh

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et al., 2015a).

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One benefit of solid-state fermentation is that this process does not require the expensive organic solvent extraction used for the purification of MK-7 because the

al

final product can be dried and pelleted as a direct supplement which itself is a food

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containing high levels of MK-7 and other nutrients (Mahanama, 2013). However, the

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process parameters of solid-state fermentation are difficult to control. The metabolic heat generated during fermentation is difficult to remove and improper humidity can greatly affect the physical properties of the solid matrix. Low humidity would lead to a decrease of substrate expansion, a reduction of nutrient solubility and an increase of osmotic pressure. Conversely, high humidity might limit the production of biomass to a large extent due to the agglomeration of particles, resulting in a decrease of MK -7 content. In addition, solid-state fermentation usually needs a large area and long cycle time, so the research direction gradually turned toward liquid fermentation.

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Liquid fermentation can improve cell growth, shorten the fermentation cycle and reduce the volume of the bioreactor. Glycerol, maltose and glucose are commonly used as carbon sources, while yeast extract and soybean peptone are the main nitrogen sources for cell growth (Wu and Ahn, 2011b). It has been reported that glucose is beneficial for cell growth and glycerol is helpful in MK-7 biosynthesis in Bacillus natto (Luo et al., 2016). Singh et al. proposed that 4% (v/w) is the optimum glycerol

oo

f

concentration and proper concentration of Ca2+ benefits MK-7 biosynthesis (Singh et

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al., 2015b). Berenjian’s group have done lots of research work about the cultivation

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optimization of menaquinone-7 production by B. subtilis natto. They used 50 g/L of

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glycerol and 189 g/L of soybean peptone for fermentation, and the yield of MK-7 reached 62.3 mg/L (Berenjian et al., 2011a). In addition, fed-batch fermentation

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strategies with secondary glycerol addition were also proposed. Adding 2% glycerol

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to the medium at 48h was beneficial for menaquinone-7 biosynthesis, leading to a

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40% increase the final yield (Berenjian et al, 2012). During the biosynthesis of menaquinone, Bacillus can also produce nattokinase (NK) at the same time. Wang et al. found a positive correlation between MK-7 and NK production (Wang et al., 2018), which also provide a new clue for enhancement of MK-7 production. 2675.73 U/mL of nattokinase was also obtained with 91.25 mg/L of MK-7 when cultivated with lactose and soybean curd residue as the carbon and nitrogen sources. 5.2.2 Optimizing environmental conditions Fermentation environmental conditions including oxygen supply, temperature and others can affect cell growth and final vitamin K2 concentration, yield and

Journal Pre-proof productivity. Bacillus can tolerate higher temperatures range from 28 to 45 0 C, but 40 0

C seems to be the optimal temperature for high vitamin K2 production (Berenjian et

al., 2011b). Bacillus is a kind of bacteria which can generate spores under the improper environmental conditions. This phenomenon largely affected the synthesis of vitamin K2. Earlier study found the slow sporulation under the static culture

f

condition is beneficial for the MK-7 production (Mahdinia et al., 2017a). However,

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other researchers demonstrated that agitated fermentation could enhance the yield of

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menaquinone significantly (Berenjian et al., 2014b). The highest MK-7 yield of 226

e-

mg/L was reported when the cells were cultivated for 5 days at an extreme high

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oxygen supply condition with 1000 rpm and 5 vvm in a 3-L fermenter. Subsequently, Luo et al reported the positive relationship between the sporulation rate and the MK-7

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titer (Luo et al., 2016). The mechanism of increased MK-7 production under high

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oxygen supply conditions was further deciphered based on the metabonomics data

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and the activities of the intracellular key enzymes in Bacillus natto (Ma et al., 2019). The high ability of MK-7 biosynthesis under high oxygen supply conditions might be related

to

high

pyruvate

kinase

activity,

low

glyceraldehyde-3-phosphate

accumulation, and a highly active TCA pathway. Additionally, some new strategies for enhancing menaquinone production were also proposed. Cell immobilization could complete the cell to a limited space and the fixed cell could be easily and repeatedly used to improve the production efficiency. Immobilization with magnetic nanoparticles has the benefit of not affecting mass transfer. Alireza et al. tired different iron oxide nanoparticles to immobilize Bacillus

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and found the novel carrier was beneficial for menaquinone-7 biosynthesis without any toxic effect on the cells (Ebrahiminezhad et al., 2016a; Ebrahimine zhad et al., 2016b).

