## Non-Isoprenoid Secondary Metabolite Production from Methane Using Methanotrophs
### Ectoine  
Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) is a substance produced by microorganisms to withstand osmotic stress in a high salt environment. It prevents water loss, regulates cell collapse, and does not interfere with cell metabolism. Ectoine can be used for cosmetic and medical purposes. At present, ectoine has a demand of 15,000 tonnes per year, the market price is $1000 USD per kg, and it is commercially produced using _Halomonas elongate_[61].  
The ectoine biosynthetic pathways have been investigated in a variety of halophilic bacteria. First, the aspartate commonly used in the synthesis of lysine, threonine and methionine is phosphorylated by aspartokinase (_ask_) to synthesize aspartyl phosphate. It is then converted to aspartate semialdehyde by aspartate semialdehyde dehydrogenase, which is used as a precursor to the ectoine synthesis pathway. Ectoine is synthesized by three enzymes, diaminobutyric acid transaminase (EtCh), diaminobutyric acid acetyltransferase (EtA), and ectoine synthase (EtCtC). The synthesis of ectoine starts with conversion of aspartate semialdehyde to 1-2,4-diaminobutyric acid (DABA) by EtCh. 1-2,4-diaminobutyric acid (DABA) is converted to acetyl-1-2,4-diaminobutyric acid by EtCh. Acetyl-1-2,4-diaminobutyric acid is synthesized as ectoine by EtCh. The genes coding this synthetic pathway have been studied in several halophilic bacteria and have been named _ectABC_, which is an ectoine synthesis cluster [62].  
Studies on the synthesis route of ectoine were also conducted for methanotrophs. _M. alcaliphlum_ 20Z, which is known to be a halophilic methanotroph, has an ectoine synthesis pathway. This pathway includes _ask_, 1-2,4-diaminobutyric acid transaminase (_ectB_), L-2,4-diaminobutyric acid acetyltransferase (_ectA_) and 1-ectoine synthase (_ectC_) (Figure 3), and it exists as one operon. Studies have been conducted to find factors that regulate the ectoine biosynthesis of _M. alcaliphlum_ 20Z. As a result, the promoters _ectAp1_ and _ectAp2_ of the _ectABAask_ operon were identified. This is similar to the \(\sigma^{70}\)-dependent promoters of _E. coli_. In addition, a MarR-like transcriptional regulator, EtR1, was identified above the gene cluster, which acts as a negative regulator of the _ectABCask_ operon. This confirmed that the purified EtCh1 specifically binds to the _ectAp1_ promoter [63; 64].  
Studies on the production of ectoine through _M. alcaliphlum_ 20Z are also underway. According to [PERSON] et al., the production of ectoine by _M. alcaliphlum_ 20Z in stirred tank reactors (non-sterile conditions) varies depending on the NaCl concentration and the stirring rates [49]. When the concentration of NaCl in the medium increased from 3% to 6%, the intracellular ectoine content increased to 37.4 mg/g cell from 16.5 mg/g cell (Table 2). A high agitation rate stresses the cells and lowers the yield of ectoine. In addition, when the concentration of copper ion in the medium was increased from 0.05 mM to 25 mM, the amount of ectoine released from the cell increased by about 20%, although it did not affect the production of the intracellular ectoine [49].  
A bio-milking process for the commercial production of ectoine was used to increase the production of ectoine from _M. alcaliphlum_ 20Z. This is a method of continuously producing ectoine by sequentially in the culture medium. _M. alcaliphlum_ 20Z was cultured in 6% NaCl and then alternately cultured in 0% controlling the NaCl concentration NaCl. As a result, it was observed that _M. alcaliphlum_ 20Z released the accumulated ectoine at a low osmotic pressure and absorbed the released ectoine at high osmotic pressure. It was confirmed that the ectoine concentration in the cell was maintained at 70.4 mg/g cell in 6% NaCl and that 70% of the ectoine was released in 0% NaCl (Table 2). This shows that it is possible to continuously produce ectoine from methane using methanotrophs [50].