Microbial production of l-ascorbic acid

Abstract

The present invention discloses an isolated polynucleotide molecule derived from a polynucleotide encoding a polypeptide having L-sorbosone dehydrogenase activity comprising a partial nucleotide sequence of at least 20 consecutive nucleotides of SEQ ID NO:1. The present invention further relates to a process for the production of L-ascorbic acid in high yield, in particular a process using resting cells of a microorganism able to convert given carbon sources into vitamin C. The thus obtained vitamin C may be further processed by purification and/or separation steps.

1 - 42 . (canceled) 
     
     
         43 . A process for the production of L-ascorbic acid comprising:
 (a) culturing microorganism under conditions which enable growth;   (b) changing the conditions such that the growth rate of the microorganism is reduced, leading to resting cells; and   (c) producing L-ascorbic acid from a substrate using the resting cells produced in step (b),   wherein the substrate is selected from the group consisting of D-glucose, D-sorbitol, L-sorbose, L-sorbosone, 2-keto-L-gulonate, D-gluconate, 2-keto-D-gluconate and 2,5-diketo-gluconate.   
     
     
         44 . The process of  claim 43 , wherein the steps of culturing and producing L-ascorbic acid are performed in 2 or more separate vessels. 
     
     
         45 . The process of  claim 43 , wherein the steps of culturing and producing L-ascorbic acid are not separated by any step of washing, any step of isolating, or both. 
     
     
         46 . The process of  claim 43 , wherein the step of producing L-ascorbic acid is performed in batch, fed-batch, continuous, or a semi-continuous mode. 
     
     
         47 . The process of  claim 43 , wherein the microorganism is grown in batch, fed-batch, continuous, or a semi-continuous mode. 
     
     
         48 . The process of  claim 43 , wherein the density of the resting cells in the medium measured as OD at 600 nm is at least 10. 
     
     
         49 . The process of  claim 43 , wherein the yield of produced L-ascorbic acid is at least 1.8 g/l. 
     
     
         50 . The process of  claim 43 , wherein the microorganism is selected from the group consisting of yeast, algae, and bacteria. 
     
     
         51 . The process of  claim 43 , wherein the microorganism is selected from the group consisting of  Candida, Saccharomyces, Zygosaccharomyces, Scyzosaccharomyces, Kluyveromyces, Chlorella, Gluconobacter, Acetobacter aceti, Pantoea, Cryptococcus, Pseudomonas  and  Escherichia.    
     
     
         52 . The process of  claim 43 , wherein the process uses a microorganism capable of producing both L-ascorbic acid and 2-keto-L-gulonic acid from a substrate and wherein the ratio between the concentration of the L-ascorbic acid and the 2-keto-L-gulonic acid is more than 0.1. 
     
     
         53 . The process of  claim 43 , further comprising:
 separating L-ascorbic acid from components in the medium using electrodialysis.   
     
     
         54 . The process of  claim 43 , further comprising:
 isolating L-ascorbic acid from the medium; and optionally, performing one or more additional purification steps.