Then

the

modified

iron

oxide

nanoparticles

coated

with

3-aminopropyltriethoxysilane or L- lysine were also introduced and generated two- fold increase in menaquinone-7 production (Ebrahiminezhad et al., 2016b;

f

Ranmadugala et al., 2017).

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5.2.3 Increasing vitamin K2 secretion

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Menaquinone, acting as a coenzyme in the respiratory chain, is present in the

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membrane or in the free form (Azarkina and Konstantinov, 2002; Kurosu and

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Begari, 2010; Melo et al., 2004). Consequently, its intracellular accumulation is not as great as that of oleaginous microorganisms, which can accumulate lipids in lipid

al

droplets (Olzmann and Carvalho, 2019; Sun et al., 2018; Yu et al., 2018). Previous

rn

studies have demonstrated that about 20-60% menaquinone-7 would be secreted into

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the broth during fermentation. Hiro and Doi (1990) proved that the secreted vitamin K2 was a soluble complex containing a specific acidic binding factor, which consisted of about 20% carbohydrate and 80% peptide. Further analysis proved that besides the MK-7, the soluble macromolecular complex also contained the 10 kDa peptide and 3 kDa amphiphilic peptides (Chatake et al., 2018). Therefore, if we can activate the secretion of vitamin K2 to stimulate the continuous production of intracellular vitamin K2 during fermentation, the total vitamin K2 production would be greatly improved. Enhancing the membrane permeability by adding the surfactants is helpful to stimulate the product secretion from the cell (Huang et al., 2019; Kang et al., 2013;

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Tamano et al., 2017; Zhang et al., 2014). Surfactants can be divided into polymeric, ionic, non- ionic and edible surfactants, which have different effects on vitamin K2 production. Hu et al. systemically studied the effects of different surfactants on the vitamin K2 secretion and proved that the edible surfactant soybean oil was an effective additive for enhancing MK-7 biosynthesis (Hu et al., 2017). The secretion

f

ratio of MK-7 increased to 61.1% by adding 20 g/L soybean oil during the logarithmic

oo

phase in Bacillus. Additionally, soybean oil also acted as an anti- foaming and

pr

extraction agent enabling 80% of the produced MK-7 to be recovered in situ.

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Berenjian et al. also used this strategy to produce 226 mg/L MK-7 in 5 days of

Pr

fermentation combined with an extreme oxygen supply process (Berenjian et al., 2014b). Another non-ionic surfactant, span 20, could also improve the product yield,

al

whereas amine surfactants such as betaine had no big effect on the secretion of MK-7.

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Small organic molecules can also change the cell membrane permeability, but

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their addition is often associated with cytotoxicity. Nevertheless, periodic addition of n-hexane into the fermentation broth enhanced the total MK-7 production by 1.7 fold (Ranmadugala et al., 2018). Interestingly, research on Flavobacterium, which produces both MK-4 and MK-6, revealed that adding a detergent such as Rikanon VA 5012 could selectively release MK-4 rather than MK-6 into the culture medium (Tani and Taguchi, 1988; Tani and Taguchi, 1989). This strategy was also used in the bioproduction of coenzyme Q10, another essential component of the respiratory chain (Zhong et al., 2009). 5.2.4 New bioreactor design

