Enzymatic and Molecular Genetic Studies of Serine Production by C_1-Microorganisms

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L-Serine-degrading activity could be suppressed by controlling the methanol concentration in L-serine synthesis using Hyphomicrobium methylovorum sp. NCIB10099 cells. Fifty-three mg/ml of L-serine were produced from 100 mg/ml of glycine and 104 mg/ml of methanol. It is believed that L-serine-degrading activity contributes to the reverse serine hydroxymethyltransferase reaction.CoASAc-independent phosphoenolpyruvate carboxylase (PEPC) was purified from a serine-producing methylotroph, H.methylovorum GM2, and compared with those from other methylotrophs. The activity of this enzyme was not affected by CoASAc, a well-known activator for enzymes from various heterotrophs, NADH and ADP,effectors for PEPC from methylamine-grown Pseudomonas MA.This suggested that the H.methylovorum enzyme should be classified into the second group. and different from that of Pseudomonas MA.The gene encoding hydroxypyruvate reductase (HPR) and its flanking regions were isolated from H.methylovorum GM2. Nucleotide sequence of the recombinant plasmids revealed that HPR gene codes the 322-amino-acid protein with calculated molecular mass 35,726Da. The amino acid sequence of the enzyme showed similarity to members of the D-isomer-specific 2-hydroxyacid dehydrogenase family. The recombinant plasmid was introduced into Escherichia coli HB101. The recombinant enzyme purified from the transformed E.coli cells was indistinguishable from the enzyme isolated from H.methylovorum GM2 by immnunological and enzymological analysis.