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Bacillus strains have a propensity to form biofilms in the broth during cultivation, which negatively affects broth viscosity and mass transfer efficiency (Dimitrov et al., 2007; Wahlen et al., 2018; Zune et al., 2017). Therefore, in addition to solid-state and traditional liquid fermentation, many new bioreactors such as biofilm reactors were also developed for vitamin K2 fermentation. Biofilm reactors, also called

f

passive immobilized cell reactors, enable microbial cells to attach themselves to

oo

supports such as lignocellulosic materials, metallic alloys, or plastic composites,

pr

helping to achieve high cell concentrations with the aid of biofilm formation (Cheng

e-

et al., 2010; Ercan and Demirci, 2015). Biofilm reactors have been used to improve

Pr

the production of a wide range of high-value-added products including antibiotics (Pongtharangkul and Demirci, 2006), biopolymers (Jiang et al., 2016), and

al

enzymes (Khiyami et al., 2006) during the past decades, which provided new ideas

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for the process improvement.

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Berenjian et al. firstly studied the relationship between biofilm formation and vitamin K2 production and proposed that they had a linear correlation in static cultures (Berenjian et al., 2013). Then, Mahdinia et al. evaluated the possibility of using a biofilm reactor to produce MK-7 by choosing different media and carrier materials to provide supports for cell attachment and biofilm formation (Mahdinia et al., 2017b). Four different types of plastic composite supports were compared to evaluate their ability to support biofilm growth, favor cell adhesion and offer high mechanical resistance to liquid shear forces. In addition, they also optimized the growth parameters in glycerol- or glucose-based media and tested batch and fed-batch

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fermentation processes in biofilm reactor (Mahdinia et al., 2018a; 2018b; 2018c; 2019a; 2019b), which finally increased the end-product concentration by 2.3 fold. The application of biofilm reactor in vitamin K2 production is still in a preliminary stage, and further improvement of new reactors need to be explored for the increase of menaquinones (Mahdinia et al., 2019c).

f

6 Downstream processes

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In fact, extraction and refining processes often account for more than half of the

pr

production cost of various biochemicals (Xu et al, 2012; Wei et al., 2018). For the

e-

vitamin K2 locating in the membrane, it is usually separated from the culture broth

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through organic solvent extraction after cell disruption. Luo et al compared different cell disruption methods to extract menaquinone-7, and they found acid heating and

al

homogenization were two effective methods to improve the extract efficiency (Luo et

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al., 2016). Then the disrupted cell would be immersed into the extract solvents.

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Usually, an organic mixed solvent composing of 2-propanol and n-hexane (1:2 v:v) was used to extract menaquinone-7 (Mahanama, 2013; Toshiro et al, 2001b; Hu et al., 2017). Similarly, Tsukamoto et al. added different solvents separately, first adding 2-propanol and mixing for 15 min, followed by the addition of hexane and renewed mixing (Tsukamoto et al., 2001). Then, the mixture was dried and dissolved in 2-propanol for HPLC analysis. Wei et al. also tried to extract different menaquinones using a single organic solvent via three successive methanol extractions (Wei et al., 2018). However, the organic solvents have to be completely removed to ensure its safety, which results in complex downstream procedures and high processing costs.

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Therefore, some researchers aimed at eliminating the use of organic solvents and use the soybean oil as the replacement. An in situ recovery process was also proposed for menaquinone-7 production, in which vegetable oil was continuously added as the anti- foaming agent and 80% of menaquinone-7 was extracted in the oil phase (Berenjian et al., 2014a). This process avoided the use of organic solvents and could

f

directly produce the oil rich in menaquinone-7.

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In the industrial vitamin K2 production (Fig. 6), the fermentation broth is treated

pr

with acid and subsequently passed through a microfiltration membrane to remove the

e-

cell debris. Subsequently, the clarified extract with vitamin K2 is concentrated using

Pr

an ultrafiltration membrane. Then, the collected concentrated mixture is extracted using soybean oil to produce the oil rich in vitamin K2. In addition, the vitamin K2

al

powder could be produced by microencapsulation with certain supplementary

rn

materials. Overall, many operations including filtration, extraction, drying and

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sub-packaging are needed in the downstream process (Akiki et al., 2006; Shin et al., 1994). Therefore, downstream processing technology has a great effect on the cost of vitamin K2 production, and

is one of the main problems affecting its

commercialization. 7 Conclusions and perspectives Vitamin K2 is an emerging nutritional supplement that can effectively prevent osteoporosis and cardiovascular disease. Since its discovery in 1929 by Dam and Diosy, research on vitamin K2 has spanned 90 years. B. subtilis natto is the main production strain for menaquinone-7. Early processes for vitamin K2 production were

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based on solid-state fermentation suffered from long cultivation periods and low volumetric productivity. Consequently, liquid fermentation was investigated to effectively increase the cell growth rate and shorten the fermentation period. Subsequently, researchers have increased the yield of vitamin K2 using mutagenesis and screening, optimization of culture conditions, and by promoting product secretion.

f

Some studies also attempted to enhance the vitamin K2 biosynthesis pathway to

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improve the productivity through metabolic engineering. However, a number of

pr

problems still need to be solved, such as the complex metabolic network of vitamin

e-

K2 biosynthesis and the low synthetic yields, which limit its industrial production.

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At present, vitamin K2 has bright market prospects, and many companies have realized the industrialization of vitamin K2 production. Future prospects of microbial

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vitamin K2 production should be discussed in light of the current progress and

rn

challenges. Firstly, the production strain is the soul of fermentation, and different

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strategies to strengthen the production capacity of vitamin K2 production strains need to be developed. Possible approaches include developing new strain mutagenesis strategies based on knowledge of the metabolic pathway, enhancing the biosynthesis pathway or inhibiting competing pathways by metabolic engineering, or stain adaption to improve the cells’ tolerance to environmental factors. Secondly, as an important oxygen regulator in the electron transport chain, menaquinone is closely related to cell respiration and oxygen utilization. It thus may be fruitful to regulate the menaquinone biosynthesis from the perspective of oxygen. In addition, new oxygen supply strategies and new bioreactor designs would also be helpful for improving

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vitamin K2 production and to resolve the foaming problems during liquid fermentation simultaneously. Finally, with the development of synthetic biology, genetic modification tools are becoming available for non- model microorganisms, which can also be used to produce different chemicals. Additionally, reconstructing the biosynthetic pathway of vitamin K2 in different model microorganisms would also

f

be an interesting research direction.

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As an emerging nutritional supplement, the safety of vitamin K2 has been

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recognized by the China Food and Drug Administration, the United States FDA, as

e-

well as the European Parliament and the European Union Council. With the

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deepening understanding of the biological processes affected by vitamin K2, its applications will be more widespread in the future. Microbial production of vitamin

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K2 has a bright future as it has high activity, good safety and is environmentally

rn

friendly. The utilization of more advance biotechnology will further enhance the

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productivity, accelerate the industrialization, and reduce the cost of microbial vitamin K2 production.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (No. 21878151), the Outstanding Youth Foundation of Jiangsu Nature Science Foundation (BK20160092), the Program for Innovative Research Teams in Universities of Jiangsu Province (2015), the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (XTE1829), and General Program on Natural Science Research Project of Higher Education of Jiangsu (18KJB530007).

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Sphingomonas sp ZUTEO3 with a coupled fermentation-extraction process. J. Ind. Microbiol. Biotechnol. 36(5), 687-693. Zune, Q., Telek, S., Calvo, S., Salmon, T., Alchihab, M., Toye, D., Delvigne, F., 2017. Influence of liquid phase hydrodynamics on biofilm formation on structured packing: Optimization of surfactin production from Bacillus amyloliquefaciens. Chem. Eng. Sci. 170, 628-638.

Product Company/Country

Method

Chemical

K2VITAL synthesis Fermentation

Gnosis/Italy

Fermentation

https://www.kappabio.com

MenaQ7

http://www.nattopharma.com

VitaMK7

http://www.gnosis-bio.com

al

NattoPharma/Norway

e-

Bioscience/Japan

Pr

Kappa

Website

pr

trademark

oo

f

Table 1. Vitamin K2 manufacturers and information

DSM/Netherlands

rn

Chemical

Quali-K

https://www.dsm.com/corporate/home.html

Fermentation

MenaquinGold

http://www.viridisbiopharma.com

Fermentation

UniK2

https://www.iff.com/en/taste/frutarom

ActivK

https://www.dupontnutritionandhealth.com

Fermentation

NattoMena

https://geneferm.en.taiwantrade.com/#

Fermentation

NattoK2

http://www.sungenbio.com/

MK-4

https://www.eisai.com/index.html

Viridis

Jo u

synthesis

Biopharma/India Frutarom/Japan

DuPont Nutrition & Health/USA GeneFerm Biotechnology/China Sungen/China

Chemical synthesis

Chemical Eisai Co. Ltd/Japan synthesis

Journal Pre-proof

Gusheng/China

Chemical

MK-4

synthesis

http://www.goodscend.com/vk2

Table 2. Different enzymes in the biosynthesis of menaquinone-7 Molecular Mass (kDa) Gene

Enzyme

Enzyme name

No.

Natur

Microorganis

Referenc

Subdoma

al

m

e

in

protei n

5.4.4.2 EC

SEPHCHC synthase

2.2.1.9 EC

menC

4.2.99.2

28

e-

SHCHC synthase

o-succinylbenzoate synthase

f

61

98

180

E. coli

1996) E. coli

menA

-

E. coli

et al., 2013) (Sharma

4.2.1.11

e A synthetase

6.2.1.26

al

EC

rn

al., 2007)

0

o-succinylbenzoyl-coenzym

1,4-dihydroxy-2-naphthoyl-

EC

CoA synthase

4.1.3.36

1,4-dihydroxy-2-naphthoate

EC

polyprenyltransferase

2.5.1.74

Jo u

menB

(Jiang et (Johnston

39

77

E. coli

3

menE

a et al.,

EC

Pr

menH

48

oo

menD

Isochorismate synthase

pr

menF

(Daruwal

EC

et al., 1993) (Sharma

49

185

E. coli

et al., 1996)

32

112

M. tuberculosis

(Truglio et al., 2003) (Shineber

-

-

E. coli

g and Young, 1976)

ubiE/men

demethylmenaquinone

G

methyltransferase

EC 2.1.1.16

(Monzin 17.4

-

3

Chorismate dehydratase

4.2.1.15

-

30.5

1 mqnB

mqnC

Futalosine hydrolase

EC 3.2.2.26

Cyclic dehypoxanthine

EC

futalosine synthase

1.21.98.

go et al., 2003)

EC mqnA

E. coli

-

-

23.8

44.7

Thermus thermophilus Thermus thermophilus

(Mahanta et al., 2013) (Hiratsuk a et al., 2009)

Streptomyces

(Cooper

coelicolor

et al.,

Journal Pre-proof 1 mqnD

2013)

1,4-dihydroxy-6-naphtoate

EC

synthase

4.1.-.-

30.0

Thermus

(Arai et

thermophilus

al., 2009)

EC mqnE

Aminofutalosine synthase

2.5.1.12

-

42.6

0 mqnP

Prenyltransferases

Prenyltransferases

-

-

31.0

-

31.0

thermophilus

2013) (Cotrim

Thermus thermophilus Streptomyces lividans

et al.,

et al., 2017) (Cotrim et al., 2017)

f

mqnP

-

(Mahanta

Thermus

Fermentation

Strain

mode

Substrate

Detail

R

pr

Strategy

oo

Table 3. Summary of the literature on the production of vitamin K2

structural analogs (HNA)

Flavobacterium meningosepticum

Mutation by

Flavobacterium

(HNMaize meal

sp.

Sulfonamide-resistant

Flavobacterium

mutation

rn

al

structural analogs hydrolysateA)

sp.

mutant

Bacillus subtilis

Jo u

Menadione-resistant

ARTP mutation and analog resistant

(HNA, β-TA, DPA) Analog resistant (HNA, pFP, mFP, β-TA) Mutation by NTG and low energy ion beam

Liquid

Pr

Mutation by

e-

Strain mutagenesis

Bacillus

amyloliquefaciens

Liquid

Liquid

Liquid

Glycerol

Polypepton

Glycerol Polypepton

1-hydroxy-2-naphthoate (HNA)

Glycerol

Derivation of sulfonamides

Peptone Soybean extract

Menadione

Glycerol Soymeal extract

Liquid

1-hydroxy-2-naphthoate (HNA)

Yeast extract

1-hydroxy-2-naphthoate (HNA), β-2-thienylalanine (β-TA),

Maize meal

Diphenylamine (DPA)

hydrolysate

Bacillus subtilis natto

Bacillus subtilis natto

Solid

Liquid

Meat extract Polypeptone

HNA,

βTA,

5

m

1

m

m

3

m

6

m

p-fluoro-D,L-

3

phenylalanine (pFP), m-fluoro- D,L-

μg/

phenylalanine (mFP),

Glycerol

N-methyl-N-nitro-N-n itroso-guanidine

Yeast extract

(NTG), low energy ion beam

Peptone

implantation

n

2

m

Genetic modification Blocking the ubiquinone biosynthesis pathway Enhancing the

Elizabethkingia meningoseptica Escherichia coli

Liquid

Glycerol-peptone

Site-directed mutagenesis of UbiA

medium Liquid

Yeast extract

Enhancing MVA pathway;

Inc

by

Iso

Journal Pre-proof biosynthesis of

Tryptone

knocked out acetate-producing genes

inc

isoprene

Glycerol

et al.

by

Glycerol

Mutagenesis of UbiA, overexpression

2

Peptone

of dxr, menA and ubiE, and

m

Yeast extract

supplementation with precursors

D

Glycerol

Overexpression of menA, dxs, dxr,

Soy peptone

yacM-yacN, glpD and deletion of

Yeast extract

dhbB

Weakening the ubiquinone pathway, co-expressing genes

Elizabethkingia meningoseptica

Liquid

in MK pathway Overexpressing the genes for MK-7 biosynthesis and reducing the use of

Liquid

Bacillus subtilis

intermediate

6

m

genes for MK-7 biosynthesis

Glycerol

Bacillus

Liquid

amyloliquefaciens

biosynthesis

Bacillus

cultivation conditions

amyloliquefaciens

rn

Bacillus subtilis natto

Jo u

approach

al

Optimizing the

methodology

Plackett-Burman

design; response

surface methodology

Bacillus subtilis natto

Bacillus subtilis

addition

natto

aeration rates fermentation

Medium optimization

menD, menE, menH and hepS

Liquid

Liquid

Tryptone

Deletion of ubiCA genes,

Yeast extract

Overexpression of menA and menD

273

D

29

M

D

Process optimization Studying different fermented soybean

1

food, optimized the temperature and

µ

carbon sources

D

Glycerol

Studying the effect of carbon source

6

Soy peptone

and nitrogen sources

m

Soybean extract

Glycerol Liquid

Sucrose Peptone

37 °C, 150 rpm, 72 h

3

m

Yeast extract

Fed-batch glycerol

High stirring and

Liquid

Pr

gene for MK-8

Response surface

Overexpression of menA, menC,

pr Escherichia coli

e-

formation of Overexpressing the

Yeast extract Soy peptone

Blocking the ubiquinone-8,

oo

Overexpressing the

f

metabolites

Bacillus subtilis natto

Bacillus subtilis natto

Yeast extract Liquid

Glycerol

Adding 2% glycerol to the medium at

8

48h

m

Soy peptone Yeast extract, Liquid

Glycerol

1,000 rpm, 5 vvm, 40 °C, 5 days

Soy peptone Soybean Solid

Glycerol Yeast extract

Optimizing pH, temperature, studying the effect of glycerol, mannitol, malt extract, yeast extract

Increasing vitamin K2 secretion

and CaCl2

m

3

μ

n

Journal Pre-proof

M Adding nonionic

Flavobacterium

detergent

sp.

Glycerol

Liquid

Polyoxyethylene oleyl ether

Peptone

18

M

11

MK Adding different surfactants

Glycerol Escherichia sp.

Liquid

Peptone

Betaine, polyoxyethylene oleyl ether, tween-80

Yeast extract

4

m

MK

6.0 Adding different

Bacillus subtilis

surfactants

natto

Glycerol

Liquid

Adding 20 g/L soybean oil

Soy peptone

Adding

4

m

Inc

organic solvents Flavobacterium

detergent

sp.

Liquid

Adding n-hexane

Glycerol

Polypepton

b

f Adding polyoxyethylene oleyl ether

39

pr

Addition nonionic

Glycerol

f

Liquid

Bacillus subtilis

oo

biocompatible

Bacillus subtilis

supports

natto

Plastic composite

Bacillus subtilis

supports

natto

Plastic composite

Bacillus subtilis

al

rn

supports

Liquid

Pr

Plastic composite

e-

New bioreactor design

natto

Bacillus subtilis

supports

natto

Jo u

Plastic composite

Liquid

Soytone

Yeast extract Glycerol Glycerol Yeast extract Soytone Glycrol

Liquid

Yeast extract

Glucose

fashion PCS formations

m

Biofilm reactors with fed-batch

2

fermentation

m

parameters

Soybean flour Yeast extract

1

Biofilm reactors with optimum growth

Soytone Liquid

Bioreactors are set up with grid-like

1

m

Selecting different

3

strains and plastic composite supports

m

Fig. 1. Different types of vitamin K Fig. 2. Different functions of vitamin K2 Fig. 3. The classic metabolic pathway of menaquinone-7 biosynthesis. Enzymes: GlpK, glycerol kinase; Dxs, 1-deoxyxylulose-5-phosphate synthase; Dxr, 1-deoxyxylulose-5-phosphate reductoisomerase; YqfP, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; YqiD, farnesyl diphosphate synthase; HepS/HepT, heptaprenyl

Journal Pre-proof

diphosphate

synthase;

MenF,

isochorismate

synthase;

MenD,

2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase; MenH, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate

synthase;

MenC,

o-succinylbenzoate synthase; MenE, o-succinylbenzoic acid-CoA ligase; MenB, 1,4-dihydroxy-2-naphthoyl-CoA

synthase;

MenA,

1,4-dihydroxy-2-naphthoate

f

heptaprenyltransferase; MenG, demethylmenaquinone methyltransferase;

HMB4PP:

1-Hydroxy-2-Methyl-2-(E)-Butenyl-4-Pyrophosphate,

pr

Pyrophosphate;

oo

Substrates: MEP: 2-C-Methyl-D-Erythritol-4-Phosphate; DMAPP: Dimethylallyl

e-

GPP: Geranyl Diphosphate; HPP: Heptaprenyl Diphosphate

Pr

Fig. 4. The alternative futalosine pathway for menaquinone biosynthesis. Enzymes: MqnA: futalosine synthase; MqnB: futalosine hydrolase; MqnC: dehypoxanthinyl

al

futalosine cyclase; MqnD: 1,4-dihydroxy-6-naphthoate synthase; MqnP: not identified,

rn

catalyzing the late step for menaquinone biosynthesis.

Jo u

Fig. 5. Key gene clusters of menaquinone biosynthesis in different strains Fig.6. Extraction process for the production of the vitamin K2 products